ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group

Transcription

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Physiology/Pharmacology
105 Inflammation and Drugs
Sunday, May 05, 2013 8:30 AM-10:15 AM
Exhibit Hall Poster Session
Program #/Board # Range: 99-125/C0104-C0130
Organizing Section: Physiology/Pharmacology
Program Number: 99 Poster Board Number: C0104
Presentation Time: 8:30 AM - 10:15 AM
Zylet (loteprednol etabonate 0.5%/tobramycin 0.3%) Compared
with Tobradex (dexamethasone 0.1%/tobramycin 0.3%) for the
Treatment of Lid Inflammation in Two Populations
Timothy L. Comstock1, Heleen H. DeCory1, Kirk Bateman2. 1Medical
Affairs, Global Pharmaceuticals, Bausch & Lomb Incorporated,
Rochester, NY; 2Statistics, Bausch & Lomb Incorporated, Rochester,
NY.
Purpose: To compare loteprednol etabonate 0.5%/tobramycin 0.3%
(LE/T) and dexamethasone 0.1%/tobramycin 0.3% (DM/T)
ophthalmic suspensions for the treatment of lid inflammation across
two patient populations.
Methods: Two multicenter, randomized, investigator-masked,
parallel-group clinical trials with similar designs were conducted. In
both studies patients aged ≥18 years with
blepharokeratoconjunctivitis in at least one eye received LE/T or
DM/T administered QID for 2 weeks. The first study was conducted
at clinical centers in the US; the second study at clinical centers in
China. Signs and symptoms were assessed at baseline and on days 3,
8, and 15. The primary endpoint in this analysis was the change from
baseline (CFB) to day 15 in composite blepharitis severity (lid
hyperemia, lid scaling or crusting, and lid margin hypertrophy, each
measured on a 5-pt scale). Safety outcomes included an assessment
of AEs, and changes in visual acuity (VA), biomicroscopy, and
intraocular pressure (IOP).
Results: A total of 273 patients (136 LE/T, 137 DM/T) and 354
patients (178 LE/T, 176 DM/T) were included in the ITT population
in the US and China studies, respectively. Patients in the US study
were primarily White (>80%); all patients in the China study were
Chinese. A significant CFB in blepharitis severity was seen in both
populations with both treatments at each follow-up visit. At day 15,
the reduction in composite blepharitis severity was 68.8% for LE/T
vs. 66.7% for DM/T in the US study and 75.0% for LE/T vs. 77.4%
for DM/T in the China study. Patients treated with DM/T experienced
a significant increase in IOP compared to those patients treated with
LE/T beginning at day 8 (US study) or Day 3 (China study)
(P≥0.0339). In both studies twice as many patients in the DM/T
group experienced IOP increases of ≥ 5 mm Hg over baseline
compared to the LE/T group. In the US study, one patient (DM/T
group), while in the China study, 19 patients (6 LE/T; 13 DM/T)
experienced an IOP increase of ≥10 mm Hg over baseline.
Conclusions: LE/T was effective in the treatment of lid inflammation
with similar results compared to DM/T in both a primarily White
population and a Chinese population. LE/T had a better safety profile
with respect to change in IOP especially in Chinese patients
considered at higher risk for steroid-induced IOP.
Commercial Relationships: Timothy L. Comstock, Bausch &
Lomb Incorporated (E); Heleen H. DeCory, Bausch + Lomb (E);
Kirk Bateman, Bausch + Lomb (E)
Clinical Trial: NCT01028027, NCT00447577
Inflammatory processes in branch retinal vein occlusion in mice
Elisa Dominguez1, 3, Sophie Cavallero1, 3, William Raoul1, 3, Sophie
Lavalette1, 3, Michel Paques1, 2, Florian Sennlaub1, 3. 1Institut de la
Vision - INSERM U 968, Paris, France; 2CHNO des Quinze-Vingts,
Paris, France; 3UPMC UMRS 968, Paris, France.
Purpose: Retinal vein occlusion (RVO) is an important cause of
blindness because of the subsequent microvascular remodelling that
may lead to a variable combination of capillary non-perfusion and
macular edema. To date, the inflammatory processes that accompany
BRVO and its role in the microvascular remodelling have received
little attention. Here, we analyzed the involvement of inflammation in
a model of laser-induced BRVO in mice.
Methods: A complete and permanent occlusion of a retinal vein of
C57Bl/6J Rj mice was obtained using 534nm laser. We followed in
vivo the development of tissue changes by funduscopy, scanning
laser ophthalmoscopy and optical coherence tomography. At different
time points after RVO, animals were sacrificed and the expression of
various genes were analyzed by real-time RT-PCR. Iba1
immunostaining was performed on the whole retina at 3 and 7 days
after RVO to evaluate macrophage/microglia responses. To quantify
cell proliferation, mice were intraperitoneally injected with the
nucleotide analog EdU and killed at 3d and 7d after RVO.
Results: Permanent occlusion of a retinal vein was obtained in all
treated eyes with minimal damage to surrounding tissue.
Subsequently, dilation of the affected vein was observed. Few retinal
hemorrhages were observed at 24h which slightly increased at day 3.
Retinal mRNA expression of the chemokine CCL2 significantly
increased 4h after RVO. The macrophage/microglia markers F4/80
and cd11b increased at 3 days. Macrophage/microglia recruitment
was confirmed at 3d by increased Iba1 positive cells throughout the
area of the occluded vein and persisted at 7d after RVO. Iba1-positive
perivascular macrophages were also more numerous around occluded
veins up to the periphery than intact veins in the same eye or in
control eyes. The macrophage/microglia response was still increased
at 7d after RVO but at lower levels. Seven days after RVO,
proliferation occurred mainly in endothelial cells of capillaries
upstream of the occluded vein.
Conclusions: Our data shows that BRVO induces CCL2 expression
and perivascular and retinal macrophage recruitement concomittantly
with retinal hemorraghe and endothelial cell proliferation distal to the
laser site. The BRVO mouse model will allow us to study the role of
inflammatory processes in vascular leakage and endothelial cell
proliferation.
Commercial Relationships: Elisa Dominguez, None; Sophie
Cavallero, None; William Raoul, None; Sophie Lavalette, None;
Michel Paques, MerckSerono (C), Roche (C), Sanofi (C); Florian
Sennlaub, None
IL-12p35 Inhibits Th1 proliferation and promotes the expansion
of Th17 and Treg
Ivy M. Dambuza, Chengrong Yu, Rashid M. Mahdi, Renxi Wang,
Sung-Hye Kim, Charles E. Egwuagu. Laboratory of Immunology,
NEI, Bethesda, MD.
Purpose: The IL-12 family cytokines have emerged as an important
regulator of host immunity and in particular T-helper lineage
commitment. It is comprised of IL-12, IL-23, IL-27, IL-35 and each
member consists of an α (p19, p28, p35) and a β (p40, Ebi3) subunit.
Although the bioactivities of each heterodimer cytokine are well
characterized, little is known about functions of the independent α
and β subunits. In this study we addressed the effects of IL-12p35
subunit in T and B cell activation and differentiation.
Methods: Full-length mouse IL-12p35 was cloned and expressed in
High Five insect cells. The secreted recombinant IL-12p35 (rIL12p35) was purified on His-Trap columns and size exclusion HPLC
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
chromatography; characterized by non-reducing PAGE and western
blotting. Naïve CD4+ T cells were cultured under Th0, Th1, Th2,
Th17, or Treg polarizing condition in presence or absence of rIL12p35. CD19+ B cells were sorted and activated with LPS in the
presence or absence of rIL-12p35. Proliferation and effector
functions of T or B cells were analyzed by [3H]-thymidine
incorporation assay, FACS and ELISA.
Results: Two forms of rIL-12p35 were generated, a 35 kDa
monomeric rIL-12p35 and a 70 kDa rIL-12p35 homodimer. Both
forms were immuno-reactive to an IL-12p35-specific antibody on
Western blot gels. Both rIL-12p35 forms inhibited proliferation of
TCR-activated CD4 T cells and LPS-activated CD19+ B cells, with
rIL-12p35 monomer displaying more suppressive activity. Their
suppressive activity correlated with up-regulation of IL-10 secretion
in the culture supernatants. Interestingly, while rIL-12p35 suppressed
proliferation of Th1 cells, it promoted the expansion of Th17 and
Treg with no effect on Th2 cells.
Conclusions: Data presented here show that rIL-12p35 has biological
activities that extend beyond its role as a component of the
heterodimeric cytokine, IL-12 or IL-35. The ability of rIL-12p35 to
suppress proliferation of activated T and B cells and induce
lymphocytes to secrete the anti-inflammatory cytokine, IL-10, makes
it an attractive therapeutic candidate for use in restraining the
excessive inflammatory responses that occur during autoimmune
diseases, such as uveitis.
Commercial Relationships: Ivy M. Dambuza, None; Chengrong
Yu, None; Rashid M. Mahdi, None; Renxi Wang, None; Sung-Hye
Kim, None; Charles E. Egwuagu, None
Anti-inflammatory Properties of Resveratrol Improve the
Treatment of Diabetic Retinopathy
Hamid Ahmadieh1, Farhad Ghadiri Soufi2, Daryoush MohammadNejad3, Ali Hafezi-Moghadam4. 1Ophthalmology, Ophthal Rsch
Ctr,Shahid Beheshti Univer. Med. Sci., Tehran, Islamic Republic of
Iran; 2Physiology, Faculty of Medicine, Hormozgan University of
Medical Sciences, Bandar-Abbas, Islamic Republic of Iran;
3
Histology, Faculty of Medicine,Tabriz University of Medical
Sciences, Tabriz, Islamic Republic of Iran; 4Radiology, Brigham and
Women's Hospital, Harvard Medical School, Boston, MA.
Purpose: Resveratrol (trans-3, 5, 4'-trihydroxystilbene) is a
nutritional supplement with anti-inflammatory properties. This work
investigates the impact of orally administered resveratrol on
inflammatory processes in streptozotocin-nicotinamid-induced
diabetic retinopathy.
Methods: Male Wistar rats were divided into four groups: normal
control, diabetic control, normal rats treated with resveratrol, and
diabetic rats treated with resveratrol. Diabetes was induced by
injection of streptozotocin (50 mg/kg; i.p.), 15 min after the
prescription of nicotinamide (110 mg/kg; i.p.) in 12 h fasted rats.
Results: Oral resveratrol administration (5 mg/kg/day for 4 months)
significantly improved glucose tolerance and alleviated
hyperglycemia and weight loss in diabetic rats. Resveratrol
administration significantly improved the elevated levels of nuclear
factor kappa B (activity and mRNA), tumor necrosis factor alpha and
apoptotic cells in the retinas of the diabetic rats. Resveratrol treated
animals showed significantly less disarrangement and reduction in
thickness of retinal layers. Compared to control, resveratrol did not
affect plasma insulin levels or retinal cyclooxygenase-2 activity.
Conclusions: Long-term resveratrol administration has beneficial
anti-inflammatory properties. Resveratrol could prove to be an
effective therapeutic supplement in the prevention and treatment of
diabetic retinopathy.
Commercial Relationships: Hamid Ahmadieh, None; Farhad
Ghadiri Soufi, None; Daryoush Mohammad-Nejad, None; Ali
Hafezi-Moghadam, None
Secretion of IL-1α, IL-1β, IL-6 and IL-8 in human keratocyte cell
cultures following photodynamic inactivation (PDI)
Berthold Seitz1, Tanja Stachon1, Jiong Wang1, 2, Nora Szentmáry1.
1
Department of Ophthalmology, Saarland University Medical Center,
Homburg/Saar, Germany; 2Department of Ophthalmology, Renmin
Hospital of Wuhan University, Wuhan, China.
Purpose: Purpose: With the increasing resistance of microorganisms
to antibiotics, photodynamic inactivation (PDI) may be an alternative
treatment option of infectious keratitis. In previous studies we
analyzed viability, apoptosis, proliferation, CD34 and alpha-smooth
actin expression of human keratocyte cell cultures after PDI using the
photosensitizer chlorin e6 (Ce6). The purpose of this study was to
determine the secretion of various interleukins by human keratocytes
following PDI, in vitro.
Methods: Methods: Primary human keratocytes were isolated by
digestion in collagenase A (1 mg/ml) from human corneal buttons,
and cultured in DMEM/Ham’s F12 medium supplemented with 10%
FCS. Using 100 nM Ce6, twenty-four hours following illumination
for 13 minutes (670 nm) the release of IL-1α, IL-1β, IL-6 and IL-8
was determined using enzyme-linked immunosorbent assay (ELISA).
Results: Results: Twenty-four hours after PDI, IL-1α (0 to 0 pg/µg
protein), IL-1β (0.19 to 0.21 pg/µg protein), IL-6 (1.17 to 1.18 pg/µg
protein) and IL-8 (6.04 to 4.59 pg/µg protein) secretion did not differ
significantly from untreated controls (p>0.05).
Conclusions: Conclusions: Photodynamic inactivation does not have
an impact on secretion of IL-1α, IL-1β, IL-6 and IL-8, twenty-four
hours after the treatment.
Commercial Relationships: Berthold Seitz, None; Tanja Stachon,
None; Jiong Wang, None; Nora Szentmáry, None
Comparison of retinal and cerebral inflammatory responses after
middle cerebral artery occlusion
Rachael S. Allen2, 3, Iqbal Sayeed1, Heather A. Cale1, Katherine C.
Morrison1, Paul H. Choi1, Jeffrey H. Boatright2, Machelle T.
Pardue2, 3, Donald G. Stein1. 1Emergency Medicine, Emory
University, Atlanta, GA; 2Ophthalmology, Emory University,
Atlanta, GA; 3Rehab R&D Center of Excellence, Atlanta VA
Medical Center, Decatur, GA.
Purpose: Ocular ischemia is often observed in cases of cerebral
stroke and atherosclerosis of the ophthalmic or carotid arteries. In
both diseases, visual function deficits are often a first symptom.
Examining both retina and cerebral responses to disease provides an
opportunity to expand our knowledge of mechanisms of disease, to
translate treatments from one tissue to the other, and to optimize
treatments effective for both tissues. Thus, we are investigating the
common mechanism of inflammation in the middle cerebral artery
occlusion (MCAO) model, a stroke model that affects both the brain
and eye.
Methods: MCAO surgery was performed on male Sprague-Dawley
rats. Under isoflurane anesthesia, a filament was inserted through an
incision in the external carotid artery and slid into the internal carotid
artery so that it blocked the right middle cerebral and ophthalmic
arteries. The filament was removed after 120 minutes to allow
reperfusion. For biomarker assays, MCAO rats were sacrificed at 6,
24, and 48 hours (n = 5/group). Western blots were performed and
densitometry was used to quantify levels of inflammatory proteins
(phosphorylated NF-kB, which indicates NF-kB pathway activity,
and IL-6, a downstream inflammatory cytokine) in the brain and
retina in MCAO and sham animals.
Results: A significant increase (41%) in levels of phosphorylated
NF-kB was observed in MCAO brains (2008.83 ± 241.89 a.u.)
compared with shams (1427.15 ± 148.76 a.u., p < 0.05). Significant
increases (69%) were observed in MCAO retinas (4940.58 a.u. ±
438.81) compared with shams as well (2924.06 ± 378.88 a.u., p <
0.05), however, we did not find a significant correlation between
brain and retina levels. We also observed a trend towards increased
levels of IL-6 in MCAO retinas (58%) and brains (45%, p = 0.051).
Conclusions: MCAO surgery increases inflammation in the retina as
it does in the brain. However, the fact that retina and brain increases
are not correlated may indicate a difference in the timing of the
response or the responsiveness of the tissue. The retina represents an
additional avenue that can be explored when using the MCAO model
to investigate stroke and its potential treatments.
Commercial Relationships: Rachael S. Allen, None; Iqbal Sayeed,
None; Heather A. Cale, None; Katherine C. Morrison, None; Paul
H. Choi, None; Jeffrey H. Boatright, None; Machelle T. Pardue,
None; Donald G. Stein, BHR Pharma (C)
Support: H. Allen and Company, Atlanta VA Rehab R&D Service
of the Department of Veterans Affairs, The Abraham J. and Phyllis
Katz Foundation, Foundation Fighting Blindness, Research to
Prevent Blindness, NIH NEI R01EY014026, R01EY016470,
R24EY017045, P30EY006360, and T32EY007092-25
Efficacy of 0.5% Indomethacin Eyedrops in Uveitic Macular
Edema: a Randomized, Double-blind, Placebo-controlled Clinical
Trial
Monica Zurria1, Pia Allegri2, Simona Peri3, Rosanna Carniglia De
Carli3, Maria Grazia Crivelli3, Silvia Compiano2, Silvia Autuori2,
Antonio Mastromarino2, Ugo Murialdo2. 1Medical Dept, ALFA
INTES Ind Ter Spl Srl, Casoria, Italy; 2Ophthalmology Dept, Uveitis
Tertiary Referral Center, Rapallo Hospital, Genova, Italy;
3
Pharmaceutical Dept, Sestri Levante Hospital, Genova, Italy.
Purpose: Cystoid macular edema as a secondary complication of
different etiology uveitis, mainly intermediate and pan-uveitis, is one
of the most important causes of visual impairment in developed
countries. Systemic and topical NSAIDs showed efficacy in the
prophylaxis and treatment of pseudophakic cystoid macular edema.
The aim of the present randomized, double-blind, placebo-controlled
clinical trial was to assess the efficacy and tolerability of 0.5%
indomethacin eyedrops in adult patients affected by uveitic cystoid
macular edema.
Methods: 46 eyes of 31 patients (20 females and 11 males), mean
age 39y (range 24-78) affected by inflammatory refractory cystoid
macular edema, were randomized to receive 0.5% indomethacin
eyedrops (INDOM®, Alfa Intes, Italy) QID (16 subjects) or placebo
(saline solution, 15 subjects) continuously during a six-month period.
Visits were at baseline and at 1, 3 and 6 months. Primary endpoint
was central foveal thickness reduction (Heidelberg Spectralis II
OCT). Secondary endpoints were visual acuity, subjective symptoms
(itching, burning, photofobia and watering, 0-3 points score) and
central volume OCT measurement.
Results: After 6 months, central foveal thickness showed a
significant decrease in treated patients by an average of 150 μ
(p<0.0001, 180days vs baseline) compared to the placebo group in
which no significant improvement of edema was seen (p=1, 180days
vs baseline). More than 50% of eyes in the placebo group got worse
after 180 days, compared to less than 10% in the treated group. Also
visual acuity was significantly improved in treated eyes (p<0.0001,
180days vs baseline) and after 6 months there was a significant
correlation between retinal thickness decrease and visual acuity
increase (R=-0.35022, p=0.0170). Not all eyes showed a complete
resolution because of central vitreo-retinal traction, frequently found
in uveitic eyes. No significant difference in subjective symptoms was
observed and no adverse event occurred except from one patient
complaining of eye burning at instillation in the treated group.
Conclusions: Topical use of 0.5% indomethacin eyedrops in eyes
affected by uveitic cystoid macular edema of different etiologies in
comparison with placebo is associated with a significant reduction in
macular edema after six months as measured by SD-OCT and an
increase of visual acuity.
Commercial Relationships: Monica Zurria, Alfa Intes Ind Ter Spl
srl (E); Pia Allegri, None; Simona Peri, None; Rosanna Carniglia
De Carli, None; Maria Grazia Crivelli, None; Silvia Compiano,
None; Silvia Autuori, None; Antonio Mastromarino, None; Ugo
Murialdo, None
Clinical Trial: 2011-001522-20
Changes in cytokine profile with liposomes sprayed on the ocular
surface and nasal mucosa
Davide Scollo1, Teresio Avitabile1, Giulia Malaguarnera2, Roberta
Amato3, Giuseppe Napolitano3, Giuseppina Marrazzo2, Michele
Reibaldi1, Antonio Longo1, Caterina Gagliano3. 1Ophthalmology,
University of Catania, Catania, Italy; 2Clinical and Molecular
Biomedicine, University of Catania, Catania, Italy; 3NEST,
Neurovisual Science Technology (NEST), Catania, Italy.
Purpose: To evaluate the effect of sprayed liposomes on signs and
symptoms of nasal, pharyngeal and ocular discomfort and to measure
the levels of inflammatory cytokines in patients with dryness and
allergies.
Methods: A randomized, placebo controlled study was conducted on
30 patients with serious symptoms of nasal, pharyngeal and ocular
discomfort due to ocular and nasal dryness and allergies. Fifteen
patients were treated with a spray liposomes solution loaded with
vitamins A and E nebulized (two sprays each) on the nasal mucosa
and eyelids, 3 times per day for 3 weeks. Fifteen control subjects
were treated with saline in nasal drops and artificial tears for the same
period of time. Expression patterns of cytokines (IL-1β, IL6, TNFα,IFN-γ) were examined in body fluids (tears, Meibomian glands
secretion, saliva, and nasal wash) every week. Brush cytology
specimens were collected at the beginning and at the end of treatment
(after 3 weeks)from nasal and conjunctival mucosa and subjected to
PAS and immune-histochemical staining to detect inflammatory cell
expression,and quantitative real-time PCR for cytokine mRNA
expression.
Results: A highly significant reduction was detected in the
inflammatory cytokine levels in tears, Meibomian glands secretion,
saliva, and nasal wash of patients treated with the spray liposomes
solution in comparison with untreated controls. Control patients
showed elevated levels for most of the tested cytokines. For example,
IL-1β was found to be elevated 2.75-fold (P<0.001) at the second
week and 3.2 at the end (P<0.001) of treatment in control group with
respect to the treatment group with spray liposomes. We also found a
significant increase (P<0.01) of inflammatory cell expression in
control group after 3 weeks.
Conclusions: This study showed the ability of spray liposomes
loaded with vitamin A and E to modify the pro-inflammatory
cytokine profile after their administration on nasal mucosa, eyelid
and ocular surface. This effect could be due to the anti-inflammatory
mechanism of the vitamins and also to the barrier effect of liposomes
on mucosal surfaces.
Commercial Relationships: Davide Scollo, None; Teresio
Avitabile, None; Giulia Malaguarnera, None; Roberta Amato,
None; Giuseppe Napolitano, None; Giuseppina Marrazzo, None;
Michele Reibaldi, None; Antonio Longo, None; Caterina
Gagliano, None
Systemic factors impacting intraocular levels of vascular
endothelial growth factor and interleukin-6 in diabetic
retinopathy
Sohee Jeon, Won Ki Lee. Ophthalmology, Seoul Saint Mary's
hospital, Seoul, Republic of Korea.
Purpose: To identify systemic factors correlating with intraocular
levels of interleukin-6 (IL-6) and vascular endothelial growth factor
(VEGF) in diabetic retinopathy (DR).
Methods: This study was a cross-sectional study. Forty-two
consecutive patients (42 eyes) undergoing pars plana vitrectomy
(PPV) for DR were enrolled. Aqueous humor was sampled just prior
to PPV for assay of IL-6 and VEGF via multiplex cytokine array.
One day before PPV, patient characteristics (age, gender, duration of
diabetes, body mass index [BMI], systolic and diastolic blood
pressure, alcohol, and smoking status) were recorded and a number of
systemic markers amassed, including fasting and postprandial
glucose, homeostasis model assessment (HOMA)-IR, HOMA-beta,
C-peptide, insulin, total cholesterol, triglycerides, high-density
lipoprotein-cholesterol, low-density lipoprotein-cholesterol,
apolipoprotein (Apo)-A, Apo-B, and lipoprotein A (Lp-A).
Relationships between systemic determinants and intraocular
cytokine levels (IL-6 and VEGF) were analyzed by univariate and
multivariate regression.
Results: Median levels of IL-6 and VEGF were 15.3 pg/mL (range,
2.4-10124.5 pg/mL) and 21.1 pg/mL (range, 3.2-766.1 pg/mL),
respectively. After adjustment for age, gender, duration of diabetes,
and BMI, multivariate analysis showed significant association of
smoking (p=0.002) and HOMA-IR (p=0.003) with intraocular IL-6
levels, while intraocular VEGF and systemic Lp-A levels correlated
significantly (p=0.032).
Conclusions: Insulin resistance (HOMA-IR) and smoking status
impacted intraocular levels of IL-6, while intraocular VEGF levels
were influenced by Lp-A. An appreciation for the relationship
between systemic factors and intraocular level of various cytokines
may help elucidate the complex pathophysiology of DR.
Commercial Relationships: Sohee Jeon, None; Won Ki Lee,
Novartis (F), Novartis (C), Bayer (C)
Support: None in the Support field below.
Effects of topical indomethacin, bromfenac, and nepafenac on
LPS-induced inflammation
Claudio Bucolo1, Giuseppina Marrazzo1, Chiara Bianca Maria
Platania1, Monica Zurria2, Filippo Drago1. 1Clinical and Molecular
Biomedicine, University of Catania, Catania, Italy; 2R&D, Alfa-Intes,
Casoria, Italy.
Purpose: To investigate the effects of topical nonsteroidal antiinflammatory drugs (NSAIDs) on retinal vascular leakage, and
inflammatory markers in endotoxin-induced uveitis (EIU) in rats.
Methods: EIU was induced in rats by single footpad injection of LPS
(350 μg/kg, Salmonella typhimurium). Animals were treated
according to the ARVO statement for the use of animals in
ophthalmology and vision research. Topical indomethacin 0.5%
(Indom™), bromfenac 0.09% (Yellox™), and nepafenac 0.1%
(Nevanac™) were given before and after LPS injection (four
administrations). Twenty-four hours after LPS injection the animals
were euthanized and retina and plasma were collected to assess PGE2
and C-reactive protein (CRP) levels using ELISA. Retinal vascular
leakage was assessed by Evans blue extravasation.
Results: Twenty-four hours after LPS injection, significant (p<0.01)
increases in retinal PGE2 levels were observed. Plasma CRP levels
were significantly (p<0.01) higher in LPS-injected rats compared to
control group. The analysis of retinal vascular leakage revealed a
significant (p<0.01) decrease after treatment with indomethacin, on
the contrary no significant changes were observed after treatment
with bromfenac and nepafenac. All NSAIDs tested significantly
prevented PGE2 synthesis with higher effect of indomethacin and
bromfenac in comparison to nepafenac. All NSAIDs tested did not
affect plasma CRP levels.
Conclusions: These results demonstrated that topical treatment with
indomethacin, bromfenac, and nepafenac has significant antiinflammatory effects. However, only indomethacin was able to
prevent vascular leakage in LPS-injected rats.
Commercial Relationships: Claudio Bucolo, Alfa-Intes (C);
Giuseppina Marrazzo, None; Chiara Bianca Maria Platania,
None; Monica Zurria, Alfa Intes Ind Ter Spl srl (E); Filippo Drago,
None
Anti-Inflammatory Effects of NOV C-ter in the EndotoxinInduced Uveitis model: Down-regulation of Pro-inflammatory
Cytokines
Chadi Mehanna1, 2, Norbert J. Minet2, Marie-Chritine Naud1, 4,
Celine Olmiere5, Jean-Louis Bourges1, 3, Francine F. Behar-Cohen1,
3 1
. CRC - UMRS 872, University of Paris Descartes, Paris, France;
2
Sisene Ophthalmology, Paris, France; 3Ophthalmology, Hotel-Dieu
Hospital, Paris, France; 4CRC - UMRS 872, University of Pierre and
Marie Curie, Paris, France; 5Laboratoires THEA, Clremont-Ferrand,
France.
Purpose: To study the possible mechanisms of action involved in the
anti-inflammatory effects observed with NOV C-ter (Sisene, France)
in the endotoxin-induced uveitis (EIU) model.
Methods: Lewis male rats (6-8 weeks old) were anesthetized and
uveitis was induced by footpad injection of 100 µl (2mg/ml) of LPS
(Sigma-Aldrich, France). Intra-vitreous injections (5µl) of the
products were performed at the same time and inflammation was
assessed at 16h by a blind observer. Rats were randomly assigned to
the following groups (3 rats each): LPS (6 eyes), NOV C-ter 1.25 µg
(6 eyes), NOV C-ter 2.5 µg (5 eyes) and NOV C-ter 5µg (4 eyes).
After the clinical grading of uveitis, animals were euthanized and
irises and ciliary bodies were extracted and snap frozen. Real-time
PCR analysis was performed to evaluate the expression of several
genes: VEGF, VEGF-R1, VEGF-R2, IL-1β, IL-6, TNF-α, MCP-1,
Cox-2, Hey-1, Notch-1 and Jagged-1. A second experiment was
performed to compare the clinical effects of sub-conjunctival
injections of NOV C-ter 15 µg (6 eyes) to intra-vitreous injections of
NOV C-ter 2.5 µg (5 eyes), dexamethasone 20 µg (6 eyes) and PBS
(6 eyes). Inflammation was assessed at 16h by a blind observer.
Results: NOV C-ter exerted a significant anti-inflammatory effect
with a U-shaped dose-response, as inflammation was lower than in
the PBS group for the doses 1.25 µg (P < 0.01) and 2.5 µg (P < 0.05).
NOV C-ter 2.5 µg was as effective as dexamethasone 20 µg. Realtime PCR analysis of the irises and ciliary bodies showed no
significant effects on the expression of VEGF, VEGF-R1, VEGF-R2,
COX-2, Hey-1, Notch-1 and Jagged-1. IL-6 was significantly
downregulated by NOV 2.5 µg (P = 0.009). IL-1β, TNF-α, and MCP1 were significantly downregulated but to a lesser extent.
Conclusions: Anti-inflammatory effects observed for NOV C-ter, in
the endotoxin-induced uveitis model, seem to be mediated by a
downregulation of pro-inflammatory cytokines, mainly IL-6, IL-1β,
TNF-α and MCP-1.
Commercial Relationships: Chadi Mehanna, Sisène (E); Norbert
J. Minet, Sisene SAS (E); Marie-Chritine Naud, None; Celine
Olmiere, Laboratoires THEA (E); Jean-Louis Bourges, None;
Francine F. Behar-Cohen, Inserm/Univesrité ParisDescartes (P)
Support: Partial support from Sisene and Laboratoires THEA
Validation of the Anterior Chamber Paracentesis Model for the
Screening of Novel Ophthalmic Anti-Inflammatory Drug
Laura Belen, Kortni Violette, Jennifer Brackett, Andy Whitlock. Ora,
Inc., Andover, MA.
Purpose: The development of novel anti-inflammatory drugs for
treatment of inflammatory conditions such as dry eye, chronic
allergy, blepharitis, uveitis, or ocular trauma is complicated by the
lack of a preclinical model with a robust, consistent inflammatory
response. The anterior chamber paracentesis model (ACP) is an acute
animal model of ocular inflammation that involves the breakdown of
the blood aqueous barrier and subsequent release of proteins and
PGE2 into the aqueous humor (AH). PGE2 up-regulation is a
common factor of many ocular surface diseases such as dry eye,
chronic allergy, and blepharitis. The purpose of this study was to
benchmark numerous classes of anti-inflammatory drugs (steroids,
NSAIDs, and cyclosporine) in an ACP model to determine if it could
serve as a surrogate for ocular surface inflammation.
Methods: Inflammation was induced in NZW rabbits by aspirating
100 µL of AH from the anterior chamber. The extent of inflammation
was assessed 2 hours following the challenge by slit lamp and postlife biochemical analysis of AH for protein exudates by BCA assay
and PGE2 levels by ELISA. Anti-inflammatory drugs tested included
Dexamethasone (Dex), Prednisolone (Pred), Keterolac, Bromfenac,
and Restasis. Balanced saline was used as a vehicle control. Four
animals (8 eyes) per treatment were dosed bilaterally four times daily
one day prior to ACP. On the day of AH collection, animals had a
clinical observation and received topical treatment 3, 2, 1 and .5
hours prior to the first AH collection. Animals received topical
treatment 30, 60 and 90 minutes post first collection.
Results: Only bromfenac and ketorolac significantly reduced AH
protein exudates in the NZW rabbits following ACP compared to
vehicle control (p < .01). Dex, bromfenac and keterolac statistically
decreased PGE2 levels compared to vehicle control (p < .01, p < .05,
p< .05). Dex and Restasis also resulted in decreased levels of PGE2
release, but the decrease was not statistically significant.
Conclusions: Both steroids and NSAIDs appear to be efficacious in
this model. While not significant, Restasis had some efficacy that
may be increased by adjusting the power of the study. The results of
this study indicate that ACP could serve as a surrogate model for
ocular surface inflammation and help further novel drug discovery
for indications such as dry eye and belpharitis.
Commercial Relationships: Laura Belen, Ora, Inc. (E); Kortni
Violette, Ora, Inc. (E); Jennifer Brackett, Ora, Inc. (E); Andy
Whitlock, Ora, Inc. (E)
Comparison of effects between glucocorticoids and mapracorat
after repeat topical ocular administration in rabbits for 28 days
Kathleen Krenzer, Sherwin Jiang, Ezra R. Lowe, Mary Richardson.
Preclinical Development, Bausch + Lomb, Rochester, NY.
Purpose: The purpose of this investigation was to assess the systemic
effects of glucocorticoids (GC) as compared to mapracorat, a new
selective glucocorticoid receptor agonist (SEGRA) after repeated
topical ocular administration for 28 days in rabbits with a 1-week
recovery.
Methods: The test articles were mapracorat (1, 3, and 6%), Maxidex
(dexamethasone sodium phosphate 0.1%), PredForte (prednisolone
acetate, 1.0%), and Durezol (difluprednate, 0.05%). The controls
were the mapracorat vehicle or saline. Seven NZW rabbits/sex/group
were used; 4/sex/group were euthanized after 28 days of dosing and
3/sex/group were euthanized after a 1-week recovery. One eye of
each animal was dosed with the respective test article QID (postoperative regimen) and the other eye with the control. In the control
group, one eye was dosed with the control and the contralateral eye
remained untreated. Animals were periodically assessed for clinical
signs, body weight, and ophthalmic findings (including intraocular
pressure). At the end of the dosing period, blood samples were
collected for toxicokinetic assessment of systemic exposure. Animals
were euthanized and blood and tissues collected for clinical
chemistry and histopathology assessments.
Results: No adverse ocular effects were observed for any of the test
articles. As compared to controls, significant systemic effects were
observed in the GC-treated animals, including low body weights and
effects on the metabolic/endocrine, cardiovascular, liver, and
immune. Partial reversibility was observed after a 1-week recovery
period. For the mapracorat-treated groups, no effects were observed
with the 1% concentration. For the 3 and 6% groups, no meaningful
effects were observed for cardiovascular or liver systems. Doserelated changes were observed for some of the metabolic (low
adrenal gland weight) and immune (low thymus weights, low
lymphocytes) systems. There was partial (adrenal gland) or full
(thymus) reversibility after a 1-week recovery period. When
observed, however, the magnitude of the effects was to lesser degree
than those observed in the GC-treated animals. No effects were
observed with mapracorat that were not within the known effects of
GCs.
Conclusions: After 28 days of repeat ocular dosing in rabbits,
mapracorat, up to 6%, demonstrates a better systemic safety profile in
rabbits than observed with traditional ocular GCs.
Commercial Relationships: Kathleen Krenzer, Bausch + Lomb
(E); Sherwin Jiang, None; Ezra R. Lowe, Bausch & Lomb (E);
Mary Richardson, Bausch and Lomb (E)
Support: None.
Efficacy of Tocilizumab, an IL-6 receptor inhibitor in ocular
inflammatory disorders: a pilot study
Vimal Sarup1, 3, Anum Butt1, 2, Kanza Aziz1, 2, C. Stephen Foster1, 3.
1
Massachusetts Eye Research & Surgery Institution, Cambridge, MA;
2
Ocular Immunology & Uveitis Foundation, Cambridge, MA;
3
Harvard Medical School, Boston, MA.
Purpose: Tocilizumab is a Humanized Anti Interleukin 6
Monoclonal antibody evaluated and used for its beneficial effects on
systemic autoimmune inflammatory disease but not for non infectious
ocular inflammation. In this pilot study, tocilizumab was evaluated to
study its effect on ocular Inflammation refractory to the extended
spectrum of conventional and immunomodulatory medications.
Methods: Eleven patients with non resolving non infectious ocular
inflammation involving anterior segment, posterior segment or both,
who had not responded completely to other medications were
evaluated. Tocilizumab was given via intravenous infusions to
augment the existing medications on a set schedule and dosage.
Detailed ocular examination with slit lamp and dilated fundus
examination was done. Ocular inflammation was graded according to
SUN criteria. A full spectrum of diagnostic testing included
fluorescein angiography, OCT, and Ultrasound B scan as necessary.
Age, sex, race, ocular and systemic side effects were noted in
addition. Systemic side effects were evaluated by blood tests and
physical examinations by general physicians or rheumatologists.
Results: Nine patients were Caucasians, eight were females, average
age was 43.8 yrs, and they had failed four systemic
immunomodulatory medications on average. There were 6 cases of
Uveitis, 3 of Scleritis, 1 orbital inflammatory disease, and 1
peripheral ulcerative keratitis. Eight patients responded favorably by
reduction in ocular inflammation including four patients who went
into remission. Two patients had infusion reactions with initiation of
treatment and the drug had to be discontinued. Two others had
systemic side effects but the ocular inflammation had shown
improvement.
Conclusions: These data indicate that tocilizumab improves anterior
and posterior segment ocular inflammation of varied non infectious
etiologies. It has the potential to not only stabilize but induce
remission of inflammation, even though other systemic medications
failed or induced systemic side effects. Further double blind
prospective trials are necessary to evaluate its role in ocular
inflammation.
Commercial Relationships: Vimal Sarup, None; Anum Butt,
None; Kanza Aziz, None; C. Stephen Foster, Abbott Medical
Optics (C), Abbott Medical Optics (F), Alcon Laboratories, Inc. (C),
Alcon Laboratories, Inc. (F), Allergan, Inc. (C), Allergan, Inc. (F),
Eyegate Pharmaceuticals, Inc. (I), Eyegate Pharmaceuticals, Inc. (F),
IOP Opthalmics (C), Ista Pharmaceuticals (C), Lux Biosciences, Inc.
(C), Lux Biosciences, Inc. (F), Novartis Pharmaceuticals Corporation
(C), Novartis Pharmaceuticals Corporation (F), XOMA Ltd (C)
Evaluation of XG-102 effects in the treatment of endotoxininduced uveitis in rats
Ikram El-Zaoui1, 2, Elodie Touchard1, 2, Marianne Berdugo Polak1, 2,
Claire Abadie4, Catherine Deloche3, Jean-Marc Combette4, Francine
F. Behar-Cohen1, 3. 1CENTRE DE RECHERCHE DES
CORDELIERS TEAM 17 UMRS 872, Paris, France; 2Université
paris Descartes Paris-5, Paris, France; 3department of ophthalmology,
Hotel Dieu,APHP, Paris, France; 4Solid Drug Development.S.A,
Geneve, Switzerland.
Purpose: To confirm the efficacy of XG-102 (JNK Inhibitor) in the
treatment of endotoxin-induced uveitis (EIU) in rats, XG-102 doseeffect was evaluated using different routes of administration.
Methods: EIU was induced in Lewis rats by LPS injection. XG-102
was administered at the time of LPS challenge either by intravenous
(IV, 3.2, 35 or 355 µg/injection), intravitreal (IVT, 0.08, 0.2 or 2.2
µg/eye) or sub-conjunctival (SCJ, 0.2, 1.8 or 2.2 µg/eye) injections.
Controls received either the vehicle or Dexamethasone phosphate
injections. Clinical effects were scored at the peak of the disease(24
hours after LPS). XG-102 targeting was controlled by Westernblotting the phosphorylated c-Jun in the “RPE-Choroid” complex.
The anti-inflammatory effects were evaluated using quantification of
cells infiltration in ocular tissues and evaluation of the expression of
inflammatory mediators(iNOS at the mRNA levels in the neuroretina.
Results: XG-102 clinically demonstrated adose-dependent antiinflammatory effect in EIU after IV and SCJ administrations. The
respective doses of 35 µg and 1.8 µg were efficient as compared with
the vehicle-injected controls, and the highest doses, respectively 355
µg and 22 µg, were as efficient as Dexamethasone. After IVT
injections, the anti-inflammatory effect of XG-102 was clinically
evaluated similar to the corticoid’s effect with all the tested doses.
Whatever the route administration tested, the lowest efficient doses of
XG-102 targeted efficiently the c-Jun N-terminal kinase; reduce cells
infiltration in the treated eyes and iNOS expression in the retina.
Conclusions: These results confirm that XG-102 has potential for
treating intraocular inflammation. The subconjunctival route will be
further investigated in humans.
Commercial Relationships: Ikram El-Zaoui, None; Elodie
Touchard, None; Marianne Berdugo Polak, None; Claire Abadie,
None; Catherine Deloche, Solid Drug Development SA (E), Xigen
SA (C); Jean-Marc Combette, Xigen (C); Francine F. BeharCohen, Inserm/Univesrité ParisDescartes (P)
Systemic symvastatin rescues death of retinal ganglion cells from
optic nerve injury possibly through suppression of glial NF-κB
activation
Seita Morishita, Hidehiro Oku, Masahiro Tonari, Teruyo Kida,
Taeko Horie, Tsunehiko Ikeda. Osaka Medical College, Takatsukishi, Japan.
Purpose: To determine whether systemic simvastatin rescues death
of retinal ganglion cells (RGCs) from optic nerve injury.
Methods: We studied the effect of systemic simvastatin on the
survival of RGCs after the optic nerve of rats was crushed.
Simvastatin (3.0 mg/kg/day) or its solvent (placebo) was given
through an osmotic mini-pump 1week prior to crushing the optic
nerves. We also performed immunohistological evaluations and realtime PCR to determine the expressions of the CD68, TNF-α, and
iNOS genes in the neuroinflammation of the crushed optic nerves.
Results: There was a recruitment of CD68 positive cells, namely
microglia/macrophages, at the crushed site. Assessment by real time
PCR showed that mRNA levels of CD68 significantly increased after
crushing the optic nerve, which peaked on day 5. Systemic
simvastatin significantly (P=0.002, ANOVA followed by Fisher)
suppressed the increase on day 3. In addition, simvastatin
significantly suppressed up-regulation of TNF-α and iNOS genes.
The mean number (± SEM) of RGCs stained by TUJ-1 antibody was
1816.3 ± 94.9/mm2 in sham operated rats (n=6), which decreased to
831.4 ± 82.6/mm2 (n = 9) on day 7 after the optic nerve was crushed
with placebo treatment. This reduction was significantly (P=0.01,
Scheffe) reduced to 1169.2 ± 82.2/mm2 (n = 9) with systemic
simvastatin treatment. We also found simvastatin (1.0 μM)
significantly suppressed NF-κB activation and iNOS expression
caused by TNF-α (50ng/ml) in cultured optic nerve astrocytes.
Conclusions: These results suggested that systemic simvastatin
suppressed the neuro-inflammtion and rescued death of RGCs after
crushing the optic nerves. One possible mechanism of the neuroprotective role is suppression of NF-κB activation of optic nerve
astrocytes.
Commercial Relationships: Seita Morishita, None; Hidehiro Oku,
None; Masahiro Tonari, None; Teruyo Kida, None; Taeko Horie,
None; Tsunehiko Ikeda, None
Infliximab Attenuates Tumor Necrosis Factor-α-Induced
Alterations in Non-Pigmented Ciliary Epithelium
Hiroshi Yamada1, Masahiko Yoneda2, Shingo Inaguma3, Masayoshi
Iwaki1, Masahiro Zako1. 1Ophthalmology, Aichi Medical University,
Nagakute, Japan; 2Biochemistry and Molecular Biology, School of
Nursing and Health, Aichi Prefectural University, Nagoya, Japan;
3
Pathology, Aichi Medical University, Nagakute, Japan.
Purpose: In human non-pigmented ciliary epithelial cells
(HNPCECs) in vitro, we investigated changes in the mRNA and
protein expression levels of matrix metalloproteinases (MMPs) and
tissue inhibitors of metalloproteinases (TIMPs) in the presence of
tumor necrosis factor-alpha (TNF-α), and examined the effects of
infliximab addition. Degradation of claudin-1 and occludin, and
permeability changes in HNPCECs were also evaluated.
Methods: HNPCECs were cultured in the presence or absence of
TNF-α, and TNF-α-exposed cells were treated with or without
infliximab. We measured the expression levels of MMP-1, MMP-2,
MMP-3, MMP-9, TIMP-1, and TIMP-2 in HNPCECs by real-time
polymerase chain reaction and by enzyme-linked immunosorbent
assay. HNPCECs and swine ciliary body were treated with MMPs,
and immunostained. Permeability of MMP-treated-HNPCECs was
measured using a cell-based permeability assay.
Results: The expressions of MMP-1, MMP-3, and MMP-9 increased
in the presence of 10 ng/mL TNF-α, and these altered expression
levels were reversed by the addition of infliximab. Immunostaining
showed that MMP-1, MMP-3, and MMP-9 degraded claudin-1 and
occludin, after which significant increases in HNPCEC permeability
were detected.
Conclusions: TNF-α increased the expressions of MMPs in cells
comprising the blood-aqueous barrier (BAB). Components of the
tight junctions of the BAB were degraded by MMPs, which increased
permeability through the cells. Infliximab was effective at attenuating
the TNF-α-induced increases in MMP expressions in cells
comprising the BAB, suggesting that such treatment may clinically
prevent anterior uveitis.
Commercial Relationships: Hiroshi Yamada, None; Masahiko
Yoneda, None; Shingo Inaguma, None; Masayoshi Iwaki, None;
Masahiro Zako, None
Ocular Pharmacokinetics of 0.2% and 0.4% Ketorolac
Tromethamine Formulated in DuraSite or DuraSite 2 Delivery
Systems Compared to Acular LS in Rabbits
Afshin Shafiee1, Lyle M. Bowman2, Eddie Hou2, Kamran Hosseini1, 3.
1
Preclinical, InSite Vision, Alameda, CA; 2Development, InSite
Vision, Alameda, CA; 3Clinical, InSite Vision, Alameda, CA.
Purpose: To compare the ocular penetration of 0.2% and 0.4%
ketorolac formulated in DuraSite or the new generation, DuraSite 2,
delivery systems to Acular LS (0.4% ketorolac).
Methods: The left eye of male and female rabbits (n=32/group)
received either a single topical instillation of ketorolac 0.2% or
ketorolac 0.4% formulated in DuraSite, or DuraSite 2, or Acular LS.
At predetermined timepoints (0.25, 0.5, 1, 2, 4, 6, 12, and 24 hours),
4 rabbits/group/timepoint were sacrificed and the ketorolac levels in
the aqueous humor (AH) were quantified using LC-MS/MS
methodology. PK parameters (Cmax, Tmax, AUC0.25-24h) were
determined.
Results: Ketorolac 0.4% formulated in DuraSite 2 achieved the
highest Cmax (1889 ± 884 ng/mL) and AUC (6836 ng/mL*h) values,
an increase of 6.9- and 4.8-fold over Acular LS which had the lowest
Cmax (275 ± 83 ng/mL) and AUC (1424 ng/mL*h) values,
respectively. Ketorolac 0.2% formulated in DuraSite 2 had Cmax
(1077 ± 415 ng/mL) and AUC (4490 ng/mL*h) values that were 3.9and 3.2-fold higher than Acular LS, respectively. Ketorolac 0.2% and
0.4% formulated in DuraSite also provided better AH
pharmacokinetics with Cmax values that were 2.9- and 4.4-fold higher
than Acular LS, respectively, and AUC values that were 2.3- and 4.0fold higher than Acular LS, respectively.
Conclusions: Ketorolac formulated in DuraSite markedly improved
drug delivery kinetics to the AH compared with Acular LS; the new
generation delivery system, DuraSite 2, showed enhanced penetration
over DuraSite. DuraSite 2 formulation may allow a major reduction
in the dosing regimen or lowering of the ketorolac levels in the
ophthalmic formulation; it may potentially lessen the side effect
profiles associated with the topical use of ketorolac.
Commercial Relationships: Afshin Shafiee, InSite Vision (E); Lyle
M. Bowman, InSite Vision (E); Eddie Hou, InSite Vision (E);
Kamran Hosseini, InSite Vision Inc. (E)
ITF2357 regulates IL-10 via JAK/STAT signaling pathway to
attenuate inflammation and fibrosis
Shyam S. Chaurasia1, 2, Yu-Chi Liu1, Alison Tan1, Rayne Lim1,
Jodhbir S. Mehta1, 3. 1Tissue Engineering and Stem Cell Group,
Singapore Eye Research Inst, Singapore, Singapore; 2Signature
Research Program in Neuroscience & Behavioral Disorder, DukeNUS Graduate Medical School, Singapore, Singapore; 3Singapore
National Eye Centre, Singapore, Singapore.
Purpose: To study the mechanism of action of a histone deacetylase
inhibitor, ITF2357 on fibrosis and inflammation in vitro and in vivo
in a rabbit PRK model of corneal wound healing.
Methods: Twenty rabbits underwent -9.0D photorefractive
keratectomy (PRK) surgery in one eye and were divided into 3
groups based on post-op treatment with a single dose of saline (3
days), ITF2357 (0.02% for 3 days) or MMC (0.02% for 60 sec postsurgery). Post-op clinical examination was made for 4 weeks using
slit lamp microscopy and in vivo confocal microscopy (IVCM).
Cultured primary human corneal fibroblasts (pHCFs) were used to
study the ITF2357-induced activation of IL-10 and JAK/STAT
signaling pathway using specific signaling inhibitors by
immunocytochemistry, western blot and ELISA.
Results: ITF2357 significantly reduced corneal haze and
extracellular matrix formation on IVCM. IL-10 expression was upregulated in the ITF2357 treated PRK corneas compared to the MMC
treated. Cultured pHCFs with ITF2357 produced elevated levels of
IL-10 in a time-dependent manner. This in turn, resulted in the
activation of pSTAT3 and downstream signaling with overexpression
of SOCS3 expression. Inhibition of recruitment of STAT3 to the
receptor complex with a specific inhibitor blocked the
phosphorylation of STAT3, preventing its nuclear entry and hence
decreased IL-10 production.
Conclusions: ITF2357 activates IL-10 levels via activation of
JAK/STAT signaling pathway to exhibits its anti-fibrotic and antiinflammatory activity in cornea wound healing.
Commercial Relationships: Shyam S. Chaurasia, None; Yu-Chi
Liu, None; Alison Tan, None; Rayne Lim, None; Jodhbir S.
Mehta, None
Support: SingHealth Grants-R729/13/2010/SHF & R756/40/2010
SHF to SSC; NMRC NIG- R751/35/2010 to SSC, TCR R620/41/2008 to JSM; BMRC TCRP - R826 to SSC
Lack of Correlation between PGE2 levels and Blood Aqueous
Barrier Inhibition in a rabbit model of ocular inflammation
L David Waterbury. Raven Biosolutions LLC, San Carlos, CA.
Purpose: In a model of ocular inflammation, several topical NSAIDs
were simultaneously tested for inhibition of PGE2 and for blood
aqueous barrier breakdown following LPS administration. The
purpose of the study was to determine if inhibition of PGE 2
concentrations was correlated with inhibition of the breakdown of the
blood aqueous barrier following the dosing with topical NSAIDs.
Methods: Topical dosing of suspensions (0.1% FR122047, 0.1%
trans-resveratrol) or solutions (0.1% amfenac, 0.09% bromfenac, 0.1
% diclofenac, 0.4% ketorolac) of NSAIDs were administered to one
eye of New Zealand White Rabbits (n=6). Rabbits received LPS
(Salmonella enterica typhimurium, 10 µg/kgm), and fluorescein
isothiocyanate-dextran (FITC-dextran), M.W. = ~70,000, 30
mg/kgm, iv, at 1 hour). FITC-dextran was used to determine the
blood aqueous leakage. At 2 hours after topical dosing , aqueous
humor samples were simultaneously analyzed for PGE2 and FITCdextran concentrations.
Results: All test drugs significantly inhibited PGE2 concentrations in
the aqueous humor. The degree on PGE2 inhibition ranged from ~ 50
to 97%, and the inhibition of FITC inhibition ranged from 7 to 98%.
Amfenac was more effective in inhibiting FITC-dextran
accumulation than PGE2 concentrations (97% vs 68% respectively).
FR122047, trans-resveratrol, and diclofenac were active in lowering
PGE2, but did not significantly affect FITC-dextran concentrations.
Both ketorolac and bromfenac were equally active with 2 hour
dosing, but with 12 hour pre-dosing, only ketorolac was active in
suppressing FITC-dextran.
Conclusions: Taking into account COX selectivity and potency,
there did not appear to be a correlation between PGE2 and FITCdextran inhibition suggesting different sites of action for both effects.
Commercial Relationships: L David Waterbury, Allergan (F),
Omeros (C), Syntex/Roche (P)
Suprachoroidal Microinjection of Triamcinolone Acetonide is
Well Tolerated in the Albino Rabbit
Rozemarijn S. Verhoeven1, Samirkumar R. Patel1, Karen ViaudQuentric2, Florian Cacciamani2, Thierry Amar2, Benjamin Yerxa1.
1
Clearside Biomedical, Alpharetta, GA; 2Iris Pharma, La Gaude,
France.
Purpose: To evaluate ocular tolerability and toxicokinetics of
suprachoroidal administration of triamcinolone acetonide (TA) using
a Clearside Biomedical proprietary microneedle in a GLP study in the
New Zealand White rabbit.
Methods: On Day 0, rabbits (5/sex/group) were administered a single
bilateral suprachoroidal injection of vehicle, 3.2 mg or 5.2 mg of TA
(Triesence®) using a 33g 750µm microneedle. Clinical observations,
body weights, food and water consumption, slit lamp biomicroscopy
with McDonald-Shadduck scoring, fundus evaluation, intraocular
pressure assessment (IOP), electroretinography (ERG), and systemic
exposure were assessed up to 17 weeks post-dose. Animals were
sacrificed on Day 1 and Week 13 for macroscopic observations,
ocular toxicokinetics, and ocular histopathology.
Results: There were no adverse effects related to test article or
method of administration on clinical observations, body weight, body
weight gain, food and water consumption, or ophthalmic
examinations. No effect on ERG a- or b-wave amplitude or implicit
time was noted in any animal. A mild, transient increase in IOP of 23 mmHg was observed in the TA groups on Days 7 and 28, which
resolved by Week 13 and was not considered adverse. Inflammatory
cells and test article were observed in the suprachoroidal space of
TA-treated animals on Day 1 but not Week 13 as assessed by
histopathology. Systemic exposure to TA was minimal. TA was
observed at high concentrations in the sclera/choroid and retina, to a
lesser extent in the iris/ciliary body, and was present only at low
concentrations in the aqueous humor, lens, and vitreous.
Conclusions: A single bilateral suprachoroidal injection of 3.2 or 5.2
mg TA using a microneedle was well tolerated in the albino rabbit.
Systemic exposure to TA was minimal, and absorption of TA into the
posterior segment of the eye was observed with minimal TA
exposure to the anterior segment of the eye. These data suggest that
suprachoroidal drug delivery is well tolerated, results in distribution
of TA to the sclera/choroid and retina, structures that are important
targets for anti-inflammatories in posterior segment disease, and
limits TA exposure in the anterior segment.
Commercial Relationships: Rozemarijn S. Verhoeven, Clearside
Biomedical (E); Samirkumar R. Patel, Clearside Biomedical (E),
Clearside Biomedical (I), Clearside Biomedical (P); Karen ViaudQuentric, Iris Pharma (E); Florian Cacciamani, Iris pharma (E);
Thierry Amar, None; Benjamin Yerxa, Clearside Biomedical (I)
Support: Georgia Research Alliance
Assessment of Efficacy and Safety of a Novel Protocol for Pulsed
Intravenous Cyclophosphamide for Recalcitrant or Severe
Ocular Inflammatory Disease
Ana M. Suelves1, 2, Cheryl A. Arcinue1, 2, Jesús María GonzálezMartín4, C. Stephen Foster1, 3. 1Massachusetts Eye Research and
Surgery Institution, Cambridge, MA; 2Ocular Immunology & Uveitis
Foundation, Cambridge, MA; 3Harvard Medical School, Boston,
MA; 4University of Valencia, Valencia, Spain.
Purpose: To analyze the success rate of pulsed intravenous (IV)
cyclophosphamide (CyP) for non-infectious ocular inflammatory
disease and to identify risk factors for failure of therapy.
Methods: Retrospective, interventional, cohort study. Through a
computer search of the Massachusetts Eye Research and Surgery
Institution’s database, we identified 65 patients (110 eyes) who were
treated with IV CyP between May 2005 and April 2012. The main
outcomes evaluated through review of the electronic health record of
each patient were clinical response, corticosteroid-sparing effect,
recurrence rate, calculated “risk factors” for failure, visual acuity and
adverse reactions.
Results: Pulsed IV CyP achieved complete remission of
inflammation (for at least 2 visits) in 54 patients (84.4%). Sustained
remission of inflammation occurred in 70% of patients within 3
months, 86.6% of patients within 6 months, and 91.7% within 9
months. The mean time to achieving quiescence was 3.5 months. The
success rate in reducing corticosteroid to prednisone 10 mg/day or
less within 6 months, while maintaining control of ocular
inflammation, was 89.7% (95% Confidence Interval (CI) 81.193.5%). The mean duration of clinical remission for those patients
who had a positive response to CyP was 32.67 months (95% CI
25.91-39.43). Relapse of vasculitis was observed in 1 patient (1.5%)
after completing the course of therapy. Early initiation of therapy
during the course of the disease was correlated with a lesser rate of
recurrence (p=0.028). The most common adverse effects were nausea
(29%) and transient lymphopenia (26%). The mean best-corrected
visual acuity (BCVA) improved from 0.59 ± 0.66 at baseline to 0.30
± 0.54 at 6 months of follow-up (p < 0.001). The mean follow-up
period was 31.61 ± 20.47 months.
Conclusions: Pulsed IV CyP employing our protocol results in an
extremely high rate of sustained complete remission in patients with
recalcitrant and fulminant, vision-threatening ocular inflammatory
disorders, with an excellent safety profile in the hands of physicians
trained and skilled in the art of this therapy. The protocol makes
possible tapering and discontinuing corticosteroids in most patients.
Early initiation of therapy may decrease the risk of relapses.
Commercial Relationships: Ana M. Suelves, None; Cheryl A.
Arcinue, None; Jesús María González-Martín, None; C. Stephen
Foster, Abbott Medical Optics (C), Abbott Medical Optics (F),
Alcon Laboratories, Inc. (C), Alcon Laboratories, Inc. (F), Allergan,
Inc. (C), Allergan, Inc. (F), Eyegate Pharmaceuticals, Inc. (I),
Eyegate Pharmaceuticals, Inc. (F), IOP Opthalmics (C), Ista
Pharmaceuticals (C), Lux Biosciences, Inc. (C), Lux Biosciences,
Inc. (F), Novartis Pharmaceuticals Corporation (C), Novartis
Pharmaceuticals Corporation (F), XOMA Ltd (C)
Acute serous retinal detachment after uncomplicated cataract
surgery
Maurizio G. Uva1, Antonio Longo1, Michele Reibaldi1, Mario D.
Toro1, Vincenza Bonfiglio1, Faro Giuseppe1, Andrea Russo1,
Caterina Gagliano2, Teresio Avitabile1. 1Institute of Ophtalmology,
University of Catania, Catania, Italy; 2Opthalmology, NEST
(Neurovisual Science Technology), Catania, Italy.
Purpose: To report some cases of acute serous retinal detachment
(ASRD) after uncomplicated cataract surgery.
Methods: In a retrospective study, were collected the data of patients
that developed an ASRD after an uncomplicated phacoemulsification
with IOL implantation.
Diagnosis was made at the first post-operative day, when all patients
had a very low best corrected visual acuity (BCVA) in spite of a good
aspect of the anterior segment, without significant keratopathy and
with only trace cells in the anterior chamber; OCT revealed a serous
retinal detachment with intraretinal fluid accumulation in macular
area. Patients received systemic treatment with indomethacin 20 mg
QD and acetazolamide 125 mg TID, and topical indomethacin 0.1%
eyedrops in addition to usual post-operative treatment (topical:
tropicamide 1% BID, steroids and antibiotics QID; oral cefixime 400
mg QD for 5 days).
Patients were examined postoperatively at 1, 3, 7, and 30 days.
BCVA and central foveal thickness (CFT), measured by a Spectral
domain Spectralis OCT, were evaluated.
It was evaluated if demographics, preoperative ocular conditions,
systemic diseases, parameters of surgery were related to the
development of ASRD.
Results: From January 2009 to June 2012, on 3900 cataract surgery
performed, an ASRD was detected in 5 patients (3m, 2f, mean age
59±9 years).
Pre-operatively, mean BCVA was 0.16±0.09 decimals.
At the first post-operative day, mean BCVA was 0.08±0.03 decimals,
and CFT was 836±96 microns; all eyes had intraretinal fluid
accumulation and serous retinal detachment.
At the following controls, BCVA improved and CFT reduced
significantly (both ANOVA p<0.001). At 3 days, mean BCVA was
0.36±0.11 decimals and mean CFT was 444±155 microns. At 7 days,
BCVA was at least 0.8 decimals in all eyes (mean 0.90±0.07), with
complete reabsorption of intra and subretinal fluid (mean CFT 210±8
microns). At 30 days, mean BCVA was 0.96±0.05 decimals and
mean CFT was 191±12 microns. In the following six months, no eye
had recurrence of macular edema.
Preoperatively, all eyes had myopic refraction (axial length range:
24.8-27.4 mm), and had no posterior vitreous detachment; no relevant
data were found on other parameters investigated.
Conclusions: Acute serous retinal detachment is a rare event that can
occur after uncomplicated phacoemulsification, that in our cases
resolved in a few days without recurrence.
Commercial Relationships: Maurizio G. Uva, None; Antonio
Longo, None; Michele Reibaldi, None; Mario D. Toro, None;
Vincenza Bonfiglio, None; Faro Giuseppe, None; Andrea Russo,
None; Caterina Gagliano, None; Teresio Avitabile, None
Clinical Outcomes Using Loteprednol 0.5% for treatment of
ocular inflammation associated with cataract surgery
Melissa M. Toyos. Discover Vision Centers, Independence, MO.
Purpose: To explore the clinical characteristics of loteprednol 0.5%
in the early postoperative period after uncomplicated
phacoemulsification by a single surgeon including average ETDRS
acuities, cell/flare scores and intraocular pressure readings.
Methods: 38 consecutive patients meeting entry criteria underwent
uncomplicated phacoemulsification with lens implantation by a
single surgeon. Patients were given loteprednol 0.5% qid for 21 days
following surgery. Participants were given topical moxifloxacin tid 3
days prior to surgery and received moxifloxiacin tid for 10 days
following surgery, loteprednol 0.5% qid and bromday qd for 21 days
following surgery. At each time point, pain was self-reported, acuities
were measured by ETDRS, inflammation was measured by slit lamp
exam and IOPs were measured by applananation. Dilated fundus
exams were performed at baseline and POD42.
Results: The average age of enrolled subjects was 68.2 All eyes were
Caucasian except 2 HIspanic and 2 African American. Average
baseline letters read were 76 and average iop was 14.5 mm Hg. At
POD1, letters read were stable at 75, pain was measured at 0.1 (0-4
0=no symptoms), k edema 0.4, conjunctival injection and chemosis
0.3, cell 1.4, flare 1 and average iop 17. POD7 letters were 81, pain
and inflammation were improved, cell 0.5, flare 0.2, iop 14. POD21
letters were 82, minimal inflammation, cell 0.4 and flare 0.2, average
12.4, 19/38 subjects have completed the study, letters read at final
visit were 81.6, with no pain, k edema, conjunctival injection,
chemosis. Cell was 0.3 and flare 0.1. Average IOP was 10.9.
Conclusions: Loteprednol 0.5% used qid after uncomplicated
phacoemulsification in conjunction with bromfenac qd is a safe and
effective method to control post surgical inflammation and pain. With
the exception of one significant spike, Iops increases following
cataract surgery were low and consistent with known data for
steroids. Data collection in this study is ongoing and the completed
data set will be reported at presentation.
Commercial Relationships: Melissa M. Toyos, alcon (C), allergan
(C), sarcode (C), bausch and lomb (C)
Support: Bausch and Lomb statistical analysis and unrestricted grant
of 10,000
Clinical Trial: 147450681
24-Hour Evaluation of the Ocular Pharmacokinetics of 14CLabeled Low-Concentration, Modified Bromfenac Ophthalmic
Solution Following Topical Instillation into the Eyes of New
Zealand White Rabbits
George Baklayan. Bausch & Lomb, Irvine, CA.
Purpose: To evaluate the 24-hour ocular distribution and
concentrations of 14C-Iabeled low-concentration, modified bromfenac
ophthalmic solution following topical instillation in New Zealand
White rabbit eyes.
Methods: Eighteen New Zealand white rabbits were randomly
assigned to 6 treatment groups (3 animals per treatment group). A
single dose of 50µL of the dosing solution was topically administered
into the conjunctival sac of both eyes of each animal. Animals were
euthanized and the aqueous humor was collected at 1 hour ± 5
minutes, 2 hours ± 15 minutes, 4 hours ± 15 minutes, 8 hours ± 15
minutes, 12 hours ± 15 minutes, or 24 hours ± 15 minutes following
dosing. The iris/ciliary body, lens, vitreous, retina, choroid, sclera,
conjunctiva, and cornea (target tissues) were also collected from each
eye for analysis. Dosing solutions were analyzed to confirm
radiochemical purity; radioactive concentration of the dosing
solutions was calculated using liquid scintillation counting (LSC).
Results: Radioactive residues of 14C-Iabeled bromfenac, expressed as
mean parts per million [(ppm) µg/g] was seen in all target tissues of
the eyes, with the highest concentrations found in the cornea,
conjunctiva, and sclera. The concentrations in the tissues diminished
to varying degrees over the 24 hour study period, with the exception
of the lens, which increased insignificantly from the 1 hour time
point. The levels detected in the lens and vitreous humor were low
and close to background levels.
Conclusions: Significant penetration and measurable amounts of
14
C-labeled bromfenac were detected in most ocular target tissues
over 24 hours, with highest levels in the cornea, conjunctiva, and
sclera. The 14C-low concentration bromfenac residues in ocular
tissues were similar to those previously reported with 0.09% 14Cbromfenac, the currently available concentration of bromfenac
ophthalmic solution.
Commercial Relationships: George Baklayan, Bausch & Lomb (E)
Comparison of Methotrexate and Mycophenolate in the
Treatment of Chronic Uveitis
Tiffany Truong1, Zvi A. Kresch1, Sanjay R. Kedhar1, 2, Vicente Diaz1,
John V. Mauro1, C. Michael Samson1, 2. 1NY Eye and Ear Infirmary,
New York, NY; 2New York Medical College, New York, NY.
Purpose: To compare efficacy and side effects of methotrexate and
mycophenolate in the treatment of chronic non-infectious uveitis.
Methods: Charts of patients seen by the New York Eye & Ear
Infirmary Uveitis Service in 2004-2005 who were treated with either
methotrexate or mycophenolate and had follow-up data for a
minimum of three years after initiation of medication were reviewed.
Statistical analysis was performed comparing aspects of efficacy and
tolerability, such as ability to control inflammation, time to control
inflammation, ability to prevent vision loss, and incidence of side
effects.
Results: A total of 95 patients were included in the study. Control of
inflammation at 1, 2, and 3 years after initiating medication was
statistically equal between the two groups, ranging from 71% to 80%
with mycophenolate, and 67% to 70% with methotrexate.
Mycophenolate achieved control of inflammation faster than
methotrexate at 6 and 9 months (p=.0179, .0485 respectively), but the
drugs evened out in the long run. Visual acuity was preserved equally
in both groups. Side effects were minimal in both groups.
Conclusions: Both methotrexate and mycophenolate have equal
ability to control inflammation and prevent vision loss in patients
with chronic uveitis who are either unresponsive or intolerant of
steroid treatment. Mycophenolate achieves control of inflammation
more quickly in the 6 to 9 month range, but the drugs are equal at 1
year and longer. Both drugs are tolerated with minimal side effects.
This is the first report of a comparison of these two drugs in a cohort
at a single treatment center.
Commercial Relationships: Tiffany Truong, None; Zvi A. Kresch,
None; Sanjay R. Kedhar, None; Vicente Diaz, None; John V.
Mauro, None; C. Michael Samson, CLS Pharmaceuticals (I),
PCAsso (I)
Randomized, Placebo-Controlled, Integrated Phase III Clinical
Trials of a Once Daily, Low-Concentration, Modified Bromfenac
Ophthalmic Solution Following Cataract Surgery: Focus on Zero
to Trace Anterior Chamber Inflammation
James A. Gow1, James D. Boyce2, Harvey J. Reiser3, Robert Berry4,
Jung T. Dao5, Simon P. Chandler1. 1Bausch & Lomb, Irvine, CA;
2
Orange County Ophthalmology Medical Group, Garden Grove, CA;
3
Eye Care Specialists, Kingston, PA; 4Eye Care Arkansas PA, Little
Rock, AR; 5Cornea Consultants of Arizona, Phoenix, AZ.
Purpose: To evaluate in a post-hoc analysis the reduction of ocular
inflammation to 0 or trace anterior chamber inflammation of lowconcentration, modified bromfenac ophthalmic solution dosed once
daily compared to placebo following cataract surgery in 2 integrated
clinical trials.
Methods: Subjects undergoing unilateral cataract surgery
(phacoemulsification or extracapsular cataract extraction) with
posterior chamber IOL implantation were randomized to either lowconcentration, modified bromfenac ophthalmic solution (n=222) or
placebo (n=218). Once daily dosing began 1 day before cataract
surgery, continued on the day of surgery, and through post-surgery
Day 14. The proportion of subjects with trace anterior chamber
inflammation, defined as a Summed Ocular Inflammation Score
(SOIS) of 0-0.5 (0-5 cells in the anterior chamber and flare grade of
0), was assessed at Days 1, 3, 8, and 15. Safety was assessed by the
incidence and frequency of ocular and systemic adverse events, and
ophthalmological evaluations (visual acuity, slit lamp examination,
intraocular pressure, and dilated funduscopic examination). Statistical
significance was determined using a Fisher’s exact test.
Results: In the intent-to-treat population, subjects had a mean age of
68.0 years, were predominantly Caucasian (74.8%), and included a
higher percentage of female subjects (65.2%). Baseline
characteristics were similar across treatment groups. A significantly
higher proportion of subjects achieved trace ocular inflammation in
the bromfenac group compared to placebo as early as Day 3 (27.9%
vs. 13.8%, p=0.0008), continued on Day 8 (55.4% vs. 24.3%, p <
0.0001), and through Day 15 (71.2% vs. 39.4%, p < 0.0001).
Compared to placebo, low-concentration, modified bromfenac
ophthalmic solution dosed once daily produced a lower overall
incidence of ocular adverse events.
Conclusions: Low concentration, modified bromfenac ophthalmic
solution dosed once daily effectively and safely reduced ocular
inflammation associated with cataract surgery.
Commercial Relationships: James A. Gow, Bausch & Lomb (E);
James D. Boyce, None; Harvey J. Reiser, Alcon (F), Alcon (C),
Bausch & Lomb (F), Insite (F); Robert Berry, None; Jung T. Dao,
None; Simon P. Chandler, Bausch and Lomb (E)
142 Drug Delivery I
Sunday, May 05, 2013 1:00 PM-2:45 PM
Presentation Time: 1:00 PM - 2:45 PM
Bioresorbable materials for ophthalmic devices
Ananth Iyer, Rick Gallagher. RT&D, DSM, Berkeley, CA.
Purpose: We report the unique properties of novel miktoarm
bioresorbable poly(ether-ester) urethane (PEEsU) accessed using
commercially available α-methoxy-ω-diol poly(ethylene glycol)
(Ymer N120) in comparison with linear PEEsU starting from
poly(ethylene glycol) (PEG diol)with potential applications as ocular
drug delivery systems, adhesives and sealants, scleral buckles,
punctal plugs, and tissue engineering applications.
Methods: Candidate PEEsU having ethylene oxide to L-lactide
(EO/LLA) ratios of 0.5, 2 and 4 were prepared using a one-pot twostep synthesis. In the first step, polyester diols were synthesized by
the ring opening polymerization (ROP) of L-lactide with the
appropriate diol using stannous octoate as the catalyst. In the second
step, the resultant polyester diol was chain extended using
hexamethylene diisocyanate (HDI) to yield the PEEsU. The
poly(ether-ester) molecular weight buildup was verified using nuclear
magnetic resonance spectroscopy (NMR). The different PEEsU were
characterized by gel permeation chromatography (GPC) and
differential scanning calorimetry (DSC). Tensile properties were
measured on dry films per ASTM D1708. The hydrolytic degradation
of these polymers were followed in a pH=7.4 phosphate buffer
solution at 37°C using protocols set in ISO10993-13.
Results: Tensile load at break for the Ymer N120 based PEEsU
ranged from 18.06N for EO/LLA of 0.5 to 0.03N for EO/LLA of 2.
PEG diol (PEG 1000) based PEEsU for EO/LLA of 0.5 was brittle
and film tensile properties were not measurable. The hydrolytic
degradation studies were followed by GPC molecular weight drop
method. Thus, during a 4 week incubation period, weight average
molecular weight (Mw) of PEEsU with EO/LLA=0.5 dropped by
25% while it dropped by 70% for EO/LLA = 2 during the same time
period. PEEsU with EO/LLA=4 completely degraded in three days.
Additionally thermal profiles of the representative PEEsU were
measured using DSC.
Conclusions: We demonstrate the successful synthesis of pendant
MPEG based hydrolytically degradable PEEsU. Different forms of
the polymer such as films, gels and viscous liquids were made by
controlling the EO/LLA ratio of the polyester segments that were
then chain extended with HDI. By controlling the EO/LA ratio in the
PEEsU, the degradation times of the polymer was manipulated from
days to weeks and may be further modified pending application
requirements.
Commercial Relationships: Ananth Iyer, DSM (E); Rick
Gallagher, DSM Biomedical Inc. (E)
PLGA capsulated porous silicon particles for sustained
intravitreal delivery of daunorubicin
Kaihui Nan1, Feiyan Ma1, Huiyuan Hou1, William R. Freeman1,
Michael J. Sailor2, 3, Lingyun Cheng1. 1Department of
Ophthalmology, Jacobs Retina Center at University of California,
San Diego, CA; 2Department of Chemistry and Biochemistry,
University of California, San Diego, CA; 3Department of
Bioengineering, University of California, San Diego, CA.
Purpose: Daunorubicin (DNR) is a FDA approved antiproliferation
agent which has been used to treat proliferative vitreoretinopathy
(PVR). However its narrow therapeutic window and short vitreous
half-life limit its intraocular application. We have shown that
adsorption loading of DNR into porous silicon (pSi) particles can
provide a 2-week release in rabbit vitreous. However, DNR release
was still fast and caused retinal toxicity. In the current study we
aimed to develop a better controlled vitreous release system using
PLGA capsulizing DNR loaded pSi particles.
Methods: Fresh etched porous silicon film was sonicated to produce
pSi microparticles. pSi particles were oxidized at 800 degree C. DNR
was absorbed into the pSi particles by soaking 10 mg pSi in 300 µL
of 5mg/mL DRN solution overnight at room temperature. The drug
loading was quantitated by thermogravimetric analysis (TGA). The
DNR loaded pSi particles were divided into two groups: group 1 with
PLGA (poly (lactic-co-glycolic acid) coating and group 2 without.
For PLGA coating, DRN loaded pSi particles were allocated into
10% PLGA dichloromethane solution and vortex for 20 min. The
mixture was dispersed into 2% PVA (polyvinyl alcohol) aqueous
solution for evaporation of dichloromethane. PLGA coated particles
were characterized under a light microscopy for PLGA capsulation.
The particles with or without PLGA were allocated each into three
closed vial with 1.5 mL of DPBS and incubated under 37°C on a
mini labroller. At designated time points, 1mL supernatant was
collected and the same amount of DPBS was added back into the
vial. DNR in the supernatant was quantitated using a fluorescence
spectrophotometer.
Results: The DNR loading into pSi particles was determined to be
32.99 µg/mg. Light microscopy showed 80% pSi particles were
capsulated by PLGA and the non-capsulated pSi particles had a mean
diagonal size of 75 µm (median 68.4 µm). DNR release from pSi
without PLGA capsulation demonstrated a predicted peak
concentration of 7200 ng/mL while only 1200 ng/mL for PLGA
capsulated pSi. The DNR release profile from pSi particles was
typical first-order kinetics while a sustained release mode was
achieved through PLGA capsulation (Figure).
Conclusions: PLGA capsulation can slow down DNR release from
pSi particles and reduce the initial burst release as well as improve
the drug release kinetics optimized toward intravitreal drug delivery
application.
Commercial Relationships: Kaihui Nan, None; Feiyan Ma, None;
Huiyuan Hou, None; William R. Freeman, OD-OS, Inc. (C);
Michael J. Sailor, Spinnaker Biosciences (I); Lingyun Cheng,
Spinnaker Biosciences (C)
Support: NIH Grant EY 020617 and NSF Grant DMR-1210417
Mechanism of ultrasound-mediated transscleral delivery:
temporary alteration of scleral structure increases permeability
of macromolecules
Ying Chau1, 3, Wai-Leung Suen1, Jun Jiang2, Yan Zeng2, Jianan Qu2,
3 1
. Chemical and Biomolecular Engineering, The Hong Kong
University of Science and Technology, Hong Kong, Hong Kong;
2
Department of Electronic and Computer Engineering, The Hong
Kong University of Science and Technology, Hong Kong, Hong
Kong; 3Division of Biomedical Engineering, The Hong Kong
University of Science and Technology, Hong Kong, Hong Kong.
Purpose: We have proposed the use of low frequency ultrasound as a
non-invasive approach to modulate the ocular barriers for transscleral
drug delivery to the posterior segment of the eye. Using this
approach, we observed significantly enhanced delivery in vivo. We
hypothesize that ultrasound increases the porosity of scleral fiber
network to allow improved diffusion of macromolecules. Here, we
aim to understand the effect of ultrasound on sclera, the first barrier
in the transscleral route of delivery.
Methods: The penetration of FITC-dextran of 70kDa through rabbit
sclera was measured ex vivo with and without ultrasound. Sonication
was applied directly above sclera at pre-designated frequency and
intensity. Diffusivity was calculated by fitting the penetration profile,
obtained by fluorescent microscopy of cryosectioned sclera, with 1-D
diffusion equation. The collagen network structure of sclera was
visualized dye-free using two-photon excitation microscopy (TPEM).
Pore size of sclera was estimated by Renkin’s restricted diffusion
model and textual analysis of TPEM images.
Results: Dextran penetrated deeper into ultrasound-treated sclera,
confirming that the barrier function of the sclera was weakened by
sonication (Figure 1a). The transscleral penetration distance increases
with decreasing frequency, suggesting the role of cavitation.
Diffusivity of dextran increased up to 8 times in sclera after low
frequency sonication. The enhancement was temporary, with the
scleral permeability restored in 3 hours (Figure 1b). TPEM image
revealed that ultrasound disrupted the ordered alignment of collagen
and increased the scleral pore size (Figure 2), agreeing with the
prediction by Renkin’s model.
Conclusions: Low frequency ultrasound alters the collagen network
structure of sclera to increase the porosity, thereby enhancing the
diffusion of macromolecules through the outmost barrier in the
transscleral route of delivery.
Figure 1. a) Penetration distance of 70 kDa dextran in 15 minutes in
sclera immediately after sonication at the indicated frequency (n=3).
b) Restoration of scleral permeability post ultrasound. Penetration
distance of 70 kDa in 15 minutes in sclera at varying time lag after
sonication at 40 kHz is shown (n=3).
Figure 2. Two-photon excitation microscopy image (100 μm x 100
μm) of sclera before and after ultrasound treatment at 40 kHz.
Commercial Relationships: Ying Chau, None; Wai-Leung Suen,
None; Jun Jiang, None; Yan Zeng, None; Jianan Qu, None
Support: Innovation and Technology Fund and Research Grants
Council of Hong Kong SAR
An aqueous clear rapamycin topical drop for retinal delivery
Kishore Cholkar1, Sriram Gunda2, Ravinder Earla1, Ashim K. Mitra1.
1
University of Missouri Kansas City, Kansas City, MO; 2PPD, Inc,
Richmond, VA.
Purpose: The objectives of this study are (i) to develop an aqueous,
clear mixed nanomicellar formulation (MNF) of rapamycin,
optimization and characterization, (ii) to determine MNF cytotoxicity
and transport across ocular cells and (iii) to determine in vivo ocular
tissue distribution of optimized novel rapamycin loaded MNF post
topical drop administration into rabbit cul-de-sac.
Methods: Polymers such as Vitamin E TPGS (D-alpha-tocopheryl
polyethylene glycol 1000 succinate), octoxynol-40 and rapamycin are
mixed in varying ratios to obtain an optimized formulation. The
novel MNF was prepared by solvent evaporation technique. In vitro
release studies were conducted with a dialysis bag method and
cytotoxicity and transport studies were conducted on HCEC and
ARPE-19 cells. In vivo studies were conducted in New Zealand
(NZW) male white rabbits.
Results: Rapamycin was loaded into MNF to generate an overall
loading of 2 and 4mg/mL. Optimized formulation was characterized
for its improvement in drug loading, entrapment efficiency, size,
polydispersity, surface charge, morphology and rapamycin release.
Optimized MNF showed a size range of 28-35 nm and encapsulation
percentage > 95% respectively. Absence of free or unentrapped
rapamycin in the MNF was confirmed by proton NMR spectroscopy.
The MNF drug release was found to be sustained. Cytotoxicity
studies on HCEC and ARPE-19 cells treated with placebo and
rapamycin loaded optimal MNF’s showed no significant difference in
cell survival relative to untreated (medium) cells. Transport studies
showed higher rapamycin permeability. In vivo rapamycin ocular
tissue distribution studies show higher rapamycin concentrations
(362.35 ± 56.17 ng/g tissue) in back of the eye tissues (retinachoroid) with no rapamycin detected in vitreous humor (VH).
Conclusions: An optimized and characterized clear aqueous MNF of
rapamycin was prepared. The novel MNF had no cytotoxic effect on
HCEC and ARPE-19 cells and had higher permeability. In vivo
ocular tissue distribution studies show that therapeutic levels of
rapamycin were observed in retina-choroid with no rapamycin in VH,
post topical drop administration. Results indicate rapamycin loaded
MNF follows conjunctival-scleral pathway to reach back of the eye
tissues (retina-choroid).
Commercial Relationships: Kishore Cholkar, None; Sriram
Gunda, None; Ravinder Earla, None; Ashim K. Mitra, None
Cell-Encapsulated Device for Intraocular Delivery of Glial-Cell
Derived Neurotrophic Factor (GDNF)
Francisca SY Wong1, Calvin CH Wong1, Barbara P. Chan2, Amy C.
Lo1, 3. 1Ophthalmology, The University of Hong Kong, Hong Kong,
Hong Kong; 2Mechanical Engineering, The University of Hong
Kong, Hong Kong, Hong Kong; 3Research Center of Heart, Brain,
Hormone and Healthy Aging, The University of Hong Kong, Hong
Kong, Hong Kong.
Purpose: While GDNF is able to exert neuroprotective effects on
photoreceptor cells, successful administration of such therapeutic
protein has been hindered by short half-life and the lack of a
sustained drug delivery method. A cell-based immunoisolated
intraocular drug delivery device for continuous GDNF release was
designed. The photoreceptor rescuing effects in a rat model with
inherited retinal degeneration were examined after the implantation
of the gel device.
Methods: HEK293 cells that overexpress GDNF were encapsulated
in a composite matrix constituted of 2mg/ml collagen and 1%
alginate. The collagen-alginate gel device was intravitreally injected
into Royal College of Surgeons rats on post-natal day 28 (P28). Rats
were randomly divided into four groups: sham, un-operated, vehicle
control, and treatment groups. Vitreous contents were collected for
GDNF content assessment on P35 and P42. Enucleation was carried
out on P56 for histological evaluations. H&E stained paraffin
sections of the retina were examined for the degree of morphological
rescue via quantifying the outer nuclear layer photoreceptors at
various retinal regions.
Results: Significant amount of GDNF was released into the vitreous
after 7 and 14 days of device implantation. Outer nuclear layer
(ONL) linings in the treatment group were better aligned at the
central retinal regions when compared to the control groups. Increase
in mean ONL cell counts were observed across the whole retina, in
particularly, the center of the inferior retina.
Conclusions: Cell growth, proliferation and sustained release of
GDNF were achieved through implanting the cell-encapsulating
collagen-alginate gel device, resulting in morphological rescue of
photoreceptor cells in vivo. This system could potentially be applied
as a sustained drug release platform of GDNF and/or other
therapeutic proteins in various ocular conditions involving similar
pathological phenotypes.
Commercial Relationships: Francisca SY Wong, None; Calvin
CH Wong, None; Barbara P. Chan, The University of Hong Kong
(P); Amy C. Lo, None
Support: Seed Funding from The University of Hong Kong
Long-lasting eye drop delivery platform for targeted ocular
delivery applications
Frank Gu1, 2, Shengyan Liu1, Lyndon W. Jones2. 1Chemical
Engineering, University of Waterloo, Waterloo, ON, Canada;
2
Optometry and Vision Science, University of Waterloo, Waterloo,
ON, Canada.
Purpose: Common topical formulations, such as eye drops or
ointments, suffer from low ocular bioavailability due to rapid
drainage through the naso-lacrimal duct, nearly constant dilution by
tear turnover, and low drug permeability across the corneal
epithelium. As a result, topical formulations are normally
administered multiple times daily in order to achieve therapeutic
efficacy, resulting in a higher potential for side effects and lower
patient compliance. Our study aims to design nano-scaled drug
carriers to overcome the rapid clearance of current eye drop solutions
thereby improving drug retention on the corneal surface.
Methods: NPs composed of poly(D,L-lactide)-b-Dextran (PLA-Dex)
surface functionalized with a mucoadhesive ligand, phenylboronic
acid (PBA), were developed as drug carriers. Using Cyclosporine A
(CycA) as a model drug, CycA molecules were encapsulated within
PLA-Dex using emulsification. Controlled drug release and
biocompatibility studies were performed in vitro and in vivo.
Results: We showed that the nanoparticle carrier can be used to
release CycA in vitro and in vivo. The average size of NPs were
found to be in the range of 25 and 28 nm. The NPs showed
encapsulated up to 13.7 wt% CycA and exhibited sustained release
for up to 5 days in vitro at a clinically relevant dose. We fine-tuned
the PBA density on the NP surface to maximize the mucin-NP
interaction without compromising the particle stability in vitro. We
showed that the NP formulation did not significantly affect the
transparency or the solution viscosity, which improves patient
compliance. The surfaces of the nanoparticles have a defined number
of ligands capable of targeting the corneal surface, allowing drugs
encapsulated in the particles to effectively circumvent the tear
turnover mechanism, and significantly improving their corneal
retention.
Conclusions: The surface of the nanoparticle formed from a linear
block copolymer poly(D,L-lactide)-b-Dextran was modified with
PBA to form a mucoadhesive nanoparticle for topical ocular drug
delivery application. The simplicity of NP design suggests it can
deliver a wide range of bioactive agents including both hydrophilic
and hydrophobic compounds. These results suggest the potential of
our nanoparticle approach can be used as a platform technology to
provide long-lasting delivery of therapeutic agents to treat anterior
eye diseases.
Commercial Relationships: Frank Gu, None; Shengyan Liu,
None; Lyndon W. Jones, Alcon (F), Alcon (R), Allergan (F), Abbott
Medical Optics (R), Bausch & Lomb (R), Ciba Vision (F), Ciba
Vision (R), CooperVision (F), Johnson & Johnson (F), Johnson &
Johnson (R)
Support: NSERC Strategic Network
Hyaluronic acid based tablet for slow release of ilomastat in
glaucoma filtration surgery
Abeer Mohamed Ahmed2, 1, Alastair Lockwood2, 1, Steve Brocchini2,
Peng T. Khaw1. 1National Institute for Health Research (NIHR)
Biomedical Research Centre at Moorfields Eye Hospital NHS
Foundation Trust and UCL Institute of Ophthalmology, London,
United Kingdom; 2UCL School of Pharmacy, London, United
Kingdom.
Purpose: Ilomastat is a matrix metalloproteinase inhibitor (MMPi)
that has been shown to inhibit fibrosis after glaucoma filtration
surgery (GFS) in a rabbit model of ocular fibrosis. To reduce scarring
and fibrosis following glaucoma surgery, a sustained dosage form
would be advantageous that allows a prolonged local concentration of
ilomastat to be maintained within the subconjunctival space.
Hyaluronic acid (HA) is used in ocular medicine. HA was examined
as a matrix to fabricate a small tablet for subconjunctival
implantation after GFS.
Methods: Ilomastat (1.0 mg) was dissolved in butanol (40% w/v).
An aqueous solution of hyaluronic acid (3.0 mg) was prepared. The
ilomastat solutions was then added to the aqueous polymer solution
and gently mixed overnight. The mixture was freeze dried. The
lyophilised powder was pressed into a tablet using 3 mm punch and
die. The release of ilomastat from the tablet in phosphate buffered
saline (PH 7.4) was determined at flow rate of 2.0 µL at 35.0 °C in a
flow rig. The concentration of the released ilomastat was measured
using by HPLC at 280 nm.
Results: A small tablet designed for ocular implantation was
successfully fabricated with a dispersion of ilomastat in both linear
and cross linked HA matrices. The weight of the tablet was in the
range of 4.5-6.5 mg. Fabrication of the ilomastat tablet using linear
HA (Healon® GV) resulted in a sustained release of ilomastat over
11 days with a total ilomastat release of 83.2±1.8% (maximum
ilomastat concentration of 246.5±32.3 µM at tmax of 31.0±18.0 h).
The release of ilomastat was more prolonged when cross linked HA
(Healaflow®) was used. A total of 101.48±4.48% was released after
26 days with a maximum concentration of 212.01±23.31 µM (t max
4.3 h). The concentration of the released ilomastat was within the
therapeutic range (10-100 µM). The total release of ilomastat from a
mixture of linear and cross linked HA (1:1) was 63.3 % (maximum
concentration of ilomastat was 199.5 µM at tmax of 33.1 h) after 14
days. The release of ilomastat was faster when more linear HA was
incorporated in the dispersion mixture.
Conclusions: Sustained release of ilomastat was achieved by its
dispersion in cross linked HA matrix up to 28 days. The prolonged
release of ilomastat from a dispersion of cross linked HA may be
suitable for a successful sub-conjunctiva implant for improvement of
the outcome of glaucoma filtration surgery.
Commercial Relationships: Abeer Mohamed Ahmed, Steve
Brocchini (WO09/063222) (P), Peng Khaw (WO09/063222) (P);
Alastair Lockwood, None; Steve Brocchini, None; Peng T. Khaw,
University College Moorfields (P)
Support: Medical research council G801650, Fight for sight, NIHR
Moorfields Biomedical Research Centre, Freemasons Grand Charity
and Helen Hamlyn Trust
NOVEL AQUEOUS NANOMICELLAR FORMULATIONS
CONTAINING FLUOCINOLONE ACETONIDE FOR THE
TREATMENT OF POSTERIOR UVEITIS
Sujay Shah, Asha Patel, Sulabh Patel, Dhananjay Pal, Ashim K.
Mitra. Division of Pharmaceutical Sciences, University of Missouri
Kansas City, Kansas City, MO.
Purpose: Diseases like posterior uveitis affect the posterior segment
of the eye and can cause partial or total vision loss. Current forms of
treatments require administration of high doses to overcome static
and dynamic barriers present in the eye. Fluocinolone acetonide (FA)
is a highly potent glucocorticoid therapeutic with anti-inflammatory
properties. Current therapy involves surgical placement of intravitreal implant of FA. This method has several disadvantages like
retinal detachment, redness, pain and discomfort - all of which can
lead to patient non-compliance. Therefore, the optimum strategy is to
develop eye drops of FA. Since FA is very poorly soluble in water, it
is difficult to prepare high concentration clear aqueous solution eye
drops of FA. Therefore, our aim in this study is to develop aqueous
nanomicellar formulations containing FA. Vitamin E TPGS (1K), a
surfactant polymer has been utilized to prepare nanomicelles. We
also synthesized polymer with higher molecular weight of PEG
(2000) (2K). This modified polymer has a very low critical micellar
concentration (CMC) which might improve the stability upon tear
dilution in eye.
Methods: Modified TPGS was synthesized by conjugation D-αtocopheryl succinate and mPEG having molecular weight of 2000.
The product was purified by dialysis method. CMC values were
calculated using standard pyrene method. Micelles were prepared by
thin film hydration technique. Box-Behnken design was used to
optimize the formulation to achieve maximum entrapment and
solubility of drug. Size and zeta potential of micelles was measured.
Cytotoxicity studies were conducted on corneal and retinal cells.
Results: 2K polymer was successfully synthesized with a yield of
65%. The CMC value obtained was 7.28μg/ml which is significantly
less than commercially available TPGS 1K. Results showed that
solubility of FA maybe increased up to 26 times with newly
synthesized 2K polymer. Entrapment efficiency greater than 90%
was achieved with 2K polymers. Nanomicelles exhibited very small
size (<20nm) and narrow size distribution with both polymers. Cell
viability on both corneal (HCEC) and retinal (D407) cells lines was
comparable to control.
Conclusions: We have successfully optimized the formulation with
respect to entrapment efficiency, aqueous solubility and no
cytotoxicity. This could be used as a potential topical eye drop
treatment for posterior uveitis.
Commercial Relationships: Sujay Shah, None; Asha Patel, None;
Sulabh Patel, None; Dhananjay Pal, None; Ashim K. Mitra, None
Support: NIH R01EY09171-16 and NIH R01EY010659-14
Topically Administered Mouse IgG1 Accumulates In the Rat
Optic Nerve And Retina
Stacy Hu, Steven B. Koevary. Biomedical Sciences and Disease, New
England College of Optometry, Boston, MA.
Purpose: Frequent intravitreal injections of monoclonal anti-VEGF
agents Lucentis and Avastin were shown to reverse the effects of wet
AMD. These injections, however, predispose patients to
endophthalmitis and stroke, among other ocular and systemic adverse
effects. Such complications, as well as patient discomfort, would be
potentially reduced or even eliminated by the delivery of these drugs
in mist or drop form. The purpose of this study was to obtain pilot
data regarding the feasibility of delivering potentially therapeutic
large, hydrophilic antibodies to the posterior pole by eye drop
application.
Methods: Female Lewis rats received a single 25 mg/10 µL topical
dose of whole mouse monoclonal IgG1 antibody in PBS without a
permeation enhancer into one eye; control rats were left untreated.
The concentration of IgG1 was determined in retinal and optic nerve
homogenates 10 to 20 minutes later using an ELISA; tissues were
homogenized in lysis buffer containing protease inhibitors. Data are
expressed as the mean concentration±SEM and were analyzed using
the Kruskal-Wallis test. Serum IgG1 levels were also determined.
Results: Mouse IgG1 levels in the optic nerve were elevated 10
(n=16) and 20 (n=15) minutes after application (11.2±1.8 and 5.8±0.8
ng/mL of clarified homogenate, respectively) relative to control rats
(n=15; 3.8±0.6 ng/mL, p=0.0004 and 0.062, respectively). Mouse
IgG1 levels in the retina were significantly elevated after an average
of 15 minutes (n=31; 3.1±0.6 ng/µg tissue) relative to control rats
(n=15; 1.3±0.2 ng/μg, p=0.02). Serum IgG1 levels 10 and 20 minutes
after application (both 0.3±0.1 ng/mL) were insignificantly different
from levels seen in control rats (0.2±0.2 ng/mL; p=0.52).
Conclusions: Mouse IgG1, applied topically in rats, accumulated in
the retina and optic nerve. As in our previous studies with insulin,
leptin, and GDNF, IgG1 levels peaked in the optic nerve earlier than
in the retina, supporting the hypothesis that topically applied
compounds traverse the sclera to reach the posterior pole. Notably,
IgG1 application did not result in appreciable uptake into peripheral
blood, suggesting that topical treatment with anti-VEGF antibodies
would not contribute to the development of systemic complications.
Commercial Relationships: Stacy Hu, None; Steven B. Koevary,
None
Support: Massachusetts Lions Eye Research Fund
A Pilot Safety Assessment of a Topical Ocular Device
(TODDD™) Intended for Sustained Drug Delivery
Charles D. Leahy1, 4, Rodney Gutner2, Whittney Varney2, Jack
Rulander4, Stephen Johnston4, Francis S. Lai4, Kathryn S. Crawford3,
Roger Albright5, Jeanne Y. Ellis1, 4, Edward J. Ellis1, 4. 1Vista
Scientific LLC, Andover, MA; 2New England College of Optometry,
Boston, MA; 3PharmOcu, Andover, MA; 4University of
Massachusetts Lowell-Massachusetts Medical Device Development
Center, Lowell, MA; 5Foresight Regulatory Strategies, Inc.,
Wilmington, MA.
Purpose: To establish safety, comfort and retention of TODDD for
continuous wear studies. Although intended to deliver drug while
worn on the superior sclera 24/7 for several months at a time, in this
early stage human study it was without drug and worn 12-15 hr/day
for 1 week.
Methods: An open label study evaluating safety, retention and
subjective symptoms of comfort of the TODDD was conducted at the
New England College of Optometry. At Visit 1 device was placed on
one eye and subjects were instructed on insertion and removal
procedures. They then wore the single device daily (12-15 hr) for 7±2
days until Visit 2. Safety evaluations on each visit included VA,
keratometry, and slit-lamp grading and any fluorescein and lissamine
green staining using the Brien Holden V. I. Grading Scales. Fit,
stability and integrity of the device were observed. Subjects reported
on comfort, positioning, and ejection of the device at each visit, and
each evening after device removal via electronic diary.
Results: 12 (11 females and 1 male, age 24-26) of 14 screened
subjects were dispensed the device and all 12 completed the study.
There were no significant adverse events or safety findings. Slit lamp
grades after 1-4 hours and 1 week of wear showed increases of 0 or 1
across all categories in either eye, and grade 0 or 1 conjunctival
staining was reported in all subjects at all examinations, but for one
subject, who had grade 3 lissamine staining of the conjunctiva at
Visit 2. There were no lid gradings above 2, and no corneal findings
above grade 1 in any subject. In the majority of subjects at all time
points, the device positioned on the superior sclera without excess
rotation, completely under the lid, with good stability and movement.
The devices remained clean and intact. At Visit 1 only mildly
decreased ocular comfort associated with some awareness of device
was reported. At Visit 2 all but 1 subject reported comfortable wear
always or most of the time. No devices spontaneously ejected from
the eye or were lost. Among prior contact lens wearers, most subjects
considered the device handling “comparable to contact lens”.
Conclusions: This preliminary study indicates that the device is welltolerated in this subject population. Retention was demonstrated and
the device produced no safety concerns after 7 days of daily wear.
Continuous wear studies are planned.
Commercial Relationships: Charles D. Leahy, Vista Scientific
LLC (I), Vista Scientific LLC (P); Rodney Gutner, Vista Scientific
LLC (F); Whittney Varney, None; Jack Rulander, Vista Scientific
LLC (F); Stephen Johnston, Vista Scientific (F); Francis S. Lai,
Vista Scientific (F); Kathryn S. Crawford, Vista Scientific, Inc. (C);
Roger Albright, Johnson & Johnson Vision Care Inc. (C),
CooperVision Inc. (C), Menicon Ltd. (C), Semprus Biosciences (C),
VISTA Scientific, LLC (C); Jeanne Y. Ellis, Vista Scientific (I),
Vista Scientific (P); Edward J. Ellis, Vista Scientific LLC (I), Vista
Scientific LLC (P)
Support: NIH Grant EY013479
Precisely Engineered Biodegradable Intraocular Implants for the
Sustained Release of Dexamethasone
Andres Garcia, Janet Tully, Benjamin Maynor, Benjamin Yerxa.
Liquidia Technologies, Durham, NC.
Purpose: The ability to fabricate biodegradable intraocular implants
with uniform size, shape and dose for the sustained delivery of
actives in multiple regions of the eye has proven elusive with current
technologies. The acceptance of intravitreal implants for the localized
treatment of multiple back-of-the-eye conditions have paved the way
for the development of a new generation of smaller intraocular
implants in the anatomically and clinically desirable, yet “hard-tomanufacture” size range of 100μm to 1,000μm. The ability to
reproducibly fabricate implants in this size range opens up a window
of opportunities for the injection and localization of implants against
multiple target tissues of the inner eye where greater spatial
constraints may exist.
Methods: We report the ability to precisely fabricate 150μm x
150μm x 500μm intraocular implants comprised of a blend of
micronized dexamethasone and poly(lactic-co-glycolic) acid (PLGA)
for the tunable release of active using the PRINT technology.
Physicochemical characterization of the implants was performed to
evaluate the overall mass uniformity range, dexamethasone content
uniformity across individual implants, and the in-vitro release of
dexamethasone from the implants in 1X PBS at 37°C.
Results: Liquidia’s PRINT technology unique ability to impart
control over size and shape (Figure 1A) allowed for the fabrication of
dexamethasone/PLGA implants (Figure 2A) with a high degree of
mass uniformity (14μg, ±1μg) and dexamethasone content (2.2μg
±0.2μg). Furthermore, PRINT implants enabled for the sustained
release of dexamethasone over therapeutically relevant timelines,
with over 40% of the initial cargo retained in the implants after 35
days in 1X PBS at 37°C.
Conclusions: The PRINT technology uniquely allows for the
fabrication of intraocular implants with uniform size, shape and dose.
We demonstrated the ability to fabricate dexamethasone/PLGA
intraocular implants in the desirable size range of 100μm to 1,000μm
for sustained release applications where anatomical constraints may
call for uniquely engineered implants.
were 6.99, 7.06, 2.51 and 2.75 days in vitrectomized and nonvitrectomized eyes, respectively.
Conclusions: Vitrectomy did not substantially affect
pharmacokinetic properties or chorioretinal concentrations of
intravitreally injected bevacizumab or ranibizumab in rabbit eyes.
Commercial Relationships: Jeeyun Ahn, None; Sunyoung Park,
None; Hyuncheol Kim, None; Ji Hyun Park, None; Ji Yeon Park,
None; Duck Jin Hwang, None; Seong Joon Ahn, None; Yong-Kyu
Kim, None; Kyu Hyung Park, None; Se Joon Woo, None
Support: National Research Foundation of Korea (NRF) funded by
the Ministry of Education, Science and Technology (No. 20110009606)
Figure 1: A) SEM image of 150μm x 150μm x 500μm
dexamethasone/PLGA PRNT intraocular implants, B) Close-up view
of the implant’s surface, showing micronized dexamethasone
embedded in PLGA matrix, C) In vitro dexamethasone release profile
from PRINT implants over 35 days.
Commercial Relationships: Andres Garcia, Liquidia Technologies
(E), Liquidia Technologies (I); Janet Tully, Liquidia Technologies
(E); Benjamin Maynor, Liquidia Technologies (E), Liquidia
Technologies (I); Benjamin Yerxa, Liquidia (E)
Intraocular pharmacokinetics of bevacizumab and ranibizumab
in vitrectomized versus non-vitrectomized rabbit eyes
Jeeyun Ahn1, 2, Sunyoung Park3, Hyuncheol Kim3, 4, Ji Hyun Park5, Ji
Yeon Park5, Duck Jin Hwang1, 5, Seong Joon Ahn1, 5, Yong-Kyu Kim1,
5
, Kyu Hyung Park1, 5, Se Joon Woo1, 5. 1Department of
Ophthalmology, Seoul National University College of Medicine,
Seoul, Republic of Korea; 2Department of Ophthalmology, Seoul
Metropolitan Government Seoul National University Boramae
Medical Center, Seoul, Republic of Korea; 3Department of Chemical
& Biomolecular Engineering, Sogang University, Seoul, Republic of
Korea; 4Interdisciplinary Program of integrated Biotechnology,
Sogang University, Seoul, Republic of Korea; 5Department of
Ophthalmology, Seoul National University Bundang Hospital,
Seongnam, Republic of Korea.
Purpose: To analyze intraocular pharmacokinetic properties of
bevacizumab and ranibizumab in vitrectomized and nonvitrectomized rabbit eyes.
Methods: Twenty-five-gauge pars plana vitrectomy without
lensectomy was performed in 35 rabbit eyes and 36 nonvitrectomized rabbit eyes served as control. Intravitreal injection of
1.25mg/0.05mL bevacizumab was performed in 17 vitrectomized
(group 1-1) and 18 non-vitrectomized (group 1-2) eyes, respectively.
Intravitreal injection of 0.25mg/0.025mL ranibizumab was performed
in 18 vitrectomized (group 2-1) and 18 non-vitrectomized (group 2-2)
eyes, respectively. Eyes were enucleated at 1 hour, 1, 2, 5, 14 and 30
days after the intravitreal injections and frozen at -80°C.
Bevacizumab and ranibizumab concentrations in the vitreous and
aqueous humor, as well as the retina/choroid, were determined using
direct enzyme-linked immunosorbent assay.
Results: Vitreous clearance of bevacizumab and ranibizumab
showed distinct patterns, consisting of 2 phases and 1 phase,
respectively. The vitreous half-life of bevacizumab and ranibizumab
Key considerations for choice of syringes for delivery of
intravitreal therapies in enhancing safety and efficacy of
therapies injected in the eye
GAUTAM SHETTY. Specialized Drug Delivery, Unilife, YORK, PA.
Purpose: Inaccuracy of dose injected into the vitreous can result in
overdosing (potentially causing increased intraocular pressure) or
underdosing (potentially reducing drug efficacy and/or increasing
drug injection frequency); this introduces unpredictability and
potential variability in clinical outcomes for treatments delivered to
treat back-of-eye disorders. Inaccuracy of dose in inherent in syringes
used currently in the delivery of injectable drugs into the vitreous of
the eye for the treatment of retinal disorders. The opportunity to
improve clinical outcomes by reducing the dose volume from the
current 50uL dose volume administered in case of anti-VEGF
treatments would further exacerbate the inaccuracy problem. Also,
syringes used to deliver off-label bevacizumab are not designed to
store the same for an extended period of time. There are currently no
guidelines from PQRI on the extractable content for drugs to be
injected inside the eye. In absence of any guidelines, understanding
the extractable content of syringes is a critical consideration in the
safety of off-label bevacizumab in the treatment of retinal disorders.
Methods: Key determinants of inaccuracy for delivery of microliter
size dose were identified in tuberculin syringes, that are currently
used to deliver intravitreal drugs. A device has been developed to
mitigate factors causing inaccuracy of a microliter sized dose. Dose
accuracy was assessed using gravimetric technique.
Extractable compounds in tuberculin and insulin syringes using water
and isopropanol as extraction media was determined by
HPLC/PDA/MS analysis.
Results: Dose accuracy of 10uL dose within 2% was shown with
coefficient of variation of 3%. Extractable compounds (volatile and
non-volatile) were determined.
Conclusions: It is possible to improve accuracy of dose injected into
the eye. With tools available, dose volumes injected in the eye can be
reduced further to further mitigate potential risk of increase in
intraocular pressure. Further research to determine toxicity of
extractable compounds from syringes used to store off-label
becaizumab are necessary to fully understand any potential risk posed
by the same.
Commercial Relationships: GAUTAM SHETTY, Unilife (E),
Unilife (P)
Freeze Drying to Develop a Bevacizumab-based Tablet for
Ocular Implantation
Garima Sharma1, 2, Ashkan Khalili2, Sahar Awwad1, 2, Kiran Malik3,
Paul Matejtschuk3, Simon Gaisford1, Steve Brocchini1, 2, Peng T.
Khaw2. 1Pharmaceutics, UCL School of Pharmacy, London, United
Kingdom; 2National Institute for Health Research (NIHR)
Biomedical Research Centre at Moorfields Eye Hospital NHS
Foundation Trust and UCL Institute of Ophthalmology, London,
United Kingdom; 3National Institute for Biological Standards and
Control, Health Protection Agency, Potters Bar, United Kingdom.
Purpose: Bevacizumab has shown the potential to control scarring
when administered into the subconjunctival space following
glaucoma filtration surgery (GFS). To overcome the problem of rapid
clearance, a solid dosage implantable tablet form of this antibody that
provides prolonged release in the bleb is being developed. In an effort
to optimise dose, we are examining each step used for tablet
fabrication. As is often the case for any protein pharmaceutical
presented in a solid reconstitutable form, a critical process step is
lyophilisation, which can cause aggregation of the protein with loss
of activity. The aim of this study is to characterise the lyophilisation
of the bevacizumab formulation used to fabricate the subconjunctival
implantable tablet. Determination of the glass transition temperature
(Tg’) and collapse temperature (Tc) during lyophilisation assists in
the development of a suitable freeze-drying cycle.
Methods: Differential Scanning Calorimetry (DSC) was used to
measure the Tg’ of the excipients alone and in combination with the
antibody. Freeze Drying Microscopy (FDM) was used to study the
collapse temperature (Tc) for the excipients (trehalose and hyaluronic
acid (HA)) and the formulation. The antibody formulation was
characterised by size exclusion chromatography (SEC) and gel
electrophoresis.
Results: DSC experiments indicate the sub-ambient glass transition
temperature (Tg’) for bevacizumab to be -28.24°C (n=2) and that of
trehalose to be -29.36°C (n=2). The melting endotherm of HA was 23.71°C (n=2) as no Tg’ could be observed. This can be attributed to
the presence of buffer salts present in the HA formulation leading to a
eutectic melt. FDM was used to monitor the progress of
lyophilisation to determine the collapse temperature of the
formulation (Fig. 1). The collapse temperature of the pharmaceutical
formulation of bevacizumab was found to be -36°C (n=2).
Conclusions: Freeze-drying is a suitable technique to obtain a solid
form of antibody that can be fabricated as a tablet. Characterisation of
freeze-drying using FDM and DSC suggested that starting the
primary drying below -36 °C may avoid collapse of the cake during
scale up for freeze drying of the bevacizumab formulation.
Fig. 1. Progression of freeze drying of bevacizumab formulation for
tablet fabrication using FDM: A) Sample frozen at -60°C B) Sample
drying at -40°C C) Sample drying with collapse at -35°C
Commercial Relationships: Garima Sharma, None; Ashkan
Khalili, University College London (P); Sahar Awwad, None;
Kiran Malik, None; Paul Matejtschuk, None; Simon Gaisford,
None; Steve Brocchini, None; Peng T. Khaw, University College
Moorfields (P)
Support: NIHR Moorfields Biomedical Research Centre, UCL
School of Pharmacy, Grand Charity, Helen Hamlyn Trust, Fight for
Sight
Efficiency of a new pre-loaded, microincision insertion system for
a 1-piece hydrophobic-acrylic intraocular lens
Scott Evans1, Don R. Nixon2. 1AMO, Santa Ana, CA; 2Northern
Ontario School of Medicine, Ontario, ON, Canada.
Purpose: To evaluate the efficiency of using a pre-loaded insertion
system for the TECNIS® 1-piece hydrophobic-acrylic IOL compared
to the standard insertion system.
Methods: Forty TECNIS® 1-piece lenses were inserted into a saline
bath under simulated operating room (OR) conditions, twenty (20)
using a standard insertion system for the lens (UNFOLDER®
Platinum inserter) and twenty (20) using a preloaded insertion system
(TECNIS iTec inserter). For consistency, one investigator (DRN)
performed all procedures. Time from opening the IOL package to
loading the IOL into the insertion system, from IOL loaded to IOL
moved to delivery position, and time to deliver the IOL into the
saline bath were recorded. IOLs evaluated were representative of the
whole diopter range; 5 D, 20 D, and 34 D. Ease of use of the two
inserters, as well as complications related to the insertion system,
were assessed.
Results: There were no damaged IOLs in either group. The
procedure time for each step and the overall combined usage time
was significantly less with the preloaded inserter compared to the
standard inserter. Time from package opening to IOL loading was 12
± 2.2 sec and 35 ± 4.5 sec (p<0.0001) for the preloaded and standard
inserters, respectively. IOL loading to IOL in the ‘ready’ position was
3 ± 0.84 sec for preloaded and 10 ± 1.82 sec for standard inserter
(p<0.0001). From IOL in the delivery position to delivery of the IOL
was 8 ± 1.43 sec for preloaded and 10 ± 1.83 sec for standard inserter
(p=0.02). Total procedure time was 23 ± 3.00 sec for preloaded and
57 ± 7.99 sec for standard inserter (p<0.0001).
Conclusions: The new TECNIS iTec preloaded inserter performed as
well as the standard inserter and took less time to prepare, increasing
the efficiency of the microincision cataract surgery procedure.
Commercial Relationships: Scott Evans, Abbott Medical Optics
(E); Don R. Nixon, Abbott Medical Optics Inc. (F), Abbott Medical
Optics Inc. (C), Allergan (C), Novartis (C), Oculus (C)
Correlation between release of rapamycin from Porous Silicon
(pSi) and the color shifting of pSi monitored by a digital camera:
a prototype of non-invasive remote monitoring system for
intravitreal drug release
Lingyun Cheng1, Huiyuan Hou1, Alejandra Nieto2, Gordon Miskelly3,
Dirk-Uwe G. Bartsch1, William Freeman1, Michael J. Sailor2.
1
Jacobs Retina Ctr at Shiley Eye Ctr, Univ of California-San Diego,
La Jolla, CA; 2Department of Chemistry and Biochemistry,
University of California, San Diego, La Jolla, CA; 3School of
Chemical Sciences, University of Auckland, New Zealand,
Auckland, New Zealand.
Purpose: pSi photonic crystals can be created through etching with a
periodically varying current to impart a color to these particles. Drug
loading and release can be measured by reflectance spectroscopy.
The current study aims to investigate the feasibility of monitoring
drug release from pSi photonic crystals by in vitro digital imaging of
the color changes associated with pSi degradation and drug release.
Methods: pSi was prepared by electrochemical etch of a silicon
wafer. pSi microparticles were prepared by ultrasonic fracture. The
pSi surface was chemically modified with undecylenic acid and then
partially oxidized before rapamycin loading through infiltration (pore
size ~15 nm). Rapamycin-loaded pSi particles were added to 4.5mL
of HBSS in a petri dish which was incubated at 37°C. At
predetermined time points, the pSi particles in the dish were
photographed using a digital camera with coaxial lighting and the
dissolution medium was sampled for rapamycin quantitation. The
photographs were imported into Image J and the color of the particles
was measured after thresholding of the images.
Results: Hydrolytic and oxidative degradation of pSi, and release of
rapamycin from pSi caused an observable change of the pSi particles
from reddish to yellowish and then to transparent. The corresponding
changes in the reflected light intensity was correlated with the
cumulative drug release (r=0.93, p<0.0001; Figure). These optical
effects result from a blue-shift in the spectral peaks displayed by the
photonic crystals during the pSi degradation/drug release process. At
the early stage the reflected light intensity increased mainly due to
the drug release while the light intensity decline at later stage mainly
from degradation of pSi.
Conclusions: Rapamycin release from pSi photonic crystals can be
quantitatively monitored with a digital camera which will allow a
non-invasive and remote monitoring for an intravitreally injected
drug and pSi delivery system.
Commercial Relationships: Lingyun Cheng, Spinnaker
Biosciences (C); Huiyuan Hou, None; Alejandra Nieto, None;
Gordon Miskelly, None; Dirk-Uwe G. Bartsch, None; William
Freeman, OD-OS, Inc. (C); Michael J. Sailor, Spinnaker
Biosciences (I)
Support: NIH EY020617; NSF DMR-1210417; Research to Prevent
Blindness (UCSD)
Porous Silicon Microparticle Formulation as an Intravitreal
Delivery System for Rapamycin
Alejandra Nieto1, Huiyuan Hou2, Michael J. Sailor1, William
Freeman2, Lingyun Cheng2. 1Chemistry and Biochemistry, University
of California San Diego, La Jolla, CA; 2Jacobs Retina Center at
Shiley Eye Center, University of California San Diego, La Jolla, CA.
Purpose: Rapamycin (RAPA) is a potent immunosuppressant
occasionally administered orally for refractory uveitis, however side
effects are associated with systemic use. Intravitreal administration
provides a means to deliver the drug to the target while limiting
systemic adverse effects. Our study aimed at developing an
intravitreally injectable rapamycin formulation based on
nanostructured porous silicon particles and evaluating the effects of
different surface chemistries on rapamycin delivery.
Methods: Porous silicon (pSi) was prepared by electrochemical etch
of a silicon wafer. Microparticles were prepared by ultrasonic
fracture. The pSi carrier prepared in this fashion had previously been
shown to be non-toxic after intravitreal injection. Porous silicon
surface was chemically modified following three different strategies.
Commercial RAPA was loaded by infiltration from concentrated
solutions into the nanopores, with diameters of ~13 nm. Rapamycin
loaded pSi particles were added to a custom designed flow cell
chamber, which duplicated the turnover rate of rabbit eye fluid. The
effluent, HBSS (Hanks Balanced Salt Solution), was sampled and
analyzed by HPLC-MS (High Performance Liquid ChromatographyMass Spectrometry) to determine RAPA and model the drug release
profile. Silicon degradation was simultaneously quantitated using
ICP-OES (Inductively Coupled Plasma-Optical Emission
Spectroscopy).
Results: Surface chemistry had an influence in the rapamycin mass
loading efficiency, being the order: 690 μg RAPA/mg pSi, 83 μg
RAPA/mg pSi and 36 μg RAPA per mg pSi for the three different
formulations. During a 14-day incubation in flow cell at 37°C, all
formulations tested showed sustained and similar cumulative percent
of RAPA released. Silicon concentration-time profile depended on
surface chemistry following the order: 7 %, 4% and 0.5 % Si released
for the three different formulations.
Conclusions: Commercial rapamycin can be loaded into nanoporous
silicon. Results demonstrated the importance of surface chemistry of
pSi on rapamycin loading and release, which can be used as a tool to
tune drug loading efficiency and modulate matrix dissolution to
control drug release rate and vitreous half-life of pSi-rapamycin
delivery system. This represents a promising sustained and tunable
intraocular RAPA delivery system for refractory chorioretinal
diseases.
Commercial Relationships: Alejandra Nieto, None; Huiyuan Hou,
None; Michael J. Sailor, Spinnaker Biosciences (I); William
Freeman, OD-OS, Inc. (C); Lingyun Cheng, Spinnaker Biosciences
(C)
Support: NIH EY020617 and NSF DMR-1210417
Uptake and Release of ciprofloxacin and dexamethasone from
commercial contact lens materials
Alex Hui2, 1, Lyndon W. Jones2, 1. 1School of Optometry and Vision
Science, University of Waterloo, Waterloo, ON, Canada; 2Centre for
Contact Lens Research, University of Waterloo, Waterloo, ON,
Canada.
Purpose: To investigate the uptake and release of a novel antibioticsteroid solution from three commercial contact lenses.
Methods: A 0.3% ciprofloxacin (CF) and 0.1% dexamethasone-23phosphate (DXP) solution was prepared in acetate buffer using
cyclodextrins to prevent precipitation. Linear standard curves were
used to quantify the concentration of the two species simultaneously
using a combination of fluorescence spectrophotometry, which is
specific to the CF species (excitation at 274 nm, emission at 419 nm),
and UV absorbance, which can be used to quantify the DXP in
solution (absorbance at 241 nm). Three commercially available
contact lenses, balafilcon A (Purevision2, Bausch+Lomb), etafilcon
A (1-Day Acuvue Moist, Vistakon) and lotrafilcon B (Air Optix,
Alcon Vision Care) were placed in 2 mL of the CF-DXP solution and
soaked at room temperature for 24 hours. The lenses were then
removed and placed in 2 mL of phosphate buffered saline, and the
release of CF and DXP into solution was monitored over 24 hours.
Statistical analysis was performed using a repeated measures
ANOVA.
Results: All lens types were able to release significant amounts of
CF and DXP (p<0.05). In agreement with previous studies, the
etafilcon A material released the largest amount of CF (p<0.05 when
compared to other lens types), while the balafilcon A material
released the most DXP but this effect was not statistically significant
(p>0.05). The absolute amount of CF and DXP released was less than
when the species were loaded into the lenses individually compared
to previous studies, suggesting a possible interaction effect. The
majority of the drug was released within the first three hours,
suggesting that specifically tailored and designed lenses are needed if
these drug delivery lenses are to be useful.
Conclusions: Antibiotic steroid combination drops are one of the
most commonly prescribed agents in the ophthalmic industry, thus
they are a natural target for those interested in developing contact
lens drug delivery devices. Further study is required to tailor the
release rates and amounts to be clinically relevant.
Commercial Relationships: Alex Hui, None; Lyndon W. Jones,
Alcon (F), Alcon (R), Allergan (F), Abbott Medical Optics (R),
Bausch & Lomb (R), Ciba Vision (F), Ciba Vision (R), CooperVision
(F), Johnson & Johnson (F), Johnson & Johnson (R)
Support: Alex Hui is supported by the Natural Sciences and
Engineering Research Council (NSERC) of Canada, the Canadian
Optometric Education Trust Fund (COETF), and a Vistakon®
Research Grant and Ezell Fellowship, both administered by the
American Optometric Foundation (AOF). This study is also
supported by the NSERC 20/20 Network for the Development of
Advanced Ophthalmic Materials.
Enhanced Topical Delivery of a Small Molecule Receptor
Tyrosine Kinase Inhibitor (RTKi) via Mucosal-Penetrating
Particle Technology
Lisa Schopf, Elizabeth Enlow Enlow, Alexey Popov, James L.
Bourassa, Hongming Chen. R&D, Kala Pharmaceuticals, Waltham,
MA.
Purpose: To improve topical ocular drug delivery, we have
developed mucosal-penetrating particles (MPP) that rapidly and
uniformly coat and penetrate mucosal barriers, including that of the
eye, providing superior drug exposure to underlying tissues. The
objective of this work was to demonstrate that MPP-based eye drops
could enable the topical delivery of a small molecule inhibitor
designed to block vascular endothelial growth factor (VEGF)
signaling, and serve as a potential therapeutic approach for AMD,
eliminating the need for more invasive treatments (ie intravitreal
injection).
Methods: Pharmacokinetic (PK) profiling of topically administered
axitinib formulated as MPP (axitinib-MPP) was performed in
pigmented and non-pigmented rabbits. Ocular irritation was assessed
via Draize scoring. PK analysis was performed using WinNonlin
software. Pharmacodynamic evaluation was carried out in a rabbit
model of VEGF-induced retinal vascular leakage. Formulations were
given topically starting 2 days prior to a VEGF injection and
continued until termination. Ocular irritation assessments were
conducted daily. Fluorescein angiograms were scored blinded to
treatment.
Results: Topical axitinib-MPP showed a 5-fold area under the curve
enhancement in retinal drug concentration over a non-MPP control in
non-pigmented rabbits. A single dose of axitinib-MPP in pigmented
rabbits sustained retinal drug levels >40-fold over the cellular IC50
value for KDR, a RTK also known as VEGFR2, out to 24hrs. MPP
formulations were well-tolerated, and drug levels peaked earliest for
cornea, plasma, and retina and later for iris-ciliary body and choroid.
Efficacy of the MPP formulation was demonstrated in a VEGFinduced retinal vascular leakage model. Both vehicle and axitinibMPP delivered topically every 4hrs over 6 days were well-tolerated.
Topically administered axitinib-MPP significantly reduced vascular
leakage.
Conclusions: Our results demonstrate that topical delivery of a small
molecule RTKi formulated as MPP greatly enhances drug levels in
the retina and could be a possibility for the treatment of AMD. These
data also validate the significant potential of the MPP technology in
creating highly effective topical treatments for a broad range of
ocular diseases.
Commercial Relationships: Lisa Schopf, Kala Pharmaceuticals (E);
Elizabeth Enlow Enlow, Kala Pharmaceuticals (E), Kala
Pharmaceuticals (R), Kala Pharmaceuticals (I), Kala Pharmaceuticals
(P); Alexey Popov, Kala Pharmaceuticals (E), Kala Pharmaceuticals
(P), Kala Pharmaceuticals (I); James L. Bourassa, Kala
Pharmaceuticals (E); Hongming Chen, Kala Pharmaceuticals (E)
Proteins in solid dosage form (implantable tablets) for ocular
delivery
Ashkan Khalili1, Garima Sharma1, 2, Alastair Lockwood1, 2, Sahar
Awwad1, 2, Steve Brocchini1, 2, Peng T. Khaw1, 2. 1NIHR Moorfields
Biomedical Research Centre & UCL Institute of Ophthalmology,
London, United Kingdom; 2UCL School of Pharmacy, London,
United Kingdom.
Purpose: Protein-based medicines (e.g. antibodies) are increasingly
being used for ocular applications with many in the developmental
pipeline. Optimised ocular pharmacokinetics are required to achieve
the best clinical efficacy possible. The risk of complicating side
effects (e.g. endophthalmitis and retinal detachment) also rises with a
higher dosing frequency. It is therefore crucial to extend the half-life
of protein therapeutics in different ocular tissues (e.g. subconjunctival
space, cornea, posterior segment).
We have developed an implantable tablet derived from bevacizumab
for subconjuctival implantation during glaucoma filtration surgery
(GFS). The tablet showed prolonged release while in vivo studies
confirmed efficacy to decrease fibrosis in a rabbit model of GFS. Our
aim now is to examine the potential for other proteins to be used in a
solid ocular implantable dosage form. This study focused on tablet
fabrication using different proteins and the physicochemical
characterisation of the proteins during tablet dissolution.
Methods: Proteins including RNase, lyzozyme, trastuzumab, holoand apo-transferrin (1.25 mg) were fabricated into solid dosage form
(diameter 3 mm) using a combination of excipients to protect the
protein during fabrication and to prolong its release. The fabricated
protein tablet was dissolved in PBS (12.5 mL) at pH 7.4,overnight at
ambient temperature. The stability of the dissolved proteins was
determined using SDS-PAGE and size exclusion chromatography
(SEC).
Results: A range protein was evaluated including, bevacizumab (149
kDa), RNase (13.7 kDa), lysosome (14.7 kDa), trastuzumab (148
kDa), holo-transferrin (≈70kDa) and apo-transferrin (≈70kDa). They
were all successfully fabricated into a solid dosage form for
implantation (diameter 3 mm). An aseptic fabrication process can be
performed when required for biological studies. Both SEC and SDS
PAGE studies showed minimum aggregation of the proteins
evaluated upon tablet dissolution after 12 h.
Conclusions: Using a bevacizumab tablet that we developed as a
template, we have found that
different proteins can be used to fabricate solid tablets for ocular
implantation. Dissolution of these tablets occurs without significant
protein aggregation.
Commercial Relationships: Ashkan Khalili, University College
London (P); Garima Sharma, None; Alastair Lockwood, None;
Sahar Awwad, None; Steve Brocchini, None; Peng T. Khaw,
University College Moorfields (P)
Support: Grand Charity, UCL School of Pharmacy, NIHR
Moorfields Biomedical Research Centre, Fight for Sight, Helen
Hamlyn Trust, Medical Research Council
Pharmacokinetics Demonstrating Sustained Dexamethasone
Delivery from a Punctum Plug in a Canine Model
Ankita Desai, Charles D. Blizzard, Michael Bassett, Amar S.
Sawhney, Peter Jarrett, Michael McGrath, Arthur Driscoll. R&D,
Ocular Therapeutix, Inc, Bedford, MA.
Purpose: To examine the dose-dependent pharmacokinetics of
dexamethasone delivered from a biodegradable hydrogel punctum
plug in a canine model.
Methods: Micronized dexamethasone (Dex) was suspended in a
multi-arm PEG solution at two different levels (high and low dose)
and injected into small bore tubing prior to cross-linking. Fluorescein
was conjugated into the hydrogel to aid plug visualization through the
tissue using the blue light from a slit lamp. The Dex hydrogel matrix
was dried and cut into punctum plugs. The Dex plugs were inserted
into the inferior canaliculus of beagles and a subset was removed
each week for imaging. Tear fluid samples were collected at weekly
intervals and dexamethasone concentration was determined by
LC/MS. The high dose was evaluated in a toxicology study and the
low dose is a clinically representative dose designed for four weeks
of tapered release that was assessed in a PK study.
Results: The Dex plug demonstrated a sustained drug release profile
in tear fluid with a tapering effect over the treatment period.
Explanted plugs (Figure Two) of the low dose formulation illustrate
the drug release over time, with full drug clearance from the plug at
four weeks.
Conclusions: Topical corticosteroids, such as dexamethasone, are
used to treat inflammation for various ophthalmic conditions
including post-operative inflammation. Many therapies require
multiple daily administrations (up to hourly for severe conditions) for
the first several weeks followed by tapering to a lesser frequency as
the inflammation subsides and the condition resolves. A single-dose
dexamethasone punctum plug which biodegrades may provide a more
convenient option to help eliminate patient compliance with stringent
dosing requirements, and help ensure appropriate treatment and
resolution of the condition.
Commercial Relationships: Ankita Desai, Ocular Therapeutix (E);
Charles D. Blizzard, Ocular Therapeutix, Inc. (E); Michael Bassett,
Ocular Therapeutix (E); Amar S. Sawhney, Ocular Therapeutix (E);
Peter Jarrett, Ocular Therapeutix (E); Michael McGrath, Ocular
Therapeutix, Inc. (E); Arthur Driscoll, Ocular Therapeutix (E)
Use of galectin-3 fusions to extend the surface residence time of
proteins topically applied to the eye
Thomas Barnes, Joseph T. Kovalchin, Allyson Masci, Michael
Schmidt, Pamela Pegman, Patricia A. Lowden, Christian
Dombrowski. Eleven Biotherapeutics, Cambridge, MA.
Purpose: One of the key limiting parameters in the penetration and
effectiveness of topically applied ocular therapeutics is their mean
residence time. Most of an instilled solution clears through drainage,
with first-order clearance about 4 times that of bulk tear flow (~5
ul/min). For protein drugs, the problem becomes more acute due to
the additional burden of reduced penetrance through the cornea and
conjunctiva. Residence time might be increased however by fusing a
protein of interest to a mucosal surface binding protein, such as
galectin-3 (gal-3). Gal-3 is a small pentameric ocular surface resident
protein that cross-links O-type mucins.
Methods: We used Gaussia luciferase (luc) as our test protein, fused
at its N-terminus to one or 2 copies of the carbohydrate binding
domain of gal-3 (gal). Fusions were expressed in HEK cells and
purified by IMAC chromatography, and evaluated mucin binding by
ELISA and by their ocular surface half-lives on mouse eyes and
rabbit corneas, using luciferase activity as the readout.
Results: We compared luc, luc-gal and luc-gal-gal in a variety of
assays. Addition of gal-3 moieties to luciferase did not affect either
the specific or relative activities of luciferase. On ELISA plates
coated with MUC1 and asialofetuin, one gal-3 copy increased the
bound luciferase by 10-20-fold, and two copies increased it by 200250-fold. Binding to isolated rabbit corneal punches after a 30-min
wash was increased by 6 and 12-fold with 1 or 2 gal copies,
respectively. 30 mins after topical instillation in mouse eyes, luc and
luc-gal had near background luciferase activity in enucleated eyes,
while luc-gal-gal was 95-fold higher. In a time-course study, luc was
lost from the surface by 15 min, luc-gal by 30 min, while luc-gal-gal
required 180 min or more to reach background levels. The t1/2 of
luc-gal-gal was at least 6-fold higher than that of luc. We also
showed that gal3 can be readily fused to therapeutic moieties,
including our novel 17kD chimeric IL-1 blocker which is the active
substance in our clinical development product EBI-005.
Conclusions: We have demonstrated that fusion of a protein to the
small sugar-binding domain of gal-3 increases the half-life of its
ocular surface residence by at least 6-fold. This might afford a means
to increase the absorption of biologics from the ocular surface,
reducing either the dose required, the frequency of administration, or
both.
Commercial Relationships: Thomas Barnes, Eleven
Biotherapeutics (E), Eleven Biotherapeutics (P), Eleven
Biotherapeutics (I); Joseph T. Kovalchin, Eleven Biotherapeutics
(E), Eleven Biotherapeutics (P), Eleven Biotherapeutics (I); Allyson
Masci, Eleven Biotherapeutics (E); Michael Schmidt, Eleven
Biotherapeutics (I), Eleven Biotherapeutics (E), Eleven
Biotherapeutics (P); Pamela Pegman, Eleven Biotherapeutics (E);
Patricia A. Lowden, Eleven Biotherapeutics (E); Christian
Dombrowski, Eleven Biotherapeutics, Inc. (E), Eleven
Biotherapeutics, Inc. (P)
Application of PRINT Microparticle and Nanoparticle
Technology Toward Preparation of Ophthalmic Suspension
Formulations with Improved Tolerability and Efficacy
Benjamin Maynor, Andres Garcia, Janet Tully, Benjamin Yerxa.
Liquidia Technologies, Research Triangle Park, NC.
Purpose: To use PRINT technology to produce micro and
nanoparticles of controlled microstructure and nanostructure that are
suitable for the preparation of aqueous ophthalmic suspension
formulations without use of solubilizing excipients (e.g. cremaphor,
oils, cyclodextrins).
Methods: PRINT technology, a novel drug/excipient micromolding
approach, was used to produce monodisperse nonspherical particles
of itraconazole, cyclosporine, and tacrolimus. Specifically, 10 micron
triangular templates, 3 micron toroids, 200 nm cylindrical and 1
micron cylindrical polymeric templates were used to prepare particles
of cyclosporine, tacrolimus, and itraconazole, respectively.
Dissolution characteristics of itraconazole suspensions were
evaluated and compared to bulk and micronized itraconazole using
standard dissolution test methods.
Results: Monodisperse, shape-specific microparticles and
nanoparticles were successfully prepared of cyclosporine, tacrolimus,
and itraconazole. Characterization of these particles using
microscopy confirms that monodisperse populations of 10 micron
triangles, 3 micron toroids, and 200 nm and 1 micron cylinders were
produced of cyclosporine, tacrolimus, and itraconazole, respectively.
The sizes and shapes of these microparticles and nanoparticles are
suitable for use in ophthalmic suspension dosage forms. Dissolution
studies of itraconazole cylinder suspensions indicate that these
particles dissolve faster under sink conditions than traditional
micronized itraconazole (50% dissolution at 5 min for PRINTitraconazole cylinders vs. 15 minutes for micronized itraconazole),
suggesting that itraconazole PRINT formulations may have greater
ocular surface bioavailability than micronized formulations.
Conclusions: We demonstrate that PRINT technology is a promising
approach for the development of improved suspension formulations
of compounds such as cyclosporine, tacrolimus, and itraconazole.
Dissolution experiments show enhanced dissolution time of these
particles compared to traditional micronized drug formulations,
without the use of excipients with poor tolerability profile.
A) Scanning electron microscopy images of PRINT microparticles
composed of cyclosporine and tacrolimus; B) Aqueous dissolution
characteristics of PRINT-itraconazole microparticles and comparison
to micronized suspensions.
Commercial Relationships: Benjamin Maynor, Liquidia
Technologies (E), Liquidia Technologies (I); Andres Garcia,
Liquidia Technologies (E), Liquidia Technologies (I); Janet Tully,
Liquidia Technologies (E); Benjamin Yerxa, Liquidia (E)
Evaluation of drug delivery and biocompatibility of
biodegradable microfilms in the anterior segment of the rat
model
Yan Peng1, Yu-Chi Liu2, 3, Nyein Chan Lwin2, Subbu S Venkatraman1,
Tina Wong2, 3, Jodhbir S. Mehta2, 3. 1School of Materials Science and
Engineering, Nanyang Technological University, Singapore,
Singapore; 2Singapore Eye Research Institute, Singapore, Singapore;
3
Singapore National Eye Centre, Singapore, Singapore.
Purpose: To develop a biodegradable, sustained-release,
prednisolone acetate (PA)-loaded poly [d,l-lactide-co-ε-caprolactone]
(PLC) drug delivery system and to evaluate its biocompatibility,
feasibility and release characteristics both in vitro and in vivo.
Methods: Blank and 40% PA-loaded PLC microfilms with thickness
of 100µm and diameter of 2mm were developed and tested in vitro
and in vivo. The degradation and drug release profiles of the
microfilms were evaluated in the in vitro and in vivo experiments.
We further implanted the microfilms to the subconjunctival space of
the rats (n=51). All eyes were monitored using slit lamp biomicroscopy with Hackett McDonald ocular scoring system and
anterior segment optical coherence tomography. Histological studies
with Hematoxylin-Eosin, Picrosirus red staining and
immunohistochemistry analysis were performed to evaluate and
compare the presence of inflammatory and fibrotic reaction in blank
and PA-loaded microfilm groups. PA concentrations in the aqueous
humor were determined by high-performance liquid chromatography
(HPLC).
Results: Subconjunctivally-implanted PA-loaded PLC microfilms
were able to deliver prednisolone acetate in a sustained manner over
3 months, with a steady rate of 0.002mg/day in vivo. Eyes with either
blank or PA-loaded implanted microfilms showed very minimal
inflammatory response at the insertion sites and mild degree of
collagen encapsulation around the microfilms, with significantly less
CD11c cells at 2 weeks (P = 0.001) and collagen extent at 2 and 4
weeks (P = 0.001 and P = 0.002) in PA-loaded microfilm group.
Desirable anterior chamber PA levels were achieved, with the
concentrations at 76.67±5.86, 69.33±2.3 and 42.67±4.1 ng/ml at 2, 4
and 12 weeks, respectively.
Conclusions: PA-loaded PLC microfilm displays good
biocompatibility, feasibility and desirable sustained drug release
profiles. This device provides a promising alternative with great
potential application in the treatments of ocular anterior segment
diseases.
Commercial Relationships: Yan Peng, None; Yu-Chi Liu, None;
Nyein Chan Lwin, None; Subbu S Venkatraman, None; Tina
Wong, 61, 250,006 (P); Jodhbir S. Mehta, None
Support: Translational and Clinical Research (TCR) Programme
(NMRC/TCR/002-SERI/2008)
Safety of DuraSite vehicle as part of a topical treatment in
cataract surgery patients
Judith Hutcheson, Lyle M. Bowman, Kamran Hosseini. InSite Vision,
Alameda, CA.
Purpose: To investigate the safety of DuraSite vehicle
(polycarbophil, sodium chloride, edetate disodium dihydrate, and
sterile water for irrigation) in post cataract surgery patients.
Methods: In a well-controlled multicenter Phase II clinical trial, 169
patients were randomized into 4 different groups to investigate
different dosing regimens of Bromfenac in DuraSite 0.075% (ISV303) compared with the non DuraSite based commercial Bromfenac
formulation (0.09%). DuraSite was the vehicle (either with or without
the drug) in three arms of the study (approximately 3/4 of the total
patients, n=127). All patients received the investigational medication
one day after cataract surgery, and self-administered the formulation
b.i.d. for a period of two weeks. Slit-lamp biomicroscopy, IOP
measurement and visual acuity test were performed on 4 clinical
visits while ophthalmoscopy was performed on Days 1 and 29. All
patients received a safety examination at Days 8, 15 and 30 post
dosing.
Results: The incidence of adverse events for all DuraSite containing
arms, ISV-303 BID, ISV-303 QD and DuraSite Vehicle BID was
comparable to the active control of commercial Bromfenac 0.09%
BID. No major differences were observed in BCVA, IOP, or
ophthalmoscopy evaluations.
Conclusions: DuraSite-containing ophthalmic formulations are well
tolerated for use in cataract surgery patients as shown in a wellcontrolled multicenter clinical trial.
Commercial Relationships: Judith Hutcheson, InSite Vision (E);
Lyle M. Bowman, InSite Vision (E); Kamran Hosseini, InSite
Vision Inc. (E)
Clinical Trial: NCT01190878
Neuroprotective effect on the retinal ganglion cells by
brimonidine loaded HSA nanoparticles in the acute optic nerve
crush model
Hyuncheol Kim1, Hyungwon Moon1, Kyoung Nam Kim2, Yu Jeong
Kim2, Jin Wook Jeoung2, Ki Ho Park2. 1Department of Chemical and
Biomolecular Engineering, Sogang University, Seoul, Republic of
Korea; 2Department of Ophthalmology, Seoul National University
College of Medicine, Seoul, Republic of Korea.
Purpose: To examine the neuroprotective effect of α2-agonist
brimonidine loaded human serum albumin (HSA) nanoparticles on
the survival of retinal ganglion cells after acute optic nerve crush.
Methods: Brimonidine loaded HSA nanoparticles were fabricated by
desolvation technique. The size distribution and zeta potential were
evaluated by dynamic light scattering method. The loaded amount
and in vitro release rate of brimonidine from the HSA nanoparticles
were determined with the HPLC. Acute optic nerve crush was
induced by clipping the optic nerve for 60s. After optic nerve injury,
brimonidine loaded HSA nanoparticles, free HSA nanoparticles, and
balance salt solution (BSS) control were adiministered intravitreally.
Retinal ganglion cell loss was evalulated by cell count in a retinal flat
mount, which was visualized by the retrograde transport of rhodamindextran 3000. Retinal ganglion cell loss was determined 5 days post
administration.
Results: The brimonidine loaded HSA nanoparticles showed narrow
size distribution and negatively charged surface to be 152.78 +/- 51.1
nm and -29.7 +/- 7.52 mV, respectively. The concentration of
brimonidine in HSA nanoparticles was 214.12 μg/mL and
consistently released for over 5 days. In the neuroprotection analysis,
compared to the uncrush retinal ganglion cells (control group), the
BSS treated group described the retinal ganglion cell loss of 66.81 +/4.34 % (P<0.05). However, retinal ganglion cell loss in the
brimonidine - HSA nanoparticle treated group showed 26.19 +/-
5.21% (P<0.05 vs. control group) at the dose of 8.78 μg
(brimonidine). Retinal ganglion cell loss was decreased more than 2
times, compared to the BSS treated group 5 days post administration.
Conclusions: Brimonidine loaded HSA nanoparticles delivered
neuroprotective glaucomatous drugs to the retina and described the
neuroprotective effect on the retinal ganglion cells effectively.
Commercial Relationships: Hyuncheol Kim, None; Hyungwon
Moon, None; Kyoung Nam Kim, None; Yu Jeong Kim, None; Jin
Wook Jeoung, None; Ki Ho Park, None
Support: the National Research Foundation of Korea (20110009606), Korean Industrial Source Technology Development
Program (10033726)
Sustained release of ovalbumin and IgG from hydrogel depots as
surrogate proteins for intravitreal injection
Rami Elhayek, Rami Elhayek, Peter Jarrett, Amar S. Sawhney, Sarah
Guedez. Ocular Therapeutix, BEDFORD, MA.
Purpose: To evaluate the sustained release of ovalbumin (OVA;
45000 g/mol) and rabbit IgG (rIgG; 150000 g/mol) from preformed
hydrogel depots with variable degradation times.
Methods: : Fine particles of OVA and IgG were formulated with a
selection of PEG hydrogels and were cured, dried and cut to form
degradable (a, b, c, d) and non-degradable (e) hydrogel depots. The
depots were examined in vitro for sustained release under accelerated
conditions. Prior to testing, acceleration rate was calculated using
degradation profiles of each hydrogel, absent of OVA and IgG, to
compare the relative degradation rate between accelerated and realtime conditions, tris buffer saline (TBS) pH8.5 and phosphate buffer
saline (PBS) pH7.4, respectively. Acceleration factor was determined
to be 16x in TBS compared to PBS. Accelerated OVA and IgG in
vitro release was conducted to determine release profiles as a
function of the hydrogel degradation over time. Concentration of
OVA and rIgG were determined by HPLC.
Results: Release profiles of OVA and rIgG shown in Figure 1 and 2
show the effect of hydrogel degradation time on sustained release of
OVA and rIgG. Slow degrading hydrogels release OVA and rIgG
over a longer time period than faster degrading hydrogels.
Comparison of OVA and rIgG release using the same hydrogels
indicate that rIgG is released slower than OVA due the difference in
size and molecular weight. In both cases, hydrogels made using nondegradable SGA linkages used as controls reach a maximum release
after diffusion of surface and free particles.
Conclusions: Monoclonal antibody fragments and fusion proteins
have been designed to inhibit vascular endothelial growth factors
(VEGF) for treatment of posterior segment diseases such as agerelated macular degeneration (AMD) and diabetic macular edema
(DME). Frequent anti-VEGF intravitreal injections have shown to
manage the progression of these diseases to prevent further vision
loss and in some cases, gain visual acuity. However, frequent
intravitreal injections can increase the risk of infection, retinal
detachment, and/or hemorrhage. The ability to tailor the release of
OVA and rIgG from hydrogel depots for sustained delivery of
proteins could provide a technology platform for use with antiVEGFs to reduce the number of yearly intravitreal injections and
potentially reduce the risk of side effects.
smooth surfaces and absence of pores for MS-A but with small
surface pores in MS-B. The inclusion of vit-E in MS-B increased the
protein entrapment from 23.4±0.5% to 30.4±1.1%. A three-phasic
sustained GDNF in-vitro release was obtained for both formulations
for 133 days. In phase-I 37 and 33 pgGDNF/mgMS/day were
obtained for MS-A and MS-B respectively. In phase-II 144
pgGDNF/mgMS/day for MS-A and 187 pgGDNF/mgMS/day for
MS-B were denoted. Finally, 8 (MS-A) and 14 (MS-B)
pgGDNF/mgMS/day were observed. Less retinal cell death (p<0.001)
was observed when cultured in presence of GDNF released from MSB (8.2%) in comparison to MS-A (15.9%).
Conclusions: Inclusion of antioxidants is a useful strategy to increase
the biological neuroprotective activity of microencapsulated
neurotrophic factors such as GDNF.
Commercial Relationships: Irene Bravo-Osuna, None; Patricia
Checa-Casalengua, None; Caihui Jiang, None; Budd A. Tucker,
None; Irene T. Molina-Martínez, None; Michael J. Young,
ReNeuron (F); Rocio Herrero-Vanrell, None
Support: MAT-2010-18242, UCM 920415 GR35/10-A, RETICS net
(RD07/0062/2002)
Commercial Relationships: Rami Elhayek, OCULAR
THERAPEUTIX INC (E); Rami Elhayek, OCULAR
THERAPEUTIX INC (E); Peter Jarrett, Ocular Therapeutix (E);
Amar S. Sawhney, Ocular Therapeutix (E); Sarah Guedez, Ocular
Therapeutix (E)
VITAMIN E INCREASES THE BIOACTIVITY IN RETINAL
CELLS OF MICROENCAPSULATED GLIAL CELL LINE
DERIVED NEUROTROPHIC FACTOR
Irene Bravo-Osuna1, Patricia Checa-Casalengua1, Caihui Jiang2,
Budd A. Tucker3, Irene T. Molina-Martínez1, Michael J. Young2,
Rocio Herrero-Vanrell3. 1Pharmacy and Pharmaceutical Technology,
School of Pharmacy, University Complutense of Madrid, Madrid,
Spain; 2Schepens Eye Research Institute, Dep. of Ophthalmology,
Harvard Medical School, Harvard University, Boston, MA; 3Institute
for Vision Research, Dep. of Ophthalmology, Carver College of
Medicine, University of Iowa, Iowa, IA.
Purpose: To evaluate the bioactivity of GDNF released form PLGA
microspheres (MS) in retinal cells in presence of vitamin E (vit-E) as
antioxidant.
Methods: GDNF-loaded PLGA MS were prepared by S/O/W
emulsion/solvent-extraction-evaporation method. 20µg of
recombinant human GDNF were suspended in the inner-phase of the
emulsion composed by PLGA/CH2Cl2 solution (MS-A) or
PLGA/CH2Cl2 solution including 20µl of vit-E (MS-B). MS were
characterized in terms of production yield, SEM, mean-particle-size
and particle-size-distribution, encapsulation efficiency (ELISA) and
in-vitro release studies. For bioassays, retinal from post-natal day-10
(B6 mice) were isolated and exposed to conditioned media released
from MS-A and MS-B. At 40h post plating, the percentage of death
cells was analysed (TUNEL).
Results: The microencapsulation method used led to production yield
of 80% for both formulations. MS (20-40µm) resulted spherical with
Pharmacokinetic Evaluation of Sustained Delivery Moxifloxacin
Punctum Plugs
Michael Bassett, Charles D. Blizzard, Peter K. Jarrett, Arthur
Driscoll, Ankita Desai, Deepa Mulani, Michael McGrath, Amar S.
Sawhney. Ocular Therapeutix, Bedford, MA.
Purpose: To assess the safety and feasibility of a moxifloxacinloaded punctum plug (MP) in a post-operative cataract surgery group.
Methods: A prospective, single-arm pharmacokinetic study was
conducted with 10 patients at the Singapore National Eye Center. The
MP was inserted into the punctum of patients following cataract
surgery. Study endpoints included ease of insertion, retention and
moxifloxacin tear fluid levels at 1h, 24h, and on days 3, 7, 10, 20 and
30. The MP is designed to deliver a bolus followed by extended
release encapsulated moxifloxacin; concentrations were targeted to
be >250ng/mL through 7 days of treatment.
Results: The average moxifloxacin levels in the tear film by LCMS/MS ranged from 2,465 to 3,236ng/mL through day 7 as shown in
Figure 1. Tear fluid concentrations were below the limit-ofquantification (<LOQ) for days 20 and 30. MP retention was 100%
through day 10. Slit lamp examinations most commonly showed
aqueous chamber cells and corneal staining/erosion, shown in Table
1. All findings were determined to be related to cataract surgery.
Plugs were well tolerated with no adverse events and no ocular
complaints or findings other than normal post-cataract symptoms.
Conclusions: The MP delivered and maintained moxifloxacin tear
fluid levels above the MIC90s for common susceptible pathogens
(250ng/mL) for 7 days in a post-cataract pharmacokinetic study. The
MP possessed a favorable safety and tolerability profile over 30 days.
Consistent and therapeutic dosing from a punctum plug may be
advantageous over patient-administered topical drops to avoid
relapse, reduce contagion, and cure the disease.
reduction in coverage area, respectively between days 14 and 112,
which was statistically significant (p < 0.001). In addition, the
number of particles decreased between days 14 and 112, as shown by
a 48%, 69%, 45% and 28% reduction in fluorescent signal intensity
of particles, which was statistically significant (p < 0.001).
Conclusions: A single hollow microneedle was able to reliably inject
50 µL of particle formulations that have a particle size up to 10 µm
within the SCS of rabbit eyes. The particles spread over a
suprachoroidal area of up to 200 mm2, which decreased by 9 - 35%
between days 14 and 112. In addition, 28% - 69% of the particles
appeared to be cleared from the SCS between days 14 and 112. We
are currently determining whether this apparent clearance is due to
removal by macrophages or simply a reduction of the fluorescence
signal intensity of the particles over time.
Commercial Relationships: Yoo C. Kim, None; Mark Prausnitz,
Clearside Biomedical (I), Clearside Biomedical (P), Clearside
Biomedical (S); Henry F. Edelhauser, Clearside Biomedical (P),
Clearside Biomedical (I), Clearside Biomedical (C)
Support: NIH Grant R24 EY017404
Commercial Relationships: Michael Bassett, Ocular Therapeutix
(E); Charles D. Blizzard, Ocular Therapeutix, Inc. (E); Peter K.
Jarrett, Ocular Therapeutix (E); Arthur Driscoll, Ocular
Therapeutix (E); Ankita Desai, Ocular Therapeutix (E); Deepa
Mulani, Ocular Therapeutix (E); Michael McGrath, Ocular
Therapeutix, Inc. (E); Amar S. Sawhney, Ocular Therapeutix (E)
Distribution And Clearance Of Microparticles And
Nanoparticles In The Suprachoroidal Space After Injection
Using Hollow Microneedles In Rabbits
Yoo C. Kim1, Mark Prausnitz1, Henry F. Edelhauser2. 1Chemical
Engineering, Georgia Tech, Atlanta, GA; 2Ophthalmology, Emory
University Eye Center, Atlanta, GA.
Purpose: The suprachoroidal space (SCS) is a virtual space between
the sclera and choroid tissues and has been shown to accommodate a
variety of fluids and particles in rabbit and porcine eyes. Drugs
delivered in this potential space can be used to treat posteriorsegment diseases such as age-related macular degeneration. This
study examines the distribution and clearance of microparticles and
nanoparticles after administration within the SCS using a hollow
microneedle up to 112 days following injection into eyes of New
Zealand White rabbit eyes.
Methods: Hollow metal microneedles 750-800 µm in length were
used to inject 50 µL of non-biodegradable, fluorescently tagged,
polystyrene particles with various sizes (20 nm, 200 nm, 2 µm, 10
µm) in balanced salt solution. A hollow microneedle was inserted 3
mm posterior to the limbus in New Zealand White rabbit eyes.
Rabbits were sacrificed 14 or 112 days after injection. The eyes were
snap frozen, dissected and imaged to visualize the spread of the
particles within the SCS followed by a homogenization process to
quantify the number of particles inside the SCS by fluorescence.
Results: Fourteen days after injection, the 20 nm, 200 nm, 2 µm and
10 µm sized particles covered an suprachoroidal surface area of
201±44 mm2, 191±25 mm2, 169±19 mm2 and 185±10 mm2,
respectively. After 112 days, these particles covered an area of
131±4.7 mm2, 135±1.3 mm2, 127±24 mm2 and 170±11 mm2,
respectively. These particles showed a 35%, 30%, 25%, and 9%
Examination of in vitro release profiles of bromfenac, diclofenac
and nepafenac as drug candidates for sustained release NSAID
punctum plug
Peter K. Jarrett, Rami Elhayek, Sarah Guedez, Amar S. Sawhney.
Ocular Therapeutix, Bedford, MA.
Purpose: To examine the in vitro release profiles of three
nonsteroidal anti-inflammatory drugs (NSAIDs): Bromfenac (BFc),
Diclofenac (DFc) and Nepafenac (NFc) from polyethylene glycol
(PEG) hydrogel punctum plugs.
Methods: BFc, DFc and NFc were each suspended with equal
loading in a multi-arm PEG solution and injected into small diameter
silicone tubing prior to cross-linking. The hydrogel NSAID matrix
confined within the silicone tubing was cut to 5mm plugs. The
NSAID release profile was determined in PBS pH7.4 at 37°C
simulating the release from a punctum plug (PP) inserted in the
canaliculus. The release media was sampled and exchanged daily.
Plugs were removed for photographic imaging to track the release of
the drug qualitatively indicated by drug clearance from both ends of
the plug. Percent drug release was determined using UV/Vis
spectrophotometry.
Results: As shown in Figure 1, each NSAID was released from the
plug at a rate relative to their aqueous solubility (BFc, DFc and NFc
having a water solubility of 53, 0.6 and 0.006 mg/mL respectively).
The most soluble BFc was released in 7 days, DFc was released in 14
days, and the least soluble NFc’s projected release was calculated at
approximately 120 days. Images of a DFc-loaded hydrogel plug
showing drug clearance over time are included within Figure 2.
Conclusions: BFc, DFc and NFc each showed variable rates of
sustained release from a PEG hydrogel plug corresponding to their
water solubility. Solubility modifiers and/or modifications to the PEG
hydrogel matrix can be employed to tailor the release profile of BFc,
DFc and NFc, depending on the drug load and duration of therapy
required. Topical NSAIDs, such as BFc, DFc and NFc are used to
treat post-surgical inflammation as well as various ophthalmic
conditions, and typically require multiple daily dose administrations
over extended periods of time. A single dose NSAID punctum plug
may provide more consistent dosing while eliminating issues of
patient non-compliance.
Commercial Relationships: Thomas Yorio, None
Program Number: 1277
Opioid and Natriuretic Peptide Axis: Nature’s Ocular
Neuroprotectants
Shahid Husain. Ophthalmology, Medical Univ of South Carolina,
Charleston, SC.
Commercial Relationships: Shahid Husain, None
Bradykinin: A Peptide for All Seasons
Naj Sharif. Pharma Regulatory Affairs, Alcon Research Ltd, Fort
Worth, TX.
Commercial Relationships: Naj Sharif, Alcon Research, Ltd (a
Novartis Co.) (E)
255 Retina/RPE: New Drugs, Mechanisms of Action, and
Toxicity
Monday, May 06, 2013 11:00 AM-12:45 PM
Contributing Section(s): Biochemistry/Molecular Biology
Commercial Relationships: Peter K. Jarrett, Ocular Therapeutix
(E); Rami Elhayek, OCULAR THERAPEUTIX INC (E); Sarah
Guedez, Ocular Therapeutix (E); Amar S. Sawhney, Ocular
Therapeutix (E)
216 Peptides and Polypeptides in Ocular Health and Dysfunction
- Minisymposium
Monday, May 06, 2013 8:30 AM-10:15 AM
618-620 Minisymposium
Program #/Board # Range: 1274-1278
Contributing Section(s): Cornea, Glaucoma, Lens, Retinal Cell
Biology
Peptides in the Eye: An Overview
Miguel Coca-Prados. Ophthalmology & Visual Sci, Yale Univ
School of Medicine, New Haven, CT.
Commercial Relationships: Miguel Coca-Prados, None
VEGF and PDGF: Roles in Physiology and Pathology
Patricia A. D'Amore. Ophthalmology, Schepens Eye Res Inst, Mass
Eye & Ear, Boston, MA.
Commercial Relationships: Patricia A. D'Amore, Valeant (C)
Endothelin: Friend or Foe?
Thomas Yorio. North Texas Eye Research Institute and
Pharmacology & Neuroscience, Univ of North Texas Hlth Sci Ctr,
Fort Worth, TX.
Presentation Time: 11:00 AM - 12:45 PM
Optimization of Cone-Directed AAV-Mediated Gene
Augmentation Therapy for CNGB3-Achromatopsia by Use of the
IRBP/GNAT2-Promoter and Intravitreal CNTF Administration
Connie Y. Yeh1, 2, Simone Iwabe1, Sanford L. Boye3, Kendra
McDaid1, Christine Harman2, Rong Wen4, William W. Hauswirth3,
Andras M. Komaromy1, 2, Gustavo D. Aguirre1. 1School of Veterinary
Medicine, University of Pennsylvania, Philadelphia, PA; 2College of
Veterinary Medicine, Michigan State University, East Lansing, MI;
3
College of Medicine, University of Florida, Gainesville, FL;
4
Bascom Palmer Eye Institute, University of Miami, Miami, FL.
Purpose: AAV5-mediated cone-directed gene therapy results in
rescue of cone function and day vision in canine models of CNGB3achromatopsia. However, the treatment success rate is <80%, and the
human red-cone opsin promoter does not target S-cones. The
objective of this study was to optimize therapeutic outcome (1) by
use of an enhanced cone transducin promoter and (2) by intravitreal
CNTF administration in non-responders.
Methods: The IRBP/GNAT2 promoter consists of the 277-bp 5'flanking sequence of the human GNAT2 promoter coupled with the
214-bp IRBP enhancer (Ying et al. 1998). The ability of this
promoter to target cone subclasses was evaluated by subretinal
injection of AAV5-IRBP/GNAT2-GFP in normal dog eyes. GFP
reporter gene expression was evaluated by observation of in vivo
fluorescence and by immunohistochemistry. Subsequently, 14 young
CNGB3-mutant dogs between 9-11 weeks of age were treated by
bilateral subretinal injection of either AAV5-IRBP/GNAT2-hCNGB3
or AAV8-IRBP/GNAT2-hCNGB3. The animals were followed for 515 months post injection by standard full-field electroretinography
and visual behavior testing in an obstacle avoidance course. Six
months post therapy, 3 “non-responder” dogs were intravitreally
injected with 12 μg of CNTF (n = 3 eyes) or PBS (n = 3 eyes) and
followed for 2 months.
Results: The IRBP/GNAT2 promoter allowed robust and specific
targeting of GFP-reporter gene expression in both cone subclasses.
Rescue of cone function and day vision was achieved with both AAV
serotypes; best results were obtained by use of ~1012 vector
genomes/mL (2/6 and 4/6 injected eyes responding with AAV5 and
AAV8 respectively). A therapeutic effect was not obtained in all
AAV-treated eyes. However, all 3 eyes treated with intravitreal
CNTF 6 months following unsuccessful gene augmentation therapy
showed robust rescue of cone function; this was not the case with
intravitreal PBS injection.
Conclusions: Gene expression can be targeted to both canine cone
subclasses and their function restored in CNGB3-achromatopsia with
the IRBP/GNAT2 promoter in either AAV5 or AAV8. It remains
unclear why some animals do not respond to AAV-mediated gene
replacement therapy alone; however, treatment outcome can be
enhanced by combination with intravitreal CNTF injection in eyes
that do not respond.
Commercial Relationships: Connie Y. Yeh, None; Simone Iwabe,
None; Sanford L. Boye, PCT/US2012/062478 (P); Kendra
McDaid, None; Christine Harman, None; Rong Wen, Neurotech
USA (C); William W. Hauswirth, AGTC (I), Bionic Sight (I),
AGTC (C), Syncona (C), RetroSense (C); Andras M. Komaromy,
None; Gustavo D. Aguirre, None
Support: NIH (EY019304, EY017549, EY006855, EY018586,
P30EY001583, P30EY008571, P30EY14801, RR007063), DoD
(W81XWH-09-1-0674), FFB, MVRF, RPB
Conversion to aflibercept for chronic refractory or recurrent
neovascular age-related macular degeneration
Yoshihiro Yonekawa1, John B. Miller1, John I. Loewenstein1, Lucia
Sobrin1, Dean Eliott1, Demetrios Vavvas1, Joan W. Miller1,
Christopher M. Andreoli1, 2, Ivana K. Kim1. 1Massachusetts Eye and
Ear Infirmary, Boston, MA; 2Harvard Vanguard Medical Associates,
Boston, MA.
Purpose: To examine the outcomes of patients with refractory or
recurrent neovascular age-related macular degeneration who were
converted from bevacizumab and/or ranibizumab to aflibercept
intravitreal injections.
Methods: This was a two-center, retrospective, interventional, noncomparative series. Treatment histories, visual acuity (VA), and
central macular thickness (CMT) on spectral-domain OCT were
collected. Patients were divided into “refractory” (those with
persistent exudation despite monthly injections) or “recurrent” (those
who required repeated injections to maintain a dry macula).
Results: 102 eyes of 94 patients were included in the study. 68 were
refractory, and 34 were recurrent. A mean of 20.4 prior
bevacizumab/ranibizumab injections and a mean of 3.8 aflibercept
injections were administered. Mean follow-up was 18 weeks. Mean
VAs were: 20/50-1 before conversion, 20/50-2 after 1 aflibercept
injection (P = .723), and 20/50+2 after the final injection (P = .253).
Subgroup analysis of refractory and recurrent cases also showed
stable VA. Of the refractory cases, mean CMT improved after 1
injection (P < .001) and the final injection (P < .001). Intraretinal (P <
.001) and subretinal (P < .001) fluid decreased after 1 injection, and
the mean injection interval could be extended from 5.2 to 6.2 weeks
(P = .003). Of the recurrent cases, mean CMT improved after 1
injection (P < .001) and the final injection (P < .001). Intraretinal (P =
.003) and subretinal (P = .046) fluid decreased after 1 injection, and
the mean injection interval could be extended from 7.2 to 9.5 weeks
(P = .001).
Conclusions: Converting patients with chronic neovascular AMD to
aflibercept may result in stabilized vision and improved anatomical
outcomes, while allowing injection intervals to be extended.
Commercial Relationships: Yoshihiro Yonekawa, None; John B.
Miller, None; John I. Loewenstein, None; Lucia Sobrin, None;
Dean Eliott, Genentech (C), Regeneron (C), Ophthotech (C), Alcon
(C), Bausch & Lomb (C), Allergan (C), Alimera (C), Acucela (C),
Arctic (C), Salutaris (C); Demetrios Vavvas, MEEI (P), Kala
pharmaceuticals (C), Roche (C), Genentech (C); Joan W. Miller,
Massachusetts Eye and Ear Infirmary (P), Novartis (I), Alcon (C),
KalVista Pharmaceuticals (C); Christopher M. Andreoli, None;
Ivana K. Kim, Genentech (F), SalutarisMD (C), Genzyme (C),
ArticDx (C), Sequenom (C)
EFFECTIVE TARGETING OF THE PI3K/AKT/mTOR
PATHWAY: A PROMISING THERAPEUTIC OPTION FOR
THE TREATMENT OF OCULAR
NEOVASCULARIZATION/INFLAMMATION/OEDEMA
Temitope Sasore, Alison L. Reynolds, Breandan N. Kennedy. Conway
Institute, University College Dubin, Dublin 4, Ireland.
Purpose: There is a clinical need to develop improved
pharmacological therapeutics for ocular neovascularization, retinal
inflammation and retinal or oedema associated with diabetic
retinopathy (DR), age-related macular degeneration (AMD) and
retinopathy of prematurity (ROP). In this study, we investigate the in
vivo anti-angiogenic efficacy of a panel of PI3K/Akt/mTOR
inhibitors, alone and in combination.
Methods: To measure the efficacy of inhibition of developmental
angiogenesis, Tg(fli1:EGFP) transgenic zebrafish were treated with
drugs from 1-5 post fertilisation and screened inhibitory effects on
intersegmental vessel (ISV) and hyaloid vessel (HV) number. Visual
motor response and optokinetic response were carried out to assess
the effects on visual behaviour. The developmental expression
pattern of PI3K subunits in the zebrafish examined using reverse
transcriptase polymerase chain reaction. Effects on human
endothelial cell number were examined using a crystal violet assay.
Results: The broad-spectrum inhibitor LY294002, followed by the
mTOR inhibitor rapamycin, ranked as the most effective PI3K
pathway inhibitors of developmental angiogenesis in zebrafish.
Additionally, there was significant anti-angiogenic effect exerted by
the dual PI3K-mTOR inhibitors, NVP-BEZ235 and PI-103.
Interestingly, some compounds that inhibit developmental
angiogenesis of the HV, are ineffective at inhibiting developmental
angiogenesis of the ISV. In agreement, the pik3r1 gene, encoding the
p85 regulatory subunit, is expressed at all developmental stages from
4 hours post fertilisation (hpf) to 5 days post fertilisation (dpf).
Whereas pik3cA, encoding the p110α subunit, is expressed only at
later developmental stages (1-5 dpf).
Conclusions: Given that the p110α isoform has been suggested to be
selectively required for angiogenesis, our results unexpectedly
suggest that mTOR, and not p110α inhibitors, are the most effective
PI3K pathway inhibitors of developmental angiogenesis in zebrafish.
In addition, the lack of expression of the p110α isoform at early timepoints in zebrafish may account for the differential effects of some
inhibitors on ISV and HV angiogenesis. Further investigations of the
PI3K pathway and drug combinations hold great promise for the
identification of better therapeutics for ocular disease.
Commercial Relationships: Temitope Sasore, None; Alison L.
Reynolds, PCT/IE2012/000002 (P); Breandan N. Kennedy,
University College Dublin (P)
Comparison of the toxicity of different drug delivery
nanoparticles in RPE and photoreceptor cells
Yueran Yan, Haijiang Lin, Hidetaka Matsumoto, Peggy Bouzika,
Joan W. Miller, Demetrios Vavvas. Massachusetts Eye and Ear
Infirmary, Harvard Medical School, Boston, MA.
Purpose: Multiple nanoparticles made of synthetic materials have
been widely used as drug delivery systems. However, their toxicity to
the retina is still not clear. In this study, three different kinds of
nanoparticles made of poly lactic-co-glycolic acid (PLGA),
polycaprolactone (PCL) and PEGylated polymer (PEG-P) were
studied and their toxic effects in RPE cells and photoreceptors were
determined in vitro and in vivo.
Methods: PLGA, PCL, and PEG-P nanoparticles were prepared by
an oil-in-water (O/W) emulsion/solvent evaporation method.
ARPE19 cells were treated with different concentrations of each of
the three nanoparticles. The toxicity observed in each case was
determined at different time points by MTT (Sigma Aldrich), LDH
(Promega), ATP (Promega) and TUNEL (Millipore) assays. For the
in vivo study, PEG-P nanoparticles were injected into C57BL/6
mouse eyes intravitreally and sub-retinally. Eyes were enucleated at
day 3 or 7. TUNEL staining was used to evaluate photoreceptor cell
death. Photoreceptor cell loss was evaluated by measuring the
thickness of outer nuclear layer (ONL). Microglias in ONL were
quantified in retinal whole mount immunolabeled with Iba-1
antibody. RPE degeneration was also assessed in RPE whole mount
immunolabeled with ZO-1 antibody.
Results: Our experiments showed that there was almost no toxicity
of PEG-P nanoparticles in ARPE19 cells. Most importantly, no
toxicity was observed when exposing the cells to a high PEG-P
concentration (200ug/ml) for up to 1 week. PLGA nanoparticles
showed greater toxicity than their counterparts. At 24 hours, PLGA
caused more than 20% cell death at a concentration of 25ug/ml and
50% cell death at 200ug/ml. These results were confirmed with
different cell death assays. The toxic effect of these nanoparticles in
the RPE cells and the photoreceptors was also determined in vivo.
Conclusions: PLGA, PCL, and PEG-P nanoparticles show different
toxicity profiles. PEG-P has the least toxicity in the RPE cells and
photoreceptors.
Commercial Relationships: Yueran Yan, None; Haijiang Lin,
None; Hidetaka Matsumoto, None; Peggy Bouzika, None; Joan
W. Miller, Massachusetts Eye and Ear Infirmary (P), Novartis (I),
Alcon (C), KalVista Pharmaceuticals (C); Demetrios Vavvas, MEEI
(P), Kala pharmaceuticals (C), Roche (C), Genentech (C)
A Wnt Inhibitor for Treatment of Diabetic Retinopathy
Danyang Chen, Erica Little. Charlesson, LLC, Oklahoma City, OK.
Purpose: Diabetic retinopathy is characterized by multiple
pathological processes which are associated with numerous
molecular pathways, such as vascular endothelial growth factor, Wnt,
inflammation and other pathways. Accumulated evidence has
demonstrated that the Wnt pathway is responsible for diabetic
retinopathy. Thus, the drugs targeting Wnt pathway would be
effective in the treatment of diabetic retinopathy.
Methods: The effects of a Wnt inhibitor (CLT-010) on the Wnt
pathway and retinal vascular endothelial cell growth were
determined. Dual-luciferase reporter assay and Western blotting were
performed to evaluate the effects of CLT-010 on the Wnt signaling
activation and Wnt-responsive gene expression, respectively. MTT
assay was used to examine the cell viability.
Results: CLT-010 attenuated the Wnt pathway over-activation by
affecting the phosphorylation of low-density lipoprotein receptor-
related protein 6 and β-catenin. The compound also down-regulated
Wnt3a-induced over-expression of Wnt-responsive gene and
inhibited the growth of retinal vascular endothelial cells.
Conclusions: CLT-010 had inhibitory effects on the over-activation
of Wnt signaling, over-expression of Wnt-responsive gene and
growth of retinal vascular endothelial cells.
Commercial Relationships: Danyang Chen, Charlesson, LLC (E);
Erica Little, Charlesson LLC (E)
Support: NIH Grant EY019417 and NIH Grant EY021683
Impact of Ocriplasmin Therapy in Symptomatic Vitreomacular
Adhesion (VMA) Patients Considered to be Clinical Candidates
for Vitrectomy
Baruch D. Kuppermann. Gavin Herbert Eye Inst Dept Ophthal,
University of California Irvine, Irvine, CA.
Purpose: To evaluate efficacy and safety of ocriplasmin versus
placebo for pharmacologic VMA resolution in the subset of eyes
considered to be clinical candidates for vitrectomy.
Methods: The ocriplasmin phase 3 program included symptomatic
patients with OCT-confirmed VMA who were randomized to receive
a single intravitreal injection of 125 µg ocriplasmin (n=464) or
placebo (n=188). Clinical criteria for consideration for vitrectomy in
this subset analysis was visual acuity (VA) of 65 ETDRS letters
(20/50) or less (n=301) or full-thickness macular hole (FTMH,
equivalent to stage II) (n=153) at baseline. A total of 127 patients met
both criteria. In patients without baseline FTMH we evaluated the
rate of VMA resolution at 28 days post-injection. In patients with
baseline FTMH, VMA resolution and FTMH closure at day 28 were
evaluated.
Results: Pharmacologic VMA resolution at day 28 was observed in a
significantly larger proportion of eyes in the ocriplasmin group
compared to placebo. Among patients with a baseline VA of 65
letters or less, 33.2% of eyes treated with ocriplasmin achieved VMA
resolution compared with 11.5% in the placebo group (P<0.001). In
patients with a baseline FTMH, these rates were 50.0% and 25.5%
(P=0.006), respectively, and correlated with a FTMH closure rate of
40.6% in the ocriplasmin group and 10.1% in the placebo group
(P<0.001). Mean baseline ETDRS scores were 53.3/54.8 (≤65
letters/FTMH) in the ocriplasmin groups and 56.3/58.7 (≤65
letters/FTMH) in the placebo groups. Greater changes in mean
BCVA were seen in both VMA and FTMH ocriplasmin groups,
+6.6/+6.8 ETDRS letters, compared placebo, +3.7/+2.3 ETDRS
letters, at month 6 (P=0.114/P=0.086). Achievement of ≥2-line
BCVA gain at month 6 occurred in a greater proportion of the
VMA/FTMH ocriplasmin groups (43.0%/44.3%) compared to
placebo (28.7%/30.4%; P=0.018/P=0.104). BCVA improvement of
≥3 lines was greater in the ocriplasmin groups (25.2%/27.4%)
compared to placebo (10.3%/13.0%; P=0.003/P=0.063). Most
suspected treatment-related adverse events were mild, non-serious,
and occurred within 7 days post-injection. No cases of
endophthalmitis were reported.
Conclusions: Treatment with a single intravitreal injection of
ocriplasmin was effective in symptomatic VMA patients who would
commonly be considered candidates for vitrectomy (VA ≤20/50 or
presence of FTMH at baseline).
Commercial Relationships: Baruch D. Kuppermann, Alimera (C),
Allegro (C), Allergan (C), Genentech (C), Glaukos (C), GSK (F),
Novagali (C), Novartis (C), Ophthotech (C), Pfizer (C), Regeneron
(C), Santen (C), SecondSight (C), Teva (C), ThromboGenics (C)
Support: ThromboGenics, Inc.
SCHISANDRIN B IMPROVES THE VISUAL MOTOR
RESPONSE AND PRESERVES PHOTORECEPTORS IN THE
ZEBRAFISH PDE6C CONE DYSTROPHY MUTANT
Yuk Fai Leung1, 3, Liyun Zhang1, Leelyn Chong1, Jin Cho1, Kam Ming
Ko2. 1Biological Sciences, Purdue University, West Lafayette, IN;
2
Division of Life Science, The Hong Kong University of Science &
Technology, Hong Kong, Hong Kong; 3Biochemistry and Molecular
Biology, Indiana University School of Medicine Lafayette, West
Lafayette, IN.
Purpose: Schisandrin B (Sch B), an active component of Fructus
Schisandrae, has been indicated in traditional Chinese medicine
(TCM) to offer eye benefits. This study investigated the extent to
which Sch B could improve retinal degeneration in the zebrafish
pde6c cone dystrophy mutant.
Methods: A titration series of Sch B was first conducted to identify a
treatment dosage that would activate G6PD, a known marker of Sch
B treatment, without affecting the gross morphology. Then, pde6c
and wild-type (WT) siblings were treated with the optimized 1.875
µM Sch B or equal amount of DMSO carrier from 3 days postfertilization (dpf) to 6 dpf. The treatment scheme was chosen because
it was theorized that most TCMs play a protective role in diseases
and thus, the best chance to determine the protective effect of the
treatment would be before any observable photoreceptor
degeneration at 4 dpf. The visual performance of the resulting larvae
was analyzed by optokinetic response (OKR) and visual-motor
response (VMR), a light-induced swimming activity (Zhang et al.,
Asia-Pac J Ophthalmol 2012; 1:374-383; Emran et al., J Vis Exp.
2008;3(20)). The VMR in this study was optimized to detect lightinduced response originated from the eyes, as the VMR was mostly
abolished in the enucleated larvae. The photoreceptors in these
embryos were also analyzed by immunohistochemistry and qPCR
Results: The pde6c larvae elicited a substantially reduced VMR;
thus, their activity profile was very different from the WT siblings.
Treating pde6c with 1.875 µM Sch B increased their VMR in
response to the light-ON but not the light-OFF stimulus and the
OKR. Interestingly, the histological analyses indicated that the rods
were preserved in the pde6c mutants and no noticeable difference in
the cones. These observations are also supported by a substantial
increase in the expression of gnat1 (rod marker) but not gnat2 (cone
marker) in the qPCR results.
Conclusions: Sch B might have improved the VMR of pde6c
mutants by the preserving their rods. Since one of the best known
functions of Sch B is its antioxidant property, oxidative stress is
implied to play a role in cell death of the rods in the pde6c mutants.
In addition, this study has established the utility of using VMR in
zebrafish for identifying drugs that may have eye benefits.
Commercial Relationships: Yuk Fai Leung, None; Liyun Zhang,
None; Leelyn Chong, None; Jin Cho, None; Kam Ming Ko, None
Phase I, Randomized, Double-Masked, Vehicle-Controlled Study
to Evaluate Safety, Tolerability and Pharmacokinetics of
Recombinant Human Nerve Growth Factor Eye Drops in
Healthy Volunteers
Ronald R. Buggage, Pier A. Ruffini, Mauro P. Ferrari. Dompe
S.p.A., Milan, Italy.
Purpose: Nerve growth factor, an endogenous protein involved in the
differentiation and maintenance of neurons, is a potential treatment
for ocular neurodegenerative conditions. A clinical trial evaluating
the safety of a topical ophthalmic formulation of recombinant human
nerve growth factor (rhNGF) in normal subjects was conducted.
Methods: Healthy adult volunteers with no notable ocular/medical
history and having normal ophthalmic and general examinations at
screening and baseline were enrolled and assigned to 1 of 3 study
phases. In Part 0, 1 eye received a single administration of rhNGF
0.5, 5 or 20 µg/ml. In Part A, 1 drop of rhNGF 20, 60 or 180 µg/ml or
vehicle was administered to 1 eye every 4 hours for 1 day while in
Part B, the same dose regimens or vehicle was administered to 1 eye
daily for 5 days. In all phases treatment was randomized and the
fellow eye received a matching vehicle regimen. Safety evaluations
included a complete ophthalmic exam, ECG, vital signs, urinalysis
and routine labs. Plasma samples for pharmacokinetics and
immnogenicity were obtained in parts A and B.
Results: Parts 0, A and Part B first cohort have been completed with
45/73 subjects treated with rhNGF. Masked review of all available
safety information reveals good tolerability to the study medication
with no clinically significant ocular or systemic changes observed.
Ocular adverse events (AEs), generally transient and mild, included
warm feeling, pressure, hazy film, increased corneal fluorescein
staining, blurred vision, upper eyelid burning, headache above the
eyes, photophobia, itching and loss of color distinction. In 3/8
subjects the AEs affected both eyes. 8 subjects reported non-ocular
AEs not related to the study treatment. No serious adverse events
were recorded.
Conclusions: NGF is a promising therapy for neurotrophic keratitis,
retinitis pigmentosa and optic neuropathies, ocular neurodegenerative
diseases for which no effective treatments currently exist. Early
findings from a first in human trial evaluating a topical ophthalmic
formulation of rhNGF suggest no local or systemic safety concerns.
Unmasked data including pharmacokinetic analysis and
immunogenicity will be presented to complete the safety profile of
rhNGF in healthy volunteers.
Commercial Relationships: Ronald R. Buggage, Dompe S.p.A.
(E); Pier A. Ruffini, Dompé S.p.A. (E); Mauro P. Ferrari, Dompé
s.p.a. (E)
Antiglycating potential of procyanidin-B2 isolated from
cinnamon bark: Prevention or treatment of diabetic ocular
complications (cataract & retinopathy)
G Bhanuprakash Reddy, Puppala Muthenna, Chandrasekhar
Akileshwari, Ganugula Raghu, Palla Suryanarayana. Biochemistry,
National Institute of Nutrition, Hyderabad, India.
Purpose: Accumulation of advanced glycation endproducts (AGE)
due to non-enzymatic glycation of proteins has been implicated in
several pathophysiologies associated with diabetic ocular
complications. Earlier we have identified a few dietary sources such
as cinnamon that have the potential to inhibit AGE formation. In this
study, we have isolated and identified procyanidin-B2 as the
antiglycating agent from cinnamon bark (Cinnamomum zeylanicum)
and investigated its potential to prevent diabetic cataract and
retinopathy in rat model.
Methods: Using bioassay-guided fractionation, procyanidin-B2 was
identified as an antiglycating agent from cinnamon and demonstrated
its antiglycating potential and mechanism of action of using in vitro
and ex vivo protein glycation systems. Further the effect of
procyanidin-B2 and its dietary source (cinnamon) to prevent diabetic
cataract and retinopathy was investigated using streptozotocininduced diabetic rat model.
Results: Under in vitro conditions, procyanidin-B2 inhibited the
protein glycation as assessed by AGE-fluorescence, SDS-PAGE and
immunodetection of specific AGE. Further, we provided insight into
the mechanism of inhibition of protein glycation that it scavenges
free radicals directly and trapping of dicarbonyls. In addition,
procyanidin-B2 inhibited the glycosylated hemoglobin formation in
human RBC under ex vivo conditions. We also demonstrated that
feeding of proycanidin-B2 to diabetic rats in the diet was effective
against development of cataract and retinopathy in streptozotocin
mainly through its antiglycating potential. Procyanidin-B2 decreased
levels of glial fibrillary acidic protein expression and vascular
endothelial growth factor and enhanced nerve growth factor
expression in diabetic retina.
Conclusions: Thus, the active principle procyanidin-B2 isolated from
dietary cinnamon might be useful for the treatment and/ or prevention
of diabetic ocular complications.
Commercial Relationships: G Bhanuprakash Reddy, Indian
Council of Medical Research (P); Puppala Muthenna, None;
Chandrasekhar Akileshwari, None; Ganugula Raghu, None;
Palla Suryanarayana, None
Support: Department of Biotechnology & Life Sciences Research
Board, India
Effects of resveratrol, epigallocatechine gallate (EGCG) and
curcumin on the proliferation of human retinal endothelial cells
in vitro
Anne F. Alex1, Manfred Spitznas2, Christian Kurts3, Nicole Eter1.
1
Department of Ophthalmology, University of Muenster Medical
Center, Muenster, Germany; 2Department of Ophthalmology,
University of Bonn Medical Center, Bonn, Germany; 3Institutes of
Molecular Medicine and Experimental Immunology, University of
Bonn Medical Center, Bonn, Germany.
Purpose: Choroidal neovascularization (CNV) is a commonly
occuring pathology in various eye diseases, e.g. in an advanced stage
of age-related macular degeneration (AMD). Vascular endothelial
growth factor (VEGF) is known to be upregulated and leads to
enhanced endothelial cell growth. The polyphenols resveratrol,
epigallocatechine gallate (EGCG) and curcumin have
antiproliferative and antiinflammatory effects. In this study, the
effects of these polyphenols on human retinal endothelial cells
(hREC) were investigated in a cell culture model.
Methods: hREC were cultured in 2% serum-containing cell culture
medium and different concentrations of resveratrol, EGCG and
curcumin were tested. Flow-cytometric analysis was performed after
24, 48 and 72 hours of incubation. Absolute cell numbers were
counted, cell proliferation was measured with the Carboxyfluorescein
succinimidyl ester (CFSE) dilution assay and dead cells were
analysed with the nuclear dye Hoechst 33258. Apoptotic cells could
be distinguished with active caspase 3 staining.
Results: All three polyphenols diminished absolute cell numbers and
the number of cell cycles in a dose-dependent manner compared to
the untreated control. 25 µM Resveratrol led to a significant
reduction in cell cycle numbers over 48 hours of nearly 40%. EGCG
also had a strong effect on the proliferation and 25 µM reduced the
accomplished cell cycles to 0,65 (control: 0,78), with 50 µM the
effect was even stronger. Curcumin showed effects already with 5
µM and these effects were enhanced with 10 µM. Only EGCG with a
concentration of 25 µM and higher slightly induced cell death,
resveratrol even significantly reduced apoptosis of hREC.
Conclusions: All three polyphenols reduced the absolute number of
hREC and had an inhibitory effect on the cell proliferation by
reducing the number of cell cycles in a dose-dependent manner. In
comparison to previously published data on retinal pigment epithelial
cells (Alex et al., 2010) the concentrations were lower to achieve
these effects on hREC. In vivo, a cell specific targeting might
therefore be achieved by dosing. Further studies are needed to
evaluate effects on other retinal cells.
Commercial Relationships: Anne F. Alex, None; Manfred
Spitznas, None; Christian Kurts, None; Nicole Eter, Novartis (F),
Bayer (R), Heidelberg Engineering (R), Sanofi Aventis (C), Allergan
(C), Bausch and Lomb (C)
Circulating miRNAs as Biomarkers of Retinal Toxicity
Qinghai Peng, Wenhu Huang, Walter Collette, Michelle Twamley,
Shirley Aguirre, Annette John-Baptiste. Pfizer, San Diego, CA.
Purpose: To examine whether circulating retinal-enriched
microRNAs were altered post treatment in a drug induced retinal
toxicity study in rat.
Methods: Wistar Hans rats were administered a single intravitreal
injection of either a pan-CDK inhibitor (10 or 30 µg/eye) or a HSP90
inhibitor (9 or 27 µg/eye). EDTA whole blood samples were
collected predose, and on days 1, 3 and 7 post dose. Retinal-enriched
miRNAs and the liver-enriched miR-192 (used as a control) were
analyzed by qRT-PCR. Electroretinography (ERG) was performed
predose and at end of study to assess retinal function. Eyes were
collected either on day 8 or at 2 weeks post dose and processed for
histopathologic evaluation.
Results: Elevations of plasma miR-124a, miR-96, and miR-183
peaked on day 3 post dose in the pan-CDKi high dosed animals
demonstrating greater than 300X, 10X and 6X fold increases
respectively in a time-dependent manner . The low dose group of the
pan-CDKi treated animals demonstrated slight elevations of miR124a, miR-96 and miR-183. Neither dose group in the pan-CDKi
treated animals demonstrated any change in miR-192 and retinal
degeneration was confirmed by ERG and histopathology.
Comparatively, the HSP90 treated group, exhibited no changes in
miRNA levels, ERG or histopathology.
Conclusions: This study demonstrated plasma miR-96, miR-124a,
and miR-183 are strong contenders as retinal toxicity biomarkers.
Although these miRNAs need additional validation whether they
truly predict retinal toxicity or not prior to histopathology, these
results provide promise for further test using additional retinal
toxicants.
Commercial Relationships: Qinghai Peng, None; Wenhu Huang,
None; Walter Collette, None; Michelle Twamley, Pfizer, Inc. (E);
Shirley Aguirre, None; Annette John-Baptiste, None
Comparison of intraocular retinal pigment epithelial (RPE) cell
injections in vitrectomized wild type pigs
Elliott H. Sohn1, 2, Chunhua Jiao2, Robert F. Mullins2, Woojin Jung2,
Stephen R. Russell1, 2, Edwin M. Stone1, 2, Budd A. Tucker2. 1Retina
Service, University of Iowa Hospitals & Clinics, Iowa City, IA;
2
Institute for Vision Research, University of Iowa, Iowa City, IA.
Purpose: Various sources of RPE cells have been proposed for cell
replacement therapy (e.g. in the subretinal space to treat geographic
atrophy) and in generating models of proliferative vitreoretinopathy
(PVR). In this pilot study we sought to determine the effects of
induced pluripotent stem cell derived RPE cells (iPS-RPE) delivered
to the subretinal space and allogeneic RPE cells injected into the
vitreous cavity of vitrectomized wild-type mini-pigs.
Methods: 23 gauge vitrectomy was performed in 12-week-old
yucatan miniature swine (n=10). Three treatment cohorts consisted of
those given blebs of balanced salt solution alone (controls); blebs
followed by intravitreal injection of porcine-derived GFP-positive
RPE cells (250,000 cells, RPE group); and subretinal blebs of
porcine-derived, GFP-positive iPS-RPE cells (250,000 cells, iPSRPE group). Indirect ophthalmoscopy, fundus photography and
spectral-domain-OCT were performed at weekly post-op intervals.
Post-op vitreous samples were screened for inflammatory cytokines
using a commercial cytokine array and total proteins were evaluated
on silver-stained SDS-PAGE gels for proteomic analysis.
Results: Vitreous membranes were seen in all eyes in the iPS-RPE
cell group but no PVR or retinal detachment was observed. Epiretinal
membranes and retinal detachment were seen in a third of eyes in the
RPE group. No PVR or vitreous membranes were seen in the saline
controls.
At post-op week 3, vitreous IL-8 levels were elevated in the iPS-RPE
group compared to the porcine RPE and saline controls (image 1);
these levels were higher than those observed in post-op weeks 1 and
2. IL-12 levels were greater in post-op week 3 compared to post-op
week 1 of the iPS-RPE group (image 2). TGF-beta levels were very
low or below limits of detection in all eyes.
Silver staining revealed a distinct subset of bands in the vitreous of
pigs that received iPS-RPE cells. MALDI-MS of one of these bands
identified several proteins including transthyretin and beta-2microglobulin.
Conclusions: Subretinal injection of iPS-RPE cells in wild-type
mini-pigs may result in an increase in inflammatory response specific
to particular cytokines. Exogenous intravitreal RPE cells may result
in a modest rate of PVR. Further research is needed to verify these
findings.
Commercial Relationships: Elliott H. Sohn, None; Chunhua Jiao,
None; Robert F. Mullins, Alcon Research Ltd (F); Woojin Jung,
None; Stephen R. Russell, IDx, LLC (I), IDx, LLC (P); Edwin M.
Stone, None; Budd A. Tucker, None
Assessment of the therapeutic value of phloroglucinol in
Stargardt’s disease
Philippe Brabet1, David Cia2, Claire Vigor3, Nathalie Jacquemot2,
Benoit Lerat1, Laurent Guillou1, Celine Crauste3, Christian P.
Hamel1, Joseph L. Vercauteren3. 1Institute for Neurosciences of
Montpellier, INSERM U1051, Montpellier, France; 2Laboratoire de
Biophysique Neurosensorielle, UMR Inserm 1107, ClermontFerrand, France; 3Institut des Biomolécules Max Mousseron
(IBMM), UMR CNRS 5247, Montpellier, France.
Purpose: Autosomal recessive Stargardt's disease is the most
common cause of macular dystrophies due to mutations in ABCA4
gene. When transporter ABCA4 activity is impaired, all-trans retinal
(atRAL) accumulates and triggers formation with
phosphatidylethanolamine of A2-PE by carbonyl and oxidative stress.
Daily photoreceptor outer segments (POS) phagocytosis by retinal
pigment epithelium (RPE) leads to A2E accumulation, causing RPE
death and progressive photoreceptor loss. Our goal is to design and
select powerful chemicals against carbonyl and oxidative stress (antiCOS).
Methods: Phloroglucinol (benzene-1,3,5- triol) was first assessed for
its efficacy as anti-COS. ARPE-19 cell line and rat RPE primary
cultures were pre-incubated with phloroglucinol before incubation
with oxidative (H2O2 and A2E) and carbonyl (atRAL) stressors. Cell
viability was measured using tetrazolium compound (MTT) assay.
Alternatively, phloroglucinol was co-incubated with atRAL. Testtube experiments were carried out to inhibit the synthesis of A2E
from atRAL and ethanolamine in the presence of phlorogucinol.
Adducts formed between phloroglucinol and atRAL were identified
by NMR and mass analysis.
Results: Treatment of RPE cells with phloroglucinol alone does not
affect the cell viability. Exposure of RPE cells to stressors caused
dose-dependent decreases in cell viability, whereas pretreatment with
phloroglucinol significantly reduced the decline. Co-treatment of
RPE cells with phloroglucinol and atRAL drastically prevented RPE
cell death. In the presence of atRAL and ethanolamine,
phloroglucinol led to complete inhibition of A2E synthesis.
Conclusions: Phloroglucinol has a dual action as anti-carbonyl stress
and antioxidant in RPE cells. The proposed mechanism for the anticarbonyl stress action is the trapping of all-trans retinal. Lower
protection observed during pre-incubation compared to co-incubation
suggests a low bioavailability of phoroglucinol.
Commercial Relationships: Philippe Brabet, None; David Cia,
None; Claire Vigor, None; Nathalie Jacquemot, None; Benoit
Lerat, None; Laurent Guillou, None; Celine Crauste, None;
Christian P. Hamel, None; Joseph L. Vercauteren, None
Support: This work was supported by private foundations (Retina
France, IRRP, UNADEV), University of Montpellier (UM1 and
UM2), and Inserm.
Recurrence of macular edema after intravitreal bevacizumab
injection in eyes with macular edema secondary to retinal vein
occlusion
young gyun Kim1, Ji young Moon1, Kyung Hoon Seo2, Seung yong
Lee3, Eung Suk Kim3, Seung young Yu2, Hyung woo Kwak2. 1Eulji
medical center, Seoul, Republic of Korea; 2Kyung Hee hospital,
Seoul, Republic of Korea; 3Eulji University Hospital, Daejeon,
Republic of Korea.
Purpose: The purpose of this study is to find out the incidence of
recurred macular edema after the intravitreal bevacizumab injection
and related factors which affects the recurrence of macular edema
during the 6 months’ follow-up.
Methods: Intravitreal bevacizumab injection was performed in 73
eyes of 73 patients who had macular edema with RVO. Best
corrected visual acuity (BCVA) and central macular thickness (CMT)
were checked for 6 months after the injection. Recurrence was
defined as a 30% increase in CMT after an initial decrease of CMT.
Factors that differed between the recurred group and the non-recurred
group were identified.
Results: Recurrence occurred in 38 of 73 eyes (49.4%) and the mean
time between injection and recurrence was 15.2 weeks. The mean
interval between the onset and the initiation of intravitreal
bevacizumab injection was 3.42 weeks in the recurred group, which
showed a significant difference compared to the non-recurred
group(P<0.05). Also, a greater decrease in CMT 1 week after
injection was associated with a decrease in recurrence(P<0.05). Other
than those, age, gender, the patients’ comorbidity, whether the
consecutive bevacizumab injection was performed or not after
resolution of macular edema, the number of injection, pre-treatment
BCVA and CMT were not associated with recurrence.
Conclusions: Recurrence rate was very high after intravitreal
bevacizumab injection in eyes with macular edema secondary to
RVO. To avoid recurrence, injection can be recommended to initiate
at least 4 weeks after the onset. A better prognosis can be expected in
patients whose CMT decreases greatly from baseline after 1 week of
treatment.
Commercial Relationships: young gyun Kim, None; Ji young
Moon, None; Kyung Hoon Seo, None; Seung yong Lee, None;
Eung Suk Kim, None; Seung young Yu, None; Hyung woo Kwak,
None
Increasing mitochondrial respiratory capacity is protective in
models of photoreceptor cell degeneration
Mausumi Bandyopadhyay1, Nathan R. Perron2, Cecile Nasarre1,
Craig Beeson2, Baerbel Rohrer1. 1Ophthalmology, Medical
University of South Carolina, Charleston, SC; 2Pharmaceutical
Sciences, Medical University of South Carolina, Charleston, SC.
Purpose: Alterations in energy metabolism are associated with rod
degeneration. An increase in glycolysis over mitochondrial
respiration has been identified prior to the onset of rod loss in the rd1
mouse model of retinitis pigmentosa (RP). We hypothesize that
preserving mitochondrial respiratory capacity (MRC) might lead to
prolonged cell survival. Hence, we sought to identify compounds,
which target MRC, for neuroprotection in the rd1 retina.
Methods: The DIVERSet library (ChemBridge), a unique, nonproprietary collection of 50,000 synthetic small molecules that covers
the maximum pharmacophore diversity with the minimum number of
compounds, was used. Three rounds of screening, increasing in
complexity, were performed. As a Primary Screen, cell survival
assays (MTS) were performed, identifying compounds that protect
against Ca2+ cytotoxicity induced by Ca2+ ionophore A23187. As a
Secondary Screen, compounds were analyzed using Seahorse
extracellular flux (XF) assays, measuring lactate secretion
(glycolysis) and oxygen consumption (mitochondrial ATP
production). As a Tertiary Screen, compounds were tested in rd1
retina/RPE explants to determine their effect on rod degeneration.
Postnatal day 10 explants were treated every 48 hrs with compounds
for a total of 10 days. Cultures were sectioned and counterstained to
count rows of photoreceptors in ten regions.
Results: A23187 was titrated to result in 50% cell death after 24 hrs.
Twelve compounds were identified to protect against Ca2+
cytotoxicity. For XF assays, IBMX was titrated to cause a 50%
decrease of the maximal (FCCP-uncoupled) oxygen consumption rate
after 24 hrs. Pretreatment with six compounds significantly preserved
uncoupled mitochondrial function. In rd1 explants, four compounds
slowed rod cell loss, resulting in 2-4-fold increase in thickness of the
outer nuclear layer after 10 days in culture.
Conclusions: These results suggest that impaired energy metabolism
in photoreceptors is a major contributor to RP; and our High-Content
Screening protocol identified novel compounds for the treatment of
RP and other forms of neurodegeneration.
Commercial Relationships: Mausumi Bandyopadhyay, None;
Nathan R. Perron, MitoChem (P); Cecile Nasarre, None; Craig
Beeson, None; Baerbel Rohrer, WO/2007/149567 (P), Colorado
University, CU3015H (P), 61/317,185 (P)
A deca-peptide inhibits retinal neovascularization by downregulation of VEGF and up-regulation of PEDF in OIR mouse
Xun Xu. Department of Ophthalmology, Shanghai First People's
Hospital, Shanghai, China.
Purpose: Neovascular retinopathies collectively comprise the most
common cause of blindness and affect millions of people from infants
to the elderly. Our previous study has demonstrated that a novel decapeptide, TKII-10, effectively inhibits angiogenesis in chick
chorioallantoic membrane and corneal neovascularization. The
purpose of this study is to investigate the inhibitory efficacy and
mechanism of TKII-10 on retinal neovascularization, in an effort to
develop a small peptide for clinical application in neovascular
retinopathies.
Methods: (1) In vitro, MTS asssy, cell migration assay with tranwell
chamber, and tube formation assay on Matrigel were carried out to
evaluate the inhibitory effect of TKII-10 on VEGF induced retinal
endothelial cell proliferation, migration, and tube formation.
Bevacizumab (Avastin) was set as positive control, and a scramble
peptide, TKII-10S, was set as negative control. (2) The
antiangiogenic effect of TKII-10 was further confirmed in retinal
neovascularization in oxygen-induced retinopathy in vivo. (3) In
oxygen-induced retinopathy model, real time PCR and western blot
assays were used to explore the influence of TKII-10 on the mRNA
and protein expression of VEGF and PEDF in mouse retina.
Results: (1) TKII-10 inhibited VEGF-induced endothelial cell
migration and tube formation dose-dependently. TKII-10 did no
inhibit endothelial VEGF-induced cell proliferation. (2) In OIR assay,
the TKII-10 group demonstrated obviously reduced nonperfused area,
less neovascular tuft at the junction, and improved vessel dilation
compared with the PBS group. There was a significant reduction in
the number of vascular cell nuclei extending from the retinal surface
into the vitreous in TKII-10 group compared with the PBS group
(p<0.01). (3) In oxygen-induced retinopathy, intravitreous injection
of TKII-10 resulted in down-regulation of VEGF mRNA and protein
expression, concomitant up-regulation of PEDF mRNA and protein
expression in the oxygen plus TKII-10 group compared with the
oxygen plus PBS group (p<0.01).
Conclusions: TKII-10 potently inhibits VEGF-induced endothelial
cell migration and tube formation in vitro while it is inactive in
inhibiting endothelial cell proliferation. TKII-10 effectively inhibits
pathological retinal neovascularization by down-regulation of VEGF
mRNA and protein expression and concominent up-regulation of
PEDF mRNA and protein.
Commercial Relationships: Xun Xu, None
Support: National Natural Science Foundation of China No.
81170862
Novel GFAP Species in Retinal Gliosis
John Wizeman, Paola Bargagna-Mohan, Sean O'Rourke, Royce
Mohan. Neuroscience, University of Connecticut Health Center,
Farmington, CT.
Purpose: Retinal gliosis occurs after injury and is an insidious
process that also undermines several leading retinal diseases. A
characteristic hallmark of gliosis is the upregulation of the type III
intermediate filaments (IF) glial fibrillary acidic protein (GFAP) and
vimentin. In injury models, the absence of GFAP and its binding
partner vimentin has been shown to be protective. We have used the
GFAP/vimentin targeting small molecule withaferin A (WFA;
Bargagna-Mohan JBC 2010) as a chemical probe of GFAP
expression in an injury model.
Methods: C57BL/6N, 129SvEV vimentin-deficient or wild type
129SvEV mice were subjected to a corneal alkali injury (1N NaOH)
followed by epithelial debridement. Mice were allowed to heal (3 to 5
days), after which retinal cups were isolated and placed into explant
organ culture in medium containing DMEM/F12 +10% FBS and
antibiotics. Retinal cultures were treated with either WFA or the
proteasome inhibitor epoxomicin for 3 days and soluble tissue
extracts (200 mM NaCl, 1% NP-40) were subjected to western blot
analysis.
Results: As expected, injury increased 52kDa soluble GFAP
expression in the retina. Unexpectedly, there was injury-related
increase in novel high molecular weight (100-200 kDa) GFAP
species in retinas from all mouse strains. This high molecular weight
GFAP species was also susceptible to perturbation by WFA, with 50
nM WFA decreasing total GFAP levels by ~2.4 fold as compared to
an injured, untreated retina. Higher concentrations of WFA had an
impact on polyubiquitination levels. At 1 μM WFA, the total amount
of ubiquitinated species was decreased ~3.8 fold compared to the
injured, untreated retina. Treatment with epoxomicin caused
accumulation of ubiquitinated GFAP species, a change that was also
modulated with the addition of WFA.
Conclusions: Collectively, our results suggest that injury causes an
induction of soluble GFAP expression in retinal Müller glia
indicative of a novel post-translational modification. Importantly,
these high molecular weight, modified GFAP species were targeted
by WFA, mediated by a mechanism independent of proteasome
inhibition. The identification of the causative modification to GFAP
could lead to a deeper understanding of the mechanisms of gliosis
caused by either injury or disease. The interaction of GFAP with the
ubiquitin proteasome pathway remains a critical mechanism that
needs to be characterized.
Commercial Relationships: John Wizeman, None; Paola
Bargagna-Mohan, UKY Research Foundation, US8283323B2 (P);
Sean O'Rourke, None; Royce Mohan, University of Kentucky
Research Foundation (P)
Support: NIH R01 EY016782; John A. and Florence Mattern
Solomon Endowed Chair
Effect of Intravitreal Injection of Iodoacetic Acid in Mice as a
Model of Pharmacological Induced Monolateral Photoreceptor
Degeneration
Sarah Roesch1, Stephan Hesse1, Christine Haselier1, Babac A.
Mazinani1, Gernot Roessler1, Christiane Pfarrer2, Peter Walter1.
1
Ophthalmic clinic, University hospital RWTH, Aachen, Germany;
2
University of Veterinary medicine Hannover, Anatomical Institute,
Hannover, Germany.
Purpose: To characterize the effect of intravitreal injection of
Iodoacetic Acid (IAA) in comparison to its systemic application as a
measure to induce monolateral photoreceptor degeneration.
Methods: Seven weeks old C57BL/6J mice received either
intravitreal injections of IAA (25 animals) in different concentrations
(between 0.1-1 mg/kg bodyweight (BW)) or systemic treatment
[intraperitoneal (8 animals, 40-70 mg/kg BW) and intravenous (3
animals, 60-70 mg/kg BW)] and were observed in the following five
weeks using ERG, OCT, and histology.
Results: Systemic application of IAA showed a mortality of 25% and
toxic systemic effects with weight loss in the first two days of 10%.
Intravenous application of IAA resulted in an extinction of the ERG
and a thinning of the retina in the OCT. Intraperitoneal injection of
IAA in mice showed no effect in the ERG or OCT for five weeks
after injection.
Animals that received intravitreal injections showed no weight loss
after injection.
Three to five days after injection the eyes developed cataracts ranging
from 20% of cases at an IAA concentration of 0.1 mg/kg BW to
100% at an IAA concentration of 0.25 mg/kg BW.
Higher intravitreal IAA concentrations lead to extinguished ERGs
from 16% of cases at concentrations of 0.2 mg/kg BW to 90% at 0.25
mg/kg BW.
Morphological signs of retinal degeneration after intravitreal
injection were not observed, neither in the OCT nor in the histology.
Conclusions: Intravitreal injection of IAA leads to dense cataracts at
lower concentrations than ERG changes occurred. ERG results must
be interpreted carefully in the presence of induced cataracts.
Morphological signs of retinal degeneration were not seen. In
summary, the intravitreal injection of IAA did not prove to be an
adequate model of monocular retinal degeneration in mice.
Commercial Relationships: Sarah Roesch, None; Stephan Hesse,
None; Christine Haselier, None; Babac A. Mazinani, None;
Gernot Roessler, None; Christiane Pfarrer, None; Peter Walter,
Novartis (R), Bayer (R), Second Sight (R), Bayer (F), Novartis (F)
Support: DFG WA 1472/6-1, DFG PAK 469
PHLOROTANNIN-RICH NATURAL EXTRACT FROM
BROWN SEAWEED ASCOPHYLLUM NODOSUM
PREVENTS IN VITRO HIGH GLUCOSE RETINAL
DAMAGES
Melody Dutot1, Roxane Fagon1, Patrice Rat2.
1
Research&Development, YSLAB, Paris, France; 2ChimieToxicologie Analytique et Cellulaire (EA 4463), Faculté de
Pharmacie, Université Paries Descartes, Paris, France.
Purpose: Activators of the class III histone deacetylase SIRT1,
which are also potent antioxidant molecules, are considered as
therapeutics for the treatment of type 2 diabetes. In this study, we
investigated the protective cellular effects of phlorotannin-rich
natural extract from brown seaweed ascophyllum nodosum against
high glucose on retinal pigmented epithelial (RPE) cells and Müller
glial cells.
Methods: RPE cells (ARPE-19 human cell line) and Müller cells
(MIO-M1 human cell line) were incubated with normal (5mM)
glucose or high (25mM) glucose for 48 hours. Reactive oxygen
species (ROS) production, mitochondrial transmembrane potential,
and SIRT1 enzymatic activity were evaluated. Phlorotannin-rich
natural extract from brown seaweed ascophyllum nodosum was
tested for its ability to inhibit oxidative damages and SIRT1 activity
alterations induced by high glucose.
Results: Phlorotannin-rich natural extract from brown seaweed
ascophyllum nodosum prevented glucose-induced ROS
overproduction and alterations of mitochondrial transmembrane
potential. We observed a significant increase in SIRT1 activity with
phlorotannin-rich natural extract from brown seaweed ascophyllum
nodosum.
Conclusions: Phlorotannin-rich natural extract from brown seaweed
ascophyllum nodosum protected retinal cells against oxidative stress,
mitochondrial damages and SIRT1 activity alteration induced by high
glucose. Ascophyllum nodosum represents a promising ingredient for
functional food supplements for diabetic patients.
Commercial Relationships: Melody Dutot, YSLAB (E); Roxane
Fagon, None; Patrice Rat, None
Support: Adebiopharm ER67
Fenretinide Inhibits Ocular Neovascularization (NV) by
Upregulation of Bone Morphogenic Protein-2 (BMP-2) and
Reduction of Inflammatory Macrophages and VEGF
Rebecca K. Stevens1, Peter A. Campochiaro1, Ji-kui Shen1, Brian C.
Oveson1, Sean F. Hackett1, Nathan L. Mata2. 1Ophthalmology, Johns
Hopkins University, Baltimore, MD; 2Acucela Inc., Seattle, WA.
Purpose: N-(4-hydroxyphenyl) retinamide (4HPR; fenretinide), a
synthetic retinoic acid derivative, is a promising chemopreventive
agent for tumors. A recent clinical trial showed that 4HPR reduced
the incidence of choroidal NV in patients with age-related macular
degeneration and geographic atrophy. We sought to investigate the
effects of 4HPR in mouse models of ocular neovascularization (NV).
Methods: The effects of intraocular and systemic 4HPR were tested
in mice with laser-induced choroidal NV, oxygen-induced ischemic
retinopathy, and rho/VEGF transgenic mice. Levels of various
mRNAs were measured by real-time RT-PCR. The effects of 4HPR
or all-trans retinoic acid (atRA) on AP1 activity was tested with a
dual luciferase expression assay using a pGL3-AP1 artificial
promoter construct in the RPE16 cell line.
Results: Oral administration of 4HPR suppressed choroidal NV and
intraocular injection of 10-7or 10-8M 4HPR significantly suppressed
NV in each of the 3 mouse models. Injection of 4HPR also reduced
the influx of macrophages into ischemic retina. Unlike atRA, 4HPR
did not stimulate expression of RARs or RXRs in the retina after
intraocular injection, nor did it reduce expression of AP1-responsive
genes. In cultured RPE cells, atRA, but not 4HPR, suppressed AP1
activity. After intraocular injection of 10-7or 10-8M 4HPR in mice
with ischemic retinopathy, there was a significant reduction in
mRNA for VEGF and CCL2, and an increase in mRNA for BMP2.
Intraocular injection of recombinant BMP2 significantly suppressed
ischemia-induced NV.
Conclusions: Systemic or intraocular 4HPR suppresses ocular NV,
not through activation of RARs or RXRs and suppression of AP1, but
rather by increasing expression of BMP2 and reducing inflammatory
macrophages with concomitant reduction in VEGF.
Commercial Relationships: Rebecca K. Stevens, None; Peter A.
Campochiaro, Advance Cell Technology (C), Aerpio (C), Elan (C),
Gene Signal (C), Genentech (C), GlaxoSmithKline (C), LPath, Inc
(C), Norvox (C), Regeneron (C), Genentech (F), Genzyme (F),
GlaxoSmithKline (F), Oxford Biomedica (F); Ji-kui Shen, None;
Brian C. Oveson, None; Sean F. Hackett, None; Nathan L. Mata,
Acucela (P)
Blocking the necroptosis pathway decreases RPE and
photoreceptor damage induced by NaIO3
Haijiang Lin, Miin Roh, Hidetaka Matsumoto, Albert H. Alhatem,
Peggy Bouzika, Yusuke Murakami, Joan W. Miller, Demetrios
Vavvas. Ophthalmology-retina, Massachusetts Eye and Ear, Harvard
Medical School, Boston, MA.
Purpose: Sodium iodate (NaIO3) has been used extensively as a
retinotoxin to induce RPE cell damage and degeneration of
photoreceptors in vitro and in vivo. RIP-Kinase dependent
programmed necrosis is an important redundant cell death pathway
that has been shown to be involved in photoreceptor cell death. We
wanted to investigate if these pathways are actively involved in RPE
and photoreceptor cell death after NaIO3 insult.
Methods: ARPE-19 cells were exposed to different concentration of
NaIO3 in the presence or absence of various concentration of RIPK
inhibitors ( Nec-1) or pan-caspase inhibitor (Z-VAD) individually or
in combination. Cell death was determined at different time points by
MTT (Sigma Aldrich), LDH (Promega), ATP (Promega) and TUNEL
(Millipore) assay. C57BL/6 and RIP3-/- mice were treated with a
peritoneal injection of NaIO3 and eyes were enucleated at day 3 or 7.
TUNEL staining was used to evaluate photoreceptor cell death.
Photoreceptor cell loss was evaluated by measuring the thickness of
outer nuclear layer (ONL). Microglias in ONL were quantified in
retinal whole mount with Iba-1 antibody. RPE degeneration was also
assessed in RPE whole mount with ZO-1 antibody.
Results: NaIO3 resulted in significant cell death of ARPE-19 cells.
Treatment with Nec1 resulted in better protection than treatment with
zVAD (P<0.01). A synergistic protective effect is observed when cotreating the cells with Nec-1 and Z-VAD. Nec-1 treatment also
decreased the ARPE-19 mitochondrial damage caused by NaIO3. In
vivo administration of NaIO3 resulted in significant RPE and
photoreceptor destruction with significant inflammatory cell
infiltration . RIP3 knockout animals displayed significantly less RPE
and photoreceptor cell loss as well as significantly less inflammation.
Conclusions: Programmed necrosis is an important cell death
pathway mediating NaIO3 RPE and photoreceptor cell toxicity.
Therefore, blocking the necroptosis pathway may serve as a novel
therapeutic target for various retinal degeneration diseases.
Commercial Relationships: Haijiang Lin, None; Miin Roh, None;
Hidetaka Matsumoto, None; Albert H. Alhatem, None; Peggy
Bouzika, None; Yusuke Murakami, None; Joan W. Miller,
Massachusetts Eye and Ear Infirmary (P), Novartis (I), Alcon (C),
KalVista Pharmaceuticals (C); Demetrios Vavvas, MEEI (P), Kala
pharmaceuticals (C), Roche (C), Genentech (C)
Impact of intravitreal dexamethasone implant (Ozurdex) on
macular morphology and function
Maria Lucia Cascavilla1, Giuseppe Querques1, 2, Rosangela
Lattanzio1, Lea Querques1, Giacinto Triolo1, Edoardo Cavallero1,
Francesco Bandello1. 1Scientific Inst San Raffaele, Segrate-Mi2,
Italy; 2University Paris-Est Creteil, Parigi, France.
Purpose: To investigate changes in macular sensitivity after
intravitreal dexamethasone implant for macular edema (ME)
secondary to retinal vein occlusion (RVO)
Methods: 19 treatment-naive patients with decreased visual acuity
due to RVO-related ME were enrolled in this prospective
uncontrolled study.Patients were treated with intravitreal Ozurdex
(and retreated on as needed basis), and followed up at 1 month 3,4,5
and 6 mos for the evaluation of morphological and functional
outcomes, by means of BCVA,microperimetry (MP), and spectraldomain OCT
Results: 19 eyes (13 central [C]RVO eyes,6 branch [B]RVO eyes)of
19 patients were included for analysis.At 1 month (19 eyes)mean
BCVA,retinal sensitivity,and central macular thickness (CMT)
improved from 0.50±0.34 LogMAR, 10.51±4.31 dB, and 762±259
μm to 0.38±0.34 LogMAR, 12.28±5.06 dB, and 385±191
μm,respectively.At 3 months (19 eyes) improvement of mean
BCVA,retinal sensitivity,and CMT decreased (0.54±0.36
LogMAR,11.62±5.05 dB, and 518±251 μm);at this time point, 2 eyes
were retreated with intravitreal dexamethasone.At 4 months (17
eyes)mean BCVA,retinal sensitivity and CMT were 0.68±0.45
LogMAR,8.64±4.45dB, and 671±239 μm respectively;at this time
point, 4 eyes were retreated.At 5 months (13 eyes) mean BCVA,
retinal sensitivity, and CMT were 0.83 ±0.42 LogMAR, 8.1±3.4 dB,
and 670±218 μm ;at this time point, 6 eyes were retreated.
Interestingly 7 eyes (4 eyes with CRVO, 3 eyes with BRVO)did not
underwent retreatment up to 6 months from first intravitreal
dexamethasone;in
these eyes mean BCVA,retinal sensitivity,and CMT changed from
0.46±0.29 LogMAR, 12.27±3.52 dB, and 715±219 μm (baseline,
versus 0.53±0.37LogMAR, 9.48±4.53dB, and 790±286μm, in eyes
that underwent retreatment <6 months),to 0.41±0.45 LogMAR,
11.33±5.32 dB, and 547±216 μm (6 months).At 6 months, 5 eyes
were retreated. 1 month after retreatment with intravitreal
dexamethasone (12 eyes) mean BCVA, retinal sensitivity, and CMT
improved from last pre-retreatment visit (from 0.93±0.77
LogMAR,8.42±3.98 dB, and 711±172 μm to
0.77±0.46LogMAR [p=0.03], 10.63 ±4.63 dB [p=0.04], and 497±262
μm [p=0.01])
Conclusions: In eyes with ME secondary to RVO, MP shows
improvement in macular sensitivity 1 month after
treatment/retreatment with intravitreal dexamethasone.Eyes that
underwent retreatment <6 months showed a worse baseline
BCVA,and macular sensitivity compared with eyes not requiring
retreatment up to 6 months
Commercial Relationships: Maria Lucia Cascavilla, None;
Giuseppe Querques, None; Rosangela Lattanzio, None; Lea
Querques, None; Giacinto Triolo, None; Edoardo Cavallero,
None; Francesco Bandello, ALLERGAN Inc. (S), NOVARTIS
PHARMACEUTICALS CORPORATION (S), FARMILA-THEA
(S), BAYER SCHERING PHARMA (S), PFIZER Inc. (S), ALCON
Inc. (S), BAUSCH AND LOMB (S), GENENTECH Inc. (S),
ALIMERA SCIENCES Inc. (S), SANOFI AVENTIS (S),
THROMBOGENICS (S)
Clinical and Diagnostic Imaging Features of Anti-Retroviral
Therapy (ART)-Associated Retinopathy
Alcides Fernandes1, John F. Payne3, Sunil K. Srivastava2, Steven
Yeh1. 1Ophthalmology, Emory University, Atlanta, GA; 2Cole Eye
Institute, Cleveland Clinic, Cleveland, OH; 3Ophthalmology,
Palmetto Retina Center, LLC, West Columbia, SC.
Purpose: The purpose of this study was to describe the clinical and
diagnostic imaging features of patients with human
immunodeficiency virus who developed retinopathy and retinal
pigment epitheliopathy in association with ART.
Methods: A retrospective review of patients with possible ARTassociated retinopathy was performed at a tertiary, university-based
academic uveitis and vitreoretinal practice. Demographic
information, CD4 counts, and past and current ART regimens (e.g.
oral nucleoside/nucleotide reverse transcriptase inhibitor [NRTIs],
non-NRTIs, and protease inhibitors [PI]) were reviewed. Visual
acuity, clinical examination findings, and functional testing
(electrophysiology and/or kinetic perimetry) were reviewed.
Diagnostic imaging including fundus photography, spectral domain
optical coherence tomography (SD-OCT), fluorescein angiography
(FA) and fundus autofluorescence (FAF) were assessed. 3 of 4
patients underwent Goldmann perimetry (GP) and 2 subjects
underwent an electroretinogram (ERG).
Results: Eight eyes of four patients with ART-associated retinopathy
were identified. The mean age at HIV diagnosis was 32 years (range
27-39). The mean initial Snellen VA was 20/125 and mean final VA
was 20/400. At least moderate visual loss of 20/40 or poorer was
observed in seven of eight eyes where the retinopathy involved the
macula. GP showed areas of central and para-central scotomata.
ERGs demonstrated mild to moderate photoreceptor dysfunction.
Fundus examination revealed a diffuse pattern of RPE mottling or
hyperplasia in four eyes of two patients, both of whom had been
treated with long-term ritonavir. SD-OCT confirmed the presence of
subretinal fluid, intraretinal cystic changes, and choroidal thinning in
these patients while FAF showed mottled hypo- and
hyperautofluorescence. One patient showed nummular,
retinochoroidal atrophy involving the peripheral and mid-peripheral
retina secondary to didanosine therapy. FAF showed nummular
hypoautofluorescence tightly corresponding to the areas of RPE loss.
Conclusions: Although ART-associated retinal toxicity is not widely
recognized, the clinical features of our findings support this
diagnosis. Consideration of ART-associated retinal toxicity should be
given to the differential diagnosis in HIV-positive patients with
retinopathy of unclear etiology.
Commercial Relationships: Alcides Fernandes, None; John F.
Payne, None; Sunil K. Srivastava, Bausch and Lomb (F), Bausch
and Lomb (C), Novartis (F), Allergan (F); Steven Yeh, Bausch and
Lomb (C)
Support: Research Prevent Blindness
S/MAR containing nanoparticles mediate long-term therapeutic
gene targeting in the RPE and rescue the rpe65-/- LCA model
Shannon M. Conley1, Adarsha Koirala1, Rasha Makkia1, Zhao Liu2,
Mark J. Cooper3, Janet R. Sparrow2, Muna I. Naash1. 1Cell Biol,
Hlth Sci Ctr-BMSB 781, Univ of Oklahoma, Oklahoma City, OK;
2
Department of Ophthalmology, Columbia University, New York,
NY; 3Copernicus Therapeutics, Cleveland, OH.
Purpose: Clinically relevant genetic therapies for chronic ocular
diseases must exhibit long-term gene expression. Our goal was to test
the ability of engineered plasmid DNA vectors, either naked or
compacted into high capacity DNA nanoparticles (NP), to mediate
long-term improvement in genetic diseases of the RPE.
Methods: Plasmid or NP vectors carrying the hRPE65 cDNA and the
RPE-specific vitelliform macular dystrophy 2 promoter were
generated (VMD2-hRPE65-S/MAR) and subretinally delivered to the
rpe65-/- mouse model of Leber’s congenital amaurosis at post natal
day 16. Outcomes were assessed at post-injection (PI)-15 months and
included gene expression and distribution, retinal structure and
function, and assessment of retinyl ester levels.
Results: At PI-15 months, hRPE65 message levels in treated eyes
were 32% of wild-type (WT) for NPs and 44% of WT for naked
DNA, and no reduction was seen from PI-6 months. Importantly,
spectral ERG demonstrated significant improvement in cone ERG
amplitudes in treated vs. untreated animals (green photopic b-waves
were 76% of WT for NPs and 79% of WT for naked DNA, vs. 39%
of WT for untreated, Fig. 1A). We also observed a reduction in the
fundus albipunctatus phenotype in NP or naked DNA treated eyes
compared to untreated (Fig. 1B). Biochemical studies showed a
significant reduction in the accumulation of retinyl esters in treated
mice (Fig. 1C), suggesting the transferred hRPE65 was functional.
Conclusions: These results indicate that both NP and uncompacted
plasmid VMD2-hRPE65-S/MAR can mediate appreciable gene
expression and improvement in an RPE-associated disease phenotype
for the life of the animal. They further highlight that nanocompaction
may not be essential for effective expression in the RPE, although
additional safety studies for uncompacted DNA will be an essential
step. These outcomes establish proof-of-concept for effective nonviral gene therapy in the RPE, and provides an excellent platform for
delivery of large genes.
aflibercept, and bevacizumab showed average dissociation
equilibrium constant (KD) values of 67, 9263, and 4456 pM,
respectively. A much slower dissociation rate constant was obtained
for ranibizumab than for aflibercept or bevacizumab, resulting in a
higher affinity for rhVEGF by ranibizumab. This observation was
consistent with data from Format 2, although the extremely slow
dissociation of ranibizumab from rhVEGF, and challenges in data
fitting for aflibercept precluded an accurate determination of kinetics
parameters for the binding of these molecules to rhVEGF. Using
Format 3, aflibercept had a KD of 1.9 pM for rhVEGF. However this
format was found to be unsuitable for evaluating ranibizumab
binding to rhVEGF because a significant amount of ranibizumab
dissociated from the capture antibody during sample analysis.
Conclusions: In a direct comparison under uniform conditions,
ranibizumab had a higher binding affinity for rhVEGF than
aflibercept. The Biacore assay format profoundly affected the binding
of aflibercept to VEGF and the indirect capture format was not
suitable for evaluating ranibizumab binding to rhVEGF. This study
highlights the importance of rigorous experimental design and careful
execution of in vitro binding studies. In addition, it suggested that
biological implications of the binding affinity/kinetics results need to
be interpreted within broader context including potency,
pharmacokinetics and clinical efficacy data.
PI-15 months improvement in the rpe65-/- phenotype. A. Green
photopic b-waves were significantly improved in naked (VMD2hRPE65-S/MAR) and NP (NP-VMD2-hRPE65-S/MAR) treated
compared to saline injected controls. Retinyl esters (B) and the
fundus albipunctatus phenotype (C) were reduced compared to
uninjected animals. * P<0.05, *** P<0.001 in 1-way ANOVA with
Bonferroni’s comparisons.
Commercial Relationships: Shannon M. Conley, None; Adarsha
Koirala, None; Rasha Makkia, None; Zhao Liu, None; Mark J.
Cooper, Copernicus Therapeutics, Inc. (E); Janet R. Sparrow,
None; Muna I. Naash, None
Support: NIH Grants EY018512 (SMC), EY018656 (MIN),
EY022778 (MIN), EY012951 (JRS), the Foundation Fighting
Blindness (MIN, JRS) and the Oklahoma Center for the
Advancement of Science and Technology (MIN, SMC)
Analysis of the binding affinity of vascular endothelial growth
factor A (VEGF) to ranibizumab, aflibercept and bevacizumab
Xiangdan Wang, Jihong Yang. Genentech, Inc., South San Francisco,
CA.
Purpose: To evaluate affinities and kinetics of recombinant human
VEGF (rhVEGF) binding to anti-VEGF therapeutics in a
comprehensive Biacore study.
Methods: The binding affinities of anti-VEGF therapeutics to
rhVEGF were evaluated using Biacore T200 at 37 oC. Three assay
formats were used (Figure). To evaluate the 3 anti-VEGF molecules
in their native conformation, rhVEGF was directly immobilized onto
a sensor chip while the anti-VEGF molecules were injected at
different concentrations as analytes in the liquid phase (Format 1). To
minimize any potential bias on the binding that may result from
rhVEGF immobilization, the anti-VEGF molecules were
immobilized as ligands and different concentrations of rhVEGF were
injected as analyte (Format 2). Finally the anti-VEGF molecules were
indirectly captured onto a sensor chip, and rhVEGF was used as the
analyte (Format 3).
Results: In the direct comparison of binding (Format 1) ranibizumab,
Commercial Relationships: Xiangdan Wang, Genentech, Inc. (E);
Jihong Yang, Genentech (E), Genentech (I)
Support: Genentech, Inc.
Low dose acetyl salicyl acid as a treatment option for central
serous chorioretinopathy
Nicole Stuebiger. Department of Ophthalmology, Charite, University
Medicine Berlin, Berlin, Germany.
Purpose: Since an increased plasminogen activator inhibitor 1 (PAJ1) was found being associated with central serous chorioretinopathy
(CSCR) it was the rationale for treating CSCR patients with acetyl
salicylic acid, which has a PAJ-1 lowering effect. We evaluated the
effectiveness of low-dose acetyl salicylic acid in patients with CSCR.
Methods: Patients with classical or multifocal CSCR were treated
with low dose acetyl salicylic acid (aspirin). The patients received
100mg per day orally for one month followed by 100mg every other
day for the following 5 months. The effect of this therapy was
evaluated in 5 patients (6 eyes). Visual acuity, metamorphopsia, and
central retinal thickness quantified by ocular coherence tomography
(OCT) were recorded at baseline and follow-up visits.
Results: After a mean follow up time of 7.8 months we could receive
improvement of visual acuity in 5 eyes, only one eye disclosed an
unchanged visual acuity (mean visual acutiy rose from 0.66 before
therapy to 0.89 at the end ot the therapeutic phase). The central
retinal sicknes was reduced during treatment and fell from a mean of
568 µm to 273 µm. There were no adverse events related to the
administration of aspirin.
Conclusions: Although there may have been a spontaneous
improvement without treatment, acetyl salicyl acid showed
effectiveness and has a plausible mechanism in CSCR.
Commercial Relationships: Nicole Stuebiger, None
Ceramide Biosynthesis Inhibition Protects the Retina from LightInduced Degeneration
Md Nawajes A. Mandal1, 2, Hui Chen1, 3, Julie-Thu Tran1, 2, Michael
H. Elliott1, 2, Richard S. Brush1, 2. 1Ophthalmology, Univ of
Oklahoma Hlth Sci Ctr, Oklahoma City, OK; 2Dean McGee Eye
Institute, Oklahoma City, OK; 3Ophthalmology, Sichuan Academy of
Medical Sciences & Sichuan Provincial People’s Hospital, Chengdu
City, China.
Purpose: Light-induced retinal degeneration (LIRD) in albino rats
causes apoptotic photoreceptor cell death. Ceramide, a sphingolipid
metabolite, is a second messenger for apoptosis. We tested whether
increases in ceramide mediate photoreceptor apoptosis in lightstressed retinas and if inhibition of ceramide formation can protect
the retina from LIRD.
Methods: Sprague Dawley (SD) rats were exposed to 2,700 lux
white light for 6 h and the retinal levels of ceramide and its
intermediary metabolites were measured by GC-MS or ESI-MS/MS.
Enzymes of the de novo biosynthetic and sphingomyelinase pathways
of ceramide generation were assayed, and gene expression was
measured by qRT-PCR. The synthetic drug FTY720, analogous to
sphingosine and known to inhibit de novo ceramide biosynthesis, was
administered intraperitoneally before light damage. The dosage and
temporal effect of FTY720 on the retina in vivo was measured by
histological and functional analyses.
Results: Retinal ceramide levels increased concurrently with the
increase of dihydroceramide and dihydrosphingosine immediately
and at various time points after light stress, well before active
apoptosis. Light stress in retina induces ceramide generation
predominantly through the de novo pathway. Intraperitoneal (IP)
injection of FTY720 (10 mg/kg) prevented ceramide generation by
the de novo pathway and protected retinal structure and function. We
determined that the neuroprotection of FTY720 was independent of
its immunosuppressive action.
Conclusions: We conclude that ceramide increase by de novo
biosynthesis mediates photoreceptor apoptosis in the LIRD model
and that inhibition of ceramide production protects the retina against
light stress.
Commercial Relationships: Md Nawajes A. Mandal, None; Hui
Chen, None; Julie-Thu Tran, None; Michael H. Elliott, None;
Richard S. Brush, OUHSC (P)
Support: NIH grants: EY022071 (NAM), EY019494 (MHE),
COBRE grant RR017703, core grant EY012190 and EY021725.
Grants from Foundation Fighting Blindness, and an unrestricted grant
from Research to Prevent Blindness, Inc.
Ocriplasmin as an Adjunct to Vitrectomy for the Treatment of
Pediatric Patients: Results of the MIC Trial
Emmanuel Chang. Ophthalmology, Associated Retinal Consultants,
Royal Oak, MI.
Purpose: The ocriplasmin MIC trial was a phase 2, single-center,
randomized, double-masked, placebo-controlled study to determine
efficacy and safety of ocriplasmin versus placebo as a preoperative
adjunct to vitrectomy in pediatric patients.
Methods: The MIC trial included pediatric vitrectomy candidates 16
years or younger (including infants) with vitreous attachment to the
posterior pole. Patients were randomized to receive a single
intravitreal injection of 175 µg ocriplasmin (n=16) or placebo (n=8)
30-60 minutes before planned start of vitrectomy. The primary end
point was total macular posterior vitreous detachment (PVD) as
assessed by masked surgeon under an operating microscope at the
beginning of vitrectomy. Selected secondary end points included
investigator assessment of vitreous liquefaction at the beginning of
vitrectomy; immediate postoperative retinal reattachment status;
presence of postoperative proliferative vitreoretinopathy and
retinopathy of prematurity (ROP) classification by fluorescein
angiography on follow-up. Selected safety assessments included
adverse event reports and B scan ultrasonography.
Results: The trial is complete with findings to be reported in early
2013. Vitreous detachment status was measured on a 3-point grade
scale with Grade 0 being no PVD at beginning of vitrectomy and
Grade 3 being total PVD without disc attachment. Vitreous
liquefaction status was measured on scale of 1 (formed gel) to 4
(water consistency). Postoperative retinal reattachment status was
scored as present or absent immediately after vitrectomy.
Postoperative ROP classification on follow-up was scored according
to a 5-stage scale with Stage 1 defined by presence of a faint
demarcation line and Stage 5 by total retinal detachment.
Conclusions: Findings from the MIC trial may be informative for
physicians seeking adjunct treatments to support vitrectomy
outcomes in pediatric patients.
Commercial Relationships: Emmanuel Chang, None
Support: ThromboGenics, Inc.
Quinic Acid Derivative, KZ-41 Protects Against RadiationInduced Retinal Endothelial Cell Dysfunction: An Early - to Late Stage Treatment of Radiation Retinopathy
Jordan J. Toutounchian1, Qiuhua Zhang2, Jayaprakash Pagadala1,
Duane D. Miller1, Jena J. Steinle2, Charles R. Yates1.
1
Pharmaceutical Sciences, Univ of Tennessee Hlth Sci Ctr, Memphis,
TN; 2Ophthalmology, Univ of Tennessee Hlth Sci Ctr, Memphis, TN.
Purpose: Radiation-induced damage to the vascularized retinal
segments of the posterior portion of the eye triggers an exuberant
pro-inflammatory response resulting in leukocyte adhesion, vessel
blockage, decreased oxygen supply, and retinal endothelial cell
(REC) death. In this study we evaluated the effect of KZ-41 on
radiation-induced leukocyte adhesion to human RECs and retinal
neovascularization (RNV) in a murine oxygen-induced retinopathy
(OIR) model.
Methods: Leukocyte Adhesion: Human primary RECs were grown
to confluence onto 75x38mm glass slides. Irradiated RECs (single
dose 30 Gy; 137Cs at ~3 Gy/min) were placed into a parallel-plate
flow chamber and treated with either KZ-41 (10μM) or vehicle
(PBS). U937 (human monocytic) cells were then perfused over RECs
and digital images were obtained every 30 min over two hours. OIR:
Mouse pups (n=5/group) were exposed to 75% oxygen at post-natal
day (P)7 for 5 days and then returned to normal oxygen (P12). Mice
received daily ocular administration of either KZ-41-loaded
nanoemulsion (100 mg/kg) or vehicle on P12-17. Eyes were
enucleated at P17 and retinal whole-mounts stained using the
endothelial cell specific marker isolectin B4-594. Images were
acquired using a Nikon Eclipse 80i confocal microscope with NikonNIS elements software. Vaso-obliteration was determined by
comparing vascular regrowth to avascular area around the optic
nerve. Image analysis was performed using Adobe Photoshop.
Results: Radiation increased leukocyte-REC adhesion as early two
hours post-exposure as compared to unirradiated controls (2 ± 2 vs.
87 ± 18 adhered cells; P < 0.05). Whereas, KZ-41 treatment reduced
leukocyte adhesion to irradiated RECs (25 ± 12 vs. 87 ± 18 adhered
cells; P < 0.05). In the murine OIR model, KZ-41 reduced the
avascular area by two-fold as compared to vehicle treated mice (P <
0.05). Histologic examination of retinal whole mounts revealed a
more organized microvasculature with less extensive neovascular
tufting in KZ-41 treated mice.
Conclusions: Leukocyte-REC adhesion is a hallmark of radiationinduced retinopathy. Subsequent vaso-occlusion and ischemia leads
to pathologic RNV. Attenuation of radiation-induced adhesion and
pathologic RNV by KZ-41 suggests that quinic acid derivatives may
have therapeutic benefit in radiation retinopathy or other
inflammatory diseases of the eye.
Commercial Relationships: Jordan J. Toutounchian, None;
Qiuhua Zhang, None; Jayaprakash Pagadala, None; Duane D.
Miller, None; Jena J. Steinle, None; Charles R. Yates, RxBio, Inc
(C), RxBio, Inc (P), RxBio, Inc (R)
Support: NEI Vision Core Grant: PHS 3P30; EY013080
Safety of intravitreal quinupristin / dalfopristin in an animal
model
Veronica E. Giordano, Sergio Hernandez-Da Mota, Jose Luis
Guerrero-Naranjo, Tania N. Adabache Guel, Samantha Salinas
Longoria, Sonia Corredor-Casas, Guillermo Salcedo-Villanueva,
Juan Manuel Jimenez-Sierra, Virgilio Morales-Canton. Retina,
Asociación para evitar la Ceguera en Mexico, Distrito Federal,
Mexico.
Purpose: To evaluate the retinal toxicity of varying doses of
quinupristin-dalfopristin when injected intravitreally in rabbits.
Methods: Design: Randomized controlled trial. Twenty-two rabbits
were used for this study and divided into four groups. Four
concentrations of quinupristin-dalfopristin were prepared: group 1:
0.01mg/0.1ml; group 2: 0.1mg/0.1ml, group 3: 1mg/0.1ml and group
4: 10mg/0.1ml.
Each concentration was injected intravitreally in one eye of each of
six rabbits of each group; 0.1 mL volume of sterile balanced saline
solution was injected into the contralateral eyes. Basal and weekly
ERG (measuring mesopic implicit times and amplitudes), fundus
photographs and macular OCT were performed and the animals were
observed for 4 weeks for signs of inflammation or toxicity. At the
end of follow-up the animals were killed. The enucleated eyes were
prepared for histologic evaluation of retinal toxicity.
Results: There was a statistically significant difference between all
groups (Friedman test, p < 0.01). In groups 1 and 2 there were no
statistically significant differences between basal and follow-up
measures of each group and between them (Wilcoxon rank-sum test,
p =1.0). In contrast, comparisons of amplitude and implicit times of
groups 3 and 4 showed high statistically significant differences
between basal and follow-up measures, as well as between follow-up
measures of injected and control eyes (p < 0.01). Groups 1 and 2
showed no ERG changes compared to groups 3 and 4 (Wilcoxon
rank-sum test, p < 0.01).
Only groups 3 and 4 revealed severe vitreous inflammation and
hemorrhage. There were no significant histopathological changes in
groups 1 and 2.
Conclusions: Intravitreal quinupristin/dalfopristin doses of
0.01mg/0.1ml and 0.1mg/0.1ml showed no signs of retinal toxicity.
Commercial Relationships: Veronica E. Giordano, None; Sergio
Hernandez-Da Mota, None; Jose Luis Guerrero-Naranjo,
Neurotech (F); Tania N. Adabache Guel, None; Samantha Salinas
Longoria, None; Sonia Corredor-Casas, None; Guillermo
Salcedo-Villanueva, None; Juan Manuel Jimenez-Sierra, None;
Virgilio Morales-Canton, Clearside Biomedical (F)
Fragment crystallizable (Fc) region results in an increased
systemic exposure with no significant difference in intra-ocular
pharmacokinetics
Jennifer Le Couter, Kapil Gadkar, J. Michael Elliott, Thomas Lee, Y.
Gloria Meng, Linda Zhang, Margaret Kenrick, Saileta Prabhu, Justin
M. Scheer. Tumor Biology and Angiogenesis, Genentech, Inc, San
Francisco, CA.
Purpose: The elimination pathways and pharmacokinetics (PK) of
monoclonal antibodies (mAb) and antigen binding fragments
following systemic administration are well characterized. Less is
known about the elimination and PK of these protein drugs following
intravitreal (ITV) administration. Molecules containing Fc region
lead to higher systemic exposure due to recycling via the neonatal Fc
receptor for IgG following ITV administration. We examined the
effect of structure on the ocular and systemic PK of targeted and
untargeted (directed against the gp120 glycoprotein expressed in the
HIV viral envelope) sets of these protein drugs in rabbits in an effort
to develop criteria for the selection of the most optimal format for the
next generation of ocular therapeutics.
Methods: Protein drugs (Figure) were administered bilaterally by
ITV injection (500 µg/50 µl/ eye) to male NZ rabbits. Each eye was
analyzed independently. Quadruplicate blood and ocular tissues
(vitreous and aqueous humor) were sampled over 28 days post-dose.
Protein drug concentrations were determined by ELISA. Individual
data points that appeared to have been affected by anti-therapeutic
antibody (ATA) in ATA-positive animals were excluded from the
analysis. Ocular and systemic PK data were analyzed using
previously established compartmental PK models for protein drugs.
Results: Vitreous levels of Fab, double-Cys F(ab’)2 , single-Cys
F(ab’)2, and mAb were not significantly different following ITV
administration. However, serum PK profiles were significantly
different between the Fab fragments and mAb. For example, the
relative areas under the curve of the concentration-time profile for the
anti-gp120 Fab, double-Cys F(ab')2 and single-Cys F(ab’)2 formats
compared to IgG were 0.007, 0.03, and 0.008 respectively.
Conclusions: Protein drug structure did not appear to dramatically
impact t1/2 in the vitreous in rabbits. As expected the systemic
exposure levels of mAb were >100-fold higher than that of Fab,
which lacks Fc region. Additionally, the apparent serum t 1/2 of mAb
was 3-fold longer than the Fab. Taken together, these data indicate
that Fc region results in an increased systemic exposure and may also
result in unwanted accumulation over long term dosing, with no
obvious therapeutic benefit to treat ocular diseases.
Commercial Relationships: SALVATORE FARO, None; Teresio
Avitabile, None; Giulia Malaguarnera, None; Franco Marco
Livio, None; Maurizio G. Uva, None; maria vittoria cicinelli,
None; Cristina Cassar Scalia, None; Elena Lionetti, None;
Caterina Gagliano, None
256 Aqueous Humor Dynamics and IOP
Monday, May 06, 2013 11:00 AM-12:45 PM
Commercial Relationships: Jennifer Le Couter, Genentech, Inc.
(E); Kapil Gadkar, Genentech (E); J. Michael Elliott, Genentech
(E); Thomas Lee, None; Y. Gloria Meng, Genentech Inc (E), Roche
(I); Linda Zhang, None; Margaret Kenrick, Genentech (E); Saileta
Prabhu, Genentech (E); Justin M. Scheer, Genentech (E)
Support: Genentech, Inc.
EFFECT OF AN ANTIOXIDANT BLEND IN DIABETIC
MACULAR EDEMA
SALVATORE FARO1, Teresio Avitabile1, Giulia Malaguarnera2,
Franco Marco Livio1, Maurizio G. Uva1, maria vittoria cicinelli3,
Cristina Cassar Scalia1, Elena Lionetti1, Caterina Gagliano3.
1
Ophthalmology, University of Catania, Catania, Italy; 2Clinical and
Molecular Biomedicine, University of Catania, Catania, Italy;
3
NEST, Neurovisual Science Technology, Catania, Italy.
Purpose: To evaluate the effect of treatment with an antioxidant,
vasoprotective and neuroprotective blend containing natural
compounds on the progression of diabetic macular edema.
Methods: Sixty patients with diabetes type I and II with nonproliferative retinopathy and macular edema in early stage were
enrolled. Twenty patients (group 1) were treated with acetazolamide
500 mg/die; twenty patients (group 2) were treated with an
antioxidant blend containing extracts of red berries, Ginkgo Biloba
and white willow bark together with carnosine and α-lipoic acid (two
tablets/die). Group 3 (twenty patients: control group) did not assume
any treatment. The observations were made at baseline, 1 and 3
months after enrollment. We measured central macular thickness
with spectral OCT. Plasma levels of glucose and glycosylated
hemoglobin (HbA1c) were also considered. Treatment doses: Group
1 took acetazolamide 500 mg/die; Group 2 took antioxidant blend: 2
capsules per day, one in the morning and 1 in the evening; Group 3
received no treatment beside the usual antidiabetic therapy.
Results: The level of plasma glucose and HbA1c were statistically
lower in the group 2 (antioxidant blend) compared to acetazolamide
and control groups (p <0.001) after one and two months of treatment.
The foveal thickness was lower in the group 2 compared to the
control group (p <0.001) at the first month of treatment and in both
groups (antioxidant blend and acetazolamide) in the second month
(p<0.001). On the contrary, control group foveal thickness at 90 days
increased significantly ( p <0.05) compared to baseline (T0). Visual
acuity improved at 90 days compared to T0 ( p <0.05) in group 2.
Conclusions: These data demonstrate that treatment with an
antioxidant blend containing extracts of red berries, Ginkgo Biloba
and white willow bark together with carnosine and α-lipoic acid may
blunt macular edema progression in the retina of diabetic patients.
A Role for Myocilin in Receptor Endocytosis
Brian S. McKay1, Nicole R. Congrove1, William M. Dismuke2, W
Daniel Stamer2. 1Ophthalmology and Vision Science, University of
Arizona, Tucson, AZ; 2Ophthalmology, Duke University, Durham,
NC.
Purpose: Myocilin has two distinct cellular distributions,
cytoplasmic and membrane-associated, yet appears extracellularly.
We have previously shown that myocilin is released from cells on
exosomes, which are a component of the endosomal pathway. In this
study we tested the hypothesis that myocilin enters the exosome
pathway during plasma membrane receptor endocytosis.
Methods: Retinal pigment epithelia (RPE) in human eye-cups was
stimulated with L-DOPA (1uM) to monitor endocytosis of GPR143
(OA1), an endogenous G-protein coupled receptor (GPCR) on the
surface of RPE. Recombinant myocilin (WT, P370L and T377M)
was heterologously co-expressed in transformed cells with GPR143
to test whether myocilin participates in receptor endocytosis and to
determine the kinetics of binding. Cell surface proteins in eye-cups
and transformed cells were biotinylated and protein trafficking and
myocilin association were monitored over time using streptavidin
beads. Finally, we tested myocilin binding to the purified cytoplasmic
domain of GPR143 using chromatography.
Results: Data obtained from both human eye-cups and cultured cells
illustrate that WT myocilin binds to biotinylated plasma membrane
proteins in a ligand-dependent manner. In cultured cells, peak
association occurs at 20 minutes after introduction of ligand, and
decreases rapidly thereafter by 60 minutes. Association of WT
myocilin with GPR143, appears to occur via its cytoplasmic domain
after ligand stimulation. Interestingly, the two mutant forms of
myocilin exhibited different binding kinetics to biotinylated plasma
membrane proteins after stimulation. P370L myocilin showed little
activity, while T377M illustrated prolonged association and disturbed
kinetics.
Conclusions: Our results suggest that myocilin enters the membrane
compartment of cells during receptor endocytosis. Targeting, sorting,
and trafficking through this pathway is likely the mechanism by
which myocilin arrives on the surface of exosomes. WT, but not
mutant myocilin matched a standard time course for endocytosis.
Taken together, these observations suggest that myocilin participates
in receptor endocytosis, and that mutations may differentially affect
receptor signaling, leading to glaucoma.
Commercial Relationships: Brian S. McKay, None; Nicole R.
Congrove, None; William M. Dismuke, None; W Daniel Stamer,
Allergan (F), Alcon (F), Acucela (C), Aerie (C), Cytokinetics (C)
Support: Research to Prevent Blindness
Differential Comparison of Aqueous Humor
Phosphatidylcholines from Control and Glaucomatous Donors
Ayman Aljohani, Yenifer C. Guerra, Maria C. Piqueras, Richard K.
Lee, Sanjoy K. Bhattacharya. Bascom Palmer Eye Institute,
University of Miami, Miami, FL.
Purpose: To compare the phosphatidylcholines (PC) of the aqueous
humor (AQH) from control and primary open angle glaucoma
(POAG) donors by mass spectrometric shotgun lipidomics. Our
hypothesis is that select PCs are absent in POAG AQH, which may
contribute to increased stiffness of trabecular meshwork in POAG.
Methods: The human AQH samples were collected adhering to
tenets of declaration of Helsinki under IRB approved protocols from
control and POAG donors of diverse race and both genders with
mean age of 55 ±8 years (n=10 each). Controls were cataract surgery
patients. AQH PCs were extracted using the Bligh and Dyer method,
re-suspended in Isoproyl, Acetonitrile solution and analyzed on a
TSQ Quantum Access Max instrument in positive mode. Precursor
ion scan (PIS) for phosphocholines (PC; product m/z of 184) were
performed using direct infusion. Ratiometric quantification was
performed for m/z range of 200-1000. The PC species of control and
glaucoma patients were identified using MZmine 2.9 version and
database from lipidmaps.org. The profiles were compared using inhouse written softwares. Further confirmatory analyses were
performed on a high resolution Q-exactive mass spectrometer.
Results: Overall total amount of PCs in POAG normalized to protein
amounts were always less than that in controls. We found 7 and 4
unique PCs in controls and POAG AQH respectively. The number of
unique PC species that showed up are 2 in control (12:0/14:1(9Z)),
PC(13:0/20:5(5Z,8Z,11Z,14Z,17Z))] and only one in POAG
[PC(12:0/14:1(9Z))] AQH.
Conclusions: Lower amounts of phosphatidylcholines were found in
POAG compared to controls. This was confirmed by high resolution
mass spectrometry. Absence of select PC species in POAG is likely
to contribute to reduced membrane fluidity and likely result in
stiffness to TM cells in glaucoma.
Commercial Relationships: Ayman Aljohani, None; Yenifer C.
Guerra, None; Maria C. Piqueras, None; Richard K. Lee, National
Eye Institute (F); Sanjoy K. Bhattacharya, None
Support: Supported by an unrestricted grant from RPB to University
of Miami, NIH grants P30EYEY014801 and R01EY016112.
Ocular Hypertension-Induced MMPs Production Within Optic
Nerve: A Regulatory Role of δ-Opioid-Receptors
Sudha Singh, Mohammad F. Pathan, Naseem Akhter, Melissa Nix,
Shahid Husain. Ophthalmology, Medical University of South
Carolina, Charleston, SC.
Purpose: This study was designed to determine the changes in the
matrix metalloproteinases (MMPs) production in optic nerve head
(ONH) and purified ONH astrocytes in response to ocularhypertension and TNF-α-induced injury, respectively. We also have
determined if δ-opioid-receptors activation counterbalances the
production of MMPs.
Methods: Brown Norway rats were used to elevate intraocular
pressure (IOP) by injecting 50 µL of 2 M hypertonic saline into the
circumferential limbal veins. Animals were treated with δ-opioidreceptor agonist, SNC-121 (1 mg/kg; i.p.), daily for 7 days. Pattern
electroretinograms (PERG) and retinal ganglion cells (RGCs) in flatmounts were counted at week-6 post injury. Primary cultures of
human ONH astrocytes were isolated and purified by
immunopanning. Cells were treated with SNC-121 (1μM), 15
minutes prior to TNF-α (25 ng/mL) treatment for 24 hours. The
expression of MMP-1, MMP-2, and MMP-3 were determined by
immunohistochemistry and Western blotting.
Results: PERG amplitudes and RGCs were reduced by 18% and
28%, respectively, in ocular-hypertensive eyes. PERG deficits and
reduction in RGCs in hypertensive eyes were significantly (P<0.05)
improved by δ-opioid-receptor agonist, SNC-121, treatment. MMP-1,
MMP-2, and MMP-3 production in ocular-hypertensive optic nerve
was increased by 51 ± 7%, 232 ± 24%, and 79 ± 8%, respectively, at
7 days post injury. The production of MMPs was fully inhibited in
the presence of SNC-121. In addition, TNF-α (25 ng/mL) treatment
of ONH astrocytes increased the MMP-1, MMP-2, and MMP-3
secretion by 124 ± 45%, 149 ± 76%, and 53 ± 5%, respectively, at 24
hours, which was completely inhibited in the presence of SNC-121.
Conclusions: These data provide concrete evidence that increase in
δ-opioidergic-receptor activity by SNC-121 provides retina
neuroprotection, as determined by PERG and RGCs counting.
Mechanistic data provide clues that MMPs are produced within the
optic nerve, in an early stage of glaucomatous injury, which will
further destabilize the optic nerve by excessive remodeling. These
detrimental signaling molecules directly and/or indirectly weaken
axons and lead to the RGC death. Delta-opioid receptor agonists
represent a novel class of drugs/agents that counteract detrimental
events within the optic nerve and prevent the loss of RGCs.
Commercial Relationships: Sudha Singh, None; Mohammad F.
Pathan, None; Naseem Akhter, None; Melissa Nix, None; Shahid
Husain, None
Support: NIH/NEI grant EY019081
Requirement for the proteasome, ATP and exportin-1 in the
nuclear export of glucocorticoid alpha (GRα) receptor following
DEX treatment in human trabecular meshwork cell-line (NTM5)
Adnan Dibas1, 2, Abbot F. Clark3, 2, Thomas Yorio1, 2. 1Pharmacology
& Neuroscience, University of North Texas Hlth Sci Ctr, Fort Worth,
TX; 2North Texas Eye Research Institute, Fort Worth, TX; 3Cell
Biology and Anatomy, Univertsity of North Texas Health Science
Center at Fort Worth, Fort Worth, TX.
Purpose: We previously showed the requirement of microfilaments
and microtubules in nuclear export of GR-alpha and in the current
study, new mechanisms involved in steroid-induced GRα receptor
nuclear export in human trabecular meshwork cells are characterized.
Methods: Stably-transfected RFP-GRα cell lines were developed.
Nuclear export of RFP-GRα in NTM5 cells treated with vehicle
(ethanol), dexamethasone (DEX) was studied using confocal
microscopy and fluorometry in isolated nuclear and cytosolic
fractions following DEX removal. Leptomycin B (an inhibitor of
exportin-1), MG132 and lactacystin (proteasomal inhibitors), and
rotenone (ATP depletor), were tested for their abilities to affect GRαtrafficking.
Results: NTM5 cells transfected with RFP-GRα showed a clear
cytosolic localization of receptor that underwent translocation to the
nucleus after DEX treatment. Surprisingly, while both proteasomal
inhibitors (MG132 and lactacystin) had no effect of nuclear import,
both suppressed nuclear export of RFP-GRα. Leptomycin while not
affecting nuclear import of RFP-GRα, also attenuated nuclear export
of RFP-GRα following DEX removal. Finally, both nuclear import
and export of RFP-GRα were abolished by rotenone.
Conclusions: While RFP-GRα nuclear import is cytoskeletonindependent, nuclear export appears to involve cytoskeletal network
rearrangement. Exportin-1 appears to mediate nuclear export of RFPGRα receptor following DEX removal. Interestingly, proteasomal
inhibitors blockade of export suggest a novel role for ubiquitination
in the export of GRα. Finally, nuclear shuttling of GRα to and from
the nucleus is absolutely ATP-dependent.
Commercial Relationships: Adnan Dibas, None; Abbot F. Clark,
Alcon Research, Ltd. (F); Thomas Yorio, None
Support: EY016242
Non-invasive Intraocular Pressure Measurements in Zebrafish
D Joshua Cameron, Pinakin G. Davey. College of Optometry,
Western University of Health Sciences, Pomona, CA.
Purpose: The zebrafish (Danio rerio) eye shares many similarities
with the human eye and is increasingly being used as model for eye
development, disease and regeneration. A mutant zebrafish, coined
bugeye because of its grossly buphthalmic eyes, has been shown to
have some similarities with human glaucoma due to its elevated
intraocular pressure (IOP) and optic nerve pathology. We
investigated whether a non-invasive clinical tonometer could measure
IOP in zebrafish and importantly, distinguish bugeye from wild-type
fish.
Methods: Adult zebrafish were anesthetized in tricaine and placed on
a styrofoam bed. A moist paper towel was placed over the fish body,
leaving the head and gills exposed. Tricaine solution was perfused
over the gills to keep the fish oxygenated and sedated. The fish’s eye
was brought up to the probe of a Paradigm blood flow analyzer
pneumotonometer and the IOP was repeatedly measured and
averaged. The average IOP of several strains of fish were then
compared to each other using analysis of variance.
Results: Bugeye eyes (n=13) were significantly larger compared to
TU/TL (n=10) strains both in size and ratio to the head (p=0.006 and
1.6E-06 respectively). The zebrafish quickly recover after IOP
measurement, returning to normal activity within minutes of being
placed in fresh water. IOP values did not significantly vary in wildtype fish over 12 months of age. Strain AB fish (n=29) had slightly
higher IOP values compared to TU/TL, but the difference was not
significant. Bugeye mutants had significantly elevated IOP values
compared to wild-type fish (bugeye 25.3 mmHg; TU/TL 14.6 mmHg;
p=0.01). However, IOP in bugeye zebrafish did not significantly
correlate with eye size or eye/head ratios.
Conclusions: A non-invasive pneumotonometer can be used to
measure IOP in adult zebrafish and distinguish bugeye fish from
wild-type strains.
Commercial Relationships: D Joshua Cameron, None; Pinakin G.
Davey, None
Characterization of TRPV4 expression & function in the ciliary
body & trabecular meshwork
Amber M. Frye1, Daniel A. Ryskamp1, 2, Peter Barabas1, Tunde
Molnar1, David Krizaj1, 2. 1Ophthalmology & Visual Sciences, Moran
Eye Institute, University of Utah School of Medicine, Salt Lake City,
UT; 2Interdepartmental Program in Neuroscience, University of Utah,
Salt Lake City, UT.
Purpose: Aqueous humor dynamics is regulated by balanced
production in the ciliary body & removal through the primary
(“conventional”) & secondary outflow pathways. Regulation of the
volume of trabecular meshwork (TM) cells thus directly impacts the
outflow facility & affects the amplitude of intraocular pressure. To
determine the molecular mechanism regulating the outflow, we
examined the expression & function of the
osmosensory/mechanosensitive TRPV4 cation channel in the anterior
chamber (AC) of the eye.
Methods: Cryosections from C57BL6 mouse eyes & human punches
from trabeculectomies were immunostained with a validated TRPV4
antibody (Ryskamp et al., 2011) & colabeled with the TM marker
aquaporin 1. Nuclei were labeled with propidium iodide & images
were acquired with a confocal microscope. Alternatively, TM tissues
were loaded with the calcium indicator dye fura-2 & the calciuminsensitive cell volume indicator calcein. Calcium levels [Ca2+]i and
cell volume were determined using high-resolution optical imaging.
To functionally map TRPV4 expression, eye sections were incubated
with 100 nM GSK1016790A (GSK), a selective TRPV4 agonist, in
the presence of extracellular agmatine (AGB 5 mM, a cation influx
marker) and/or the TRPV4 antagonist HC067047 (HC 1 µM). Ocular
slices were then fixed & immunostained for AGB. To assess the
impact of maximal TRPV4 activation on histology, 75 µM GSK was
injected into the AC. Eye slices were processed for H&E, apoptosis
markers, metabolic markers, & EM ultrastructure.
Results: TRPV4 immunoreactivity was observed confirming its
expression in the pars plicata of the fluid producing, non-pigmented
epithelial cells lining the ciliary processes, but was absent from the
pigmented epithelial cells of the ciliary body. Both mouse & human
TM were labeled by the TRPV4 antibody. The functional presence of
TRPV4 in these cells was confirmed by GSK- & HC-dependent
changes in [Ca2+]i & AGB influx. In contrast to the pronounced
proapoptotic effects of GSK in retinal cells, saturating TRPV4
activation induced minimal apoptosis & histological changes within
the ciliary body & TM cells.
Conclusions: Our data provide molecular & physiological evidence
of TRPV4 localization in the ciliary body, trabecular meshwork &
the posterior eye and suggest a novel mechanism that could
potentially regulate the volume of cells within the conventional
outflow pathway.
Commercial Relationships: Amber M. Frye, None; Daniel A.
Ryskamp, None; Peter Barabas, None; Tunde Molnar, None;
David Krizaj, None
Support: NIH, DOD, FFB, University of Utah and an unrestricted
award from RPB to the Moran Eye Institute.
Ocular Hypertension-Induced Changes in the Anterior Segment
of Cynomolgus Monkeys
Shenouda Yacoub, Quinn Sessums, Byron H. Li, Ganesh Prasanna.
Glaucoma Research, NIBR-Ophthalmology, Fort Worth, TX.
Purpose: To compare the corneal thickness (CT), corneal curvature
(CC), anterior chamber depth (ACD), iridocorneal angle (ICA), and
corneal endothelial cell density (ECD), in naïve and chronic ocular
hypertensive (OHT) cynomolgus monkeys.
Methods: Chronic OHT of various durations (0.7-12 y) was induced
in the right eye (OD) of 52 cynomolgus monkeys by argon laser
trabeculoplasty. The left eye (OS) with normal intraocular pressure
(IOP) was untreated. An additional 23 naïve animals served as
controls. The central corneal thickness (CCT), para-central corneal
thickness (PCCT), peripheral corneal thickness (PCT), CC, ACD and
ICA were evaluated with a Galilei Dual Scheimpflug Analyzer
(GDSA, SIS, Switzerland). The ECD at central corneal area was
evaluated with a SP2000 Specular microscope (Topcon).
Results: In the 23 naïve 4-yo monkeys, there were no differences in
CT, CC, ACD, ICA, and ECD between OD and OS. In 20 OHT 4-yo
monkeys (OHT duration in OD = 0.7 y), the differences of CT, CC,
ACD and ICA between OD and OS were insignificant. However,
significant ECD reduction in OD (-17.2%, P < 0.01) was detected
compared to the OS. In a second group of 32 OHT monkeys (age = 716 y; OHT duration = 2-12 y), the mean CCT of OD (490 ± 23 µm)
was thicker (11 µm, 2.4%, p < 0.05) than the contralateral control
eyes (479 ± 20 µm). The ECD of OD was reduced (-20%, p < 0.001)
compared to OS. There was no significant difference in PCCT, PCT,
ACD, and ICA between OD and OS.
Conclusions: Chronic OHT in monkeys caused a duration-dependent
reduction in ECD in the central corneal area. Longer duration of IOP
elevation correlated with a more severe reduction, concomitant with a
small increase of CCT. There was no difference in CC, ACD, and
ICA between OHT and normal eyes. The thickening of CCT and
progressive decrease of ECD observed in monkey eyes with
increased duration of OHT is similar to the disease progression in
primary open angle glaucoma patients.
Commercial Relationships: Shenouda Yacoub, Novartis (E);
Quinn Sessums, None; Byron H. Li, Novartis (E); Ganesh
Prasanna, Alcon Research Ltd (E), Novartis Institute of Biomedical
Research (E)
Regulation of Mammalian Sympathetic Neurotransmitter
Release and Intraocular Pressure by Hydrogen Sulfide Donor,
GYY 4137
Ankita Salvi1, Pratik Bankhele1, Jamal Jamil1, Ya Fatou Njie-Mbye2,
Madhura S. Kulkarni2, Sunny E. Ohia2, Catherine A. Opere1.
1
Creighton University, Omaha, NE; 2Texas Southern University,
Houston, NE.
Purpose: We have evidence that hydrogen sulfide (H2S) donors can
regulate sympathetic neurotransmission and intraocular pressure
(IOP) in the mammalian anterior uvea. In the present study, we
investigated the effect of a slow releasing H2S donor, GYY 4137 on
electrically evoked [3H]NE release in isolated superfused, bovine irisciliary bodies (ICB), in vitro and on IOP in male New Zealand White
rabbits, in vivo.
Methods: Isolated bovine ICB were incubated in oxygenated Krebs
solution containing 2.5 μCi/ml of [ 3H]NE and then prepared for
neurotransmitter release studies. Release of [3H]NE was elicited by
two (S1 and S2) electrical pulses (300 d.c electrical pulses) applied 27
min apart. For IOP studies, a single drop of GYY 4137 (0.1-2%) and
vehicle were instilled into the right and left rabbit eyes, respectively.
IOP measurements were made using a pneumatonometer (model 30
classic; Reichert Ophthalmic Instruments, Depew, NY) until baseline
readings resumed.
Results: GYY 4137 (1-30μΜ) attenuated field-stimulated [3H]NE
release in bovine ICB in a concentration-dependent manner,
achieving an inhibition of 20.8% (n=3; p<0.05) at 30 µM. Although
cystathionine β-synthase inhibitor, aminooxyacetic acid (3 mM) and
the ATP-sensitive potassium channel (KATP) inhibitor, glibenclamide
(300 µM) had no effect (p>0.05) on [ 3H]NE release, they both
reversed the inhibitory action of GYY 4137 (10-30 µM) on the
neurotransmitter release. GYY 4137 (0.1-2%) reduced IOP in both
treated and untreated eyes for a duration of 7-9 h, exhibiting a
maximum effect after 5-6 h. For instance, GYY 4137 (2%) elicited a
maximum IOP inhibition of 27.76 % (n=5; p<0.001) 6 h after
treatment in normotensive rabbits.
Conclusions: The H2S-donor, GYY 4137 attenuates both
sympathetic neurotransmitter release and IOP in mammalian anterior
uvea. The inhibitory action on neurotransmission is partially
dependent on in situ release of H2S and on the activation of KATPchannels.
Commercial Relationships: Ankita Salvi, None; Pratik Bankhele,
None; Jamal Jamil, None; Ya Fatou Njie-Mbye, None; Madhura
S. Kulkarni, None; Sunny E. Ohia, None; Catherine A. Opere,
None
Support: NIH Grant NEI EY022215
Episcleral Venous Pressure Elevation in Untreated Open Angle
Glaucoma
Arthur J. Sit1, Nitika Arora1, Jay W. McLaren1, Mehrdad Malihi1, 2,
Lilit Voskanyan3. 1Ophthalmology, Mayo Clinic, Rochester, MN;
2
Ophthalmology, University of Medicine and Dentistry of New
Jersey, Newark, NJ; 3Malayan Ophthalmology Centre, Yerevan,
Armenia.
Purpose: The contribution of episcleral venous pressure (EVP) to the
elevation of intraocular pressure (IOP) in open angle glaucoma
(OAG) is unknown. Previous studies of EVP in OAG have been
contradictory. In this study, we used a new automated
venomanometer to investigate differences in EVP between untreated
OAG patients and normal individuals. We also evaluated
relationships between EVP and other ocular and systemic variables in
OAG patients.
Methods: EVP was measured by using a computer-controlled
venomanometer (pressure-chamber method) in one eye each of 101
subjects with untreated OAG (mean age, 64 years; range 24 to 83
years) and 191 eyes of 100 healthy volunteers (mean age, 48 years;
range 19 to 81 years). We also measured intraocular pressure (IOP),
axial length (AL), central corneal thickness (CCT), systolic and
diastolic blood pressure (SBP and DBP respectively), height and
weight, and calculated body mass index (BMI). Descriptive statistics
were calculated for IOP and EVP, and differences between groups
were examined by using generalized estimating equation (GEE)
models. Relationships between EVP and IOP, SBP, DBP, BMI, AL,
and CCT in glaucomatous subjects were assessed by using Pearson
correlation and significance was determined by using GEE models.
Results: IOP of normal eyes and eyes with OAG was 13.7 ± 3.0
mmHg (mean ± SD) and 27.4 ± 8.0 mmHg respectively (p<0.001).
EVP of normal eyes and eyes with OAG was 6.9 ±1.9 mmHg and 7.7
± 2.0 mmHg respectively (p=0.003).
In OAG patients, there were no significant correlations between EVP
and any of the physiologic variables assessed (Table 1).
Conclusions: EVP in OAG is elevated by a small amount compared
with normal subjects, and although the increase could contribute in a
small part to the elevation of IOP, it is not a primary cause of high
IOP in these patients. EVP in OAG is not related to age, CCT, IOP,
axial length, BMI, or blood pressure.
Commercial Relationships: Arthur J. Sit, Glaukos, Corp. (F),
Alcon Laboratories, Inc. (C), Allergan, Inc. (C), Glaukos, Corp. (C),
Sensimed, AG (C); Nitika Arora, None; Jay W. McLaren, None;
Mehrdad Malihi, None; Lilit Voskanyan, Glaukos corporation (E)
Support: American Health Assistance Foundation, Glaukos Corp.,
Mayo Foundation for Medical Education and Research, Research to
Prevent Blindness
Expansion of Schlemm’s Canal by Travoprost in Healthy
Subjects determined by Fourier-Domain Optical Coherence
Tomography
JUNYI CHEN1, Haili Huang1, Shenghai Zhang1, Xueli Chen1,
Xinghuai Sun1, 2. 1Ophthalmology, Eye & ENT Hospital of Fudan
University, Shanghai, China; 2State Key Laboratory of Medical
Neurobiology, Institutes of Brain Science, Fudan University,
Shanghai, China.
Purpose: To determine the effect of travoprost 0.004% on
Schlemm’s canal (SC) in healthy human eyes using Fourier-domain
optical coherence tomography (FD-OCT).
Methods: Twelve healthy volunteers were recruited for a doubleblind, placebo-controlled, randomized, and paired comparison study.
Right eyes of subjects were randomly assigned to receive either
travoprost 0.004% or placebo; the contralateral eye received the other
treatment. FD-OCT imaging of SC and measurements of intraocular
pressure (IOP) were carried out beforeand at 1, 2, 4, 6, 8, 12, 24, 36,
48, 60, 72 and 84 hours after eye drop instillation.
Results: After instillation of travoprost eye drops, IOP gradually
reduced, and the SC lumens expanded, while those values remained
unchanged in placebo treated eyes. At 8 hours after the travoprost
administration, the mean SC area increased 90.30% and 90.20%,
respectively, in nasal and temporal quadrant of the treated eyes as
compared to the placebo group. The SC area and IOP showed a
similar pattern of changes at most time points examined. In
travoprost-treated eyes, a statistically significant correlation between
SC area and IOP is observed (r= -0.2817; p=0.0004). Measurements
of the SC area showed sufficient repeatability and reproducibility.
Conclusions: SC can be noninvasively imaged and quantitatively
assessed in the living healthy human eye by FD-OCT. Travoprost
treatment leads to SC lumen expansion accompanied by a drop of
IOP in the healthy eye, likely as a result of the enhancement of
pressure sensitive trabecular meshwork outflow induced by
travoprost.
Commercial Relationships: JUNYI CHEN, None; Haili Huang,
None; Shenghai Zhang, None; Xueli Chen, None; Xinghuai Sun,
None
Support: the Key Clinical Program of the Ministry of Health(201136),and the National Natural Science Foundation of China
(NSFC81020108017)
Thinning of RNFL is correlated with declining of PhNR ERG
amplitude in glaucomatous monkeys
Byron H. Li, Nalini V. Rangaswamy, Steven Q. Sessums, Richard L.
Ornberg, Ganesh Prasanna. NIBR Ophthalmology, Novartis, Fort
Worth, TX.
Purpose: To understand the structure-function relationship between
retinal nerve fiber layer (RNFL) thickness and ERG in the nonhuman primate model of glaucoma.
Methods: Chronic ocular hypertension (OHT) of various durations
(1.6-13 y) was induced in the right eye (OD) of 43 cynomolgus
monkeys by argon laser trabeculoplasty. The left eye (OS) was
untreated with normal intraocular pressure (IOP). An additional 25
naïve animals served as controls. The juxtapapillary RNFL thickness
in the normal and experimental eyes was evaluated with Spectralis
SD-OCT (Heidelberg Engineering, Germany). The photopic ERG
responded to 8 ascending luminous intensities of brief-(≤ 5 msec) and
long-(200 msec) durations red Ganzfeld flashes on a rod-suppressing
blue-adapting background were recorded with Espion ERG System
(Diagnosys, MA, USA).
Results: In the 25 naïve 5-yo monkeys, there was no difference in
RNFL thickness and ERG amplitudes between OD and OS. In 23
OHT 5-yo monkeys (OHT duration = 1.6 y), significant thinning of
OD RNFL (-16 ± 5 µm, -11%, p < 0.05) was detected compared to
the OS in the nasal-inferior (NI) section. This early structural change
did not significantly alter ERG but a trend was observed in photopic
negative response (PhNR). In a second group of 20 OHT monkeys
(age = 6-17 y; OHT duration = 2-13 y), Global RNFL thickness of
OD (82 ± 4 µm) was significantly reduced (-17%, p < 0.001) than
contralateral control eyes (99 ± 2 µm). Only the ERG PhNR but not
the a- and b-waves response was significantly reduced in the OHT
eyes. Brief flash stimulation was more sensitive to detect PhNR
amplitude changes than long duration flash. There was a significant
negative correlation (R2=0.8304) between RNFL thickness and
PhNR by comparing the difference of eyes (OS-OD) in each measure
in the 43 OHT monkeys.
Conclusions: Initial thinning of RNFL was detected in the NI section
of the eyes with 1.6 y of OHT. This early structural change did not
alter ERG but a trend was observed in PhNR. A longer duration
(7.5±3.7 yo) of IOP elevation significantly reduced RNFL thickness
and PhNR in lasered monkey eyes with strong correlation.
Progressive thinning of RNFL and reduction of PhNR amplitudes
observed in monkey eyes with different duration of chronic OHT is
likely indicative of progressive loss of retinal ganglion cells and is
similar to the disease progression in POAG in humans.
Commercial Relationships: Byron H. Li, Novartis (E); Nalini V.
Rangaswamy, Novartis Institute for Biomedical Research (NIBRFW) (E); Steven Q. Sessums, None; Richard L. Ornberg, Alcon
Research (E), Novartis Institute for Biomedical Research (E);
Ganesh Prasanna, Alcon Research Ltd (E), Novartis Institute of
Biomedical Research (E)
In Vivo Agreement of Intraocular Pressures Simultaneously
Measured Using Tonometer and Manometers Placed in Anterior
and Vitreous Cavity under General Anesthesia
Hyun Seung Yang, June-Gone Kim. Department of Ophthalmology,
Asan Medical Center, Seoul, Republic of Korea.
Purpose: To analyze the agreement between three intraocular
pressure (IOP) measurements obtained using tonometer and
manometers placed in anterior chamber (AC) and vitreous cavity
(VC) in various vitreous conditions and their association with ocular
properties.
Methods: Sixty nine patients who were planning to have vitreous
surgery for epiretinal membrane or macular hole (ERM and MH;
n=26), vitreous hemorrhage (DMVH; n=24), or silicone oil removal
(n=19) were included consecutively in this prospective study. One
examiner, who was blinded to the patient’s information, performed a
complete ophthalmologic examination, including the corneal
thickness (CT), anterior chamber depth (ACD), and axial length
(AL). Another examiner simultaneously measured IOP using
Tonopen AVIA® and 2 automated transducers during the surgery
(Fig. 1).
Results: The mean POAIOP, ACIOP and VCIOP were 16.7±3.7
mmHg, 16.7±3.8 mmHg and 16.0±4.6mmHg, respectively. The
correlations between POAIOP and ACIOP, between ACIOP and
VCIOP, and between POAIOP and VCIOP were all very good
(R=0.92, 0.65, and 0.76, respectively). However, the VCIOP was
significantly higher compared with POAIOP or ACIOP in the ERM
and MH group (by 1.4 and 1.5 mmHg, respectively), butsignificantly
lower in the DMVH (by -1.0 and -1.1 mmHg, respectively) and the
silicone oil groups (by -2.8 and -2.7 mmHg, respectively) (Fig.2). In
the multivariate analysis, only CT among ocular properties was
significantly correlated with POAIOP (p<0.037) and the increase in
POAIOP per 100 um of CT was about 2.7 mmHg.
Conclusions: Generally, transducers in AC and VC provide IOP
measurements that correlate well with those offered by tonopen and
are not affected by the patient’s individual ocular properties such as
CT, ACD and AL, though CT affected Tono-pen AVIA®
measurement. However, the VCIOP using transducer overestimated
the ACIOP or POAIOP in normal vitreous condition, whereas
significantly underestimated depending on the associated pathologic
vitreous conditions especially in silicone-oil filled eyes. Thus, IOP
measured by VC transducer in certain vitreous conditions needs
further validation about the real IOP difference or measurement error.
Fig 1. Schematic drawing of setup for the simultaneous measurement
of IOP.
Fig 2. Box plots of IOP measured by tonopen and two transducers
located in AC and VC in 3 groups.
Commercial Relationships: Hyun Seung Yang, None; June-Gone
Kim, None
Support: I have no grants or support to list
Clinical Trial: S2012-1157-0002
Regulatory Roles of Anoctamin-6 in Human Trabecular
Meshwork Cells
Juni Banerjee1, Ang Li1, 3, Chi Ting Leung1, Kim Peterson-Yantorno1,
W Daniel Stamer4, Mortimer M. Civan1, 2. 1Physiology, Univ of
Pennsylvania Perelman Sch of Med, Philadelphia, PA; 2Medicine,
Univ of Pennsylvania Perelman Sch of Med, Philadelphia, PA;
3
Anatomy, University of Hong Kong- (LKS) Faculty of Medicine,
Hong Kong SAR, China; 4Ophthalmology, Duke University,
Durham, NC.
Purpose: Cell volume of trabecular meshwork (TM) cells is linked
with aqueous humor outflow resistance. TM-cell volume regulation
depends on swelling-activated Cl- channels (ICl,swell) of unknown
molecular identity. ICl,swell in some cells has been reported
modulated by anoctamins. Most tissues express anoctamins Ano1-2,
which are Ca2+-activated Cl- channels (CaCCs), and Ano6, which is
a scramblase, but the full functions and sites of this protein family are
uncertain. We are testing if anoctamins regulate TM cell volume
regulation.
Methods: Explant-derived human TM (HTM) cells, transformed
normal human TM5 and glaucomatous GM3 TM cells and HEK293
cells were studied. Gene expression was measured by reversetranscription PCR (RT-PCR) and real-time PCR (qPCR), protein
product by western blots, membrane currrents by whole-cell
ruptured-patch recording, cell volume by electronic cell sizing and
Ca2+ concentration by fura-2.
Results: All cells strongly expressed Ano6. Ano1 was present in
HEK293 cells, very much less in HTM cells and undetected in GM3
and TM cells (N=3). Expression of Ano1 by qPCR was much lower
(<0.1%) than that of Ano6 in HTM cells. RT-PCR detected Ano2
only in GM3 and HEK293 cells but not in HTM and TM5 cells
(N=3). Increasing intracellular Ca2+ with 5 μM ionomycin strongly
activated CaCCs in HTM (N=10), TM5 (N=3) and GM3 (N=5) cells.
The nonspecific CaCC inhibitor tannic acid (TA, 100 μM) blocked
these currents by ca. 80% in all TM cells (N=3 each). HTM CaCC
was also reduced (52±6% inhibition, N=4) by the nonselective CaCC
inhibitor, CaCCinh-AO1 (50 μM) but not by the selective inhibitor of
Ano1-2 channels, T16Ainh-A01 (20 μM), (N=3). ICl,swell and the
regulatory volume decrease (RVD) following hypotonic swelling
were also inhibited by non-selective CaCC blockers (TA > CaCCinhAO1) and not by the selective T16Ainh-A01. SiRNA knockdown
(80%) of Ano6 inhibited CaCCs of TM5 cells (N=3) and the RVD of
HTM (N=3) and TM5 (N=4) cells.
Conclusions: Normal human TM cells display undetectable/minor
expression of Ano1-2 documented to underlie CaCCs. Nevertheless,
non-selective CaCC blockers inhibit CaCCs, ICl,swell and the RVD
of TM cells and knockdown of highly expressed Ano6 inhibits
CaCCs and the RVD of TM cells. We suggest that Ano6 may
regulate both CaCC and ICl,swell through its scramblase activity,
modulating signal transduction.
Commercial Relationships: Juni Banerjee, None; Ang Li, None;
Chi Ting Leung, None; Kim Peterson-Yantorno, None; W Daniel
Stamer, Allergan (F), Alcon (F), Acucela (C), Aerie (C),
Cytokinetics (C); Mortimer M. Civan, None
Support: NIH Grants EY13624 (M.M.C.) and Core Grant EY 01583
Aqueous Humor Dynamics of the Water Drinking Test in
Healthy Individuals
Nitika Arora1, Jay W. McLaren2, Arthur J. Sit2. 1Ophthalmology,
Mayo Sch of Grad Med Education, Rochester, MN; 2Ophthalmology,
Mayo clinic, Rochester, MN.
Purpose: The magnitude and duration of the intraocular pressure
(IOP) increase after water drinking test have been used to assess the
risk of developing glaucoma. However, the mechanism of the rise in
IOP after water drinking is poorly understood. In this study we
examined the effect of the water drinking test on aqueous humor
dynamics (IOP, outflow facility, episcleral venous pressure (EVP),
and aqueous humor flow) in healthy individuals.
Methods: Sixteen eyes of 8 healthy participants (age 27-61; mean
37.8 years) were studied. Variables were measured in two visits.
During one study visit, IOP was measured in the sitting position by
pneumatonometry, outflow facility was measured by digital Shiøtz
tonography, and EVP was measured by using an automated
venomanometer, based on the pressure-chamber method. After
baseline measurements, participants drank 15 ml/kg of water within
10 minutes. Variables were re-measured at 10 min, 30 min, and 60
min after ingestion of water. During another study visit, baseline
aqueous humor flow rate was determined during a one hour interval
by fluorophotometry. Participants were then asked to drink same
amount of water within 10 minutes. Aqueous humor flow was remeasured on the intervals 0-10 minutes, 10-30 minutes, and 30-60
minutes after water drinking. Aqueous humor flow rate, outflow
facility, IOP, and EVP after drinking water were compared to the
same variables at baseline by using generalized estimating equation
(GEE) models. The increase in IOP after water drinking was
compared to the change in EVP by using GEE models.
Results: EVP and IOP increased after water drinking by 3 mmHg at
30 minutes (Table 1, see p-values in table). Outflow facility did not
change after water drinking. Aqueous humor flow decreased during
the 10-minute interval after water drinking but was not different from
baseline during any other interval. The rise in IOP was not different
from the rise in EVP at 10 minutes and 30 minutes (p=0.06 and p=0.8
respectively). At 60 minutes, the difference in IOP from baseline was
less than the difference in EVP from baseline (p=0.004).
Conclusions: IOP rises after the water drinking test primarily
because of the increase in EVP. Changes in outflow facility and
aqueous humor flow do not contribute to the rise in IOP. The
elevation in IOP recovers earlier than the elevation in EVP after the
water drinking test.
Commercial Relationships: Nitika Arora, None; Jay W.
McLaren, None; Arthur J. Sit, Glaukos, Corp. (F), Alcon
Laboratories, Inc. (C), Allergan, Inc. (C), Glaukos, Corp. (C),
Sensimed, AG (C)
Support: American Health Assistance Foundation, Mayo Foundation
for Medical Education and Research, Research to prevent blindness
(Schaub Special Scholar Award and departmental grant)
Foreign-body-reaction-induced chronic ocular hypertension in
rat
Bing Li1, Yu Wang2, Shutong Cao2, Olga Kraszewska2, Richard
Ornberg2, Ganesh Prasanna1. 1Glaucoma Research, NIBR, Novartis,
Fort Worth, TX; 2Retina Research, NIBR, Novartis, Fort Worth, TX.
Purpose: To develop a chronic ocular hypertension model in rats by
inducing foreign body reaction (FBR) and scar tissue formation in
Schlemm’s canal (SC) and trabecular meshwork (TM) following
insertion of human hair into the SC .
Methods: A human hair was inserted around SC of one eye in rats;
the other eye was untreated. Intraocular pressure (IOP) was
monitored weekly for eight months with a TonoLab. Retinal
functional changes were assessed using photopic negative response
(PhNR) elicited using green Ganzfeld flashes (22.76cds/m2).
Immunohistochemical analysis was performed on anterior segments
of treated and control eyes. Sections were immunolabeled with Ilba1
antibody for macrophages and stained with Trichrome collagen.
Retinal flat-mounts were prepared with Brn-3a immunolabeling to
count retinal ganglion cells (RGCs).
Results: Hair inserted into the rat SC produced a gradual, chronic
IOP elevation lasting at least eight months. At week 31, a 25%
increase in IOP was observed in the experimental eye (IOPexp eye =
20 ± 1 mmHg; mean ± SEM; IOPcontralateral eye = 16 ± 1 mmHg; p
= 0.039). Six months post-procedure, granuloma, increased
macrophages and foreign body giant cells, and dense fibrosis based
on collagen staining were observed in the angle around the inserted
hair of the experimental eye. The figure shows positive staining of
CD3 immunoreactivity was mainly detected in the SC and TM area
around the inserted hair, which confirm the T cell infiltrating.
Preliminary data in FBR eyes indicate a trend toward reduction in
PhNR post 8 months (35 ± 2.8%, p = 0.333), indicating reduced
retinal ganglion cell (RGC) activity. In retinal sections, Brn-3a
labeled RGCs were reduced 26% ± 19 (P = 0.001) at six months and
37 ± 16% (P = 0.001) at eight months compared to contralateral eyes.
Conclusions: In rats, a FBR model produces stable, moderately
elevated IOP which results in functional and morphological changes
to the retina, probably via changes in RGC density.
Induced immune-based inflammation
Commercial Relationships: Bing Li, Novartis (E); Yu Wang,
Novartis, Alcon (E); Shutong Cao, nibr (E); Olga Kraszewska,
Novartis (E); Richard Ornberg, Novartis Institute for Biomedical
Research (E); Ganesh Prasanna, Alcon Research Ltd (E), Novartis
Institute of Biomedical Research (E)
Effect of the Water Drinking Test in Subjects with Pure
Autonomic Failure and Normal Tension Glaucoma
William M. Watkins1, David Robertson2, Karen M. Joos1. 1Vanderbilt
Eye Institute, Vanderbilt University Medical Center, Nashville, TN;
2
Clinical Pharmacology Division/Medicine, Vanderbilt University
Medical Center, Nashville, TN.
Purpose: Intraocular pressure (IOP) is influenced by systemic
vascular control. Pure autonomic failure (PAF) patients have
elevation of blood pressure (BP) and symptomatic improvement
following water consumption. This study examines the effect of
water consumption on BP, IOP, and central choroidal thickness
(ChT) in subjects with (PAF) or normal tension glaucoma (NTG).
Methods: This study was approved by the Vanderbilt University IRB
(ClinicalTrials.gov NCT00338065) and informed consent was
obtained from all subjects. PAF (n=10), NTG (n=10), and control
(n=10) subjects were evaluated by baseline mean arterial systemic
blood pressure (MAP), Goldmann and Tono-pen (Reichert
Technologies, NY) tonometry, and optical coherence tomography
(OCT, Spectralis, Heidelberg, CA) central choroidal thickness (ChT).
The subjects then consumed 500 ml of room-temperature water
within 5 minutes. MAP, tonometry, and ChT measurements were
obtained 15 minutes after water consumption. The method to obtain
ChT was adapted from Mwanza, et al with the Spectralis OCT
enhanced depth imaging protocol.
Results: Baseline MAP (mm Hg) for each group was 68+14.5 (PAF),
97.7+8.2 (NTG), and 97.8+8.8(Control). After water consumption,
the MAP increased to 87.1+11.2 (PAF,P<0.001), 100.7+13.5
(NTG,P=0.09), and 101.26+8.4(Control,P=0.03). Baseline IOP (mm
Hg) for each group was 14.5 +3.5 (PAF), 13.4+2.9(NTG), and
16.8+1 .4(Control). After water consumption, the IOP increased to
19.5+3.0 (PAF,P< 0.001), 15.2 +3.4 (NTG,P< 0.001), and 17.4+1.4
(Control,P<0.004). The 15-minute mean IOP was greater in the PAF
than the control group (P<0.001, Mann-Whitney Rank Sum Test ).
Regarding ChT, the increase following water consumption in all three
groups was not statistically significant. PAF subjects demonstrated
the greatest change in ChT (5.1+3.4%,P=0.06) followed by NTG
subjects (4.8+3.0%,P=0.18), and controls (2.7+5.0%,P=0.36).
Conclusions: This study demonstrated a statistically significant
increase in IOP in all three subject groups with the greatest increase
occurring in the PAF group. A statistically significant change in
MAP was seen in both the control group as well as the PAF group
following water consumption. However, no subjects demonstrated a
significant change in ChT. This study provides evidence that water
consumption may influence IOP as well as MAP in patients with
PAF.
Commercial Relationships: William M. Watkins, None; David
Robertson, None; Karen M. Joos, Vanderbilt University (P)
Support: CTSA award UL1TR000445 from the National Center for
Advancing Translational Sciences, Joseph Ellis Family, William
Black, and Renette Corenswet Glaucoma Research Funds, and an
Unrestricted Departmental Grant to Vanderbilt Eye Institute from
Research to Prevent Blindness, Inc., N.Y.
Suction-cup oculopression offers minimal-invasive opportunities
of arbitrary IOP elevations in rats
Theresa Lueckner, Oliver W. Gramlich, Maren Kriechbaum, Julia
Teister, Norbert Pfeiffer, Franz H. Grus. Department of
Ophthalmology, University Medical Center, Experimental
Ophthalmology, Mainz, Germany.
Purpose: Patients suffering from primary open angle glaucoma show
fluctuating intraocular pressure (IOP) profiles within circadian
variations and pressure peaks. To investigate the pathological role, an
animal model with selectable IOP adjustment is needed. The
presented study aimed for establishing a minimal-invasive method for
controlled IOP elevation in rats based on suction-cup oculopression
and examined basic properties of IOP dynamics and glaucomatouslike alterations.
Methods: Suction-cup oculopression method reduces the globe range
by sucking the cornea into a cup (Ø 0.4 mm). IOP elevation
theoretically depends on cup diameter and correlates to the applied
negative pressure in a mercury column. Manipulations were
performed unilateral in nine Long-Evans rats at -100, -200, -300, 400 and -500 mmHg under Medetomidine sedation. To investigate
IOP dynamics, IOP values in mmHg were recorded every five
minutes during one hour. Eyes were examined for global
glaucomatous alterations, retinal thickness and morphological
changes of the optic nerve head via Spectralis OCT®. Post-Hoc tests
of IOP data were performed.
Results: IOP could easily be elevated using the cup-suction method.
The observed averaged IOP of 16.5±3.4 at -100 mmHg negative
pressure showed a significant increase compared to basic IOP values
of 12.2±1.8 under sedation (p<0.02). Further pressure elevation to 200 mmHg lead to significant IOP elevation of 20.8±5.6 (p<0.0001),
likewise -200 to -300, IOP was 27.6±6.7 and p<0.0001. Again,
elevation to -400 revealed a significant IOP addition to 30.9±10.6
(p=0.01). Exertion of -500 mmHg did not cause further IOP
elevation. IOP values of each elevation up to -400 are linear related
to the negative pressure level with y=0.0485x+11.91, R^2=0.99. Eye
examination exposed glaucomatous-like characteristics as fundus
alteration, slight iris and ciliary alteration. Even after ten IOP
elevation cycles, OCT examination showed a decrease of 1.6% of the
retinal thickness between manipulated eye (188,2±13.5 µm) and the
untreated (191.2±14.4 µm, p=0.13).
Conclusions: This minimal-invasive model is suitable to simulate
arbitrary profiles of IOP elevation with glaucomatous effects.
Pressure elevation depends on cup size and negative pressure level,
while Ø 0.4 mm cup works sufficient up to -400. Specific setups
changes elicited phenotypic acute angle closure glaucoma with high
IOP values and ciliar flushes.
Commercial Relationships: Theresa Lueckner, None; Oliver W.
Gramlich, None; Maren Kriechbaum, None; Julia Teister, None;
Norbert Pfeiffer, Sensimed AG (F), Sensimed AG (R), MSD (F),
MSD (R), Alcon (F), Allergan (F), Novartis (F), Novartis (R), Bayer
(F), Heidelberg Engineering (F), Bausch&Lomb (F), BoehringerIngelheim (F), Carl Zeiss Meditech (F), Chibret (F), Nidek (F), Pfizer
(F), Santen (F), Santen (R), Topcon (F), Ivantis Inc (F), Ivantis Inc
(R); Franz H. Grus, None
Comparative IOP lowering from single dose studies of
latanoprost, timolol and ONO-9054 in Dogs and Monkeys
Kazufumi Nagai, Shinsaku Yamane, Kazumi Moriyuki, Tomohiro
Karakawa, Shintaro Nakao, Tsutomu Shiroya, Yutaka Shichino. ONO
PHARMACEUTICAL CO., LTD, Osaka, Japan.
Purpose: ONO-9054 (Ono Pharmaceuticals, Osaka Japan) is a novel
prodrug compound. ONO-9054 is an isopropyl ester derivative of the
free acid ONO-AG-367 that has been classified as a dual FP/EP3
agonist that may be effective in lowering intraocular pressure in
humans. The purpose of this study was to investigate the effect of
ONO-9054 on IOP in normotensive dogs and monkeys following a
single ocular administration compared with that produced by
latanoprost (a prostaglandin FP receptor agonist), timolol (a betaadrenergic receptor antagonist) and a fixed combination of
latanoprost / timolol.
Methods: IOP lowering effects of ONO-9054 (1, 3 and 10 µg/mL),
latanoprost (50 µg/mL), timolol (5000 µg/mL) and a fixed
latanoprost / timolol combination were examined in male beagle
dogs. IOP was measured before and at 2, 4, 6, 8, and 24 hours after
administration with an applanation pneumatonometer. In a second
study the IOP lowering effect of ONO-9054 (0.1, 0.3, 1, 3, 10, 30
µg/mL) and latanoprost (50 µg/mL) were examined in male
cynomolgus monkeys. The IOPs were measured before and at 4, 8,
12, 24, 48, 72 and 96 hours after administration. Compounds were
administered topically in a 30 µL volume once into the right eye.
Results: In dogs, ONO-9054 lowered IOP in a dose-dependent
manner, and the reduction of IOP persisted for at least 24 hours after
administration. ONO-9054 at 3 µg/mL or more showed a more potent
and longer-lasting reduction of IOP than latanoprost, ONO-9054 at
10 µg/mL showed more potent and longer-lasting reduction of IOP
than the fixed latanoprost / timolol combination. In monkeys, ONO9054 lowered IOP in a dose-dependent manner, and the reduction of
IOP at 3 and 10 µg/mL was observed for up to 48 hours after
administration and IOP at 30 µg/mL persisted for up to 72 hours after
administration. The IOP reduction of ONO-9054 at 1 µg/mL was
comparable to that of latanoprost and showed more potent and
longer-lasting IOP-reducing effects at concentrations of 3 µg/mL or
more.
Conclusions: The IOP-lowering effects of the FP/EP3 agonist ONO9054 were more potent and longer-lasting than those produced by an
FP receptor agonist, a beta-adrenergic receptor antagonist and a fixed
combination FP agonist/beta-adrenergic antagonist. These results
suggest that ONO-9054 should be considered further in clinical trials
in patients with elevated intraocular pressure due to glaucoma.
Commercial Relationships: Kazufumi Nagai, ONO
PHARMACEUTICAL CO.,LTD (E); Shinsaku Yamane, ONO
PHARMACEUTICAL CO., LTD. (E); Kazumi Moriyuki, ONO
Pharmaceutical Co Ltd (E); Tomohiro Karakawa, ONO
PHARMACETICAL CO., LTD (E); Shintaro Nakao, ONO
Pharmaceutical Co.,LTD (E); Tsutomu Shiroya, ONO
Pharmaceutical.Co.Ltd. (E); Yutaka Shichino, ONO Pharmaceutical
Co Ltd (E)
BAICALEIN LOWERS INTRAOCULAR PRESSURE IN
GERBILS
Chi-wai W. Do1, Chi-ting T. Leung1, Ho Lung Henry Chan1,
Mortimer M. Civan2, Chi-ho To1. 1School of Optometry, The Hong
Kong Polytechnic University, Hong Kong, Hong Kong; 2Department
of Physiology, University of Pennsylvania, Philadelphia, PA.
Purpose: Currently, no clinical treatments are available to prevent
the glaucomatous optic neuropathy except for lowering intraocular
pressure. Baicalein is a common traditional Chinese medicine that
has no reported toxicity. We have previously demonstrated that
baicalein inhibits chloride secretion and net fluid transport across the
excised ciliary epithelium, potentially reducing aqueous humor
formation and intraocular pressure. The aim of this study is to
determine the changes in intraocular pressure by baicalein in living
animals.
Methods: Gerbils (sand rats) were used in the experiments. Baicalein
was administered intra-peritoneally to the animals in the treatment
group once every two days for two weeks, while saline solution was
used in the control animals. Measurements of the intraocular pressure
were conducted under anesthesia with a rebound tonometer
(TonoLab) prior to drug administration.
Results: Intra-peritoneal injection of baicalein (100mg/kg)
significantly lowered IOP in gerbils. The maximum hypotensive
effect was achieved two days after the first injection and then
maintained at almost the same level throughout the experimental
period. After two weeks, baicalein-treated animals had a lower
intraocular pressure of -3.92 ± 0.89 mmHg (N = 4, p < 0.05) as
compared to the saline-treated controls, accounting for a 23-26%
reduction of intraocular pressure from the baseline measurements. At
a lower concentration of baicalein (4mg/kg), a hypotensive effect of
15% was also observed (N = 5).
Conclusions: Baicalein appears to function as an ocular hypotensive
agent. Its site and mode of action remain to be determined.
Commercial Relationships: Chi-wai W. Do, None; Chi-ting T.
Leung, None; Ho Lung Henry Chan, None; Mortimer M. Civan,
None; Chi-ho To, None
Support: General Research Fund (B-Q34C), NIH Grant (EY13624),
PolyU Niche Fund (1BB8W) and PolyU Internal Grants (A-PL45, GYK88, G-U585 and G-U858)
Definition of Normal Ophthalmic Measures in the African Green
Monkey
Robin J. Goody, Wenzheng Hu, Steve D. Whittaker, Steve T. Henry,
Rohn Brookes, Michael J. Struharik, Erich Hechanova, Matthew S.
Lawrence. RxGen, Inc, Hamden, CT.
Purpose: Clinical trials require assessment of patients to confirm
suitability for study enrollment. Similar considerations have to be
made preclinically for efficacy and safety studies, particularly in
large animal models where smaller cohort sizes are often employed.
Defining the normal range of a test species, whether ophthalmic
parameters or otherwise, can facilitate study recruitment and support
evaluation of adverse or therapeutic responses to treatment. We
conducted ophthalmic examinations in a population of healthy
African green monkeys to define the normal range of these
parameters in this species.
Methods: Ophthalmic examinations were performed on healthy adult
male and female African green monkeys. Animals were sedated with
ketamine and xylazine and endpoints including laser flare
photometry, pachymetry, tear film break up, corneal staining and
anatomic dimensions were evaluated. The normal range was defined
as mean values plus (upper value) or minus (lower values) the
standard deviation multiplied by two. All studies were conducted in
compliance with the ARVO Statement for the Use of Animals in
Ophthalmic and Vision Research.
Results: The normal range of laser flare in healthy eyes was 1.5 to
10.9 photons/msec, with an average of 5.4 photons/msec. No
significant differences in laser flare were observed between male and
female eyes. Central corneal thickness measures were significantly
higher in males (471±4.7 μm) than females (453±4.5 μm, p<0.05).
The normal pachymetry range was 406 to 535 μm in males and 393
to 514 μm in females. No sex differences were evident from tear film
break up time, fluorescein or lissamine corneal staining or from
(TFBUT) or Schirmer’s test analyses. Additional measures, including
tonometry and various anatomical dimensions, have also been
examined and normal ranges defined.
Conclusions: Our studies have established normal limits for a range
of ophthalmic measures and supported use of rigorous animal
enrollment and exclusion criteria for nonhuman primate studies,
which has the potential of reducing inter-animal variability and
increasing the opportunity for statistically significant responses to
therapeutic intervention. Assessments of other ophthalmic parameters
in the African green monkey are underway and will further enhance
the value of this species for ophthalmic disease modelling.
Commercial Relationships: Robin J. Goody, RxGen, Inc (E);
Wenzheng Hu, RxGen (E); Steve D. Whittaker, RxGen Inc (E);
Steve T. Henry, Rxgen.inc (E); Rohn Brookes, RxGen, Inc (E);
Michael J. Struharik, Rx-Gen (E), St. Kitts Biomedical Research
Foundation (E); Erich Hechanova, RxGen, Inc. (E); Matthew S.
Lawrence, RxGen (E)
BK2A77: A novel non-peptide bradykinin B2 agonist lowers
intraocular pressure in ocular hypertensive cynomolgus monkeys
Ganesh Prasanna1, Naj Sharif1, Byron H. Li1, Curtis Kelly1, Shouxi
Xu1, Linya Li1, Daniel Scott1, Rad Daly1, Mark Hellberg2, Keith
Combrink2. 1Glaucoma Research, Novartis Institute of Biomedical
Research (NIBR) Ft. Worth/Alcon Research Ltd, Fort Worth, TX;
2
Global Discovery Chemistry, Novartis Institute of Biomedical
Research (NIBR) Ft Worth/Alcon Research Ltd, Fort Worth, TX.
Purpose: Bradykinin (BK), a nonapeptide has been shown to
regulate intraocular pressure (IOP) in different species including
rabbits and monkeys. Presently we intended to characterize the IOP
lowering effects of BK2A77, a novel and selective non-peptide
bradykinin B2 receptor agonist in ocular hypertensive (OHT)
cynomolgus monkeys.
Methods: BK2A77 was evaluated in several in-vitro efficacy and
receptor binding assays using human cloned B1 and B2 CHO cells
and in human ciliary smooth muscle (HCM) cells. Intracellular
calcium mobilization ([Ca2+]i) was assessed using FLIPR assay and
prostaglandin (PG) release was measured using an EIA assay. Ocular
safety assessments including slit lamp examinations were performed
in monkeys following topical ocular application of BK2A77.
BK2A77 or vehicle-induced IOP changes were measured using an
Alcon computerized pneumatonometer in normal and hypertensive
eyes of cynomolgus monkeys.
Results: BK2A77 is a selective B2 receptor agonist with EC50 values
of 13 ± 5 nM (Emax = 92 ± 1%; BK response was 100%) in [Ca2+]i
assay and 12 ± 7 nM (Emax = 100 ± 14%) in the PG release assays
respectively. BK2A77 exhibited comparable high affinity binding to
B2 receptors (Ki = 3 - 10 nM) with no detectable affinity towards B1
receptors. Topical ocular dosing of BK2A77 (3 μg x 3 times, 1 hour
apart) to sedated cynomolgus monkeys caused no flare or cells to
appear in the anterior chamber as observed up to 24h post-dose. No
other adverse effects were noted. A single topical ocular application
of BK2A77 (0.03 - 3 μg) caused a dose-dependent IOP reduction up
to 25% from baseline between 6 - 24h post-dose in the hypertensive
eyes of conscious cynomolgus monkeys. Maximal percent IOP
reduction of 25% was observed at 0.9 - 3 μg doses. While the
magnitude of IOP reduction with BK2A77 appeared to be similar to
that observed with travoprost, the duration of action appeared to last
>24h for BK2A77.
Conclusions: Unlike the issues surrounding topical ocular
application of BK peptide, including non-selectivity against B1 and
B2 receptors, poor ocular penetration and susceptibility to rapid
degradation by angiotensin converting enzyme, BK2A77 offers
several new therapeutic advantages. BK2A77 is a selective nonpeptide B2 receptor agonist capable of robust and long lasting IOP
reduction that appears to also be well tolerated following topical
ocular dosing in OHT monkeys.
Commercial Relationships: Ganesh Prasanna, Alcon Research Ltd
(E), Novartis Institute of Biomedical Research (E); Naj Sharif,
Alcon Research, Ltd (a Novartis Co.) (E); Byron H. Li, Novartis (E);
Curtis Kelly, Alcon / Novartis Institutes for Biomedical Research
(E); Shouxi Xu, Novartis (E); Linya Li, Alcon Labs (E); Daniel
Scott, NIBR at ALCON (E); Rad Daly, Novartis Institute of
Biomedical Research (E); Mark Hellberg, NIBR (E), Alcon (P);
Keith Combrink, None
Evaluation of Changes in Intraocular Pressure Immediately
After Intravitreal Injection of Anti-VEGF Medication
Julio C. Chora, Victor H. Gonzalez. 19204, Valley Retina Institute,
McAllen, TX.
Purpose: To determine changes in the intraocular pressure (IOP) in
patients receiving intravitreal injection of 4 Anti-VEGF Medication
(bevacizumab, pegaptanib, ranibizumab, aflibercept).
Methods: This was a retrospective chart review of eighty five
consecutive patients who received intravitreal Anti-VEGF medication
for various conditions such as exudative age-related macular
degeneration, venous occlusions, proliferative diabetic retinopathy
and diabetic macular edema.
IOP was measured using a Reichert Tono-Pen® XL Applanation
Tonometer before and 1 minute after intravitreal injection.
Results: Out of the 85 patients 70% were female, and 30% were
male with an average age of 74.4 years. 50% of the patients were
being treated for exudative age-related macular degeneration, diabetic
macular edema 27%, proliferative diabetic retinopathy 13%, and vein
occlusions 10%. 56% were injected with ranibizumab, 20% with
pegaptanib, 19% with bevacizumab and 5% with aflibercept.
Baseline median IOP was 15 mmHg (Max Value 22 mmHg and Min
Value 12 mmHg).
1 minute after the injection the IOP rose from a high value of 89
mmHg to a low of 15 mmHg with a median of 60 mmHg.
With ranibizumab the IOP rose from a a high value of 89 mmHg to a
low value of 24 mmHg with a median of 59.50 mmHg, with
bevacizumab the IOP rose from a high value of 87 mmHg to a low of
15 mmHg with a median of 55 mmHg, with aflibercept the IOP rose
from a high value of 75 mmHg to a low value of 54 mmHg with a
median of 58.50 mmHg and with pegaptanib the IOP rose from a
high value of 89 mmHg to a low value of 33 mmHg with a median of
86 mmHg.
Conclusions: All medications had a significant elevation of IOP with
pegaptanib having the highest elevation probably related to the higher
volume of pegaptanib that needs to be injected (0.09 ml).
The elevation in IOP demonstrated in this study is significant enough
to justify a focused study on possible long-term side effects
secondary to this acute IOP elevation.
Commercial Relationships: Julio C. Chora, None; Victor H.
Gonzalez, Genetech (C), Regeneron (C), Pfizer (C), Valiant (C),
Alimera (C)
Efficacy and safety of Polyquad-preserved Travoprost in Ocular
Hypertensives and Open Angle Glaucoma patients: an open
label, observational, 6-month, switch study
Teresa Rolle1, Rachele Penna1, Alessandro G. Actis1, Luigia
Scudeller3, Gianmaria Pasinetti4, Gemma C. Rossi2. 1Clin
Physiopathol-Section of Opht, University of Torino, Torino, Italy;
2
Eye Clinic, University of Pavia, IRCCS Policlinico San Matteo
Foundation, University of Pavia, Pavia, Italy; 3Clinical Epidemiology
and Biometric Unit, Scientific Direction, IRCCS Policlinico San
Matteo Foundation, University of Pavia, Pavia, Italy; 4Eye Unit,
Istituto Beato Palazzolo, Bergamo, Italy.
Purpose: To evaluate the clinical benefit of eliminating BAK from
prostaglandin analog therapy examining the safety and efficacy of
polyquad -preserved travoprost ophthalmic solution compared to
previous use of latanoprost monotherapy.
Methods: This was an observational study. Consecutive adults with
open-angle glaucoma or ocular hypertension treated with latanoprost
monotherapy who were going to change brand therapy to the generic
one, were switched to travoprost BAK-free ophthalmic solution. All
patients were submitted to an ophthalmic examination, IOP
measurement and ocular surface status (BUT and corneal staining)
evaluation. Patients' discomfort was evaluated with the Ocular
Surface Disease Index (OSDI). All examinations were performed at
baseline and 6 months later. Descriptive statistics were produced for
demographic and clinical characteristics of cases. Median and
interquartile range are presented for non-normally distribuited
variables. For group comparison, parametric and non-parametric tests
were used for quantitative variables and Pearson’s χ2 test for
categorical variables. All analysis refer to right eye, left eye’s data
are similar.
Results: 44 patients were enrolled and treated with polyquadpreserved travoprost once a day. TF-BUT changed from 8 [IQR 6-10]
sec at baseline to 10 [IQR 8-12] sec at 6 month (p<0.0001). No eye
developed corneal staining that statistically improved after switching
monotherapy: punctatae keratitis was absent in 13 (29.5%) patients at
baseline and in 31 (70.4%) after 6 months. OSDI was (median [IQR])
16 [10-30] at baseline and 9 [2-20] at 6 months (p=.18). The median
[IQR] baseline IOP was 18 [15.5-21] mmHg and 16 [14-17] mmHg
(p<.0001) after 6 months. At baseline, 18 (40.9%) patients had an
IOP value < 18 mmHg, 11 (25%) < 16 mmHg, 2 (4.3%) < 14 mmHg
and 1 (2.3%) < 12 mmHg, 6 months later the proportions were as
follows 36 (81.8%) (p<.0001), 21 (47.7%) (p=.0129), 8 (18.2%)
(p=.0313) and 6 (13.6%) (p=.065), respectively.
Conclusions: No patient switched from BAK-preserved latanoprost
to polyquad-preserved travoprost developed ocular surface disease
after 6 months. Ocular surface status statistically improved when
examined by BUT and corneal staining. Many patients reached a
lower IOP. Polyquad-preserved travoprost is therefore an effective
drug that is safe for the ocular surface status.
Commercial Relationships: Teresa Rolle, THEA (C); Rachele
Penna, None; Alessandro G. Actis, None; Luigia Scudeller, None;
Gianmaria Pasinetti, None; Gemma C. Rossi, None
Ocular Hypotensive Activity Of A New Melatonin Analog OPD11, In Normotensive Rabbits
Maria Caballo Gonzalez1, Carmen del Campo2, Loreto Salazar2,
Vanessa Andres-Guerrero1, Marta Vicario de la Torre1, Rocio
Herrero-Vanrell1, Irene T. Molina-Martínez1. 1Pharmacy and
Pharmaceutical Technology, Faculty of Pharmacy/Complutense
Univ., Madrid, Spain; 2Pharmaceutical and Organic Chemistry,
Faculty of Pharmacy/Complutense Univ., Madrid, Spain.
Purpose: The main risk factor in the development of glaucoma is an
elevated intraocular pressure (IOP). It has been shown that the topical
application of melatonin analogs, such as 5-MCA-NAT, can
effectively reduce IOP. OPD-11 is a new melatonin analog able to
decrease IOP in normotensive rabbits. The role of dissacharides, as
protectant compounds against cells dissecation, has been previously
investigated. The aim of this work was to prepare new ophthalmic
formulations of OPD-11 (100 µM) containing fructose (F) or
trehalose (T) to be administered by topical route. In vitro tolerance
and in vivo efficacy of these formulations were also determined.
Methods: Two formulations were prepared. OPD-11 was first
dissolved in propylene glycol and then diluted with an isotonic
solution with F or T. Final formulations were composed of OPD-11
(100 µM) and F 2% or T 2% (OPD-11/F and OPD-11/T,
respectively). Osmolarity, viscosity, surface tension and pH were
assessed. In vitro cytotoxicity was evaluated in normal human
conjunctival cells at different exposure times (15 min, 1 h and 4 h).
The ocular hypotensive effect was evaluated after instillation (25 µL)
in normotensive rabbits (n=20 eyes) for 8 h. As control, animals
received vehicle without OPD-11. All the in vivo experiments were
conducted in compliance with the ARVO statements for the Use of
Animals in Ophthalmolgy and Vision Research.
Results: OPD-11/F and OPD-11/T showed pH values nearly neutral,
were within the range of isotonicity and satisfied the requirements of
surface tension and viscosity for ophthalmic solutions. Cytotoxicity
assays showed good tolerance in conjunctival cells for both
formulations (cell viability>80% at all exposure times). OPD-11/F
and OPD-11/T were able to maintain a hypotensive activity providing
similar effects. Non significant differences were found between the
maximum IOP reduction reached by both formulations (p>0.05;
OPD-11/F: 21.32 ± 4.7%, OPD-11/T: 20.70 ± 3.7%). The
hypotensive activity lasted 5h and 6h for OPD-11/F and OPD-11/T,
respectively. The time of maximum effect was 3 hours in both cases.
Conclusions: Formulations containing the new agent OPD-11 (100
µM) and fructose or trehalose (2%) showed suitable properties for
topical ophthalmic application. Besides, both formulations were
capable of providing a hypotensive effect in normotensive rabbits.
OPD-11 should be considered potentially useful in the treatment of
glaucoma.
Commercial Relationships: Maria Caballo Gonzalez, None;
Carmen del Campo, None; Loreto Salazar, None; Vanessa
Andres-Guerrero, None; Marta Vicario de la Torre, None; Rocio
Herrero-Vanrell, None; Irene T. Molina-Martínez, None
Support: Research group UCM 920415, RETICS RD07/0062, FIS
PI10/00645 and FIS PI10/00993.
Comparison of Rebound Tonometry in Sedated and Non-Sedated
Non Human Primates (NHP)
Mark Vezina1, Sylvie Wise1, Kelly Tenneson1, Martin Bussieres2,
Timothy Bryant1, Elridge Edwards1. 1Ocular And Neuroscience,
Charles River, Senneville, QC, Canada; 2V&O Services, St. Lazare,
QC, Canada.
Purpose: The Tono-Vet® rebound tonometer is commonly used to
measure IOP in lab animals due to its portability, ease of use, quick
measurements and no topical anesthesia is required for its use. On
ocular toxicology studies, intraocular pressure (IOP) measurements
of non-human primates (NHP) are normally performed in sedated
animals in order to safely handle them for the procedure. The purpose
of this study was to assess the feasibility of obtaining consistent IOP
measurements from conscious animals.
Methods: Ten conscious male Cynomolgus monkeys (n=20 eyes;)
were acclimated to tonometry procedure over a period of five days by
holding or placing them in a sling, touching their face repeatedly
using leather/kevlar gloves and bringing the instrument near the face.
IOP measurements were recorded on three of these acclimation
occasions. Baseline measurements were then performed 8x/day on
two separate occasions. Values were then compared to those obtained
from sedated animals (n=172 eyes).
Results: Mean IOP values in conscious animals were consistent over
all occasions (19.78 - 20.13 mm Hg) and variability decreased as
animals acclimated to the procedure (±3.42 to ±2.60 mm Hg). As
expected, IOP values were higher in conscious animals, as compared
to values obtained from sedated animals. An average pressure
increase of 15% was observed. The variability in IOP readings
between conscious and sedated animals was very similar (within ±
3.5%), despite the difference in the number of eyes compared (20
eyes vs. 172 eyes, respectively). When tonometry was performed
8x/day, mean pressure values were highest during the first daily
timepoint. No technicians were injured during data capture on
conscious animals.
Conclusions: These results demonstrate that consistent IOP values
can be safely obtained from conscious acclimated animals using a
Tono-Vet®. This is particularly useful in assessing potential
glaucoma therapies, allowing multiple IOP measurements to be
performed daily without the pressure decreases associated with
sedation and removing the anesthesia-related risk and side effects,
reducing the anesthesia-related risk to the animals.
Commercial Relationships: Mark Vezina, Charles River
Laboratories (E); Sylvie Wise, Charles River (E); Kelly Tenneson,
Charles River (E), Eleven Biotherapeutics (E); Martin Bussieres,
Charles River Laboratories (E), V&O Services Inc (C); Timothy
Bryant, Charles River Laboratories (E); Elridge Edwards, CRL (I)
Expression of Circadian Rhythm Genes in the Mouse Iris-Ciliary
Body Complex
Jeffrey J. Dunmire1, Lauren Dalvin1, Rachida Bouhenni1, Deepak P.
Edward2, 3. 1Ophthalmology, Summa Health System, Akron, OH;
2
Ophthalmology, King Khaled Eye Specialist Hospital, Riyadh, Saudi
Arabia; 3Ophthalmology, Wilmer Eye Institute, Johns Hopkins
University, Baltimore, MD.
Purpose: It is well known that intraocular pressure (IOP) exhibits a
circadian rhythm. However, the molecular basis for this circadian
pattern of IOP remains to be understood. We hypothesized that a
local circadian clock exists in the iris-ciliary body complex, where
clock gene expression may contribute to daily IOP variation. We
investigated the temporal and spatial expression of circadian clock
genes in iris-ciliary body of mice.
Methods: C57BL/6J mice were entrained to a 12-hour light-dark
cycle for two weeks and then sacrificed at Zeitgeber Times (ZT) 2, 6,
10, 14, 18, and 22 (n=4 per group), where ZT 12 is the beginning of
the dark cycle. Eyes were enucleated and either flash-frozen or fixed
in 10% neutral-buffered formalin. Following dissection of iris-ciliary
body tissues, RNA was extracted and assessed by real-time PCR in
an array format profiling 84 circadian clock or clock-controlled
genes. Immunohistochemistry (IHC) was performed on formalin
fixed eyes to confirm protein expression and identify localization.
Results: Among the 84 genes tested in the PCR array, 35 showed
expression with varying degrees of circadian oscillation. The
circadian clock genes Bmal1, Clock, Cry1, Cry2, Per1, Per2, and
Per3 were all found to be rhythmically expressed. Expression levels
were highest for Bmal1 and Clock, with peak expression between ZT
2-6, and their oscillation was antiphase to Cry1, Cry2, Per1, Per2, and
Per3. Other well known clock-controlled genes that showed a clear
pattern of rhythmic expression included Dbp, Rev-erbA-α, Rev-erbβ, Tef, and Rorc. The amplitude of oscillation was greatest for Dbp,
with a 17-fold difference in expression between ZT 10 and ZT 22.
IHC showed strong staining of CLOCK and BMAL1 at ZT 14 in the
non-pigmented ciliary epithelium. Expression was also detected in
the anterior surface of the iris, but to a much lesser degree. Protein
localization was both nuclear and cytoplasmic, with increased
distribution to the cytoplasm at ZT 14 compared with ZT 2.
Conclusions: The components of an intrinsic circadian clock were
identified in the mouse iris-ciliary body complex with temporal
changes in clock gene expression and immunolocalization of their
protein products. This finding is a first step toward elucidation of
clock-controlled pathways that may be involved in aqueous humor
dynamics and an understanding of diurnal IOP variation.
Commercial Relationships: Jeffrey J. Dunmire, None; Lauren
Dalvin, None; Rachida Bouhenni, None; Deepak P. Edward, None
Support: Summa Foundation
Sphingosine-1-Phosphate signaling in cultured human trabecular
meshwork cells
Sietse T. Braakman1, Kristin M. Perkumas2, Darryl R. Overby1,
David F. Woodward3, W Daniel Stamer2. 1Bioengineering, Imperial
College London, London, United Kingdom; 2Ophthalmology, Duke
University, Durham, NC; 3Biol Sci RD-2C, Allergan, Inc, Irvine, CA.
Purpose: Sphingosine-1-Phosphate (S1P) is a bioactive lipid that
decreases conventional outflow facility in human, porcine and mouse
eyes, likely by decreasing the permeability of Schlemm’s canal
endothelium (SCE). Located upstream of SCE, the trabecular
meshwork (TM) is a prime candidate for S1P synthesis. In this study,
we test the hypothesis that human TM cells synthesize S1P as a
possible mechanism to regulate facility. We also measure S1P levels
in human aqueous humor (AH).
Methods: TM cell lines were isolated from non-glaucomatous human
donors and differentiated by allowing confluent cultures to mature for
1 week in DMEM containing 10% fetal bovine serum (FBS) followed
by 1 week in 1% FBS. After differentiation, medium was refreshed
(1% FBS) and conditioned by the cells for 2 hours, before being
sampled. AH samples were obtained from 5 non-glaucomatous
patients during cataract surgery. Conditioned medium, fresh medium
containing 1% FBS, and pooled AH were analyzed for S1P and
prostaglandin E2 (PGE2) using LC-MS/MS. Cell viability was
measured by lactate dehydrogenase (LDH) activity using a
colorimetric NAD assay. Cell lysates were analyzed by western blot
for sphingosine-kinase 1 (SphK1).
Results: S1P in fresh medium (1% FBS) was measured to be 3.5 nM,
which decreased to 1.4±0.3nM (mean±SD, n=29) after 2h of cell
conditioning. In contrast, PGE2 levels remained constant (65±22nM
vs. 60nM, n=29, p=0.6). Ceramides and sphingosine, substrates for
S1P synthesis, were present in conditioned media (26±9.7 and
5.0±1.4nM, n=29). In AH, S1P was below the detection limit, while
PGE2 was measured to be 28.8nM. There were no signs of cell death,
as measured by LDH (6.3-14.3 mIU/mL vs. 1139mIU/mL in positive
control). SphK1 was detected by western blot in both TM and SC
cells.
Conclusions: S1P was undetectable in AH and conditioned media
from TM, however both TM and SC cells expressed the enzymes and
substrates necessary for S1P generation and receptors for signaling.
Since S1P agonists/antagonists alter outflow facility, endogenous S1P
generation must be local (autocrine/paracrine), stimulated and/or
short lived.
Acknowledgements: Lipidomics Core Facility MUSC
Commercial Relationships: Sietse T. Braakman, None; Kristin M.
Perkumas, None; Darryl R. Overby, Allergan, Inc. (F), Allergan,
Inc. (C); David F. Woodward, Allergan Inc. (E); W Daniel Stamer,
Allergan (F), Alcon (F), Acucela (C), Aerie (C), Cytokinetics (C)
Support: Stichting Dondersfonds (STB), NIH EY022359, Research
to Prevent Blindness
Shear Stress Stimulation of NO release from Schlemm’s Canal
Cells
Nicole E. Ashpole1, Darryl R. Overby2, C R. Ethier3, W Daniel
Stamer1, 4. 1Biomedical Engineering, Duke University, Durham, NC;
2
Biomedical Engineering, Imperial College, London, United
Kingdom; 3Biomedical Engineering, Georgia Institute of Technology,
Atlanta, GA; 4Ophthalmology, Duke University, Durham, NC.
Purpose: Nitric Oxide (NO) has important physiological effects,
including increasing endothelial permeability and smooth muscle
relaxation. In vascular endothelia, NO is produced by endothelial NO
synthase (eNOS), whose activity and abundance are regulated by
shear stress. In Schlemm’s Canal (SC) shear stress is calculated to be
comparable to those in large arteries (2-20 dynes/cm^2). Here we
investigate the relationship between NO production and shear stress
in cultured human SC cells.
Methods: Two strains of human SC endothelial cells isolated from
non-glaucomatous donor eyes were seeded into Ibidi flow chambers
at confluence, allowed to acclimate for at least 7 days and subjected
to continuous shear stress (0.1, 10 and 15 dynes/cm^2) for 7 days.
Cell alignment was assessed using phase-contrast microscopy
coupled with Cell Profiler analysis (Broad Institute). NO production
was measured by two methods: (i) DAF-FM Fluorescence
(Invitrogen) monitored using ImageJ analysis, giving a qualitative
measure of NO concentration; and (ii) Griess Reagent reaction
(Invitrogen), which measures nitrite concentration, a product of the
spontaneous oxidation of NO. HUVECs were used as a positive
control.
Results: SC cells, like HUVECs, aligned with the direction of flow, a
behavior that was both time and shear-dependent. While HUVECs
aligned within hours, SC cells took days. NO production by SC cells
increased with shear stress as assayed by both DAF-FM Fluorescence
and Griess Reagent. A similar effect was seen in HUVECs (Table).
Conclusions: Human SC cells respond to shear stress similar to other
vascular endothelia, aligning with flow and increasing NO production
in a shear-dependent manner. When IOP increases (and SC collapses,
thus increasing shear stress in SC), our data suggest that NO
production by SC cells will increase. This could relax trabecular cells
and increase permeability of the inner wall to increase outflow
facility. It will be important to see if the shear-eNOS response is
compromised in glaucomatous SC cells.
Commercial Relationships: Nicole E. Ashpole, None; Darryl R.
Overby, Allergan, Inc. (F), Allergan, Inc. (C); C R. Ethier, None; W
Daniel Stamer, Allergan (F), Alcon (F), Acucela (C), Aerie (C),
Cytokinetics (C)
Support: Research to Prevent Blindness and National Institutes of
Health (Grant #EY017007 and #EY022359).
Peptide and Non-Peptide Bradykinin (BK) Agonists and
Antagonists Help Define Functional BK Receptors in Human
Trabecular Meshwork and Ciliary Body
Naj Sharif1, Parvaneh Katoli1, Curtis R. Kelly1, Linya Li1, Shouxi
Xu1, Yu Wang1, Ganesh Prasanna1, Keith D. Combrink1, Mark
Hellberg1, Shahid Husain2. 1Ophthalmology Research, Alcon
Research, Ltd-Novartis Institute of Biomedical Research (NIBR),
Fort Worth, TX; 2Ophthalmology, Medical University of S. Carolina,
Charlston, SC.
Purpose: 1) to examine bradykinin (BK) receptor system in human
trabecular meshwork (h-TM), human ciliary muscle (h-CM) and
ciliary process (CP) by immunohistochemistry; 2) to
pharmacologically characterize the associated signaling mechanisms
in isolated cells from these tissues using peptide and non-peptide BK
agonists (Compounds 1 & 2) and antagonists; 3) to study the
intraocular pressure (IOP)-lowering effects of Compound 1 (CMPD1)
Methods: Published methods were utilized throughout
Results: Human and Cynomolgus monkey TM, CM and CP
expressed a high level of B2-receptor protein immunoreactivity. In
isolated h-TM, h-CM and human non-pigmented epithelial (h-NPE)
cells, BK and related analogs (e.g. Lys-BK; Hyp3-BK) exhibited a
high potency (EC50 = 2-8 nM, n = 3-6), while Des-Arg9-BK (B1receptor agonist) was much weaker (EC50= 4.2 µM), at stimulating
intracellular Ca2+ ([Ca2+]i) mobilization. Two non-peptide B2receptor agonists (CMPD-1 & 2) had lower potencies (CMPD-1
EC50s = 150-276 nM; CMPD-2 EC50s = 25-74 nM; n = 3-29) than
BK in these assays. While BK peptides and CMPD-2 were full
agonists, CMPD-1 was a partial agonist (Emax = 38% in NPE; 64% in
CM; 80% in TM cells). These effects of BK, CMPD-1 & 2 were
blocked by HOE-140 (peptide B2-antagonist; Ki = 1-8 nM; n = 4-6)
and WIN-63448 (non-peptide B2-antagonist; Ki = 157 - 450 nM, n =
4-5) in all these ocular cells. While h-CM and h-TM cells responded
to BK, CMPD-1 & 2 by secreting prostaglandins (PGE2, PGF2α) (e.g.
EC50s = 4 - 61 nM in h-TM cells, n = 3-5; CMPD-1 Emax = 28%),
NPE cells failed to release any PGs. The latter PG secretion effects of
the BK agonists were also blocked by HOE-140 and WIN-63448, and
were attenuated by cyclooxygenase inhibitors (bromfenac and
flurbiprofen). BK, CMPD-1 & 2 also increased secretion of ProMMP-3 by 1.3-1.6 fold above basal levels in h-CM cells 24 hr post
incubation. CMPD-1 lowered IOP in conscious ocular hypertensive
Cynomolgus monkeys (e.g. 37.7 ± 5.4% with 30 µg, 24 hrs post
topical ocular dosing).
Conclusions: These data support the existence of functionally active
B2-receptors in h-TM, h-CM and h-NPE cells that mediate [Ca2+]i
mobilization, PG secretion (not in NPE cells) and pro-MMP-3 release
(h-CM). These data help explain the potent, efficacious, and
prolonged IOP-lowering effects of CMPD-1.
Commercial Relationships: Naj Sharif, Alcon Research, Ltd (a
Novartis Co.) (E); Parvaneh Katoli, Alcon Labs (E); Curtis R.
Kelly, Alcon / Novartis Institutes for Biomedical Research (E);
Linya Li, Alcon Labs (E); Shouxi Xu, Alcon, Novartis (E); Yu
Wang, Novartis, Alcon (E); Ganesh Prasanna, Alcon Research Ltd
(E), Novartis Institute of Biomedical Research (E); Keith D.
Combrink, Novartis (E); Mark Hellberg, NIBR (E), Alcon (P);
Shahid Husain, None
Effect of ONO-9054 on Aqueous Humor Dynamics in Monkeys
Tomohiro Karakawa, Shinsaku Yamane, Kazufumi Nagai, Shintaro
Nakao, Tsutomu Shiroya, Yutaka Shichino. ONO
PHARMACEUTICAL CO., LTD, Mishimagun, Japan.
Purpose: ONO-9054 (Ono Pharmaceuticals, Osaka Japan) is a novel
prodrug compound. ONO-9054 is an isopropyl ester derivative of the
free acid ONO AG-367 that has been classified as a dual FP/EP3
agonist that may be effective in lowering intraocular pressure in
humans. The purpose of this study was to investigate the effect of
ONO-9054 on the aqueous humor dynamics in cynomolgus monkeys.
Methods: Aqueous humor flow was measured with a
fluorophotometer in monkeys. Ophthalmic vehicle, timolol (5000
μg/mL), latanoprost (50 μg/mL) and ONO-9054 (3 μg/mL) were
administered topically into the eye, and aqueous humor flow rate and
intraocular pressure (IOP) measurements were conducted. Outflow
facility was measured by a two-level, constant-pressure perfusion
method in monkeys. Latanoprost (50 μg/mL) and ONO-9054 (3 and
30 μg/mL) were administered to the eye unilaterally, and the
contralateral eye was administered ophthalmic vehicle solution.
Measurements of outflow facility and IOP were conducted.
Results: Timolol, latanoprost, and ONO-9054 reduced IOP. Timolol
decreased the aqueous humor flow (0.93 ± 0.16 μL/min) relative to
vehicle (1.88 ± 0.13 μL/min). On the other hand, ONO-9054 at 3
μg/mL (2.06 ± 0.24 μL/min) and latanoprost (2.05 ± 0.08 μL/min)
had no effect on aqueous humor flow. When compared with the
contralateral vehicle-treated eyes (0.62 ± 0.10 μL/min/mmHg),
latanoprost had no effect on the outflow facility (0.62 ± 0.11
μL/min/mmHg). Likewise, ONO-9054 at 3 μg/mL had no effect on
the outflow facility (0.56 ± 0.08 μL/min/mmHg). On the other hand,
ONO-9054 at 30 μg/mL slightly increased outflow facility (0.87 ±
0.09 μL/min/mmHg) relative to contralateral eyes (0.66 ± 0.08
μL/min/mmHg).
Conclusions: ONO-9054, a dual agonist for prostaglandin FP and
EP3 receptors, did not have any effect on aqueous humor flow. The
present results suggest that the mechanism of IOP-lowing for ONO9054 appears to involve an enhancement of uveoscleral outflow,
similar to the mechanism reported for pure FP receptor agonists, and
an effect on conventional outflow pathways.
Commercial Relationships: Tomohiro Karakawa, ONO
PHARMACETICAL CO., LTD (E); Shinsaku Yamane, ONO
PHARMACEUTICAL CO., LTD. (E); Kazufumi Nagai, ONO
PHARMACEUTICAL CO.,LTD (E); Shintaro Nakao, ONO
Pharmaceutical Co.,LTD (E); Tsutomu Shiroya, ONO
Pharmaceutical.Co.Ltd. (E); Yutaka Shichino, ONO Pharmaceutical
Co Ltd (E)
Cabergoline and IOP: implications for structure-based drug
discovery of selective dopaminergic ligands
Filippo Drago, Chiara Bianca Maria Platania, Giuseppina
Marrazzo, Gian Marco Leggio, Claudio Bucolo. Clinical and
Molecular Biomedicine, University of Catania, Catania, Italy.
Purpose: To investigate the role of cabergoline, an anti-Parkinson
agent, on intraocular pressure regulation using wild type (WT) and
D3R knockout (KO D3R-/-) mice. Further, to assess the precise role
of dopaminergic and serotonergic systems on IOP modulation, a
computational structure-based study was also carried out.
Methods: WT and KO D3R-/- C57BL6J mice were used. Both mice
were used with normal eye pressure or steroid-induced ocular
hypertension. All animals were treated according to the ARVO
statement for the use of animals in ophthalmic and vision research.
Mice were treated with cabergoline at different concentration (0.01%,
0.1%, 1%, 5%) and IOP measured by tonometer. We modeled and
optimized the structures of hD3, h5-HT1a, h5-HT2a, h5-HT2b, h5HT2c receptors by homology modeling and by molecular dynamics
respectively. Next we docked, using AutoDock 4.2, cabergoline into
the binding sites of these receptors, and rescored the binding modes
with DSX-score.
Results: Topical application of cabergoline significantly (p<0.01)
decreased, in a dose-dependent manner, the intraocular pressure in
WT mice both in an ocular normotensive group and an ocular
hypertensive group. No change of intraocular pressure was observed
after topical application of cabergoline in KO D3R-/- mice. High
correlation (R2=0.92, Pearson=0.94 p=0.02) of DSX-scores
compared to experimental Ki was obtained. Cabergoline binds better
to the D3 receptor than to the analyzed serotonergic receptors both in
computational and experimental studies.
Conclusions: The present study highlighted the dopaminergic
system, particularly D3R subtype, as the major target of cabergoline
to decrease IOP over the serotonergic system with relevant
implications for structure-based drug discovery of selective
dopaminergic ligands.
Binding modes of cabergoline interacting with hD3 and h5HT2a
receptors. AU = arbitrary unit
Commercial Relationships: Filippo Drago, None; Chiara Bianca
Maria Platania, None; Giuseppina Marrazzo, None; Gian Marco
Leggio, None; Claudio Bucolo, Alfa-Intes (C)
Support: Chiara B. M. Platania was supported by the International
Ph.D. Program in Neuropharmacology, University of Catania, Italy.
The authors wish to thank the ‘‘HPC Cineca" Italy for the
computational hours.
Twenty-four hour Variations in Ocular Biometric Parameters in
Patients with Ocular Hypertension
Shan Fan, Vikas Gulati, Donna G. Neely, Nathan V. Harms, Carol B.
Toris. Ophthalmology, Univ of Nebraska Medical Ctr, Omaha, NE.
Purpose: The overall refractive status of the eye is determined by the
corneal power, anterior chamber depth, lens thickness and axial
length. Intraocular pressure (IOP) has the potential to affect these
parameters. Over a 24 hour period the changes in these parameters
need to be complimentary to each other to keep vision clear. To
determine the extent of the ocular physical changes and whether they
correlate with IOP, this study evaluates biometric parameters
throughout a day and night in patients with ocular hypertension
(OHT) treated with brimonidine or vehicle.
Methods: Thirty patients with OHT (58.6±9.2 years of age) were
enrolled in this randomized, double-masked, crossover study.
Participants self-administered 0.2% brimonidine or vehicle three
times daily for 6 weeks. At the end of each 6 week period,
measurements of habitual (seated during the day and supine at night)
intraocular pressure (IOP), central cornea thickness (CCT), anterior
chamber depth (ACD), axial length (AXL) and lens thickness were
made. The results were compared by two way ANOVA, one way
ANOVA and post hoc testing. P values <0.05 were considered
statistically significant.
Results: Time of measurement had a significant effect on CCT, ACD
and AXL. In vehicle-treated eyes, CCT was thicker at 3 AM than any
other time (p<0.01), ACD and AXL were larger at 3 AM and 8 PM
than 3 PM (p<0.01) and lens thickness did not change (p=0.40).
Supine IOP at 3 AM was greater than seated IOPs during the day
(p<0.01). Brimonidine, with a mean (±standard deviation) habitual
IOP decrease of 1.09 ± 3.72 mmHg during the day and 0.33 ± 4.33
mmHg during the night, did not alter these patterns. Brimonidine
lowered IOP during the day but not at 3 AM. The shortest AXL and
ACD were temporally close to the lowest habitual IOP during the
day.
Conclusions: The increase in axial length at night (approximately 75
µm) can be attributed to an increase in anterior chamber depth
(approximately 90 µm). The increase in anterior chamber depth is
independent of any change in lens thickness. These potentially IOPmediated changes appear complimentary towards maintenance of
refractive status of the eye, demonstrating inherent homeostatic
mechanisms of the eye despite significant changes in the IOP.
Brimonidine use does not alter the normal diurnal rhythm of ocular
biometric parameters but a more potent ocular hypotensive drug may.
Commercial Relationships: Shan Fan, None; Vikas Gulati, None;
Donna G. Neely, None; Nathan V. Harms, None; Carol B. Toris,
Alcon (F), Allergan (F)
Support: AGS MAPS grant (VG); Research to Prevent Blindness
Voltage-coupled single-needle constant-pressure anterior
chamber perfusion in live mice
MinHee K. Ko1, Aleksandr Yelenskiy2, Jose M. Gonzalez1, James C.
Tan1. 1Ophthalmology, University of Southern California, Los
Angeles, CA; 2School of Medicine, University of Wisconsin,
Madison, WI.
Purpose: To describe voltage-coupled single-needle constantpressure anterior chamber perfusion in live mice
Methods: We established apparatus in which transduced pressure
was coupled to a microperfusion pump to automatically modulate
flow rate to maintain a steady pressure and measure outflow facility
by constant pressure perfusion. All perfusion was performed by 35G
single-needle anterior chamber cannulation in anesthetized mice. We
characterized the following: (i) perfusion pressure stability; (ii)
outflow facility; (iii) presence of “washout”; (iv) effect of different
perfusates such as phosphate buffered saline with Ca2+and Mg2+ (PBS
w Ca/Mg), PBS without Ca/Mg (PBS w/o Ca/Mg), and Barany’s
solution. H&E staining was performed on frozen sections following
perfusions.
Results: Twenty nine live C57BL/6 mice underwent perfusion.
Constant pressure was attained within seconds, stably maintained,
and not significantly affected by different perfusates (p>0.05).
Relationship between flow rate and pressure fit a linear function for
perfusion between 15-35mmHg (R2>0.9). Outflow facility
determined by 1-level constant pressure perfusion was similar
irrespective of perfusate (p>0.5): 0.0095 μl/min*mmHg for PBS w
Ca/Mg (n=10); 0.0123 μl/min*mmHg for PBS w/o Ca/Mg (n=10);
0.0074 μl/min*mmHg for Barany’s solution (n=9). Needle resistance
was negligible relative to physiologically relevant perfusion. 2-level
constant pressure perfusion over 2 hours showed no washout
phenomenon. Histological disruption of drainage tissue following
perfusions was not seen.
Conclusions: Stable constant pressure perfusion under
physiologically relevant conditions was achieved by perfusion using
single-needle cannulation, which is well-suited to the tiny mouse eye.
The washout phenomenon was not seen. Our methods are potentially
applicable to live mouse studies.
Commercial Relationships: MinHee K. Ko, None; Aleksandr
Yelenskiy, None; Jose M. Gonzalez, None; James C. Tan, None
Support: NIH grant EY020863, NIH EY03040, Career Development
Award from Research to Prevent Blindness (JCHT), and an
unrestricted grant from the Research to Prevent Blindness
Effects of Nitric Oxide Donor on Outflow Facility in Mice
Jason Y. Chang1, Catherine M. Marando1, C R. Ethier1, 2, W Daniel
Stamer3, Darryl R. Overby1. 1Bioengineering, Imperial College
London, London, United Kingdom; 2Biomedical Engineering,
Georgia Institute of Technology, Atlanta, GA; 3Ophthalmology,
Duke University, Durham, NC.
Purpose: Overexpression of endothelial nitric oxide synthase
(eNOS) has been shown to increase conventional outflow facility and
lower intraocular pressure (IOP) in transgenic mice (Stamer et al.
IOVS 2011 52:9438). To better understand how nitric oxide (NO)
contributes to the regulation of IOP in non-transgenic animals, we
examined the effects of a NO-donor on conventional outflow facility
in wild-type mice.
Methods: Enucleated eyes from C57BL/6 mice (6-8 week old
females) were perfused at pressures of 8, 15, 20 and 25 mmHg using
a computerized system. Eyes were perfused at 35°C in a bath of
isotonic saline with Dulbecco’s phosphate buffered saline + 5.5mM
glucose (DBG) supplemented with either a light-activated NO-donor
(100µM S-nitroso-N-acetylpencillamine, SNAP; N = 4) or inactive
donor (100µM N-acetylpencillamine, NAP; N = 4). Eyes were
perfused either immediately after enucleation or after 4 hours storage
at 4°C, with the treatments assigned randomly. The conventional
outflow facility was defined as the slope of the linear regression
through the flow rate-pressure data. NO release from SNAP and NAP
was characterized using an NO-sensitive probe (WPI; ISO-NO II)
that was calibrated following the manufacturer’s protocol. Light
exposure levels were measured using a luminometer (Mastech).
Results: SNAP was highly light sensitive: a brief initial light
exposure (10 min; 680-800 lumens/m2) followed by darkness caused
a burst of NO release, however continued light exposure led to an
initial increase followed by a rapid NO depletion. Under conditions
of initial light exposure followed by darkness, NO release from 100
µM SNAP (10 mL) increased over 1 hr to reach a peak of 140 ± 20
nM (mean ± SD), while NO release from 100 µM NAP was
negligible (0 ± 4 nM). Conventional outflow facility in SNAP-treated
eyes following brief light exposure was 64% greater than in NAPtreated eyes (0.0570±0.0147 vs. 0.0348±0.0085 µL/min/mmHg; p <
0.05).
Conclusions: A NO-donor increased conventional outflow facility by
64% in wild-type mice, similar to the 2-fold increase previously
reported in transgenic mice that over-express eNOS. These data
further support the role of NO in the regulation of IOP by modulation
of conventional outflow facility.
Commercial Relationships: Jason Y. Chang, None; Catherine M.
Marando, None; C R. Ethier, None; W Daniel Stamer, Allergan
(F), Alcon (F), Acucela (C), Aerie (C), Cytokinetics (C); Darryl R.
Overby, Allergan, Inc. (F), Allergan, Inc. (C)
Support: EY022359
Tracking protein endocytosis and trafficking to exosomes
released from trabecular meshwork cells
W M. Dismuke1, Brian S. McKay2, Aaditya Khatri1, Kristin M.
Perkumas1, W Daniel Stamer1. 1Ophthalmology, Duke University,
Durham, NC; 2Ophthalmology, University of Arizona, Tucson, AZ.
Purpose: Aqueous humor and conditioned media from cultured
human trabecular meshwork (TM) cells contain exosomes,
extracellular vesicles with an unclear physiological role. Exosomes
have been shown to act as signal transducers and vehicles for
delivery of small RNAs and enzymes to adjacent cells. However,
regulation of exosomal cargo loading is poorly understood. Here we
test the hypothesis that plasma membrane proteins from human TM
cells are trafficked to exosomes in response to external stimuli.
Methods: To monitor plasma membrane protein trafficking to
exosomes, we utilized a novel experimental paradigm. Cultured
human TM cells were serum starved and biotinylated on their cell
surface. Cells were then incubated with or without fetal bovine serum
(10% v:v) for various lengths of time. Following serum stimulation,
media was collected over time and exosomes were isolated by serial
ultracentrifugations and characterized by established techniques.
Exosome proteins were separated by SDS-PAGE and biotinylated
proteins were detected with strepavadin conjugated HRP.
Results: Proteins accessible to the extracellular surface of human
trabecular meshwork cells were exclusively labeled with biotin, as
indicated by lack of cytoplasmic actin biotinylation . As early as 4
hours after media change, biotinylated proteins were detected on
exosomes released from human TM cells, regardless of the presence
of serum. Notably, we observed the appearance of several unique
biotinylated proteins in exosomes from cells that were stimulated
with serum. In particular, a ~65kDa, biotinylated exosome protein
was reproducibly observed. Treatment of cells with the Ca++
ionophore, ionomycin (10μM, 10 minutes), at the end of the 4 hour
serum stimulation increased the abundance of the observed
biotinylated exosome proteins.
Conclusions: Our data demonstrates that plasma membrane proteins
from human TM cells are endocytosed, trafficked to, incorporated in
and are constitutively released with exosomes. Additionally, we
detected a unique set of proteins trafficked to exosomes upon ligand
stimulation. These findings suggest that the endocytic pathway is
very active in TM cells and that exosomal trafficking responds to
environmental cues. Both are potentially involved in the regulation
receptor signaling, outflow facility and thus intraocular pressure.
Commercial Relationships: W M. Dismuke, None; Brian S.
McKay, None; Aaditya Khatri, None; Kristin M. Perkumas,
None; W Daniel Stamer, Allergan (F), Alcon (F), Acucela (C),
Aerie (C), Cytokinetics (C)
Support: Research to Prevent Blindness
PEDF Effects on Outflow Facility
Morgan E. Rogers1, Iris Navarro1, Kristin M. Perkumas1, R Rand
Allingham1, Pratap Challa1, Craig E. Crosson2, W Daniel Stamer1.
1
Duke University, Durham, NC; 2Medical University of South
Purpose: Preliminary data from our laboratory show that pigment
epithelium derived factor (PEDF) increases transendothelial electrical
resistance of human SC and porcine AAP endothelial monolayers in a
time and dose-dependent manner. In this study, our goal was to
determine PEDF levels in human aqueous humor, and the effect of
PEDF on outflow function in enucleated mouse eyes.
Methods: Aqueous humor was obtained from consented patients
during ocular surgery. Fresh samples were immediately placed on dry
ice and kept frozen at -80C until an ELISA PEDF assay (Chemikine)
was performed. Eyes from culled C57BL/6 mice were enucleated and
cannulated for ex vivo perfusion using a computer-controlled
perfusion system optimized for mouse eyes. Purified PEDF (1μg/ml)
was perfused at four different pressures (4, 8, 15, 25 mmHg),
measuring flow to determine outflow facility (slope of flow/pressure
relationship). Data were compared to eyes perfused with vehicle
alone (negative control) and two positive controls to establish range
for detection of changes in outflow facility: dithia PGE-1 (10 nM)
and sphingosine-1-phosphate (S1P, 5μM).
Results: Eighteen human aqueous humor samples were examined
and found to contain PEDF at levels ranging from 0.12-10.72 μg/mL
(1.519 ± 2.492). We did not detect a relationship between PEDF level
and gender, race or donor glaucoma status. We perfused a total of 27
C57BL/6 mouse eyes. Compared to vehicle-perfused controls, we
observed an 86% increase in outflow facility for dithia PGE-1 (0.035
vs. 0.019 μl/min/mmHg), and a 20% decrease in outflow facility for
S1P (0.017 vs. 0.022 μl/min/mmHg), similar to values previously
reported. We perfused 7 eyes with PEDF and observed an average
outflow facility of 0.018 μl/min/mmHg compared to 0.019
μl/min/mmHg for controls. Interestingly, there appeared to be two
sets of responses within our data: 4 eyes having mean outflow facility
of 0.010 ± 0.005 μl/min/mmHg, and the other 3 with mean of 0.028 ±
0.001 μl/min/mmHg.
Conclusions: PEDF is present in human aqueous humor at
physiologically relevant concentrations. On average PEDF did not
significantly affect outflow facility. However, the data set was
bimodal with 4 eyes decreasing outflow facility by 50%, and the
other 3 increasing outflow facility by 67%. We do not presently
understand this phenomenon but have only tested one concentration
of PEDF which was effective in vitro but may be too high ex vivo.
We plan to perform a full concentration-response.
Commercial Relationships: Morgan E. Rogers, None; Iris
Navarro, None; Kristin M. Perkumas, None; R Rand Allingham,
New World Medical (C); Pratap Challa, None; Craig E. Crosson,
Alimera Sciences (C), Lexicon Pharmaceuticals, Inc (R); W Daniel
Stamer, Allergan (F), Alcon (F), Acucela (C), Aerie (C),
Cytokinetics (C)
Support: EY022359
Ocular hypertensive effect of pilocarpine in the anesthetized rat
Jeffrey W. Kiel, Alma L. Maldonado. Ophthalmology, Univ of Texas
Hlth Sci Ctr SA, San Antonio, TX.
Purpose: A previous study by Pang et al. (Exp Eye Res 80: 207-214,
2005) reported that pilocarpine tended to increase IOP in conscious
rats one hour after dosing but the effect was not significant. The
present study sought to determine if continuous IOP measurement
before and after application would detect a more robust and rapid
response.
Methods: In rats (n=14) anesthetized with ketamine plus xylazine,
we measured femoral mean arterial pressure (MAP) and IOP by
direct cannulation, carotid blood flow (BFcar) by transit time
ultrasound and heart rate (HR) by a digital cardiotachometer. The
protocol entailed 10 min of baseline followed by 20 min of
measurement after topical application of pilocarpine (1%). The data
(mean +/- standard error) were analyzed by paired t-test.
Results: The systemic parameters were unchanged (p>0.05 for MAP,
BFcar and HR) before and after pilocarpine (MAP: 96 +/- 4 Vs 92 +/4 mmHg; BFcar: 4.2 +/- 0.3 Vs 4.0 +/- 0.3 ml/min; HR: 268 +/- 9 Vs
268 +/- 9 bpm). IOP increased significantly from 12.6 +/- 0.7 to 14.8
+/- 1.0 mmHg (p<0.01). The IOP response began within minutes
after application and began to decline slowly after 30-45 min.
Conclusions: In contrast to its hypotensive effect in humans and
primates, pilocarpine causes a rapid, transient increase in IOP in
anesthetized rats. The rapidity of the IOP response suggests an
episcleral venous pressure mediated mechanism that warrants further
investigation.
foundation for the development of novel therapeutic agents and
diagnostic tests.
Commercial Relationships: Jonathan D. Nussdorf, None; Janet
Manalac, None; Yan Lu, None; Song Hong, None; Nicolas G.
Bazan, None
280 AMD and Drugs
Monday, May 06, 2013 2:45 PM-4:30 PM
618-620 Paper Session
Commercial Relationships: Jeffrey W. Kiel, None; Alma L.
Maldonado, None
Support: NIH Grant EY09702, van Heuven endowment
Poly-Unsaturated Fatty Acids in Human Aqueous Humor
Jonathan D. Nussdorf1, 2, Janet Manalac1, Yan Lu3, Song Hong3,
Nicolas G. Bazan3. 1Department of Ophthalmology, Ochsner Clinic
Foundation, New Orleans, LA; 2University of Queensland School of
Medicine, Brisbane, QLD, Australia; 3Neuroscience Center,
Louisiana State University Health Science Center, New Orleans, LA.
Purpose: The purpose of this study is to determine whether lipids
derived from omega-3 and omega-6 polyunsaturated fatty acids
(PUFA) are present in human aqueous humor.
Methods: Undiluted aqueous humor samples were collected from 10
patients who underwent either elective cataract surgery and/or nonelective glaucoma surgery. Samples were removed from the anterior
chamber following the initial paracenthesis and prior to any further
intervention. The samples were immediately packed in dry ice and
stored at -80 °C. The samples were analyzed in a masked fashion for
the presence of poly-unsaturated fatty acids. Lipids were extracted
from aqueous humor by solid phase exaction, and analyzed via liquid
chromatography tandem mass spectrometry (LC-MS/MS, Thermo
Scientific) in negative-ion mode. Deuterium-labeled prostglandin D2
(PGD2-d4) and/or docosahexaenoic acid (DHA-d5) were added to
each sample as the internal standard for quantification.
Results: The analysis of ten samples of aqueous humor demonstrated
the presence of the omega-6 fatty acid linoleic acid (LA), the omega3 fatty acid-alpha linolenic acid (ALA), with its metabolites DHA
and EPA. A one-way repeated measures ANOVA demonstrated a
significant difference between the concentration levels of the 8 lipids
identified in the aqueous humor [F (7, 27) = 634.2, p<.0001. A Tukey
post-hoc pairwise comparison analysis demonstrated that LA was
significantly highest in concentration. The mean concentrations were
0.88 ng/uL for LA, 0.09 ng/uL for ALA, and 0.10 ng/uL for both
DHA and EPA. There were no intra-operative or post-operative
complications related to aqueous humor sample collection.
Conclusions: We demonstrate the presence of omega-3 and omega-6
PUFAs in human aqueous humor. The omega-6 fatty acid, linoleic
acid, is highest in concentration of the lipids we identified. The
omega-6 metabolic pathway gives rise to arachidonic acid proinflammatory bioactive lipids. The omega-3 PUFAs, DHA and EPA,
are identified in our analysis and serve as precursors for a novel class
of mediators termed Resolivins and Protectins possessing pro-antiinflammatory, pro-resolving and neuro-protective properties. The
identification of PUFAs in human aqueous humor helps build a
A Phase 2b Study of Fovista™, a Platelet Derived Growth Factor
(PDGF) inhibitor in combination with a Vascular Endothelial
Growth Factor (VEGF) inhibitor for Neovascular Age-Related
Macular Degeneration (AMD)
David S. Boyer. Ophthalmology, Retina Vitreous Assoc Med Group,
Los Angeles, CA.
Purpose: To assess the safety and efficacy of Fovista™ in
combination with ranibizumab compared to ranibizumab
monotherapy in a large (449 patients) randomized Phase 2b trial in
neovascular AMD patients.
Methods: 449 patients with neovascular AMD were randomized in a
prospective, controlled, superiority trial to receive one of the
following treatment regimens administered every 4 weeks for 24
weeks: Fovista™ 0.3 mg in combination with ranibizumab 0.5 mg;
Fovista™ 1.5 mg in combination with ranibizumab 0.5 mg; or sham
in combination with ranibizumab 0.5 mg.
Results: The combination of Fovista™ (1.5 mg) with ranibizumab
met the pre-specified primary endpoint of superiority in mean visual
acuity gain compared to ranibizumab monotherapy (10.6 ETDRS
letters at week 24, compared to 6.5 letters, p=0.019). An additional
62% benefit from baseline was noted in the Fovista™ (1.5 mg)
combination therapy arm over ranibizumab monotherapy. A classic
dose-response curve was observed. Enhanced visual outcomes for
Fovista™ 1.5 mg combination therapy compared to ranibizumab
monotherapy were present at every monthly timepoint. The relative
magnitude of visual benefit increased over time. The superiority of
Fovista™ 1.5 mg combination therapy over ranibizumab
monotherapy was consistent across all subgroups including those
analyzing baseline vision, lesion size, and central retinal thickness.
Fovista™ 1.5 mg combination was superior to ranibizumab
monotherapy across multiple treatment endpoints including 3, 4 and 5
lines of vision gain (ETDRS chart). OCT and fluorescein
angiography analysis showed patients receiving Fovista™ 1.5 mg
combination therapy had greater reduction in neovascular
size compared to those receiving ranibizumab monotherapy. No
significant safety issues were observed for either treatment group in
the trial.
Conclusions: The combination of Fovista™ 1.5 mg and ranibizumab
demonstrated alpha protected, statistical superiority in terms of mean
vision gain when compared to ranibizumab monotherapy in this large
controlled phase 2b trial of eyes with neovascular AMD. The
increasing divergence of efficacy curves over time implies a benefit
to continued anti-PDGF combination therapy. Combination therapy
was well tolerated.
Commercial Relationships: David S. Boyer, Alcon (C), Allegro
(C), Allergan (C), Bayer (C), Genentech (C), Glaukos (C), GSK (C),
Neurotech (C), Optos (C), Regeneron (C)
MULTIFUNCTIONAL ANTIOXIDANTS PROTECT CELLS
FROM MITOCHONDRIAL DYSFUNCTION AND ABETA
NEUROTOXICITY
Hiroyoshi Kawada1, Peter F. Kador1, 2. 1Pharmaceutical Sciences,
University of Nebraska Medical Center, Omaha, NE;
2
Ophthalmology, University of Nebraska Medical Center, Omaha,
NE.
Purpose: We have synthesized orally active multifunctional
antioxidants (MFAOs) possessing distinct free radical scavenging
activity and independent metal attenuating activity and demonstrated
in rats that they delay cataract formation and protect the
photoreceptor layer against light damage. Since mitochondrial
dysfunction and amyloid beta (Aβ) neurotoxicity are associated with
age-related retinal changes, the effect of MFAOs on these factors
have been investigated.
Methods: Human neuroblastoma (SH-SY5Y) cells and retinal
pigmented epithelial (RPE) cells were pre-incubated with medium
containing 1 mM/10 µM of select MFAOs along with their respective
nonfunctional parent compounds as controls, and clioquinol (CQ).
Mitochondrial viability was assessed after 3-hr exposure to
manganese chloride with/without the presence of drugs using
Hoechst 33342 and rhodamine 123 staining. The neurotoxic potential
of Zn promoted Aβ aggregation on these cells was evaluated with
Zinquin staining (10 µM) which visualizes the cellular levels of labile
Zn released from the Aβ:Zn complex by MFAOs, their respective
nonfunctional parent compounds or CQ. Cellular fluorescence was
evaluated using Zeiss confocal scanning laser microscopy (LSM).
Results: The mitochondria of cells stained with rhodamine fluoresce
and exposure to manganese chloride induces mitochondrial
dysfunction which results in loss of fluorescent staining. Cells treated
with MFAOs or CQ in the presence of manganese chloride retained
fluorescence while fluorescence was lost when the cells were treated
with the nonfunctional parent compounds. Zinquin staining of cells
exposed to Aβ:Zn aggregates and MFAOs or CQ demonstrated the
presence of labile Zn suggesting that these compounds have a metal
attenuating effect on the Aβ:Zn aggregate. Similar Zinquin staining
was not observed in cells similarly exposed to the Aβ:Zn aggregates
and treated with the nonfunctional parent compounds.
Conclusions: MFAOs protect both human neuroblastoma and RPE
cells against manganese chloride induced mitochondrial dysfunction
and neurotoxicity of Zn promoted Aβ aggregation by releasing Zn
from the Zn-Aβ complex. Since mitochondrial dysfunction and ZnAβ complex are both present in age-related macular degeneration,
MFAOs may have therapeutic potential.
Commercial Relationships: Hiroyoshi Kawada, None; Peter F.
Kador, Aventix Animal Health (C), Aventix Animal Helth (F),
Aventix Animal Helth (R), Therapeutic Vision, Inc (F), Therapeutic
Vision, Inc (F), Therapeutic Vision, Inc. (R), Threapeutic Vision, Inc
(S), US 20090105269 (P)
Support: Alzheimer's Grant 20110702 and Therapeutic Vision, Inc.
Integrin Peptide Therapy: The First Wet AMD Experience
Peter K. Kaiser1, David S. Boyer2, Peter A. Campochiaro5, Jose Luis
Guerrero-Naranjo8, Jeffrey S. Heier7, Julia Kornfield6, Baruch D.
Kuppermann3, Hugo Quiroz-Mercado4, Samantha Salinas Longoria8,
Shulamit Schwartz4. 1Division of Ophthalmology, Cole Eye Institute,
Cleveland, OH; 2Retina Vitreous Associates, Los Angeles, CA; 3Dept
of Ophthalmology, Unversity of California, Irvine, Irvine, CA; 4Dept
of Ophthalmology, Unversity of Colorado, Denver, Denver, CO;
5
Dept of Ophthalmology, Wilmer Eye Institute - Johns Hopkins,
Baltimore, MD; 6Dept of Chemical Engineering, California Institute
of Technology, Pasadena, CA; 7Ophthalmic Consultants of Boston,
Boston, MA; 8Dept of Ophthalmology, Association Para Evitar La
Ceguera, Mexico City, Mexico.
Purpose: ALG-1001 is a synthetic anti-integrin oligopeptide. ALG1001 inhibits integrin receptors in vitro and arrests aberrant blood
vessel growth in vivo meditated by integrins αvβ3, αvβ5, and α5β1 sites that are expressed in neovascular ocular tissue in wet AMD and
diabetic retinopathy. ALG-1001 has demonstrated a statistically
significant reduction in CNV, ROP, and vascular permeability in
mouse models conducted by Dr. Peter Campochiaro. A 15 subject
human phase 1 DME study demonstrated nearly 55% of the study
subjects improving 3 lines or more in BCVA with at least a 30%
reduction in OCT CMT with ALG-1001 mono-therapy.
The purpose of this study is to evaluate the safety and dose ranging of
intravitreal ALG-1001 in subjects with CNV due to wet AMD with a
primary endpoint of observing for any dose limiting toxicity.
Methods: Key inclusion criteria included: Baseline BCVA between
20/50 and 20/320 and CNV due to AMD with no previous antiVEGF treatment within 45 days of enrollment. A combination of
treatment naïve and previously treated subjects were enrolled. All
patients received a loading dose of 3 monthly intravitreal injections
of either 1.5mg, 2.5mg, or 4.0mg ALG-1001 and were followed for
an additional 4 months off treatment. Safety measurements included
BCVA, slit lamp, fundus exam, IOP, OCT, FA, and fundus photos.
Results: To date, 15 subjects with neovascular AMD have been
enrolled in this ongoing open label safety study. There have been no
SAEs reported to date. AEs have primarily been limited to injection
related events. While this is an ongoing study with 15 subjects
enrolled to date, there are already clear improvements in BCVA and
macular anatomy by OCT in this mono-therapy study in
approximately 40% of the study subjects with clinical benefits lasting
at least 60 days off treatment after a 3 monthly injection loading
dose.
Conclusions: This was the first clinical trial of ALG-1001 in wet
AMD and the first clinical study evaluating the dose ranging safety in
human subjects. To date, there has been consistency in the lack of
toxicity across all study metrics at doses up to 4.0 mg. Despite the
small study size and open-label monotherapy dosing regimen,
clinically relevant indicators of efficacy are apparent with
improvements in BCVA alongside anatomic improvements in OCT
CMT that persist at least 60 days past the last intravitreal treatment.
Further studies will evaluate the further efficacy of this therapy.
Commercial Relationships: Peter K. Kaiser, Allegro Ophthalmics
(C), Alcon (C), Novartis (C), Bayer (C), Regeneron (C), Genentech
(C), Ophthotech (C); David S. Boyer, Alcon (C), Allegro (C),
Allergan (C), Bayer (C), Genentech (C), Glaukos (C), GSK (C),
Neurotech (C), Optos (C), Regeneron (C); Peter A. Campochiaro,
Advance Cell Technology (C), Aerpio (C), Elan (C), Gene Signal
(C), Genentech (C), GlaxoSmithKline (C), LPath, Inc (C), Norvox
(C), Regeneron (C), Genentech (F), Genzyme (F), GlaxoSmithKline
(F), Oxford Biomedica (F); Jose Luis Guerrero-Naranjo,
Neurotech (F); Jeffrey S. Heier, Acucela (C), Aerpio (C), Alimera
(F), Allergan (C), Bayer (C), Forsight Labs (C), Fovea (F),
Genentech (C), Genzyme (C), Genentech (F), Genzyme (F),
Thrombogenics (C), Sequenom (C), Notal Vision (F), Novartis (F),
Ophthotech (F), Ophthotech (C), Oraya (C), Paloma (F), Regeneron
(F), Regeneron (C); Julia Kornfield, Allegro Ophthalmics (C);
Baruch D. Kuppermann, Alimera (C), Allegro (C), Allergan (C),
Genentech (C), Glaukos (C), GSK (F), Novagali (C), Novartis (C),
Ophthotech (C), Pfizer (C), Regeneron (C), Santen (C), SecondSight
(C), Teva (C), ThromboGenics (C); Hugo Quiroz-Mercado, Allegro
Pharmaceutical (C); Samantha Salinas Longoria, None; Shulamit
Schwartz, None
Topical Pazopanib for the Treatment of Previously Untreated
Choroidal Neovascularization due to Age-related Macular
Degeneration
Rishi Singh1, John I. Wurzelmann2, Li Ye3, Michael A. Fries4, John
Norris2, Mohammad Hossain4, Trupti M. Trivedi4, Deborah S. Kelly4.
1
Cole Eye Institute, Cleveland Clinic, Cleveland, OH;
2
GlaxoSmithKline, Research Triangle Park, NC; 3GlaxoSmithKline,
Upper Providence, PA; 4GlaxoSmithKline, King of Prussia, PA.
Purpose: To evaluate the potential of topical ocular pazopanib, a
tyrosine kinase inhibitor, to reduce retinal edema and improve visual
acuity (VA) in subjects with previously untreated subfoveal choroidal
neovascularization (CNV) secondary to age-related macular
degeneration (AMD) over 4 weeks and to characterize the
safety/tolerability of the study drug over 12 weeks.
Methods: In a multicountry Phase 2a open-label trial, 19 eligible
subjects were treated with pazopanib eye drops 10 mg/mL (1 drop), 4
times daily (QID) for 12 weeks. VA, optical coherence tomography
(OCT), and safety were assessed weekly in Month 1, biweekly in
Month 2, and once in Month 3. The primary assessments were the
changes from baseline after 4 weeks of treatment in central retinal
thickness (CRT) as measured by OCT, and best-corrected visual
acuity (BCVA) as determined by the electronic ETDRS method.
Results: There were no meaningful changes from baseline at Week 4
in mean (standard deviation [SD]) CRT (38 [90] µm) or mean (SD)
BCVA (0 [10] letters) (primary endpoint). Similarly, there were no
meaningful changes from baseline in any of the additional parameters
measured by OCT, or in CNV or total lesion size as measured by
fluorescein angiography. There were no obvious differences observed
for changes from baseline in BCVA or CRT between subjects with
the CFH Y402H T allele (CT and TT genotypes combined) and
subjects with the CC genotype. In the study eye, 8 of 19 subjects
experienced 9 ocular adverse events (AEs), 1 of which was severe
(macular edema due to progression of the underlying disease). Nine
subjects discontinued due to protocol-defined stopping criteria and
received rescue medication. Nine subjects reported nonocular AEs, 1
of which was severe. No deaths or serious AEs were reported.
Steady-state concentrations appeared to have been reached by Week
2 after administration of study drug.
Conclusions: In subjects with previously untreated neovascular
AMD, pazopanib eye drops 10 mg/mL QID as monotherapy did not
appear to improve BCVA or decrease CRT. There was no meaningful
change from baseline in CRT, retinal morphology, CNV size, or total
lesion size. Pazopanib eye drops 10 mg/mL were generally safe and
well tolerated when instilled QID for 12 weeks.
Commercial Relationships: Rishi Singh, Genentech (C), Alcon (C),
Bausch and Lomb (R), Zeiss (R), Quark Pharmaceuticals, Inc. (F);
John I. Wurzelmann, GlaxoSnithKline (E); Li Ye, None; Michael
A. Fries, GSK (E); John Norris, GlaxoSmithKline (E); Mohammad
Hossain, GlaxoSmithKline (E); Trupti M. Trivedi,
GlaxoSmithKline (E); Deborah S. Kelly, GlaxoSmithKline (E)
Support: Clinical trial support from GSK
Clinical Trial: 2011-000243-24
IKK2 Inhibition Using TPCA-1/PLGA Microspheres Attenuates
the Laser Induced Choroidal Neovascularization
Qiutang Li1, 2, Subhash Gaddipati1, 2, M. Clarke Miller2, John O.
Trent2, 3, Henry J. Kaplan1, Qingxian Lu1, 2. 1Department of Ophthal
and Visual Science, University of Louisville, Louisville, KY; 2JG
Brown Cancer Center, University of Louisville, Louisville, KY;
3
Department of Medicine, University of Louisville, Louisville, KY.
Purpose: IKK2 is a key kinase in activation of transcriptional factor
NF-kappaB that regulates multiple cellular processes including
inflammation, stress response, cell death and angiogenesis.
Neovascularization is a hallmark of wet AMD. The present study
aims to assess the therapeutic effect of IKK2 inhibitor, TPCA1, on
the laser-induced choroid neovascularization (CNV) using
biodegrable poly-lactide-co-glycolide (PLGA) microsphere delivery
vehicle.
Methods: (1) The water-insoluble small molecule, TPCA-1, was
loaded into PLGA microspheres by packaging 10 mg of TPCA-1 into
100 mg of PLGA, average MW 7,000-17,000 lactide:glycolide
(50:50). The sphere size and TPCA-1 encapsulation efficiency were
measured to meet the pharmaceutical applicable standard. (2) TPCA1 release from polymers in vitro was tested by dialysis of TPCA-1PLGA polymers (10 mg/0.5 ml PBS-Tween80) against 50 ml of
external media that were replaced daily. The released TPCA-1 in the
dialysis medium was extracted with dichloromethane and quantified
by HPLC. (3) In vivo releasing, tissue distribution, safety, and
inflammatory response to TPCA-1 was tested on the wild type
C57BL/6 mice after bilateral retrobulbar injections of TPCA-1PLGA (10 mg of microspheres loaded with 1 mg TPCA-1 suspended
in 100 μL PBS) and sham-loaded PLGA microspheres (10 mg of
microspheres suspended in 100 μL PBS). (4) The development of
CNV after laser photocoagulation in TPCA-1-PLGA treated mice and
controls was quantified by scoring the fluorescence leakage and
isolectin-B4-594 stain areas.
Results: TPCA-1 encapsulation efficiency into the microspheres
reached to more than 95%, with average PLGA bead size of 2
microns in diameter. TPCA-1-loaded beads showed consistent
cumulative drug release in vitro and in vivo for up to a month.
Histologic analysis and OKR testing showed no cellular and
functional toxicity. In addition, laser-induced choroid
neovascularization was significantly attenuated by retrobulbar
injection of TPCA-1-PLGA microspheres.
Conclusions: Retrobulbar injection of small molecular IKK2
inhibitor, TPCA-1, delivered by biogradable PLGA microsphere, can
achieve a sustained and controllable drug release into choroid/retina
tissues and attenuate the laser-induced CNV development without the
systemic toxicity. Our results suggest IKK2 inhibition is an
innovative therapeutic approach for treating wet AMD.
Commercial Relationships: Qiutang Li, None; Subhash
Gaddipati, None; M. Clarke Miller, None; John O. Trent, None;
Henry J. Kaplan, None; Qingxian Lu, None
Effects of intravitreally injected ranibizumab and aflibercept on
retina and choroid of monkey eyes
Ulrich Schraermeyer, Sylvie Julien. Experimental Vitreoretinal
Surgery, Centre for Ophthalmology, Tubingen, Germany.
Purpose: Because there is evidence that the Fc domain of anti-VEGF
drugs may cause unexpected effects in retinal and choroidal vessels,
the effects of intravitreal ranibizumab and aflibercept on monkey
eyes were investigated.
Methods: Four Cynomolgus monkeys were intravitreally injected
with 0.5mg ranibizumab (Lucentis®) and another four with 2mg
aflibercept (Eylea®). All eyes underwent fluorescein angiography
(FA) and OCT before injection as well as before sacrifice (1 or 7
days post-injection). Three untreated monkeys and one injected with
aflibercept vehicle served as controls. The eyes were inspected by
light and electron microscopy and the choriocapillaris lumen width
was measured by morphometry.
Results: Neither FA nor OCT showed any effects of drug treatment.
Focal thrombocyte activation, fibrin formation and microparticles
were seen in choroidal vessels 24 hours after injection of either drug,
but more often with ranibizumab. Hemolysis in the choriocapillaris
was observed in all eyes even vehicle-injected. In aflibercept-treated
eyes swelling of endothelial cells was observed and individual RPE
cells were completely filled with hemoglobin indicating cell death
(Fig.1b). Deposits on the endothelial cells of choroidal vessels were
denser after treatment with aflibercept compared to ranibizumab or
control. The choriocapillaris lumen width was significantly reduced
after treatment with either drug. After aflibercept, individual
capillaries collapsed completely (Fig.1a).
Conclusions: OCT and FA are not suitable to detect
microangiopathy. Both drugs reduced similarly the choriocapillaris
lumen. The cause of hemolysis is not clear, as it can also be induced
by fluorescein. After aflibercept treatment, microangiophathy
(Fig.1a) and RPE cell death (Fig.1b) were observed. The dense
material attached on choroidal vessel walls indicates protein complex
formation as observed after bevacizumab treatment1. Whether these
results are related to aflibercept’s Fc domain or to other
characteristics of the molecule remain to be investigated.
1
Schraermeyer & Julien (2012) 10.1517/14712598.2012.748741
[doi]
Fig.1a: After aflibercept treatment endothelial cells (arrow) show
microangiopathy and the lumen (asterisk) of the choriocapillaris is
completely collapsed. Fig.1b: A dead RPE cell with damaged
mitochondriae (arrowhead) is filled with hemoglobin (asterisk) after
aflibercept treatment. B = Bruch’s membrane
Commercial Relationships: Ulrich Schraermeyer, Novartis
Pharma AG (F), Novartis Pharma AG (R), Acucela (C); Sylvie
Julien, Novartis Pharma AG (F), Novartis Pharma AG (R)
Support: Novartis Pharma AG
Development and Implementation of an ELISA to Detect “AntiRanibizumab” Immunity in Age-Related Macular Degeneration
Patients
Aaron L. Magno1, 2, May Lai1, 2, Cora Pierce1, Kathleen M. Davern3,
Matthew E. Wikstrom4, Thomas W. Chalberg5, Ian Constable2,
Elizabeth P. Rakoczy1, 2. 1Molecular Ophthalmology, Lions Eye
Institute, Perth, WA, Australia; 2Centre for Ophthalmology and
Visual Sciences, The University of Western Australia, Perth, WA,
Australia; 3Monoclonal Antibody Facility, Western Australian
Institute for Medical Research, Perth, WA, Australia; 4Centre for
Experimental Immunology, Lions Eye Institute, Perth, WA,
Australia; 5Avalanche Biotechnologies, San Francisco, CA.
Purpose: To determine if there is a correlation between the nonresponsiveness of patients to ranibizumab and the presence of antiranibizumab antibodies in patients with exudative age-related
macular degeneration (AMD). In order to achieve this we have
developed an enzyme-linked immunosorbent assay (ELISA) to detect
anti-ranibizumab antibodies in serum of patients who are responders
and non-responders to ranibizumab.
Methods: The assay we developed uses a homogeneous biotindioxigenin based bridging ELISA format. Briefly, patient serum was
simultaneously incubated with both a ranibizumab-biotin conjugate
and a ranibizumab-dioxigenin conjugate. Following incubation this
mixture was loaded into a streptavidin coated well and complexes of
anti-ranibizumab antibodies with the biotin-dioxigenin conjugates
was detected using a horseradish peroxidase-conjugated antidioxigenin antibody. Mice were immunised with ranibizumab to
generate a positive control for this assay. A cohort of 48 AMD
patients was recruited from the clinic at the Lions Eye Institute,
Western Australia. The cohort included 8 patients participating in an
anti-VEGF gene therapy study currently being conducted by our
group. All patients had received multiple injections of ranibizumab
prior to sampling of their serum.
Results: Mouse serum containing anti-ranibizumab antibodies was
used to successfully validate this assay. While the mouse serum
generated a robust response no anti-ranibizumab antibodies were
detected in the 48 patient serum samples screened.
Conclusions: In this initial limited study we have found no
correlation between patient responsiveness to ranibizumab and antiranibizumab antibodies. However, having successfully generated a
robust assay capable of detecting anti-ranibizumab antibodies within
sera this study will be expanded to 300 patients and will continue to
include further patients from our anti-VEGF gene therapy study. A
greater understanding of anti-ranibizumab antibodies will assist in the
development of guidelines to match the treatment strategies to the
responsiveness of patients.
Commercial Relationships: Aaron L. Magno, Avalanche
Biotechnologies, Inc. (F), Avalanche Biotechnologies, Inc. (R); May
Lai, Lions Eye Institute (P); Cora Pierce, None; Kathleen M.
Davern, None; Matthew E. Wikstrom, None; Thomas W.
Chalberg, Avalanche Biotechnologies, Inc. (E), Avalanche
Biotechnologies, Inc. (I), Avalanche Biotechnologies, Inc. (P),
Avalanche Biotechnologies, Inc. (S); Ian Constable, Lions Eye
Institute (P), Avalanche Biotechnologies (C); Elizabeth P. Rakoczy,
Avalanche Biotechnologies (C), Lions Eye Institute (P)
Support: National Health and Medical Research council of
Australia: 1010405
312 Gene Therapy
Tuesday, May 07, 2013 8:30 AM-10:15 AM
Program #/Board # Range: 2707-2746/A0151-A0190
Program Number: 2707 Poster Board Number: A0151
Corrective Gene Therapy for RPGR-XLRP Rescues a Canine
Model at Mid-Stage Disease
William A. Beltran1, Artur V. Cideciyan2, Alfred S. Lewin3, Simone
Iwabe1, Sanford L. Boye4, William W. Hauswirth4, Samuel G.
Jacobson2, Gustavo D. Aguirre1. 1Clinical Studies, School of
Veterinary Medicine, University of Pennsylvania, Philadelphia, PA;
2
Ophthalmology, Scheie Eye Institute, School of Medicine,
University of Pennsylvania, Philadelphia, PA; 3Molecular Genetics &
Microbiology, University of Florida, Gainesville, FL;
4
Ophthalmology, University of Florida, Gainesville, FL.
Purpose: Mutations in the RPGR gene are the most common cause
of X-linked RP in man, a condition that is currently still incurable.
However, we have recently shown in two canine models that AAVmediated gene transfer of human RPGRORF15 cDNA can rescue
photoreceptors when delivered prior to the onset, or at an early stage
of degeneration. We now investigate whether gene therapy delivered
at a more advanced stage of disease can still provide a positive
outcome.
Methods: An AAV2/5 vector construct (titer: 1.51 x 1011 vg/ml)
carrying full-length human RPGRORF15 cDNA under the control of
a hIRBP promoter was injected subretinally in three 12-wk-old
XLPRA2 dogs. At that age, there is on-going cell death and the ONL
thickness is reduced by ~ 40%. In addition, one XLPRA2 dog was
injected shortly after the onset of disease (5.1 wks of age) as an early
disease stage control. Contra-lateral eyes were either injected with
BSS, or received a similar dose of viral construct intravitreally.
Photoreceptor structure and function was assessed by means of noninvasive retinal imaging (cSLO/ SD-OCT) and ERG at 39 and 42
weeks of age, respectively.
Results: In vivo retinal imaging showed preserved ONL thickness in
the treated retinal areas. ERG function was greater in treated than in
control eyes, with more than an 8-, 1.2-, and 1.6-fold difference in the
amplitudes of rod, mixed rod-cone, and cone 29 Hz flicker responses,
respectively. ONL thickness was better preserved in the animal
treated at 5.1 weeks than in the 3 dogs injected at 12 weeks of age,
and ERG amplitudes were in average 1.6-fold higher.
Conclusions: These results expand our recently published findings
by showing that a sustained and beneficial effect on photoreceptor
structure and retinal function can be achieved even when delivering
RPGR gene augmentation at a mid-stage of XLRP disease. This has
important translational application given that most patients are likely
to have relatively advanced disease at the time of treatment.
Commercial Relationships: William A. Beltran, None; Artur V.
Cideciyan, None; Alfred S. Lewin, University of Florida (P);
Simone Iwabe, None; Sanford L. Boye, PCT/US2012/062478 (P);
William W. Hauswirth, AGTC (I), Bionic Sight (I), AGTC (C),
Syncona (C), RetroSense (C); Samuel G. Jacobson, None; Gustavo
D. Aguirre, None
Support: NIH EY-06855, -17549, -021721, -022012, Foundation
Fighting Blindness, Macula Vision Research Foundation, Research to
Prevent Blindness, Inc.
RECOVERY OF VISUAL FUNCTION FOLLOWING GENE
THERAPY IN A LARGE ANIMAL MODEL OF CNGA3
ACHROMATOPSIA
Edward Averbukh1, Ron Ofri2, Elisha Gootwine3, Raaya Ezra-Elia2,
Hen H. Honig3, Alexander Rosov3, Esther Yamin1, Alexey
Obolensky1, William W. Hauswirth4, Eyal Banin1. 1Ophthalmology,
Hadassah Hebrew University Medical Center, Jerusalem, Israel;
2
Koret School of Veterinary Medicine, Hebrew University of
Jerusalem, Jerusalem, Israel; 3Department of Ruminant Research,
Agricultural Research Organization, the Volcani Center, Bet Dagan,
Israel; 4Department of Ophthalmology, University of Florida,
Gainesville, FL.
Purpose: Recently, we reported on novel hereditary dayblindness in
sheep caused by a mutation in the CNGA3 gene (Reicher et al.,
Genomics 95:101-104, 2010). Since mutations in this gene can also
cause achromatopsia in humans, we decided to use these sheep as a
cone-enriched, large animal model to evaluate safety and efficacy of
CNGA3 gene therapy.
Methods: The unique anatomical features of the ovine eye required
development of a surgical procedure for subretinal delivery.
Subsequently, different types of Adeno-Associated Viral (AAV)
vectors carrying the intact human or mouse CNGA3 gene were
delivered unilaterally into the subretinal or vitreal space of affected
sheep. Animals were electrophysiologically and behaviorally
assessed preoperatively and 2 and 6 months post-operatively. A subgroup of animals were also tested 12 months after treatment. Cone
function was measured by electroretinography (ERG) following light
adaptation (10 min., 30 cd/m2). Responses to photopic flash and
flicker (10-80Hz) stimuli were recorded at 4 intensities (1-10 cd x
sec/m2). Behavioral assessment included scotopic and photopic maze
testing under standardized conditions. Passage times and number of
collisions were recorded. Age-matched normal and day-blind sheep
were similarly assessed as controls.
Results: Cone function as measured by ERG was significantly
depressed in affected sheep prior to surgery. Following surgery, there
was significant improvement in eyes treated by either the human or
the mouse CNGA3 gene under control of the red-green Opsin
promoter. Behaviorally, there were no differences between dayblind
and normal controls in scotopic testing, but dayblind animals failed to
navigate the maze under photopic conditons. Following delivery of
either the human or the mouse CNGA3-carrying vector, the ability of
previously dayblind sheep to navigate the photopic maze without
collisions improved dramatically, approaching that of normal
controls. The electrophysiological and behavioral improvement in
operated sheep persisted for at least 1 year post-op without affecting
the animals' health.
Conclusions: AAV-mediated gene therapy improves cone-dependant
visual function in CNGA3 dayblind sheep, with a good safety profile.
The long-term electrophysiological and behavioral improvement in
this naturally-occurring large animal model may pave the way to
application of similar treatment in human achromatopsia patients.
Commercial Relationships: Edward Averbukh, None; Ron Ofri,
None; Elisha Gootwine, None; Raaya Ezra-Elia, None; Hen H.
Honig, None; Alexander Rosov, None; Esther Yamin, None;
Alexey Obolensky, None; William W. Hauswirth, AGTC (I),
Bionic Sight (I), AGTC (C), Syncona (C), RetroSense (C); Eyal
Banin, None
Support: Israeli Ministry of Health Grant 3-00000-8290, the Macula
Vision Research Fund (MVRF) and the Yedidut 1 Research Grant
Phase I Gene Therapy Trial in Israeli Patients with Leber
Congenital Amaurosis Caused by a Founder RPE65 Mutation:
Safety and Efficacy Update with Up to Two Years of Follow-up
Eyal Banin1, Alexey Obolensky1, Yitzhak Hemo1, Devora MarksOhana1, Malka Sela1, Esther Yamin1, William W. Hauswirth2, Samuel
G. Jacobson3, Dror Sharon1. 1Ophthalmology, Hadassah-Hebrew
Univ Med Ctr, Jerusalem, Israel; 2Ophthalmology, University of
Florida at Gainsville, Gainsville, FL; 3Scheie Eye Institute,
University of Pennsylvania, Philadelphia, PA.
Purpose: Gene therapy of human patients with Leber congenital
amaurosis (LCA) due to mutations in the RPE65 gene became a
reality following demonstration of safety and efficacy in RPE65mutant dog and mouse models. Our phase I clinical gene therapy trial
in Israeli patients, launched in February 2010, was the fourth of its
kind worldwide (NCT00821340). The purpose of this report is to
describe the results in the first three patients treated, with up to two
years of follow-up.
Methods: Gene therapy was performed in three LCA patients (ages
21-29 years) who harbor a homozygous splicing mutation (c.952A>T; IVS2-2A>T) in the RPE65 gene. Following vitrectomy,
subretinal injection of an AAV2-hRPE65 viral vector carrying the
normal human RPE65 gene was carried out in one or two sites,
avoiding the foveal area. Ocular and systemic safety parameters were
monitored closely, including resolution of the subretinal blebs,
possible viral spread and immune response to the vector. Visual
function and structure were evaluated repeatedly as per protocol by
clinical eye exams, computerized light- and dark-adapted perimetry,
Goldmann perimetry and non-invasive color, infrared, OCT and
autofluorescence imaging.
Results: Two years of follow-up data are available for the first two
patients, and 18 months for the third. No toxicity or complications
were observed to date in any of the patients. Post-operative data
indicates stable visual acuity and increased sensitivity to light in the
treated regions in all patients, to varying degrees. In the first patient,
up to 100-fold increases persisted through the two year exam, and
interestingly, he began to use these extra-foveal areas as his preferred
locus for fixation. The third patient also reports and objectively
shows significant functional improvement. The treatment effect in the
second patient was slow to occur and is less pronounced.
Conclusions: Magnitude of the treatment effect varies between
patients, but previous studies by others as well as the present study
attest to the safety and efficacy of gene therapy for treatment of
RPE65 LCA.
Commercial Relationships: Eyal Banin, None; Alexey Obolensky,
None; Yitzhak Hemo, None; Devora Marks-Ohana, None; Malka
Sela, None; Esther Yamin, None; William W. Hauswirth, AGTC
(I), Bionic Sight (I), AGTC (C), Syncona (C), RetroSense (C);
Samuel G. Jacobson, None; Dror Sharon, None
Support: The Macula Vision Research Foundation (MVRF), The
Yedidut 1 Research Grant and NIH U10-EY13729
AAV Mediated Gene Transfer Restores Retinal and Visual
Function in Lebercilin-/- (LCA5) Mice
Daniel C. Chung, Jeannette Bennicelli, Adam Wojno, Nicoletta A.
Commins, Robert W. Bloom, Daniel J. Bennett, Thu T. Duong, Meera
Sivalingam, Arkady Lyubarsky, Jean Bennett. FM Kirby Center for
Molecular Ophthalmology, Scheie Eye Institute, University of
Pennsylvania Perelman School of Medicine, Philadelphia, PA.
Purpose: Lebercilin, encoded by the LCA5 gene, is a ciliary protein
found in the connecting cilia of photoreceptors in vivo and in the
microtubules, centrioles and primary cilia of mammalian cells
cultured in vitro. It is widely expressed in human tissues throughout
development. Since loss of lebercilin function is associated with
Leber Congenital Amsurosis (LCA), we used a gene augmentation
strategy to test the ability to correct retinal degeneration in vivo in
knockout (Lca5-/-) mice.
Methods: Cohorts of genotyped neonatal (P1-2) Lca5-/- mice
received intravenous injections of 15 ul of
AAV2/9.CBA.lebercillin.flag via the right palpebral vein. Some
animals were co-injected with a smaller dose of AAV2/9.eGFP to be
able to monitor transduction efficiency non-invasively in vivo.
Littermates were injected with AAV2/9.eGFP as control.
Retinal/visual function testing was performed at 28-35 days post
injection by pupillary light response, water maze testing, and
electroretinograms. Ophthalmoscopy, fundus photography and SDOCT imaging was used for retinal structure analysis. Animals were
euthanized and eyes were sectioned for immunofluorescence analysis
for gene expression and retinal structure.
Results: Mice injected with AAV2/9.CBA.Lebercilin.flag,showed
improvements in speed and accuracy in the water maze test, and
improved pupillary light reflexes and electroretinograms, when
compared to control animals. Ophthalmoscopy and fundus photos of
animals receiving injection of AAV.EGFP revealed widespread GFPpositive retinal cells. SD-OCT imaging documented an increase in
outer nuclear layer (ONL) thickness in experimental versus control
animals. Analysis of the experimental and control AAV-injected eyes
showed expression of the transgene in the inner and outer retina.
Immunofluorescence analyses in AAV2/9.CBA.lebercilin.flaginjected mice revealed flag-positive cells. The ONL was thicker in
AAV2/9.CBA.Lebercilin.flag-injected mice than in untreated
littermates.
Conclusions: AAV mediated gene augmentation therapy via an
intravenous route can restore retinal function in Lca5-/- mice.
Improvements in retinal function were demonstrated using behavioral
and physiologic testing and correlated with retinal structure and
transgene expression. The proof-of-concept data from these studies
will expedite the process of moving forward with a human clinical
trial.
Acknowledgements: In collaboration with the LCA5 consortium.
Commercial Relationships: Daniel C. Chung, None; Jeannette
Bennicelli, None; Adam Wojno, None; Nicoletta A. Commins,
None; Robert W. Bloom, None; Daniel J. Bennett, None; Thu T.
Duong, None; Meera Sivalingam, Canon Inc (F); Arkady
Lyubarsky, None; Jean Bennett, Gensight Biologics (S)
Support: Foundation Fighting Blindness, Foundation for Retinal
Research, F.M. Kirby Foundation, Research to Prevent Blindness,
Mackall Foundation Trust, Hope for Vision Foundation
AAV-Mediated Expression of Secreted Human Ciliary
Neurotropic Factor (hCNTF) Leads to Long-Term Preservation
of Cone Photoreceptors in an Autosomal Recessive Model of
Retinitis Pigmentosa
Daniel M. Lipinski1, Alun R. Barnard1, Mandeep S. Singh1, Edward
Lee2, Robert E. MacLaren1, 2. 1Nuffield Lab of Ophthalmology,
University of Oxford, Oxford, United Kingdom; 2Moorfield Eye
Hospital and University College London Institute of Ophthalmology
Biomedical Research Centre, London, United Kingdom.
Purpose: Loss of cone photoreceptors secondary to advanced rod
degeneration leads to a decline of central vision in retinitis
pigmentosa (RP). hCNTF has previously been shown to preserve
retinal neurons, but its long-term neuroprotective effect in vivo
remains poorly characterised. Herein, we assess the effect of hCNTF
in a rhodopsin knockout mouse model of RP with transgenic
fluorescence in cone photoreceptors, allowing cone survival to be
quantified longitudinally against a background of rod-specific
degeneration by fundus imaging.
Methods: rAAV2/2 vector expressing secreted hCNTF was injected
intravitreally (2µl) at high (1x10^10gp/µl), medium (1x10^9gp/µl) or
low (1x10^8gp/µl) titre at postnatal week (W) 4 in Rho-/-TgOPN1EGFP+/- mice (n=5-8 per group); the contralateral eye received 2µl
PBS. Intrinsically fluorescent cones were quantified by in vivo
scanning laser ophthalmoscopy (SLO) at W8, 10, 12, 15, 18, 21, 24
and 30. Function was assessed by electroretinography (ERG) at W8,
10, 12 and 15, and visual acuity determined at W30 by optomotor
response (OMR) and laser speckle Doppler flowometry.
Immunohistochemistry (IHC) and total RNA sequencing was
performed at W30.
Results: IHC showed transduction of ganglion cells and Müller glia
after intravitreal injection of rAAV2/2.hCNTF vector. In vivo
quantification of fluorescent cones demonstrated preservation for 30
weeks in high and medium titre groups (p<0.001, 2-way ANOVA,
n=5-6); cones were absent by W18 in sham eyes. Histology
confirmed preservation of cones and 1-2 outer nuclear rows. Photopic
b-wave was suppressed by hCNTF and absent beyond W15 in all
groups. Square-wave, high-contrast grating elicited OMR responses
(head tracking) only in hCNTF treated eyes at W30.
Conclusions: AAV-mediated delivery of hCNTF anatomically
preserved cone photoreceptors for the duration of the 7 month study.
Despite suppression of the photopic ERG, other assessments (OMR,
light-evoked cortical blood flow changes) showed maintenance of
functional vision in treated eyes, indicating utility of hCNTF for the
treatment of end-stage RP presenting with secondary cone loss.
hCNTF secreted from inner retinal cells provides global trophic
support, potentially allowing protection of central/foveal cones to be
achieved without subfoveal gene delivery, thus limiting surgical risk.
Commercial Relationships: Daniel M. Lipinski, None; Alun R.
Barnard, None; Mandeep S. Singh, None; Edward Lee, None;
Robert E. MacLaren, None
Support: Fight for Sight, the Wellcome Trust, the Health
Foundation, the Medical Research Council, the Royal College of
Surgeons of Edinburgh, the Oxford Stem Cell Institute and the NIHR
Ophthalmology and Oxford Biomedical Research Centers
Ultrahigh Resolution Mouse Optical Coherence Tomography to
Aid Retinal Gene Therapy Research
Mark C. Butler1, 2, Tiffany A. Kolniak3, Jack Sullivan1, 4. 1Research
Service, Veterans Administration Western NY Healthcare System,
Buffalo, NY; 2Ophthalmology, SUNY at Buffalo, Buffalo, NY;
3
Program in Neuroscience, SUNY at Buffalo, Buffalo, NY;
4
Ophthalmology, Pharm/Tox, Phys/Biophys, SUNY Eye Institute,
SUNY at Buffalo, Buffalo, NY.
Purpose: Apply ultrahigh resolution spectral domain optical
coherence tomography (UHR-SD-OCT) to assess both the in vivo
progress of outer retinal photoreceptor degeneration in mouse
models, and to exploit the instrument to capacity to aid in delivery of
gene therapy vectors into the subretinal space.
Methods: We developed a parafocal, paraxial Greenough-type stereo
microscope as an optimized non-contact retinal imaging system
(RIS) for real time subretinal surgery in the anesthetized mouse. The
RIS is optimized for the small mouse pupil, operates in various
modes (bright field, fluorescence, infrared) with color CCD camera
image capture, and has long working distance for surgical
manipulations. The UHR-SD-OCT (Bioptigen Inc) operates with near
infrared light (~850 nm), has optics optimized for the mouse eye, and
resolution of 2x2x2 micron voxels. Different mouse retinal
degeneration models and controls were used. Subretinal injections of
AAV vector or nanoparticles (NP) are accomplished with pulled
glass microneedles advanced with micromanipulator by trans-scleral
and trans-choroidal approach and positioned beneath the retina before
a calibrated pressure injector delivers into the subretinal space.
Results: The RIS presents a narrow beam through the dilated pupil to
illuminate the retina, and the optic nerve, vasculature, and posterior
pole are crisply visualized, while the entire anterior retina can be
observed through eye positioning. Normal and degenerative retinal
phenotypes are followed over time by microscopic direct fundoscopic
imaging. The RIS and micromanipulator-based delivery system
allows real-time visualization and tight control over subretinal
injections in neonatal (~P14) or adult mice. The UHR-SD-OCT is
used to immediately determine success of a subretinal injection. Gold
nanorods, useful for infrared OCT imaging, mark regions expected to
be transduced by viral vector. UHR-SD-OCT permits visualization of
all retinal layers, and allows rigorous statistical quantitation of outer
nuclear layer (ONL) thickness as measure of photoreceptor vitality to
assess degeneration or rescue.
Conclusions: Advanced imaging devices (RIS, UHR SD-OCT) are
powerful tools to assist real time subretinal surgery in mice eyes, and
to assess the state of subretinal injection, and vitality of
photoreceptors over time in animal models of retinal degeneration or
in preclinical gene therapy trials.
Commercial Relationships: Mark C. Butler, None; Tiffany A.
Kolniak, None; Jack Sullivan, None
Support: NIH/NEI Grant EY013433; VA Merit Award
1I01BX000669; Research to Prevent Blindness Unrestricted Award
Human MiniPromoters with Restricted-Retinal Expression when
Docked in the Mouse Genome Show the Same Restricted
Expression when Delivered in Adeno-Associated Virus (AAV)
Elizabeth M. Simpson1, 2, Charles N. de Leeuw1, 2, Frank M. Dyka3,
Sanford L. Boye3, Michelle Zhou1, Lisa Borretta1, Robert Holt4, 5,
Daniel Goldowitz1, 2, William W. Hauswirth3, Wyeth Wasserman1, 2.
1
Centre for Molecular Medicine and Therapeutics at the Child and
Family Research Institute, University of British Columbia,
Vancouver, BC, Canada; 2Department of Medical Genetics,
University of British Columbia, Vancouver, BC, Canada;
3
Department of Ophthalmology, College of Medicine, University of
Florida, Gainesville, FL; 4Canada's Michael Smith Genome Sciences
Centre, British Columbia Cancer Agency, Vancouver, BC, Canada;
5
Department of Molecular Biology and Biochemistry, Simon Fraser
University, Vancouver, BC, Canada.
Purpose: The development of small promoters that can drive celltype restricted gene expression will be critical for the future success
of human gene therapy. In this work, we have focused on the
development of such gene-therapy promoters for the retina. First, we
tested the hypothesis that the human Pleiades MiniPromoters selected
for expression in the brain, would be a rich source of new promoters
for the retina. Second, we tested the hypothesis that brain-restricted
promoters such as the Pleiades Promoters, which were developed
using single-copy site-specific knock-in to the mouse genome, would
function predictably when used in adeno-associated virus (AAV). We
provide data in support of both hypotheses.
Methods: Promoters were bioinformatically designed, generated by
fusion PCR from human BACs, cloned to drive reporters EGFP
and/or lacZ, and targeted 5’ of Hprt in mouse embryonic stem cells
from which mice were derived; as described in Portales-Casamar et
al., P.N.A.S., 2010. Expression analysis was in male mice at
embryonic day 12.5 or adulthood. Recombinant AAV plasmids
containing MiniPromoters driving hGFP were packaged in
AAV2(Quad Y-F) viruses (Petrs-Silva et al., Mol Ther., 2011) and
injected intravitreally into four-week old C57BL/6J mice.
Results: We have developed 18 new MiniPromoters capable of
driving expression in the brain when docked single-copy sitespecifically in the mouse genome. In addition, we have further
characterized the expression pattern of 15 MiniPromoters. Together
we have identified 17 new MiniPromoters with eye expression. Of
these, three with restricted expression in the ganglion-cell layer were
tested driving expression from an AAV genome and found to
maintain that restricted expression.
Conclusions: We conclude that the Pleiades MiniPromoters, which
were initially selected for brain expression, represent a rich resource
of promoters able to express in the retina. We further conclude that
either the computational method of MiniPromoter design, and/or the
characterization of those promoters using single-copy site-specific in
the mouse genome, renders them unusually suitable for use in AAV
and perhaps other viruses. Finally, the MiniPromoters are all humanDNA sequence and so should be particularly useful for clinical
applications.
Commercial Relationships: Elizabeth M. Simpson, Pleiades
Promoter Project: S100B MiniPromoters (P), Pleiades Promoter
Project: OLIG1 MiniPromoters (P), Pleiades Promoter Project:
PITX3 MiniPromoters (P), Pleiades Promoter Project: CLDN5
MiniPromoters (P), Pleiades Promoter Project: MKI67
MiniPromoters (P), PCP2 Minipromoters (P); Charles N. de Leeuw,
UBC (P); Frank M. Dyka, 61/560,437 (P), PCT/US2012/062478
(P); Sanford L. Boye, PCT/US2012/062478 (P); Michelle Zhou,
None; Lisa Borretta, None; Robert Holt, None; Daniel Goldowitz,
None; William W. Hauswirth, AGTC (I), Bionic Sight (I), AGTC
(C), Syncona (C), RetroSense (C); Wyeth Wasserman, None
Support: Operating - Genome Canada, Pleiades Promoter Project;
Genome British Columbia, Pleiades Promoter Project, SoF, and
CanEuCre; GlaxoSmithKline R&D Ltd., Pleiades Promoter Project;
BC Mental Health and Addiction Services, Pleiades Promoter
Project; Child and Family Research Institute, Pleiades Promoter
Project; University of British Columbia (UBC) Institute of Mental
Health, Pleiades Promoter Project; UBC Office of the Vice President
Research, Pleiades Promoter Project. Salary - Canadian Research
Chairs (to D.G. and E.M.S.); Canadian Institutes of Health Research
New Investigator Award (to W.W.W.); Canadian Institutes of Health
Research Canada Graduate Scholarships (to C.N.d.L.); Michael
Smith Foundation for Health Research Awards (to C.N.d.L., R.A.H.,
and W.W.W).
Sustained Transgene Expression with Non-viral Gene Transfer
Following Chitosan Mediated Delivery
Ana Vanessa Oliveira1, 4, Gabriela A. Silva2, 3, Daniel C. Chung4.
1
Doctoral Program in Biomedical Sciences, Department of
Biomedical Sciences and Medicine, University of Algarve, Faro,
Portugal; 2Centre for Molecular and Structural Biomedicine
(CBME/IBB, LA), University of Algarve, Faro, Portugal;
3
Department of Biomedical Sciences and Medicine, University of
Algarve, Faro, Portugal; 4F.M. Kirby Center for Molecular
Ophthalmology, Scheie Eye Institute, University of Pennsylvania
Perelman School of Medicine, Philadelphia, PA.
Purpose: A successful ocular gene therapy strategy requires efficient
gene transfer and stable transgene expression. Recently, several
strategies have been evaluated to promote safe, site-specific
integration, long-term gene expression and the ability to mediate
large gene transfer. One of the most promising technologies exploits
a site-specific recombinase, the phage phiC31 integrase. We aim to
develop a novel system for sustained large gene transfer by coupling
hybrid polymeric vectors with phiC31-integrase to simultaneously
promote enhanced delivery and transgene integration, therefore
obtaining sustained transgene expression.
Methods: Chitosan-pDNA and chitosan/hyaluronic acid-pDNA
nanoparticles were prepared using a NH3:PO4 ratio of 15:1. Particles
were produced using either pGFPattB, pCEP290attB (>8kb) with or
without pCMVINT at a molecular ratio of 2:1. These nanoparticles
were used for pDNA encapsulation and transfection studies on
cultured HEK293T cells. Co-transfection with pCMVINT was done
by delivering the two plasmids in the same particle or separately.
Transgene expression was evaluated by fluorescence microscopy and
western blot analysis.
Results: Both types of particles encapsulated pDNA efficiently, even
when two plasmids were complexed simultaneously. Transfection
studies indicate that transfection efficiency and transgene expression
is affected by polymer size and type of polyplex used, as well as the
way integrase is delivered with the polyplexes. Transgene expression
decreased over time, however GFP expression was still detected 14
weeks after transfection. Over-expression of the large transgene
CEP290 following delivery by chitosan polyplexes was detected at
least 14 days post transfection.
Conclusions: We have shown that long-term transgene expression
can be achieved with a non-viral approach using hybrid polymer
polyplexes, as well as its ability to deliver large genes. The combined
strategy of polymers and integrase is more efficient than nonintegrative strategies. Long-term transgene expression will also be
evaluated in vivo in mouse models of retinal pathologies, with further
transfection optimization.
Commercial Relationships: Ana Vanessa Oliveira, None;
Gabriela A. Silva, None; Daniel C. Chung, None
Support: Fundação para a Ciência e Tecnologia (PTDC/SAUBEB/098475/2008 to G.A.Silva, SFRH/BD/70318/2010 to
A.V.Oliveira), Marie Curie Reintegration Grant (PIRG-GA-2009249314 to G.A.Silva) under the FP7 program and Hope for Vision, F
M Kirby Foundation to D.C.Chung.
AAV-mediated Lpcat1 gene replacement therapy rescues retinal
degeneration in rd11 mice
Xufeng Dai1, Juanjuan Han1, Zi-Bing Jin1, Yan Qi1, Jie Li2, Wen-Tao
Deng2, Bo Chang3, William W. Hauswirth2, Ji-Jing Pang1, 2. 1The Eye
Hospital, School of Ophthalmology and Optometry, Wenzhou
Medical College, Wenzhou, China; 2University of Florida,
Gainesville, FL; 3The Jackson Laboratory, Bar Harbor, ME.
Purpose: Retinal degeneration 11 (rd11) mice are a newly
discovered naturally occurring animal model with early and rapid
photoreceptor dysfunction/degeneration caused by a spontaneous
mutation in the Lpcat1 gene encoding lysophosphatidylcholine
acyltransferase. This study is to test whether highly efficient tyrosine
mutant AAV-mediated gene replacement therapy can restore the
retinal function/structure in rd11 mice.
Methods: At postnatal day 14, 1 μl of AAV8 (Y733F)-smCBALpcat1 vector (1013 viral genome containing particles/ml) was
injected subretinally into one eye of 20 rd11 mice. Uninjected
contralateral eyes were used as controls. Ten weeks after injection,
dark- and light-adapted ERGs were recorded. Then both treated and
control eyes were harvested for immunohistochemical and
morphological studies.
Results: In treated eyes, ERG signals were recorded 10 weeks after
treatment; both dark- and light-adapted ERG amplitudes were about
70% of the normal level seen in wild type C57 mice. Dark- and lightadapted ERGs were unrecordable in untreated rd11 eyes. More than
half of the outer nuclear layer (ONL) was preserved in treated eyes,
while only one row of ONL nuclei remained in untreated eyes.
Lpcat1 expression was observed in the outer retina, particularly in
photoreceptor outer segments in treated eyes by
immunohistochemistry, but not in untreated eyes. Both M- and Scone opsin were observed extensively in treated eyes; in contrast,
cone opsin was nearly undetectable in untreated eyes.
Conclusions: Tyrosine-capsid mutant AAV mediated Lpcat1
expression restores/maintains rod and cone function/structure for at
least 10 weeks in the rd11 mice, a retinal degeneration model with
early and rapid photoreceptor degeneration.
Commercial Relationships: Xufeng Dai, None; Juanjuan Han,
None; Zi-Bing Jin, None; Yan Qi, None; Jie Li, None; Wen-Tao
Deng, None; Bo Chang, None; William W. Hauswirth, AGTC (I),
Bionic Sight (I), AGTC (C), Syncona (C), RetroSense (C); Ji-Jing
Pang, None
Support: NIH grants, EY021721, EY019943, FFB, MVRF, and
RPB, Inc.
Enhancement of AAV2-mediated retinal transduction in diabetic
rats
Nundehui Díaz-Lezama1, Zhijian Wu2, Mayda Ramírez1, Bibiana
Moreno-Carranza1, Gonzalo Martinez de la Escalera1, Peter Colosi2,
Carmen Clapp1. 1Instituto de Neurobiología, Universidad Nacional
Autónoma de México (UNAM), Querétaro, Mexico; 2Neurobiology,
Neurodegeneration and Repair Laboratory, National Eye Institute,
National Institutes of Health, Bethesda, MD.
Purpose: Ocular gene therapy based on the adeno-associated virus
(AAV) vector-mediated delivery of antiangiogenic molecules offers
considerable promise for the treatment of diabetic retinopathy. AAV
type-2 (AAV2) uses cell surface heparan sulphate proteoglycans
(HSPG) as the primary receptors for cell entry. HSPGs are present on
retinal ganglion cells, and likely mediate vector transduction. Here,
we compared the transduction of retinal ganglion cells in diabetic and
normal rats following intravitreal delivery of AAV2 vectors, and
investigated whether such transduction correlates with the level of
HSPG expression in the respective retinas.
Methods: An AAV2 CMV EGFP reporter vector was delivered by
intravitreal injection (2.8e10 vg/eye) to adult non-diabetic rats
(controls) and diabetic rats treated two weeks prior with a single
injection of streptozotocin. One month after vector administration,
retinal flatmounts were examined for EGFP expression, by confocal
microscopy, and expression of the HSPGs syndecan, glypican, and
perlecan, by quantitative PCR.
Results: In normal rat retinas, transduction was limited to occasional
cells in the ganglion cell layer. In the diabetic rats, the transduction
was enhanced more than 4-fold in ganglion cell somas and processes.
The expression of mRNAs for syndecan and glypican was elevated 2and 1.5-fold in the diabetic rat retina, respectively, whereas that of
perlecan was not modified.
Conclusions: Retinal transduction by AAV2 vectors is enhanced in
diabetes and may be mediated by elevated expression of HSPGs on
ganglion cells. The diabetes-induced factors responsible for these
changes warrant further study. AAV2 vectors may be desirable for
gene therapeutics targeting in diabetic retinopathy.
Commercial Relationships: Nundehui Díaz-Lezama, None;
Zhijian Wu, None; Mayda Ramírez, None; Bibiana MorenoCarranza, None; Gonzalo Martinez de la Escalera, None; Peter
Colosi, None; Carmen Clapp, None
Support: CONACYT grant S0008-161594 (C.C.) and National Eye
Institute, NIH, USA (P.C.).
Ex vivo gene transfer of anti-angiogenic soluble Flt-1 to corneal
epithelial cells sheets suitable for ocular surface reconstruction
Jingbo Liu, Victor H. Guaiquil, Aihong Liu, Mark Rosenblatt.
Margaret M. Dyson Vision Research Institute, Weill Cornell Medical
College, New York, NY.
Purpose: To evaluate ex vivo gene transfer of anti-angiogenic
soluble Flt-1 (sFlt1) to corneal epithelial cell sheets transplanted in a
rabbit model of total limbal stem cell deficiency (TLSCD).
Methods: Mouse sFlt1 was cloned into pLenti6.3/V5-DEST vector
using the Gateway systems. The vector was cotransfected with viral
packaging plasmids into 293FT cells and the produced viruses were
harvested and titrated. Primary rabbit corneal epithelial cells (RCEC)
were transduced with the lentivirus and the sFlt1 mRNA expression
and protein levels were analyzed by qRT-PCR, immunofluorescence
staining and ELISA, respectively. Functional assays using aortic ring
were performed to evaluate the inhibitory effect of sFlt1 on
VEGF164 induced endothelial cell proliferation. RCEC transduced
with sFlt1 were grown as sheet on decellularized amniotic
membranes and transplanted on the rabbit corneas with TLSCD. The
biocompatibility of the cell sheet was monitored by slit lamp
biomicroscopy.
Results: Lentiviruses containing msFlt1 gene were successfully
produced. qRT-PCR analysis showed high level of expression of
msFlt1 mRNA in the transduced RCEC. The sFlt1 protein was
detected by immunofluorescence staining in the cytoplasm and up to
one week by ELISA in the culture medium. The aortic ring assays
showed that sFlt1 inhibits the VEGF164 induced proliferative effect
of aortic endothelial cells (p<0.01). Transduced corneal epithelial
cells sheets were well tolerated on the limbal stem cell depleted
rabbit corneas.
Conclusions: We have successfully created and transplanted cells
sheets expressing heterologous anti-angiogenic molecules.
Genetically modified cultivated epithelial stem cell transplants
provide a basis for ex vivo corneal gene therapy and could be used to
treat corneal disease.
Commercial Relationships: Jingbo Liu, None; Victor H.
Guaiquil, None; Aihong Liu, None; Mark Rosenblatt, None
Support: Research to Prevent Blindness Career Development
Award, NYSTEM, and Tri-Institutional Stem Cell Initiative
Comparison of Adeno-Associated Viral Vector Serotypes for
Gene Transfer To Corneal Endothelial Cells
Thomas A. Fuchsluger1, Christian Mueller2, Reza Dana3.
1
Department of Ophthalmology, Heinrich-Heine-University Hospital,
Dusseldorf, Germany; 2Pediatrics and Gene Therapy Center,
University of Massachusetts Medical School, Worcester, MA; 3Dept.
of Ophthalmology, Schepens Eye Research Institute, Boston, MA.
Purpose: Due to the anatomical location and its monolayer layout the
corneal endothelium (EC) is an ideal target for gene therapy. The aim
of this study was to determine the efficacy and efficiency of the nonpathogenic recombinant adeno-associated viral vectors (rAAV) for
gene transfer to EC. Therefore, different rAAV pseudotypes were
compared to assess the expression and kinetic patterns of a reporter
gene (GFP).
Methods: A comparison of GFP expression and kinetics after EC
transduction using a AAV 2/1, 2/2, 2/5, 2/8, 2/9, 2/10 was performed
on both murine EC (Balb/C) and human EC (corneas, cell line and
primary cells). In addition, the effects of different vector
concentrations (3x10^3/10^4, 10^5, 10^6, 10^7, 10^8IU/µl) were
investigated. Analyses were performed using laser scanning
microscopy and flow cytometry.
Results: We detected significant differences between human and
murine EC as well as primary and immortalized EC. Whereas in
murine corneas transduction of EC with AAV2/2, 2/5 and 2/9
resulted in considerable protein expression, AAV 2/5 did not show
considerable expression in human corneas. We also detected a slow
onset of GFP expression both in immortalized and primary EC,
leading to peak and stable expression around 2-3 weeks. However,
peak expression was reached earlier in primary EC and immortalized
murine EC compared to immortalized human EC. In addition, the
overall plateau of expression was highest in primary EC (~80%).
Conclusions: Recombinant AAV pseudotypes differ in their tropism
and transduction efficiency between murine and human corneas. In
general high levels of gene expression can be obtained with several
of these pseudotypes. Characteristic slow-onset kinetics of protein
expression have to be taken into account when applying AAVs in
translational applications, e.g. in corneal storage in eye banks.
Commercial Relationships: Thomas A. Fuchsluger, None;
Christian Mueller, None; Reza Dana, Allergan (C), Alcon (C),
Bausch & Lomb (C), Eleven Bio (I), GSK (F), Novabay (C),
Revision Optics (C), Novaliq (C), RIgel (F)
Support: Eye Bank of America Research Grant
Effect of intraviteal injection of AAV9.TGFβ2 on the expression
of anterior segment and intraocular pressure in rabbits
Guilin Zhan, Chris Long, Allan Shepard, Nasreen Jacobson, Richard
Ornberg, Yu Wang, Shutong Cao, Ganesh Prasanna. Ophthalmology
Res./Glaucoma Res., Novartis Institutes for Biomedical Res., Fort
Worth, TX.
Purpose: TGFβ2 levels are elevated in aqueous humor (AH) of
primary open angle glaucoma patients and have been associated with
ultrastructural changes to trabecular meshwork (TM) leading to
elevated intraocular pressure (IOP). This study is to explore viral
transduction and overexpression of TGFβ2 on IOP in New Zealand
rabbits.
Methods: Eight rabbits received unilateral intravitreal injection of
50µl of AAV9-mut446-sc-smCBA-hTGFβ2 (1.25E12 vg/mL) and
other four received AAV9-mut446-sc-smCBA-hGFP (6.4E12
vg/mL). The injections were repeated one week following the initial
injection. IOP was measured at day 0 and once or twice per week
using a pneumatonometer on day 6-62 post second-injection. Ocular
inflammation including flare, cells and fibrin in anterior chamber and
GFP expression were examined by slit lamp (SL) beginning one day
after injection. The expression of GFP in the anterior segment of
rabbits was assessed by immunohistochemistry (IHC). Total and
bioactive TGFβ2 in rabbit AH were detected by ELISA 62 days postinjection.
Results: GFP expression was detected in 75% of four AAV9.GFP
treated rabbits by SL beginning 14 days post-injection and 50% of
these injected eyes showed a positive expression of GFP at 49 and 62
days post-injection by IHC in anterior segment of eye. Compared
with uninjected (UI) eyes, total TGFβ2 in AH showed an increasing
trend (P=0.1) in AAV9.TGFβ2 (2314 ± 316 vs UI eye: 1664 ± 198
pg/mL), while was significantly different in AAV9.GFP treated
group (2412 ± 246 vs UI eye: 1972 ± 185 pg/mL; p=0.035).
Bioactive TGFβ2 levels in AAV9.TGF β2 eyes were 41 ± 9 pg/mL
(UI eye: 42 ± 9 pg/mL) while that in AAV9.GFP eyes was 16 ± 8
pg/mL (UI eye: 41 ± 21 pg/mL). No statistically significant
difference in total and bioactive TGFβ2 levels were found between
AAV9.TGFβ2 and AAV9.GFP injected groups. Inflammation could
be detected in 50% of eyes in both groups lasting for 2-3 weeks.
Compared with pre-injection baseline, IOP was not significantly
increased in either AAV9.TGFβ2 (BL: 20 ± 0.8 mmHg; Day 62: 23 ±
0.5 mmHg) or AAV9.GFP treated eyes (BL: 17 ± 0.5 mmHg; Day
62: 19 ± 1.5 mmHg).
Conclusions: Our results indicate that AAV9.GFP can be transduced
in the ocular anterior segment of rabbits. However, TGFβ2 levels in
AH did not result in elevated IOP in rabbits and could be due to
species differences and/or insufficient expression of TGFβ2 with
these vectors.
Commercial Relationships: Guilin Zhan, Novartis Institution for
Biomedical Research (E); Chris Long, None; Allan Shepard, None;
Nasreen Jacobson, Novartis Institute Biomedical Res (E); Richard
Ornberg, Novartis Institute for Biomedical Research (E); Yu Wang,
None; Shutong Cao, nibr (E); Ganesh Prasanna, Alcon Research
Ltd (E), Novartis Institute of Biomedical Research (E)
Changes in S and L/M cone opsin expression in the RPE65 dog
model following AAV mediated gene addition therapy
Knut Stieger1, Daniela Klein1, Alexandra Mendes-Madeira2,
Fabienne Rolling2, Silke Haverkamp3, Birgit Lorenz1. 1Department of
Ophthalmology, Justus-Liebig-University Giessen, Giessen,
Germany; 2Institut UMR 1089, Institut de Recherche Thérapeutique
1, Nantes, France; 3Max Planck Institute for Brain Research,
Frankfurt, Germany.
Purpose: The Swedish Briard dog containing a null mutation in the
RPE65 gene represents a naturally occurring animal model for early
onset severe retinal dystrophy (EOSRD) in humans. Adenoassociated virus (AAV) mediated gene therapy in these dogs resulted
in tremendous treatment benefits in terms of restoration of function in
both, rods and cones. However, treatment benefit in humans is less
pronounced. The aim of this study was to examine changes in cone
opsin expression in RPE65 -/- dogs following gene therapy in order
to further elucidate the treatment effect.
Methods: Retinae from 9 affected dogs (n=18) were used in this
study, four of them treated unilaterally, at the age of 7-12 months,
with an AAV vector carrying the human RPE65 gene. Dogs were
sacrificed at various ages, between 2 and 4.5 years. Antibodies
against S-opsin and L/M-opsin were used to study opsin
delocalisation on cryosections and to quantify cone density on
flatmount preparations.
Results: Following gene therapy, opsin delocalisation was reduced in
both L/M and S cones. The total number of opsin stained S cones
increased by up to 40% in treated areas, compared to non-treated
areas in the same eye. L/M cone opsin staining did not increase
following gene therapy.
Conclusions: The remarkable increase in S cone staining and
reduced delocalisation of S cone opsin indicates a strong treatment
effect on this photoreceptor subtype. In contrast, L/M cones seem to
benefit less efficiently from gene therapy, given only the reduction of
opsin delocalization. These results may have consequences for the
analysis of gene therapy results in humans, where central visual
acuity is L/M cone dependent.
Commercial Relationships: Knut Stieger, None; Daniela Klein,
None; Alexandra Mendes-Madeira, None; Fabienne Rolling,
None; Silke Haverkamp, None; Birgit Lorenz, Optos (F)
Support: University Medical Center Giessen and Marburg Grant
Natural History of the CNGB3/NRL Double Knock-Out Mouse
William W. Hauswirth, Seok-Hong Min, Sanford L. Boye, Daniel
Kasuga, Qing Ruan, Jingfen Sun, Mai Tram Phan, Shannon E. Boye,
Christine N. Kay. Dept. of Ophthalmology, University of Florida
College of Medicine, Gainesville, FL.
Purpose: Achromatopsia (ACHM) is a congenital cone
photoreceptor disorder with an incidence of 1:30,000. Cyclic
nucleotide gated channel beta 3 (CNGB3) accounts for approximately
50% of this autosomal recessive disease. CNGB3 knock-out (KO)
mice have been successfully used as an animal model for gene
replacement therapy. However, the mouse retina lacks a cone-
dominant macula and is comprised of less than 3% cones, compared
to cone rich human macula, making clinical translation difficult for
any cone-related retinal degeneration. To address this problem of rodbias in the mouse, we generated a cone exclusive mouse model
lacking CNGB3 by crossing CNGB3 KO mice with mice lacking
Neural retina leucine zipper (NRL KO) and characterized the natural
history out to four months postnatal.
Methods: CNGB3/NRL double KO (DKO) mice were generated by
crossing NRL KO mice with CNGB3 KO lines. We followed the
natural history of CNGB3/NRL DKO mice with photopic
electroretinography (ERG) at 0.5, 1, 2, 3 and 4 months of age,
respectively. Responses were elicited at four increasing light
intensities (1.25 cds/m2, 5cds/m2, 10cds/m2 and 25cds/m2). For
comparison, ERG measurements were also recorded from agematched CNGB3 KO and NRL KO mice (data collection ongoing).
Following ERG, one animal from each age group was sacrificed and
their retinas collected for histological analysis.
Results: Photopic recordings at the highest flash intensity revealed
that cone amplitudes of CNGB3 KO mice were ~50 µV at 16 days
and remained low throughout the following 3 months. In contrast,
ERG amplitudes of CNGB3/NRL DKO mice increased from ~50 µV
at 16 days to 150 µV at 2 months and remained stable thereafter.
There was a significant difference in the CNGB3 and CNGB3/NRL
model at 1, 2, and 3 months (p<0.001). We are currently processing
retinas from both CNGB3 KO and CNGB3/NRL DKO.
Conclusions: ERG data with CNGB3 KO mice confirmed previous
findings that CNGB3 KO mice do not completely lose cone-mediated
ERG function. Residual cone-mediated ERG was further increased
when CNGB3 mice were crossed with NRL-/- mice, reaching to 160
µV at 3 month post-natal age. The CNGB3/NRL DKO mouse model
may provide insight into the disease progression of ACHM in the
cone rich human fovea and perhaps serve as a model for evaluating
gene replacement based therapies.
Average photopic b-wave amplitudes of CNGB3 and CNGB3/NRL
mice at 0.5, 1, 2 and 3 months of age (means±SE).
Commercial Relationships: William W. Hauswirth, AGTC (I),
Bionic Sight (I), AGTC (C), Syncona (C), RetroSense (C); SeokHong Min, None; Sanford L. Boye, PCT/US2012/062478 (P);
Daniel Kasuga, None; Qing Ruan, None; Jingfen Sun, None; Mai
Tram Phan, None; Shannon E. Boye, None; Christine N. Kay,
None
Support: Foundation Fighting Blindness, Career Development
Award (PI: Kay) (August 1, 2001 - July 31, 2006). Foundation
Fighting Blindness grant C-GT-071-0536-UFL04 (PI: Hauswirth)
7/01/11-6/30/16. Unrestricted grant to department of ophthalmology
from Research to Prevent Blindness.
Subretinal Injection of AAV2-CMV-hEPO to Diabetic Retina is
Protective and Safe
Hua Xu1, 3, Jingfa Zhang1, 2, Limei Zhang2, Limin Gu1, 3, Lixia Lu1, 2,
Weiye Li1, 4, Guo-Tong Xu1, 2. 1Department of Ophthalmology of
Shanghai Tenth People’s Hospital, and Tongji Eye Institute, Tongji
University School of Medicine (TUSM), Shanghai, China;
2
Department of Regenerative Medicine and Stem Cell Research
Center, Tongji University School of Medicine (TUSM), Shanghai,
China; 3Tongji University School of Life Sciences and Technology,
Shanghai, China; 4Department of Ophthalmology, Drexel University
College of Medicine, Philadelphia, PA.
Purpose: Erythropoietin (EPO), a hormone known to protect both
blood-retinal barrier (BRB) and neurons in early diabetic rat retina,
was reported to be beneficial for patients with chronic diabetic
macular edema who were unresponsive to current therapies. To
maintain a long-term effect and to avoid side effects due to repeated
intraocular injections, the efficacy and safety of adeno-associated
virus, serotype 2 (AAV2) mediated human EPO expression (AAV2CMV-hEPO) were studied on experimental diabetic rats after
subretinal administration.
Methods: Male Sprague-Dawley (SD) rats, Dark Agouti (DA) rats
and C57BL/6 mice were used in this study. Hematocrit (Hct) was
measured to prove the erythropoietic function of hEPO after
intramuscular injection of the virus. The hEPO concentration was
measured by ELISA. Diabetes was induced by intraperitoneal
injection of streptozotocin (STZ, 60 mg/kg BW). Electroretinography
(ERG) was applied to evaluate the retinal function in different
intervals after the virus administration. Two weeks after diabetes
onset, the subretinal injection of AAV2-CMV-hEPO or shame buffer
(PBS) was performed. Four weeks later, Evans blue permeation
(EBP) was determined to test BRB breakdown. Retinal cell apoptosis
was measured by TUNEL. Retinal thickness and cell counts were
examined under a light microscope. Neovascularization in retina and
choroids were evaluated by both fluorescein angiography (FA) and
immunostaining for flatmount.
Results: The serum hEPO was elevated 2 weeks after intramuscular
injection of AAV2-CMV-hEPO, and Hct began to rise after 4 weeks.
In the subretinal injection group, the expression of hEPO in aqueous
humor, vitreous and retina follows both a dose- and time- dependent
manner. In AAV2-CMV-hEPO treated diabetic group, BRB
maintained, and retinal cell apoptosis was significantly reduced.
Long-term expression of AAV2-CMV-hEPO did not induce
neovascularization. The ERG results also demonstrated that the
retinal function was not affected for at least one year after subretinal
injection of AAV2-CMV-hEPO.
Conclusions: The present data demonstrate that the gene therapy
with AAV2-CMV-hEPO delivered into subretinal space showed the
long-term protection of BRB and retinal neurons in experimental
diabetic rats. This therapy can safely maintain normal
electrophysiologic function and be void of neovascularization
formation in retina.
Commercial Relationships: Hua Xu, None; Jingfa Zhang, None;
Limei Zhang, None; Limin Gu, None; Lixia Lu, None; Weiye Li,
None; Guo-Tong Xu, None
AAV-mediated gene therapy restores cone function in the
Cnga3/Nrl double knockout mouse
Ji-Jing Pang1, Ye Tao1, 2, Sanford L. Boye1, Jie Li1, Wen-Tao Deng1,
Xi-Qin Ding3, Stylianos Michalakis4, Martin Biel4, Shannon E. Boye1,
William W. Hauswirth1. 1Ophthalmology, University of Florida,
Gainesville, FL; 2Fourth Military Medical University, Xi'an, China;
3
University of Oklahoma Health Sciences Center, Oklahoma City,
OK; 4University of Munich, Munich, Germany.
Purpose: Deletion of neural retina leucine zipper in mice (Nrl−/−)
results in the complete absence of rods and an all-cone retina. Nrl−/−
mice lack a scotopic response and have an enhanced photopic ERG
amplitude. Mutations in the gene encoding the alpha-subunit of the
cone cyclic nucleotide-gated (CNGA3) channel cause cone function
loss in mammals and are associated with achromatopsia 2 in humans.
Cnga3/Nrl double knockout (Cnga3/Nrl DKO) mice have been bred
in part to mimic the cone-rich central retina in human achromatopsia
2 with CNGA3 mutations. We therefore tested whether AAVmediated Cnga3 gene therapy targeting cones can restore cone
system function/structure in this model.
Methods: At postnatal day 14, one μl of AAV5-IRBP/GNAT2humanCnga3 vector (1013 particles/ml) was injected subretinally into
one eye of 20 Cnga3/Nrl DKO mice. The other eye was uninjected as
control. Dark- and light-adapted ERGs were recorded from 6 weeks
to 6 months after injection. Six months after treatment visual acuity
and contrast sensitivity were measured optokinetically. Treated and
control eyes were harvested for immunohistochemical studies.
Results: In treated eyes, restored light-adapted ERG waveforms were
recorded 6 weeks after injection and remained stable for at least 6
months. Cone mediated ERG amplitudes were similar as those of
Nrl−/− mice 6 months after treatment; light-adapted ERGs were
unrecordable from untreated eyes. Dark-adapted ERGs were absent
in both eyes. Behavioral tests showed that improved visual acuity and
contrast sensitivity were found in treated eyes, compared to untreated
eyes under a light environment. In untreated eyes, cone opsin staining
was greatly attenuated with remaining cone opsins mis-localized to
the inner retinal layers. In contrast, both M- and S-opsins were
readily observed in the outer segments of treated eyes although these
outer segments were much shorter than normal C57 BL/6J.
Conclusions: AAV mediated gene replacement therapy restores cone
system function and halts cone degeneration in the Cnga3/Nrl DKO
mouse, a cone dominant model. These results suggest that this model
will be useful in testing approaches for rescuing cone function in the
cone-rich macula/fovea of human achromatopsia.
Commercial Relationships: Ji-Jing Pang, None; Ye Tao, None;
Sanford L. Boye, PCT/US2012/062478 (P); Jie Li, None; Wen-Tao
Deng, None; Xi-Qin Ding, None; Stylianos Michalakis, None;
Martin Biel, None; Shannon E. Boye, None; William W.
Hauswirth, AGTC (I), Bionic Sight (I), AGTC (C), Syncona (C),
RetroSense (C)
Support: NIH grants, EY021721, FFB, MVRF, and RPB, Inc.
Gene Delivery of ATP6 by A Mitochondrial Targeting Sequence
Modification of AAV Capsid VP2 Rescues Cells with Mutated
T8993G MtDNA Responsible for Neuropathy Ataxia and
Retinitis Pigmentosa
Huijun Yuan1, Hong Yu1, John Guy1, 2. 1University of Miami Miler
School of Medicine, Miami, FL; 2Bascom Palmer Eye Institute,
Miami, FL.
Purpose: Two mitochondria diseases, neuropathy, ataxia, retinitis
pigmentosa (NARP) and maternally inherited Leigh's syndrome
(MILS) are caused by a T8993G mutation in mtDNA encoding
subunit 6 of ATP synthase that is complex V. Our goal is to rescue
the consequences of this mutation that result in blindness and death.
Methods: The human ATP6 gene was packaged into AAV serotype
2 or AAV serotype 9 to which the cytochrome oxidase subunit 8
(COX8) leader sequence was inserted into the VP2 capsid.
Expression of ATP6 was under the control of the human
mitochondrial heavy strand promoter (HSP). NARP cybrid cells with
100% mutated T8993G mitochondrial DNA were infected at different
MOI with self-complementary (sc) AAV9 or AAV2 containing
scAAV-HSP-ATP6 with FLAG. After 48 hrs, high glucose media
was exchanged for glucose free galactose media for 5 days that
forced the cells to rely on oxidative phosphorylation to produce ATP.
Cell survival was assessed by the MTT assay. ATP synthesis was
measured using a luciferin-luciferase assay. Expression of mRNA
and protein were respectively assessed by qRT-PCR,
immunofluorescence and immunoblotting.
Results: After infection of rAAV with scAAV-HSP-ATP6, cell
survival increased in a dose-dependent matte with an absorbance
0.305, 0.372, and 0.4055 for MOI of 0, 500, and 5,000 respectively.
Relative to no infection, differences were statistically significant with
P value of 0.007 and 0.0009.Relative to uninfected NARP cells, the
rate of ATP synthesis increased 41% (p< 0.01). Compared to
uninfected, infected cybrids ATP6 RNA transcripts were elevated 9
fold and 34 fold with AAV2 and AAV9 infection (p<0.05). Flag
tagged ATP6 surrounded DAPI stained nuclei and exhibited the
punctate perinuclear distribution of mitochondrial expression with
colocalization to MitoTracker Green. Western blotting showed that
FLAG tagged ATP6 was detected at day 8. Blue native PAGE
revealed wild-type ATP6 integrated into complex V.
Conclusions: MTS modified AAV2 or AAV9 restores ATP synthesis
and prevents cell death. Without an animal model it is unclear
whether intraocular injection (AAV2) may save the visual loss of
adults with NARP or with systemic injection (AAV9) the lives of
children with MILS, half of whom die by age 3.
Commercial Relationships: Huijun Yuan, None; Hong Yu, None;
John Guy, None
Support: R24EY018600 and R01 EY017141
Effective delivery of small interfering RNA to mouse cornea by
eye drops
Kaili Wu, Zhongting Li, Fang Duan, Jingyu Liao, Qiang Huang.
Zhongshan Ophthalmic Center, Sun Yat-Sen Univ, Guangzhou,
China.
Purpose: The transfection of siRNA in vivo is essential for gene
therapy. However, the successful delivery of siRNA in ocular surface
is still very difficult to achieve. In the current study, we sought to
investigate the efficacy of deliver small interference RNA (siRNA) to
the mouse cornea epithelium by eye drops that contain siRNA and
different cationic complexing agents.
Methods: The mouse (BALB/c) corneal epithelial layer was
mechanical damaged in 2 mm diameter in the center. After 12 hours,
eye drops, including 20μmol/l cy3 labeled siRNA (cy3-siRNA) with
different transfection reagents (Lipofectamine 2000, EntransterTM-in
vivo, PEI, in vivo-jetPEI and PEO-PPO-PEO polymeric micelles),
were applied for various times (4 times a day for two days and 8
times a day for one or two days). The transfection efficiency was
examined by fluorescence microscopy of the corneal flat mount and
frozen section. In addition, the transfection of the infected-cell
polypeptide 4-targeting small interfering RNA (ICP4-siRNA) in
mouse herpes simplex keratitis (HSK) was examined using the above
method. The antiviral effects were evaluated by in vivo obsevation,
microscopy examination and reverse transcription polymerase chain
reaction (RT-PCR).
Results: Compared with the other transfection reagents, 0.75mg/ml
PEI with cy3-siRNA complex that was instilled 8 times daily for two
days revealed the highest fluorescence in cornea epithelial cells
(p<0.01). In mouse HSK, application of ICP4-siRNA+PEI eye drops
reduced the damage of cornea epithelia, leading to recovery of cornea
ulcer, and decreased the viral VP16 expression in cornea tissue (P <
0.05). Meanwhile, we also found that cornea became cloudy in the
eye instilled with PEI.
Conclusions: PEI can effectively deliver the siRNA into the cornea
epithelium by eye drops. ICP4-siRNA transfection can inhibit HSV-1
replication in vivo
Commercial Relationships: Kaili Wu, None; Zhongting Li, None;
Fang Duan, None; Jingyu Liao, None; Qiang Huang, None
Support: NNSFC81070765
Targeted Suppression of Human Adenovirus DNA-polymerase
Gene by RNA Interference
Natalia Nikitenko1, Anastasia Klimova1, Pavel Spirin1, Peter Groitl2,
Thomas Speiseder2, Vladimir Prassolov1. 1Engelhardt Institute of
Molecular Biology Russian Academy of Sciences, Moscow, Russian
Federation; 2Heinrich Pette Institute - Leibniz Institute for
Experimental Virology, Hamburg, Germany.
Purpose: Epidemic keratoconjunctivitis is a hyperacute and highly
contagious infection of the eye caused by human adenovirus types
within species D, in particular, serotypes D8, D19, and D37.
Currently there is no causally directed treatment available that is
effective against epidemic keratoconjunctivitis. The E2B region of
the adenovirus genome encodes for DNA polymerase (pol) that is
required for adenoviral DNA replication. We assume that blocking of
pol mRNA translation will stop the adenoviral infection. The purpose
of our study was to develop a model system for studying DNApolymerase inhibitors and elaborate an approach for significant
reduction of pol expression by RNA interference (RNAi).
Methods: To express the gene pol of human adenovirus D36 (polD36) in the model cell lines a set of recombinant lentiviral vectors
was constructed, containing pol-D36 and tdTomato (tandem tomato
fluorescent protein) cDNAs separated by an IRES sequence. The
transgenic cell lines expressing pol-D36 and the fluorescent marker
gene were obtained from human lung carcinoma cell lines A549,
H1299 transduced with the corresponding recombinant lentiviruses.
We designed 3 types of small interfering RNAs (siRNA)
complementary to the pol mRNA sequences of serotypes D8, D19,
D36, and D37. In addition, based on the sequence of the siRNA with
the highest level of gene inhibition, a small hairpin RNA (shRNA)
encoding vector was designed.
Results: Inhibition of pol-D36 expression was observed within 48 h
post transfection of 3 siRNAs types by flow cytometry and qPCR.
The fluorescence signal of tdTomato decreased 2.5, 2.0 and 1.5 fold
respectively corresponding to control cells. The amount of pol-D36
mRNA decreased 5.0, 2.5 and 1.7 fold respectively. The progress of
the shRNA biological activity was monitored 14 days post
transduction of transgenic cells A549, H1299 by qPCR and Western
blot. The amount of pol-D36 mRNA decreased 4.5 and 3.5 fold
respectively, compared to the control cells transduced with shRNA
expressing lentiviral vectors targeting nonspecific sequences. The
gene suppression was confirmed by Western blot and reduced steady
state protein levels.
Conclusions: We proposed a powerful model system to test viral key
factor, i.e. DNA-polymerase, as putative target to develop a highly
efficient antiviral therapy based on the inhibitory effects of siRNAs
and showed significantly reduced expression levels of the
corresponding polymerase.
Commercial Relationships: Natalia Nikitenko, None; Anastasia
Klimova, None; Pavel Spirin, None; Peter Groitl, None; Thomas
Speiseder, None; Vladimir Prassolov, None
Evaluation of Influenza Associated Virus (IAV)-based
replication-incompetent for Ocular Delivery of microRNAS
Coralia C. Luna1, Benjamin tenOever2, Guorong Li1, Sonja Schmid2,
Pratap Challa1, David L. Epstein1, Jianming Qiu1, Pedro Gonzalez1.
1
Ophthalmology, Duke University, Durham, NC; 2Microbiology,
Mount Sinai School of Medicine, New York, NY.
Purpose: Safe and efficient delivery to cells of interest in the eye
remains a significant challenge to the therapeutic potential of
microRNAs (miRNAs) for the treatment of glaucoma. IAV-based
replication-incompetent vectors are a promising new class of vectors
that avoids the toxicity associated with saturation of the endogenous
miRNA biogenesis system. Our objective was to evaluate the
potential of these vectors for in vivo delivery of miRNAs to ocular
tissues relevant to glaucoma.
Methods: To generate IAV-based replication-incompetent vectors
expressing miR-124 or GFP, fibroblasts were transfected with
plasmids encoding the RNA dependent RNA polymerase components
(PA, PB1, PB2 and NP), hemaggluttinin (HA) and the eight viral
RNA segments. The supernatant from these cells was transferred to a
Madin-Darby Canine Kidney cell line that expresses the HA protein
of IAV. Vectors were then purified through 30% sucrose. IAV-based
vectors expressing miR-124 were applied to primary human
trabecular meshwork (TM) cell cultures at a 1:1 ratio and miRNA
expression was evaluated by Q-PCR, in situ hybridization and
Northern blot (NB). To evaluate the ability of these vectors to infect
cells from different ocular tissues, mixed rat retina cell cultures were
treated with 10E7 MOI/mL expressing GFP, and mice eyes were
inoculated intravitreally with 10E6 MOI/mL of the same vector. GFP
expression was evaluated by fluorescent microscopy.
Results: IAV-based vectors at a 1:1 ratio of vector to TM cells
demonstrated robust delivery of miR-124 as measured by Q-PCR,
RNA in situ hybridization, and NB. Similarly, this vector generated
high levels of expression of the reporter gene GFP in retinal ganglion
cells in culture (fig.1a). Intravitreal injection of IAV-based vectors
expressing GFP in mice eyes resulted in high levels of GFP
expression in TM, iris (fig.1b); retinal ganglion cell layer (RGL),
inner nuclear layer (INL), and outer nuclear layer (ONL) in the retina
(fig.1c); as well as in the lamina cribosa (LC) and meningeal cells
(MC) of the optic nerve (fig.1d).
Conclusions: IAV-based vectors are effective for in vivo delivery of
miRNAs to multiple cell types relevant to the pathophysiology of
glaucoma. Future studies with these vectors expressing specific
miRNAs relevant to glaucoma pathogenesis will help to evaluate the
safety and efficacy of these vectors as potential therapeutic agents in
glaucoma.
Commercial Relationships: Coralia C. Luna, None; Benjamin
tenOever, None; Guorong Li, None; Sonja Schmid, None; Pratap
Challa, None; David L. Epstein, None; Jianming Qiu, None;
Pedro Gonzalez, None
Support: NEI EY01894, NEI EY016228, NIH P30 EY-005722, and
Research to Prevent Blindness
LONG-TERM PROTECTION BY COMPLEMENT
REGULATORY PROTEIN CRRY IN A MOUSE MODEL OF
LIPOFUSCIN-MEDIATED RETINAL DEGENERATION
Shanta Sarfare, Anita Kim, Zhichun Jiang, Marcia B. Lloyd, Shannan
R. Eddington, Samer Habib, Dean Bok, Steven Nusinowitz, Gabriel
H. Travis, Roxana A. Radu. Ophthalmology, Jules Stein Eye Institute,
Los Angeles, CA.
Purpose: Mutations in the human ABCA4 gene cause recessive
Stargardt disease (STGD1) and predispose to the development of
age-related macular degeneration (AMD). Mice with a knockout
mutation in the abca4 gene (abca4-/- mice) accumulate A2Elipofuscin in the retinal pigment epithelium (RPE) and exhibit slow
photoreceptor degeneration. Recently, we described complement
activation and increased oxidative stress in abca4-/- mice due to A2E
accumulation. In this study we tested the hypothesis that increasing
expression of complement regulatory proteins (CRP’s) in the RPE
would protect these cells from complement attack and hence protect
photoreceptors from degenerating.
Methods: We generated a recombinant adeno-associated virus
containing mouse CRRY (rAAV8-CRRY), which we injected
subretinally into one-month-old albino abca4-/- and BALB/c mice.
Negative control mice received AAV8-null or rAAV8-GFP viruses.
Fundus imaging was performed at different times following the
injections. Expression of CRRY was determined by qRT-PCR,
immunoblotting and immunocytochemistry. Spectral-domain opticalcoherence tomography (sdOCT) and electroretinography (ERG) were
obtained as previously described. Visual retinoids and lipofuscin
pigments were quantified by high-performance liquid
chromatography.
Results: At 12-months post-injection, mice that received rAAVCRRY showed 2 to 7-fold increased expression of CRRY by qRTPCR and quantitative immunoblotting. Fundus images of these mice
were normal, suggesting no detrimental effects of the injections. The
mean scotopic a-wave and b-wave ERG’s show no significant
difference between the CRRY and Null-treated eyes. We observed
two-fold decreased A500-lipofuscin and a three-fold decreased A2E
levels in CRRY-treated abca4-/- mice at 11-months post-injection.
sdOCT showed a limited maintenance in overall retinal thickness in
CRRY-treated compared to Null virus-treated mice, which correlated
with enhanced preservation of photoreceptors and increase in 11-cisretinal chromophore levels.
Conclusions: Aberrant complement activation is strongly linked to
the development of AMD. For the first time, we show that inhibiting
complement-convertase activity by gene therapy with a virus that
increased CRRY expression may be useful in treating age-related
retinal degenerations.
Commercial Relationships: Shanta Sarfare, None; Anita Kim,
None; Zhichun Jiang, None; Marcia B. Lloyd, None; Shannan R.
Eddington, None; Samer Habib, None; Dean Bok, None; Steven
Nusinowitz, None; Gabriel H. Travis, None; Roxana A. Radu,
None
Support: NIH/NEI 2P30 EY00331-45
Testing siRNAs for use in a rapidly progressing model of
autosomal dominant retinitis pigmentosa
Brian P. Rossmiller, Haoyu Mao, Alfred S. Lewin. Department of
Molecular Genetics and Microbiology, University of Florida,
Gainesville, FL.
Purpose: We are interested in characterizing and treating the rapidly
degenerating autosomal dominant retinitis pigmentosa (ADRP)
mouse model, T17M RHO. Photoreceptors in this transgenic strain
are highly susceptible to light damage. I hypothesize that the
knockdown of transducin in rods, will reduce rod degeneration and
preserve cone function. It is, therefore, the purpose of this study to
determine if siRNA mediated knockdown of transducin can inhibit
the phototransduction cascade in rod cells preventing apoptosis
resulting from exposure to bright light.
Methods: gene (GNAT1). Two small hairpin RNAs (shRNAs) were
designed to target homologous regions between mouse and dog
GNAT1. Transfections were done in HEK293 cells (n=6) using
CMV-GNAT1 and H1-shRNA GNAT1 given at 1:0, 1:2, 1:4, and 1:6
ratios. Using a plasmid with a control miRNA, total amount of DNA
transfected at each ratio was kept constant. The amount of transducin
produced was observed at 24 and 72 hours post transfection using
western blot with a mouse transducin specific antibody. The percent
knockdown was then calculated using tubulin as the loading control
and normalized against the control ratio which received only CMVGNAT1 and CBA-GFP plasmids. Following the in vitro knock down
assay, each shRNA was inserted into an a plasmid containing GFP
under the expression of a mouse opsin promoter and packaged into
adeno associated virus serotype 8 with a capsid modification, AAV8
(Y733F).
Results: siRNA GNAT1(a) showed the greatest knockdown with
76%, 71.4% and 69.1% of the GNAT mRNA remaining at the 1:2,
1:4 and 1:6 ratios, respectively, after 72 hours.
Conclusions: We demonstrated siRNA GNAT1(a) to be a viable
siRNA for testing in T17M RHO transgenic mice. Both shRNAs (a)
and (b) targeting GNAT1 are being packaged into AAV8 (Y733F) for
in vivo analysis in mice. Once we verify reduction of transducin-α in
these mice, we will expose them to clinical light levels and measure
retinal preservation. Since the siRNAs also target dog transducin,
they will be tested in the T4R dog model of ADRP which is also
unusually sensitive to light damage.
Commercial Relationships: Brian P. Rossmiller, None; Haoyu
Mao, None; Alfred S. Lewin, University of Florida (P)
Support: T32-EY07132 Training grant, Foundation Fighting
Blindness grant TA-GT-0611-0526-UFL, NIH grant R24EY022012,
and the Vision Core grant EY021721
Gene Therapy for CEP290-associated LCA in Patient-Derived
Induced Pluripotent Stem Cells
Erin R. Burnight1, Emily E. Kaalberg1, Mari E. Eyestone1, Jeremy
Hoffman1, Christine M. Haas1, Robert F. Mullins1, Edwin M. Stone1,
2
, Budd A. Tucker1. 1Ophthalmology and Visual Sciences, University
of Iowa, Iowa City, IA; 2HHMI Investigator, Iowa City, IA.
Purpose: Mutations in the CEP290 gene are major contributors to
Leber Congenital Amaurosis (LCA), the most severe form of
inherited retinal degenerative disease for which there is currently no
cure. Autosomal recessive CEP290-associated LCA is a good
candidate for gene-replacement therapy and reprogrammed somatic
cell technologies provide researchers with the ability to study human
disease and therapeutic gene correction in vitro. The purpose of this
study was to develop lentiviral vectors containing human CEP290
and to use these vectors to deliver wild type CEP290 to induced
pluripotent stem cell (iPSC)-derived photoreceptor precursor cells
(PRPCs) from mice and humans with CEP290-associated LCA.
Methods: Fibroblast-derived iPSCs were generated from the retinal
degenerative mouse model CEP290rd16 and patients with molecularly
confirmed CEP290-associated LCA. Mouse and human iPSCs were
differentiated into photoreceptor precursor cells using our previously
developed step-wise differentiation protocol. An HIV-1 lentiviral
vector containing human CEP290 under the control of the CMV
promoter was packaged and used to transduce PRPCs from rd16 mice
and LCA patients. Vector-derived mRNA and protein were detected
via RT-PCR and western blot, respectively.
Results: To evaluate CEP290 gene transfer we initially transduced a
surrogate cell line, JK1, derived from murine testicular stromal cells
that do not express the human CEP290 transcript. RT-PCR analysis
using human-specific primers demonstrated a dose-dependent
increase in CEP290 expression post-transduction. However, cell
viability was significantly reduced in cultures transduced with the
highest dose. Using results from these pilot experiments, we
delivered a lower dose of CEP290 vector to iPSC-derived murine and
human PRPCs. RT-PCR analysis and western blotting indicated
vector-derived expression in these cells.
Conclusions: We show successful gene transfer of human CEP290
to iPSC-derived photoreceptor precursor cells. Cell viability assays
suggest that overexpression of CEP290 is cytotoxic. Thus, it will be
important to carefully titrate vector dosage when developing gene
replacement strategies for this disease. This work will contribute to
our overall goal of vision restoration in patients with LCA through
gene and patient-specific photoreceptor cell replacement.
Commercial Relationships: Erin R. Burnight, None; Emily E.
Kaalberg, None; Mari E. Eyestone, None; Jeremy Hoffman, None;
Christine M. Haas, None; Robert F. Mullins, Alcon Research Ltd
(F); Edwin M. Stone, None; Budd A. Tucker, None
Support: NIH EY022834; Grousbeck Family Foundation
AAV8 mediated gene therapy for corneal cystinosis
Duy H. Nguyen1, Hongjun Du1, Matthew Bedell1, Seanna Grob1, Jing
Luo1, John L. Quach1, Peter Shaw1, Stephanie Cherqui2, Kang
Zhang1. 1Ophthalmology, University of California San Diego, La
Jolla, CA; 2Molecular and Experimental Medicine, Scripps Research
Institute, La Jolla, CA.
Purpose: Cystinosis is a lysosomal storage disorder that results from
mutations in the gene CTNS, which encodes cystinosin, the
lysosomal membrane transporter for cystine. Corneal accumulations
of intracellular cystine crystals lead to debilitating ocular
complications. The aim of this study is to deliver an adeno-associated
virus (AAV8)-Ctns vector into the cornea of Ctns-/- mice to study the
efficacy of AAV8-mediated CTNS gene transfer.
Methods: 1.5 to 4.5-month old Ctns-/- mice from the Cherqui
Laboratory at The Scripps Research Institute and 2-month old
wildtype C57BL6 mice were used. AAV8 vectors cloned with either
green fluorescent protein (GFP) or CTNS were delivered by single
intrastromal corneal injections of 1.5 µl (vector titer= 2x1011) with a
Hamilton micro-syringe. Successful injection was gauged by
observing that ≥70% of the cornea became whitened and edematous
immediately following the injection. The non-injected contralateral
eyes were used as controls. At 11 to 15 months post-injection, mice
were sacrificed and eyes collected. CTNS mRNA expression was
measured using real-time polymerase chain reaction (q-PCR). Optical
coherence tomography and corneal histology were used to evaluate
changes in corneal crystal content.
Results: AAV8 vector had a high transduction efficiency that
resulted in high levels and uniform expression of reporter gene GFP
in the cornea, starting from day 3 and lasted up to 11 months at the
point of eye collection. At 12 to 15 months post-injection, there was a
significant difference in mRNA expression of CTNS as measured by
qPCR in AAV8-Ctns eyes (n=4) when compared to that in the
untreated eyes (n=4) (2225±1102.02 fold increase vs. 138.5±241.33
fold increase respectively, P-value = 0.0051).
Conclusions: AAV8 is a reliable vector with high transduction
efficiency for gene transfer in Ctns-/- mice, producing extended
expression of cystinosin for 12 to 15 months and likely longer after a
single injection. Current study is aiming at characterizing corneal
histology and biochemistry in treated and untreated mice.
Commercial Relationships: Duy H. Nguyen, None; Hongjun Du,
None; Matthew Bedell, None; Seanna Grob, None; Jing Luo,
None; John L. Quach, None; Peter Shaw, None; Stephanie
Cherqui, None; Kang Zhang, Acucela (F), Genentech (F),
Thrombogenics (F)
Support: Cystinosis Research Foundation Grant, NEI/NIH Grants,
Research to Prevent Blindness, BWF Clinical Scientist Award in
Translational Research
Tropisms of AAV for Subretinal Delivery to the Neonatal Mouse
Retina and Its Application for In Vivo Rescue of the Crx
Knockout Retina
Satoshi Watanabe1, 2, Rikako Sanuki1, Shinji Ueno3, Takahisa
Furukawa1. 1Laboratory for Molecular and Developmental Biology,
Institute for Protein Research, Osaka University and JST, CREST,
Suita, Japan; 2Department of Molecular Genetics, Kyoto University
Graduate School of Medicine, Kyoto, Japan; 3Department of
Ophthalmology, Nagoya University Graduate of School of Medicine,
Nagoya, Japan.
Purpose: Adeno-associated virus (AAV) is well established as a
vehicle for in vivo gene transfer into the mammalian retina. This virus
is promising not only for gene therapy of retinal diseases, but also for
in vivo functional analysis of retinal genes. Previous reports have
shown that AAV can infect various cell types in the developing
mouse retina. However, AAV tropism in the developing retina has
not yet been examined in detail. Thus, we examined the tropisms of
seven AAV serotypes (AAV2/1, 2/2, 2/5, 2/8, 2/9, 2/10, and 2/11) for
subretinal delivery into the P0 mouse retina.
Methods: We subretinally delivered seven AAV serotypes of AAVCAG-mCherry into P0 mouse retinas, and quantitatively evaluated
the tropisms of each serotype by its infecting degree in retinal cells.
After subretinal injection of AAV into postnatal day 0 (P0) mouse
retinas, various retinal cell types were efficiently transduced with
different AAVs. To confirm the usefulness of AAV-mediated gene
transfer into the P0 mouse retina, we performed AAV-mediated
rescue of the Cone-rod homeobox gene knockout (Crx KO) mouse,
which exhibits an outer segment formation defect, flat
electroretinogram (ERG) responses, and photoreceptor degeneration.
We subretinally injected an AAV expressing Crx under the control of
the Crx 2kb promoter into the neonatal Crx KO retina.
Results: Photoreceptor cells were efficiently transduced with
AAV2/5. Retinal cells, except for bipolar and Müller glial cells, were
efficiently transduced with AAV2/9. Horizontal and/or ganglion cells
were efficiently transduced with AAV2/1, AAV2/2, AAV2/8,
AAV2/9 and AAV2/10. We showed that AAV-mediated Crx
expression significantly decreased the abnormalities of the Crx KO
retina by histological and physiological analyses.
Conclusions: In the current study, we report suitable AAV tropisms
for delivery into the developing mouse retina. Using AAV2/5 in
photoreceptor cells, we demonstrated the possibility of gene
replacement for the developmental disorder and subsequent
degeneration of retinal photoreceptors caused by the absence of Crx.
Commercial Relationships: Satoshi Watanabe, None; Rikako
Sanuki, None; Shinji Ueno, None; Takahisa Furukawa, None
Accessibility of Retinal Cells to Different AAV Serotypes After
Intravitreal Injection in Old Versus New World Primates
Maureen Neitz1, Anita E. Hendrickson1, Michael Lukason2, Jay
Neitz1, Daniel E. Possin1, Gelareh Abedi3, Jing Huang1, Abraham
Scaria2, Sam Wadsworth2. 1Ophthalmology, University of
Washington, Seattle, WA; 2Gene Therapy, Genzyme-Sanofi,
Framingham, MA; 3Ophthalmology & Optometry, University of
Texas, San Antonio, TX.
Purpose: Adeno-associated virus (AAV) is a promising vehicle for
ocular gene therapy but little is known about what cells types are
accessible to virus injected intravitreally (IVT) in primate. Here we
evaluated this issue for AAV serotypes 2 and 5.
Methods: 10 animals each received a single IVT injection containing
an admixture of two AAVs: one carrying the gene for GFP the other
carrying the gene for a chimeric soluble VEGF receptor, sFLT02. All
vectors had a ubiquitous chicken beta actin promoter. For each
animal, the same serotype, 2 or 5, was injected in both eyes but AAV
injected into the right eye had a wild type (wt) capsid, AAV injected
into the left eye had a capsid modification (see Table 1). Macaques
received 1x1010 vector genomes (vg) of each vector in 50 µL.
Marmosets received 8x109 vg of each vector in 10 µL. GFP
expression was evaluated histologically, sFLT02 transgene product
was measured using a commercially available ELISA.
Results: In macaques, sFLT02 expression was similar for wild type
AAV2 and modified AAV2 capsids, and expression with AAV2
vectors was higher than for AAV5; however, AAV5 with modified
capsids gave stronger expression than wt AAV5 capsids. ELISA
results for sFLT02 in marmosets were inconsistent across animals for
AAV5 vectors but modified AAV2 gave higher sFLT02 levels than
wt AAV2. All but one macaque eye injected with AAV2 gave strong
GFP labeling of ganglion cells around the foveal pit with fewer
foveal amacrines, horizontals, bipolars and cones. Scattered labeled
ganglion cells and many Mueller cells were present in the mid to far
periphery. Pars plana epithelial cells were strongly labeled. The
distribution of GFP was similar for eyes treated with wt versus capsid
modified AAV5. In marmosets, GFP labeling for eyes treated with
AAV2 was similar to macaque but labeling at the fovea was much
less pronounced. AAV5 virus gave weak GFP labeling in marmosets,
with little or no foveal labeling.
Conclusions: IVT administration of AAV vectors transduces a
variety of cell types within the eye including ganglion, amacrine,
bipolar, horizontal cells, and to some extent, photoreceptors. Tropism
differs among serotypes with AAV2 transducing more cell types than
AAV5. While the capsid modifications examined in these
experiments did not appear to alter tropism, overall transgene
expression level was affected.
Commercial Relationships: Maureen Neitz, Genzyme (F), Alcon
(F), Alcon (P); Anita E. Hendrickson, None; Michael Lukason,
Genzyme (E); Jay Neitz, Alcon (F), Alcon (P); Daniel E. Possin,
None; Gelareh Abedi, None; Jing Huang, None; Abraham Scaria,
Genzyme Corporation (F), Genzyme Corporation (P); Sam
Wadsworth, Genzyme (E)
Support: NIH Grants EY09303, EY016861, EY01730, RPB
Transfer of TACSTD2 Gene into Corneal Epithelial Cells of
Gelatinous Drop-Like Corneal Dystrophy and Its Functional
Expression
Toru Matsunaga1, 2, Koji Kitazawa3, Kenta Yamasaki4, Takao Sato1, 2,
Yasuo Watanabe1, 2, Toshinari Funaki1, Akira Matsuda1, Nobuyuki
Ebihara1, Satoshi Kawasaki3, Akira Murakami1. 1Ophthalmology,
Juntendo University School of Medicine, Tokyo, Japan; 2Reserch and
Development, SEED Co., Ltd., Saitama, Japan; 3Ophthalmology,
Kyoto Prefecutural University School of Medicine, Kyoto, Japan;
4
Biomedical Engineering, Faculty of Life and Medical Sciences,
Doshisha University, Kyoto, Japan.
Purpose: Gelatinous drop-like corneal dystrophy (GDLD) is a
refractory disease that deposits amyloid into the subepithelium
through increased intercellular permeability in the corneal epithelium.
As a novel treatment for the disease, we have been investigating the
transfer of TACSTD2 gene that is responsible for the disease, into a
contact lens (CL) to use as the device. For the purpose of this study,
we have used corneal epithelial cells that were obtained from a
GDLD patient and investigated the transfer and functional expression
of TACSTD2 gene.
Methods: Corneal epithelial cells that had been obtained from a
GDLD patient were immortalized with the lentiviral transduction of
the simian virus 40 (SV40) large T antigen and human telomerase
reverse transcription (hTERT) genes. CL that had phosphate groups
as its side chains was synthesized as the gene transfer device to allow
it to form a complex, mediated by calcium ions, with plasmid DNA
obtained through implanting pLenti6.3_V5-TOPO vector with the
TACSTD2 gene (TACSTD2-V5). Using this DNA-CL, gene transfer
was performed into the immortalized corneal epithelial cell line from
the GDLD patient (imHCE-T_GDLD) cells, followed by observation
for expression of TACSTD2 as well as claudin (CLDN).
Results: The imHCE-T_GDLD cells processed with DNA-CL
observed fluorescence in the cellular cytoplasm in the
immunostaining with an anti-TACSTD2 antibody. Localization of
CLDN1 and CLDN7 were also observed. Western blotting
recognized protein expression as well.
Conclusions: Transfer into imHCE-T_GDLD cells and functional
expression of TACSTD gene recognized through the use of DNA-CL
has indicated that gene transfer can be achieved by non-invasive
treatments.
Commercial Relationships: Toru Matsunaga, SEED Co.,Ltd. (E),
JP5132958 (P); Koji Kitazawa, None; Kenta Yamasaki, None;
Takao Sato, SEED Co., Ltd. (E); Yasuo Watanabe, None;
Toshinari Funaki, None; Akira Matsuda, None; Nobuyuki
Ebihara, None; Satoshi Kawasaki, None; Akira Murakami,
SEED(Japan) JP4855782 (P), SEED(Japan) JP5132958 (P)
Support: Health and Labour Sciences Research Grant on Intractable
Diseases H23-Nanchi-Ippan-084
Clinical Trial: UMIN000007228
Transduction Profile of Anterior Chamber-injected Capsid
Mutated Self-complementary Adeno-Associated Virus Type 2 in
Rodents
Barbara Bogner1, Sanford L. Boye2, Seok-Hong Min2, James J.
Peterson2, Qing Ruan2, Vince Chiodo2, Renee C. Ryals2, Herbert A.
Reitsamer1, William W. Hauswirth2, Shannon E. Boye2.
1
Ophthalmology/Optometry, Paracelsus Medical University,
Salzburg, Austria; 2Ophthalmology, University of Florida,
Gainesville, FL.
Purpose: The surface properties of the adeno-associated virus (AAV)
capsid are essential for cellular tropism and efficiency of transgene
expression. The transduction properties of AAV2-based capsid
mutants have not been investigated in the anterior chamber (AC). In
this study we investigate transduction profiles of AAV2 mutants
containing self-complementary AAV (scAAV) genomes coding for
green fluorescent protein (GFP) by localizing GFP in AC-injected
mice and rats. scAAV2 mutants contain single or combinations of
triple and septuple tyrosine (Y) to phenylalanine (F) mutations in the
highly conserved surface-exposed capsid tyrosine residues.
Methods: AC injections were performed in C57BL/6 mice (1 µl) and
Wistar rats (2 µl). After general and topical anesthesia, the peripheral
cornea was punctured (33G needle) anterior of the iridocorneal angle.
scAAVs expressing GFP under the constitutive small CMV-chicken
β-actin (smCBA) promoter and vehicle control (BSS+0.014% Tween
20) were infused slowly using a micropump. 4 weeks post-injection,
animals were sacrificed and GFP-expression was evaluated by
immunohistochemistry and confocal microscopy. In Table 1
experimental groups and titers for scAAVs are listed.
Results: scAAV-injected eyes showed intense GFP-signals in the
corneal endothelium, the ciliary non-pigmented epithelium (NPE),
the chamber angle and the iris. Depending on the scAAV mutant, the
expression pattern ranged from mosaic-like to homogenous. Both,
vehicle control and control without injection revealed no
immunopositivity. Table 1 summarizes the localization and
semiquantitative evaluation of the GFP-signal in injected eyes.
Conclusions: scAAVs bypass rate-limiting second-strand DNA
synthesis to obtain the transcriptionally active AAV genome. This
results in earlier onset of transgene expression and facilitates the
transduction of difficult-to-transduce cells (e. g. trabecular
meshwork). This study shows that mutagenesis of surface-exposed
tyrosine residues affects the tropism and transduction efficiency of
scAAV2 in the AC. While single and triple mutants show enhanced
transduction efficiency, the septuple mutant reveals no improvement
compared to scAAV2 WT. These results are of interest for the
development of transgene-based models to investigate pathological
processes in the AC and for the development of gene-based therapies
targeted to the trabecular meshwork.
Commercial Relationships: Barbara Bogner, None; Sanford L.
Boye, PCT/US2012/062478 (P); Seok-Hong Min, None; James J.
Peterson, None; Qing Ruan, None; Vince Chiodo, None; Renee C.
Ryals, None; Herbert A. Reitsamer, None; William W.
RetroSense (C); Shannon E. Boye, None
Support: Austrian Academy of Sciences, PMU-FFF, Fuchs
Endowment, Adele Rabensteiner Endowment, Lotte Schwarz
Endowment
Glycosidic Enzymes Enhance Retinal Transduction Following
Intravitreal Delivery of AAV2
Jasmina Cehajic-Kapetanovic1, 2, Annette Allen2, Paul N. Bishop1,
Robert Lucas2. 1Institute of Human Development, University of
Manchester, Manchester, United Kingdom; 2Faculty of Life Sciences,
University of Manchester, Manchester, United Kingdom.
Purpose: At present only limited retinal transduction can be achieved
following intravitreal delivery of unmutated AAV vectors. We
hypothesized that the inner limiting membrane and extracellular
matrix proteoglycans act as a barrier to AAV vector entry into and
movement across the retina. In this study we investigated the effects
of enzymatic digestion of these barriers on the depth of vector
penetration into the retina.
Methods: The green fluorescent protein (GFP)-expressing AAV2
vector was co-injected intravitreally into adult mice with various
glycosidic enzymes and their combinations. The efficacy of the virus
transduction was evaluated by quantitative analysis of GFP positive
cells in histological cross-sections, using fluorescent microscopy. We
also analyzed safety of these treatments and retinal function using
electroretinography.
Results: Glycosidic enzymes namely Chondroitin ABC lyase,
Heparinase III and Hyaluronan lyase, both singly and in
combinations, led to a significant improvement in retinal transduction
following intravitreal delivery. Electroretinograms survived at much
higher doses of enzymes than were needed for optimal retinal
transduction.
Conclusions: AAV2-mediated retinal transduction is improved by
co-injection of glycosidic enzymes into the vitreous. This approach
may allow intravitreal injection to become the preferred route for
delivering gene therapy to the retina in both preclinical and clinical
settings.
Commercial Relationships: Jasmina Cehajic-Kapetanovic, None;
Annette Allen, None; Paul N. Bishop, None; Robert Lucas, None
Support: Medical Research Council
Antisense oligonucleotide-based therapy for CEP290-associated
LCA
Alejandro Garanto, Frans P. Cremers, Rob W. Collin. Human
Genetics, Radboud University Nijmegen Medical Centre, Nijmegen,
Netherlands.
Purpose: The most frequent genetic cause of Leber congenital
amaurosis (LCA) is an intronic change in CEP290, that results in the
insertion of a cryptic exon (exon X) into the CEP290 mRNA.
Recently, we have shown promising results of antisense
oligonucleotide (AON)-based therapy for this disorder, since
transient transfection of AONs to cultured lymphoblastoïd cells of
these patients almost completely rescued the aberrant splicing of
CEP290 mRNA that is caused by the intronic mutation. The aim of
this work is to further develop this therapeutic strategy, by employing
other cell types, generate viral vectors that express AONs and by
characterizing a CEP290 knock-in mouse model that carries part of
the human CEP290 gene including the intronic mutation, specifically
designed for this purpose.
Methods: Skin biopsies from LCA patients homozygously carrying
the intronic CEP290 mutation allowed the generation of fibroblast
cell lines. These fibroblasts were transfected with either naked AONs
or AONs cloned into viral constructs. CEP290 transcripts were
analyzed by RT-PCR, and CEP290 protein levels via Western blot
analysis. In addition, cilium length was assessed by immunostaining.
Two knock-in models have been generated, and initially
characterized at transcriptional and morphological levels, by RT-PCR
and immunohistochemistry. Animal work was performed according
to the ARVO statement for the use of animals in ophthalmic and
vision research.
Results: Our data have proved the efficacy of the naked AONs in
fibroblasts, restoring the correct splicing of the transcript. We are
currently working on the validation of these results, by measuring
CEP290 protein levels and cilium length in AON-treated fibroblasts.
We are also testing viral constructs to further application in cell lines
and animal models. The initial characterization of the humanized
models at transcriptional level, has shown an unexpected splicing
pattern in the humanized mice. This new splicing event results in the
insertion of a second cryptic exon (Y) in part of the CEP290
transcripts. Ongoing work will determine the presence/absence of the
human aberrant exon (X) and exon Y, as well as whether the splicing
events are tissue or mouse-strain-specific.
Conclusions: Together, this work will be instrumental for the
development of AON-based therapy for CEP290-associated LCA.
Commercial Relationships: Alejandro Garanto, None; Frans P.
Cremers, None; Rob W. Collin, Radboud University Medical
Centre (P)
Support: FFB USA Individual Investigator Grant Award: TA-GT0912-0582-RAD
Mitochondrial Gene Therapy for G11778A LHON: Safety of
Human ND4 in Nonhuman Primates and Expression in Ex Vivo
Human Eyes
Rajeshwari D. Koilkonda1, David T. Tse1, William W. Hauswirth2,
Vince Chiodo2, Sanford L. Boye2, Martha Neuringer3, Tim Stout3,
John Guy1. 1Ophthalmology, Bascom Palmer Eye Institute,
University of Miami-Miller School of Medicine, Miami, FL;
2
Department of Ophthalmology, University of Florida, College of
Medicine, Gainesville, FL; 3Oregon Health and Science University,
Beaverton, OR.
Purpose: To demonstrate the safety and expression of allotopic
human ND4 to be used for the Leber’s hereditary optic neuropathy
gene therapy trial.
Methods: We modified ND4 in the nuclear genetic code for import
into mitochondria. The protein was targeted into the organelle by
ATPc (P1) sequence. The gene was packaged into selfcomplementary AAV2 packaged with triple Y-F capsid mutant
(Y444F+Y500F+Y730F) VP3 then injected into rodent, nonhuman
primate and ex vivo human eyes. Expression and integration into
complex I of rodents and ex vivo human eyes was tested by
immunohistochemistry and blue native (BN) PAGE. Three rhesus
macaques (1m, 2f, ages 3-7 yrs) were injected intravitreally (iv) with
scAAV2(Y444,500,703F)-smCBA-P1ND4. The vector titer was
1.23x1011 vg/ml, and a volume of 200 μL was injected for a total
dose of 2.46x1010 vg. We next tested for transgene expression in two
normal human eyes that were surgically removed for control of
periocular spread of cancers. After incising the cornea, eyes were
immersed immediately in tissue culture media. One eye received iv
injection of 120 μL of scAAV-ND4FLAG (1.0 x 1012 vg/ml) the
other 20 µL (4.0 x 1012 vg/ml).
Results: Rodents.2-D BN PAGE with the anti-FLAG antibody
reacted against infected retinal mitochondrial extracts from rats
1month post injection (PI) of scAAV-ND4FLAG showed a distinct
band of FLAG, absent in uninfected control retinal mitochondria.
Antibodies against NDUFA9, NDUFS3, NDUFS4, NDUFB8 or
NDUFA6 detected these nuclear encoded complex I subunits. Human
Eyes. 3days PI, ND4FLAG co-localized with mitochondrial porin.
Quantitative analysis revealed, injection of more than 10 11 vg, 84% of
RGCs expressed ND4FLAG. Nonhuman Primates. Clinical
examinations and retinal fundus photography done at baseline, 5, 8
and 15days PI revealed no clinical signs of inflammation, such as
haze or presence of inflammatory cells in the anterior chamber or
vitreous and looked similar to the uninjected left eye. One animal
developed mild vitritis ~2m after AAV injection. OCT images of the
retina and optic nerve head showed no structural changes from
baseline in all 3 animals, but cells were present in the vitreous of one
animal.
Conclusions: Expression of allotopic ND4 in the ex vivo human eye,
safety of the test article in rodent and primate visual systems and the
severe irreversible loss of RGCs in LHON support clinical testing.
Commercial Relationships: Rajeshwari D. Koilkonda, None;
David T. Tse, None; William W. Hauswirth, AGTC (I), Bionic
Sight (I), AGTC (C), Syncona (C), RetroSense (C); Vince Chiodo,
None; Sanford L. Boye, PCT/US2012/062478 (P); Martha
Neuringer, Pfizer (F), Applied Genetic Technologies Corporation
(F); Tim Stout, Clayton Foundation (P), Oxford Biomedica (C),
AGTC (F), Peregrine Pharmaceuticals Inc (C), Stem Cells Inc (C);
John Guy, None
Support: R24EY018600
Dual Adeno-associated virus vectors for large cDNA gene
replacement therapy
Frank M. Dyka, Sanford L. Boye, Vince Chiodo, Shannon E. Boye,
William W. Hauswirth. Ophthalmology, University of Florida,
Gainesville, FL.
Purpose: Adeno-associated virus (AAV) is emerging as the preferred
vector to mediate gene replacement therapy due to its low
immunogenicity, ability to efficiently transduce a wide variety of cell
types and demonstrated efficacy in clinical trials. One of its major
drawbacks is a relatively small packaging capacity, ~4.8 kb. Retinal
disorders such as recessive Stargardt disease and Usher syndrome 1B
are caused by mutations in large genes (ABCA4, MYO7A
respectively) that exceed this limit. Alternatively the use of
Lentivirus or Adenovirus is problematic due to low transduction
efficiency of mature photoreceptors or immunogenicity. After viral
entry into the host cell nucleus, dual AAV vectors (cDNA split
between two defined vectors) or fragmented (packaging of randomly
truncated genomes) AAV vectors recombine to reconstitute full
length cDNA. Capitalizing on this we sought to develop defined dual
AAV vector systems capable of generating full length cDNAs of
large genes in vitro and in vivo.
Methods: The coding sequences of MYO7A and ABCA4 were cloned
in AAV vector pairs, where one vector contains a CMV/chicken
βactin (CBA) promoter and the 5’ portion of the cDNA sequence and
a second vector contains the 3’ portion followed by a polyA signal.
One vector pair shares a central cDNA sequence of 1.3 kb (simple
overlap vector). Another pair utilizes splice donor and acceptor sites
(trans-splicing vector) and the third pair utilizes these splice sites in
combination with an efficiently recombinogenic overlapping
sequence (hybrid vector). All vectors were packaged in AAV2 for
transduction of HEK293 cells (10,000 MOI) or retina after subretinal
injection into null ABCA4 or MYO7A mice. Cells were collected at
3 days post infection and retina at 6 weeks post infection for analysis.
Results: Co-infection of HEK293 cells with simple overlap, transsplicing or hybrid AAV vector pairs expressed full length protein in
vitro, all with equal or higher efficiency than that of the fragmented
vector. Of the three dual vector systems, the hybrid system was the
most efficient at expressing full length protein. In-vivo analysis is ongoing.
Conclusions: Large cDNAs exceeding the size limitations of
conventional AAV can be expressed with high efficiency and
specificity using optimally designed dual AAV vector systems. Our
results suggest that a dual AAV vector system may be a viable option
for the treatment of large gene disorders of the retina.
Commercial Relationships: Frank M. Dyka, 61/560,437 (P),
PCT/US2012/062478 (P); Sanford L. Boye, PCT/US2012/062478
(P); Vince Chiodo, None; Shannon E. Boye, None; William W.
RetroSense (C)
Support: NIH grant EY021721, Foundation Fighting Blindness,
Macula Vision Research Foundation, and Research to Prevent
Blindness
A comparison of the effects of subretinal injection of scAAV2CMV-GFP on retinal structure, visual function, and GFP
expression by two injectors
Hui Li, Stephen H. Poor, Chad E. Bigelow, Vivian Choi, Shawn
Hanks, Joanna Vrouvlianis, Michael Maker, Steve Louie, Sha-Mei
Liao, Bruce D. Jaffee. Novartis Institutes for Biomedical Research,
Cambridge, MA.
Purpose: To compare the effects of subretinal (SR) injection of
adeno-associated virus (AAV) expressing GFP and saline by two
scientists, using optical coherence tomography (OCT) and
electroretinography (ERG) and RPE/Choroid tissue flat mount for
GFP expression.
Methods: 1μl of scAAV2-CMV-GFP (5.67x108 DNase resistant
particle/μl) or saline, both mixed with sodium fluorescein was
injected subretinally into the eyes of C57Bl/6 mice (16 eyes
/treatment group/operator). Injection quality was evaluated at the
time of the procedure using a 3-level scoring system (1= no
hemorrhage minimal intravitreal backflow, score 2 = minor
hemorrhage and mild backflow, score 3 = moderate hemorrhage,
significant backflow). Fundus images were acquired immediately
after the SR injection. OCT was performed 1 week post-injection and
ERG recordings were performed 2 weeks post-injection. RPEchoroidal flatmounts were made from ocular tissues collected 3
weeks post-injection. Levels of GFP expression were quantified
using Axiovision software to delineate fluorescent area on the flat
mounts.
Results: 59 of 64 injections were scored a “1”, 3 injections were
scored “2”, and 2 injections were scored “3”. Eyes with an injection
score of 1 or 2 were used for OCT and ERG quantification. Retinal
structure 1 week post subretinal injection was overall similar by OCT
examination with a slight increase in incidence of injection site
trauma and reduced media clarity for one operator. Photoreceptor
function measured with a single flash intensity ERG showed an
average drop of approximately 18% vs. uninjected controls (no
statistically significant) for both operators and injection materials. No
statistically significant difference in function was detected between
groups injected by each operator or between materials injected (AAV
vector vs. saline). GFP expression per percentage of flatmount area
for score 1 was 74%, score 2 was 60%, and score 3 was 25%. No
significant difference in GFP coverage was observed between
operators.
Conclusions: Subretinal injections disrupted retinal structure to a
similar degree for both injection materials (vector and saline). ERGs
from injected eyes were reduced in amplitude vs. uninjected controls,
but the reduction was independent of individual operator or injection
material. Poor quality injections predicted a smaller area of GFP
expression.
Commercial Relationships: Hui Li, None; Stephen H. Poor,
Novartis Institue of Biomedical Research (E); Chad E. Bigelow,
Novartis (E); Vivian Choi, Novartis Institute for BioMedical
Research (E); Shawn Hanks, Novartis Institutes for Biomedical
Research (E); Joanna Vrouvlianis, None; Michael Maker, Novartis
Institutes for Biomedical Research (E); Steve Louie, Novartis (E);
Sha-Mei Liao, Novartis (E); Bruce D. Jaffee, Novartis (E)
Glaucoma-GT, a novel gene therapy treatment for primary openangle glaucoma
Scott Ellis, Katie M. Binley, Vicky Scripps, Sharifah Iqball, Stuart M.
Naylor, Kyriacos Mitrophanous. Oxford BioMedica (UK) Limited,
Oxford, United Kingdom.
Purpose: Glaucoma is the second leading cause of blindness
worldwide, affecting around 70 million people, over 2.5 million
people in the USA alone. It is characterized by irreversible
degeneration of the optic nerve, usually associated with an elevated
intraocular pressure (IOP). Prostenoid analogues such as Latanoprost
are a first-line treatment for glaucoma due to their high level of
efficacy and low risk of side-effects, but fail to halt disease
progression in many patients due to non-adherence, which can be
>50%. Surgery is often required, which is expensive and only
partially effective. There is therefore a real medical need for a novel
drug that overcomes this issue of poor compliance.
Initial proof-of-concept for this approach has been demonstrated
(Barraza et al, 2010), and we are now moving this into a translational
gene therapy platform for clinical evaluation. Glaucoma-GT is a gene
therapy product aimed at lowering IOP via the prostenoid pathway: a
single transcorneal administration leads to the expression of human
cyclooxygenase-2 (COX-2) and prostaglandin F (FP) receptor in the
front of the eye. COX-2 is a rate-limiting enzyme in the biosynthesis
of prostaglandins, and both COX-2 and the FP receptor are downregulated in glaucomatous eyes. Expression of both genes increases
both prostaglandin 2α (PGF2α) biosynthesis and signaling, increasing
aqueous outflow and reducing longterm IOP.
Methods: Gene transfer following transcorneal administration of
EIAV-GFP vector was characterised in animal models. Various
vector genome configurations were assessed for the production of
PGF2α and activation of the FP receptor in in vitro assays. Further
preclinical studies are currently ongoing and data from these will be
presented.
Results: Transcorneal injection of EIAV vector led to the
transduction of cells of the trabecular meshwork and corneal
endothelium in vivo. Different configurations of expression cassette
gave a broad range of PGF2α and FP activation, with CMV-COX-2IRES-FP having the highest activity.
Conclusions: We have demonstrated significant gene transfer
following transcorneal administration of EIAV-GFP vector in animal
models, and have optimised the therapeutic expression cassette to
produce high levels of prostaglandin 2α and enhanced FP activation
in in vitro studies. These vector configurations will be assessed for
IOP lowering in a relevant animal model.
Commercial Relationships: Scott Ellis, Oxford BioMedica (E);
Katie M. Binley, Oxford BioMedica (UK) Ltd (E); Vicky Scripps,
Oxford BioMedica (E); Sharifah Iqball, Oxford BioMedica (UK)
Ltd (E); Stuart M. Naylor, Oxford BioMedica (UK) Ltd (E);
Kyriacos Mitrophanous, Oxford BioMedica (UK) Ltd (E)
Functional Biomarkers For Successful Molecular Therapy Of
The Outer Retina In Retinal Degenerations
Vithiyanjali Sothilingam1, Marina Garcia Garrido1, Christina Seide1,
Susanne Koch2, Stylianos Michalakis2, Martin Biel2, Naoyuki
Tanimoto1, Mathias W. Seeliger1. 1Division of Ocular Degeneration,
Ctr for Ophthal Inst for Ophthalmic Rsch, Tuebingen, Germany;
2
Department of Pharmacy-Centre for Drug Research, Center for
Integrated Protein Science Munich (CIPSM), Munich, Germany.
Purpose: Retinal degenerations are severe and frequently blinding
human diseases. Several current therapeutic approaches have
demonstrated long-lasting amelioration in respective disease models
and show promise for a successful translation to human patients.
Here, we focus on electroretinographic (ERG) features in the
experimental data in order to identify informative biomarkers for
upcoming clinical trials.
Methods: Core of this work is the electrophysiologic evaluation of
treated mouse models representing achromatopsia (Cnga3-/-) or
retinitis pigmentosa (Cngb1-/-). Mice of these lines received adenoassociated virus (AAV)-mediated gene replacement therapy designed
to express mouse CNGA3 or CNGB1 under control of respective
photoreceptor-specific promoters (mouse S opsin or rhodopsin).
Treatment success was monitored with different single flash and
flicker protocols of in vivo electrophysiology. The findings were
compared to immunohistochemistry, in vivo imaging, and behavioral
assays.
Results: In both disease models, the therapeutic effects were well
detectable with several of the explorative ERG protocols used in this
study. However, the signal-to-noise ratio varied between the
particular tests, depending on the nature of the underlying disease
(rod- or cone-specific origin). On this basis, options for biomarkers
were developed for potential use in human trials.
Conclusions: The ERG is a very effective method to assess outer
retinal functionality. We show here that specific ERG protocols are
well suited to follow therapeutic effects in retinal degenerations. Such
tests may thus serve as functional biomarkers for molecular therapy
in upcoming clinical trials.
Commercial Relationships: Vithiyanjali Sothilingam, None;
Marina Garcia Garrido, None; Christina Seide, None; Susanne
Koch, None; Stylianos Michalakis, None; Martin Biel, None;
Naoyuki Tanimoto, None; Mathias W. Seeliger, None
Support: DFG Se837/6-2, Se837/7-1 (KFO 134), Kerstan
Foundation/RD-CURE
In Vivo Quantification Of Photoreceptor Transduction Efficiency
Using Novel Modified AAV Capsids
Renee C. Ryals, Christine N. Kay, Sanford L. Boye, Seok-Hong Min,
Andrea E. Ayala, William W. Hauswirth, Shannon E. Boye.
Purpose: Because the majority of inherited retinal diseases are
caused by mutations in photoreceptor (PR)-specific genes, there is
great need to develop PR-targeted gene therapies. Development of a
less invasive surgical procedure is also warranted, particularly when
an underlying genetic defect results in fragile retina prone to further
damage upon surgically induced retinal detachment. Retinal
degeneration patients would benefit from vectors that possess an
enhanced ability to transduce photoreceptors following intravitreal
delivery. The purpose of our study was to develop an in vivo assay to
quantify the relative abilities of novel AAV vectors to transduce
photoreceptors following intravitreal delivery to mouse.
Methods: One month old heterozygote Rho-GFP mice received
trans-scleral, intravitreal injections (1.5 ul, 1E12vg/ml) of AAV2,
AAV5 or AAV8-based vectors (with or without tyrosine/threonine
capsid mutations). 4 weeks post injection, mCherry expression was
visualized by fundoscopy, retinas were dissociated and FACS
analysis was used to quantify the percentage of cells that were GFP
positive (rods), mCherry positive (any retinal cells transduced with
AAV) and both GFP and mCherry positive (rods transduced by
AAV). Percentage of mCherry positive photoreceptors= (% of cells
both GFP and mCherry positive cells/% of total photoreceptors).
Results: Fundoscopy and FACS showed that mCherry expression
varied with the serotype injected. Photoreceptor transduction was
enhanced following injection with tyrosine/threonine capsid mutants.
Retinas injected with AAV2 had 1.7% mCherry positive PRs, retinas
injected with AAV2 Y-F QM and AAV2 Y-F QM + T-V had 6.1%
and 21.8% mCherry positive PRs, respectively.
Conclusions: We have established a reliable in vivo assay for scoring
the relative photoreceptor transduction efficiencies of novel AAV
vectors following delivery to the vitreous. This assay will be used to
determine the optimal serotype(s) with which to treat models of
inherited retinal disease. Furthermore, it will support development of
AAV-based clinical vectors for the treatment of various forms of
photoreceptor-mediated inherited retinal disease following a
surgically less invasive intravitreal injection.
Commercial Relationships: Renee C. Ryals, None; Christine N.
Kay, None; Sanford L. Boye, PCT/US2012/062478 (P); Seok-Hong
Min, None; Andrea E. Ayala, None; William W. Hauswirth,
AGTC (I), Bionic Sight (I), AGTC (C), Syncona (C), RetroSense (C);
Shannon E. Boye, None
Support: FFB Indivdual Investigator Award, Reseach to Prevent
Blindness
The LentiVector® Gene Therapy Platform for Ocular Disease: a
clinical update
Kyriacos Mitrophanous, Scott Ellis, James Miskin, Katie M. Binley,
Michelle Kelleher, Cherry A. Lucas, Stuart M. Naylor. Oxford
BioMedia (UK) Ltd, Oxford, United Kingdom.
Purpose: Oxford BioMedica has developed ocular gene therapies
using its proprietary LentiVector® platform which is based on
recombinant Equine Infectious Anaemia Virus (EIAV). Currently we
have three ocular therapies in clinical development: RetinoStat®,
StarGen™ and UshStat®, for the treatment of age-related macular
degeneration, Stargardt macula dystrophy and Usher Syndrome 1B
respectively. Regulatory approval in the US and France has been
received for RetinoStat® and StarGen™, and UshStat® has been
approved in the US. Phase I (RetinoStat®) and Phase I/IIa (StarGen™
and UshStat®) clinical evaluations are currently underway.
Methods: The RetinoStat®, StarGen™ and UshStat® trials consist of
a dose-escalation phase followed by an expanded final cohort at the
highest safe and tolerated dose. Safety and signs of clinical benefit
will be assessed throughout these trials. Additionally, the secreted
nature of the transgenes in the RetinoStat® trial has meant that
transgene expression from aqueous tap samples can also be
quantified over time at each dose.
Results: The dose-escalation phase of the RetinoStat® trial has been
completed. For StarGen™ and UshStat® the dose-escalation phase is
in progress. Subretinal administration of all three products has been
well tolerated, causing no ocular inflammation or immune responses
in any patients. There have been no SAEs related to product. All
three products have been safe and well tolerated thus far.
Transduction of the retina following subretinal injection of
RetinoStat® rapidly produced high levels of both transgenes
detectable in the aqueous. These levels are substantial, show dose
dependence.
Conclusions: Ocular gene therapies based on the LentiVector®
platform continue to show good safety following subretinal delivery
into patients for three different ocular indications. The platform has
proved to be a highly effective delivery system for relatively large
genes into target retinal cells, resulting in stable and long-term
expression.
Commercial Relationships: Kyriacos Mitrophanous, Oxford
BioMedica (UK) Ltd (E); Scott Ellis, Oxford BioMedica (E); James
Miskin, Oxford BioMedica (UK) Ltd (E); Katie M. Binley, Oxford
BioMedica (UK) Ltd (E); Michelle Kelleher, Oxford BioMedica
(UK) Ltd (E); Cherry A. Lucas, Oxford BIoMedica Ltd (C); Stuart
M. Naylor, Oxford BioMedica (UK) Ltd (E)
Assessment of Intravitreal AAV-TEAD4 Isoforms in the OIR
Model
Matthew Hartzell, Andrew J. Stempel, Trevor J. McFarland, Binoy
Appukuttan, Tim Stout. Opthalmology, Oregon Health & Science
Univ, Portland, OR.
Purpose: Purpose: The mouse model of oxygen-induced retinopathy
(OIR) has been used extensively as a model of retinopathy of
prematurity to study retinal neovascularization (NV). We have shown
that the transcription factor TEAD4, and its isoforms, can influence
VEGF expression; a determining factor driving tuft formation in this
model. The purpose of this study was to observe the effects of
TEAD4 isoforms on retinal NV within this model.
Methods: Methods: Postnatal day 7 (P7) C57BL/6 mice were
injected intravitreally with AAV-DJ containing either the TEAD4
1305 or 447 isoforms (n=4), AAV-DJ containing GFP (n=4) or PBS
(n=4). The mice were then introduced to a 75% oxygen environment
for five days before recovering in room air. All mice were sacrificed
at P17 and eyes were enucleated. One eye from each mouse was
enucleated, fixed in 10% neutral buffered formalin and paraffin
embedded. Tissue was sectioned at 5uM and sequential slides were
stained for hematoxylin and eosin. Neovascular tuft nuclei were
manually counted (15 slides/eye). The retina from the contralateral
eye was dissected and used for western blot analysis using antiTEAD4 antibodies to confirm viral introduction of human isoforms.
Results: Results: Western blotting confirmed the presence of the
exogenous TEAD4 1305 and 447 isoforms. The AAV 447, 1305 and
GFP injected eyes averaged 3.2, 1.6 and 5.1 tuft nuclei per section
respectively. PBS injected eyes had an average of 11.75 tuft nuclei
per section.
Conclusions: Discussion: Successful ocular transduction of P7 OIR
mice was achieved with AAV-DJ viruses encoding the TEAD4 1305
and 447 isoforms. Neovascular tuft nuclei counts suggest no
stimulatory effect with the introduction of stimulatory TEAD4
isoforms in this model. The presence of elevated levels of TEAD4 in
this model may influence the expression of VEGF, VEGF receptor
and/or other angiogenic factors, perhaps leading to the decrease in
overall tuft formation in the treated groups.
Commercial Relationships: Matthew Hartzell, None; Andrew J.
Stempel, None; Trevor J. McFarland, None; Binoy Appukuttan,
None; Tim Stout, Clayton Foundation (P), Oxford Biomedica (C),
AGTC (F), Peregrine Pharmaceuticals Inc (C), Stem Cells Inc (C)
Support: NIH;NEI;RO1 EY019042
AAV-mediated Combination Therapy of Neurotrophic and AntiApoptotic Factors in a Mouse Model of Inherited Retinal
Degeneration
Cecile Fortuny1, Leah C. Byrne1, Deniz Dalkara2, Trevor S. Lee1,
Bilge Esin Ozturk1. 1HWNI, Flannery Lab, University of California,
Berkeley, Berkeley, CA; 2Institut de la Vision, Paris, France.
Purpose: Many inherited retinal degenerative diseases, such as
retinitis pigmentosa, result in blindness as a result of photoreceptor
cell death. Since numerous different genetic mutations are involved
in these disorders, it is advantageous to develop general therapies that
promote photoreceptor survival regardless of the underlying
mutation. In a mouse model, we investigated the therapeutic potential
of a combination cell survival therapy using intravitreal injections of
two serotypes of adeno-associated virus (AAV) to express a secreted
trophic factor, glial-derived neurotrophic factor (GDNF) in Müller
glia, and an anti-apoptotic factor, X-linked inhibitor of apoptosis
protein (XIAP), in photoreceptors.
Methods: C57BL/6 mice were co-injected intravitreally with
different concentrations of two engineered adeno-associated virus
(AAV) variants: ShH10, an AAV6 variant which selectively targets
Müller glia, and 7m8, an AAV2 variant capable of extensive
photoreceptor transduction from the vitreous. Fluorophores mCherry
and GFP were respectively expressed under the control of the CAG
promoter and the photoreceptor-specific rhodopsin promoter (Rho).
Fundus fluorescence imaging was performed before eyes were
enucleated four weeks post-injection.
Dark reared rd10 mice were injected intravitreally in one eye at p15
with (1) 7m8.Rho.XIAP, (2) ShH10.CAG.GDNF or (3) a mix of both
XIAP/GDNF. The contralateral control eyes were sham injected with
PBS. Electroretinograms (ERGs) were recorded to quantify rescue of
rod and cone-mediated responses at p45 and p55.
Results: Imaging of retinal sections showed that, at a variety of
concentration ratios, co-injection of 7m8 and ShH10 led to
simultaneous and strong expression of GFP in photoreceptors and
mCherry in Müller glia. Co-injection of vectors encoding XIAP and
GDNF led to a significant increase in amplitude of the ERG A-wave
and B-wave.
Conclusions: We showed that co-injected 7m8 and ShH10 remained
cell-specific and transduced their intended targeted cells without
significant competitive inhibition. The combination therapy codelivering XIAP and GDNF showed a significant effect in slowing
the progression of retinal degeneration in a rodent model of retinitis
pigmentosa and represents a promising gene therapy approach to treat
inherited diseases.
Commercial Relationships: Cecile Fortuny, None; Leah C. Byrne,
None; Deniz Dalkara, None; Trevor S. Lee, None; Bilge Esin
Ozturk, None
Support: The Foundation Fighting Blindness
333 Drug Delivery II
Tuesday, May 07, 2013 11:00 AM-12:45 PM
Deslorelin and transferrin mono- and dual- functionalized
nanomicelles for drug delivery to the anterior segment of the eye
Trivedi Ruchit1, Puneet Tyagi1, Sunil K. Vooturi1, Uday B. Kompella1,
2 1
. Pharmaceutical sciences, University of Colorado Anschutz
Medical Campus, Aurora, CO; 2Ophthalmology, University of
Colorado Anschutz Medical Campus, Aurora, CO.
Purpose: To determine whether encapsulation in novel micelles
enhances delivery of hydrophobic molecules to the anterior segment
of the eye after topical administration.
Methods: Deslorelin and transferrin were conjugated with PLGA
polymer using N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide.
Conjugation was confirmed using infrared spectroscopy (FTIR) and
MALDI-TOF mass spectrometry. Micelles were formed by the
addition of conjugates in dichloromethane to water, and characterized
for appearance, size, zeta potential, and CMC. Uptake of Nile red
(NR) and pazopanib (anti-angiogenic drug) loaded micelles following
topical administration was evaluated in an ex-vivo bovine eye model
at 5 and 60 minutes post administration using fluorescence
spectroscopy for NR and mass spectroscopy for pazopanib.
Results: FTIR and MALDI-TOF confirmed conjugate formation.
Sizes were 89.0, 74.3, and 59.4 nm, CMCs were 2.5, 100, and
2.5µg/ml, and zeta potentials were 1.0, 21.5, and 11.5 mV for
deslorelin-PLGA, transferrin-PLGA, and mixed micelles,
respectively. Pink colored, uniform appearance indicated NR loading
in micelles. Pazopanib loading was 57, 83, and 70 µg/mg micelles,
while NR loading was 5, 6, and 5 µg/mg micelles for deslorelinPLGA, transferrin-PLGA, and mixed micelles, respectively. NR
uptake in epithelium was 0.2, 16.1, 14.4, and 19.3%, at 5 minutes and
0.2, 20.8, 20.3, and 24.9 % at 60 minutes for plain NR, deslorelinPLGA, transferrin-PLGA, and mixed micelles, respectively. Addition
of excess deslorelin and transferrin reduced epithelium uptake to
0.31, 10.1, 9.49, and 11.77%, at 5 minutes and 0.25, 13.53, 14.61,
and 15.01% at 60 minutes for plain NR, deslorelin-PLGA,
transferrin-PLGA, and mixed micelles respectively. Pazopanib
uptake in epithelium was 0.4, 14.6, 11.9, and 15.8 % at 5 minutes and
1, 19, 14.9, and 18 % at 60 minutes for plain pazopanib, deslorelinPLGA, transferrin-PLGA, and mixed micelles, respectively. Stroma
showed ≤ 1% uptake for micelles and ≤ 0.03% for plain drug
suspension. Endothelium and aqueous humor uptake was negligible
for both NR and pazopanib. Micelles increased corneal epithelial
delivery by 80-120 fold for Nile red and by 15-39 fold for pazopanib.
Conclusions: Functionalized micelles are promising delivery systems
for the delivery of poorly soluble hydrophobic drugs to the anterior
segment of the eye.
Commercial Relationships: Trivedi Ruchit, None; Puneet Tyagi,
None; Sunil K. Vooturi, None; Uday B. Kompella, University of
Colorado Denver (P), PCAsso Diagnostics (C), NanoTrans
Technologies, Inc. (F), Univesity of Nebraska Medical Center (P)
The Influence of Formulation Factors on Transscleral
Iontophoretic Delivery of a Macromolecule in Vitro and in Vivo
Sarah A. Molokhia1, 2, Kongnara Papangkorn1, Charlotte Butler1,
Donald Mix1, John Higuchi1, Balbir Brar1, S Kevin Li3, William I.
Higuchi1, 4. 1Aciont Inc, Salt Lake City, UT; 2Ophthalmology, Moran
Eye Center, University of Utah, Salt Lake City, UT; 3College of
Pharmacy, University of Cincinnati, Cincinnati, OH; 4College of
Pharmacy, University of Utah, Salt Lake City, UT.
Purpose: To study the effects of ionic strength and pH on the
transscleral anodal iontophoresis (AI) delivery of Immunoglobulin G
(IgG) and to evaluate these effects on the amount and distribution of
IgG delivered into the rabbit eye.
Methods: IgG formulations (MW~150 kDa, a surrogate for
bevacizumab) of different ionic strengths and pH were investigated in
vitro (n=3 per group). In vitro AI experiments were conducted with
conjunctiva + sclera membranes of New Zealand white (NZW)
rabbits in a side-by-side diffusion cell with the IgG formulation in the
donor chamber and PBS in the receiver chamber. Silver and silver
chloride electrodes were used as the conductive elements for the
iontophoresis experiments. For the in vivo experiments, an
iontophoretic applicator (Visulex-I) was applied on the rabbit eye
under optimized formulation conditions. After 20 minutes of 4 mA
AI delivery, the rabbits were sacrificed and the eyes enucleated. The
eyes were dissected into cornea, anterior chamber, vitreous,
conjunctiva, sclera and choroid/retina and the tissues were analyzed
for IgG content by ELISA.
Results: The in vitro permeability coefficient (P) value determined
for conjunctiva+sclera membrane increased approximately 2.7 fold
when decreasing the formulation ionic strength by approximately 13
fold of a prior formulation. This corresponds to a 2.7 fold increase in
total amount delivered across the membrane. A decrease in the
formulation pH from 6.5 to 2.5 resulted in a 40 fold decrease of the P
value. An increase in the total amount of IgG delivered in vivo was
also observed at the lower ionic strength. The total amount of IgG
delivered in vivo into the eye and into the retina/choroid with our
optimized low ionic strength formulation was approximately 302.2 ±
30.0 μg and 5.2 ± 2.4 μg, respectively. The retina/choroid amount is
of the same order of magnitude of levels delivered to the
retina/choroid from an IVT injection of bevacizumab in rabbits
(literature reported Cmax value of 94 μg/g, which is approximately
15 μg).
Conclusions: Electroosmosis can be compromised by a low pH but
can be enhanced by utilizing a low formulation ionic strength. An
optimized Visulex-I applicator and formulation can be expected to
deliver to the eye a therapeutically relevant dose of an
antibody/macromolecule such as Avastin.
Commercial Relationships: Sarah A. Molokhia, Aciont Inc (C);
Kongnara Papangkorn, Aciont Inc (E); Charlotte Butler, Aciont
Inc (E); Donald Mix, Aciont, Inc (E); John Higuchi, Aciont Inc.
(E); Balbir Brar, Aciont Inc (C), Atheronova Inc (C), Imprimis
Pharma (C); S Kevin Li, Aciont Inc (C); William I. Higuchi, Aciont
Inc. (F)
Support: NEI, Grant no. 2R44EY014772-02A1
Versatile time-release technology reducing frequency of invasive
injections in treating retinal diseases
Chi-Chun Lai1, 3, Ling Yeung2, 3, Lan-Hsin Chuang2, 3, Yih-Shiou
Hwang1, 3, Chih-Chiang Tsai4, Po-Chun Chang4, Luke S.S. Guo4, 5,
Yun-Long Tseng4, Sheue-Fang Shih4, Keelung Hong4, 5.
1
Ophthalmology, Chang Gung Memorial Hospital - Kaohsiung,
Kwei-Shan, Taiwan; 2Ophthalmology, Chang Gung Memorial
Hospital, Keelung, Taiwan; 3Chang Gung University College of
Medicine, Kwei-Shan, Taiwan; 4Taiwan Liposome Company, Taipei,
Taiwan; 5TLC Biopharmaceutical, Inc., california, CA.
Purpose: To design a controlled release system capable of prolong
effective therapeutic agents against chronic eye diseases by
intravitreal injection.
Methods: In this study we report that a versatile drug delivery
platform, namely BioSeizer, prolong effective therapeutic agents
against chronic ocular diseases. This technology is directly applicable
to existing drug products through a modified dehydration-rehydration
vesicles (DRV) method. A small molecule drug dexamethasone
sodium phosphate (DSP) or a macromolecular protein drug
(bevacizumab) or both was formulated with this BioSeizer. To assess
the sustained release capability of BioSeizer, in vitro and in vivo
pharmacokinetic studies were done. The DSP or
bevacizumab/BioSeizer were injected separately into the vitreous
(IVI) of rabbits. The animals were sacrificed at each time point and
the eyes enucleated for the assessment of vitreous concentration of
drugs. In another experiment, to assess the effectiveness of this slow
release system, a rabbit model of blood retinal barrier (BRB)
breakdown was used to test the prolong effect of IVI lipid based drug
with DSP.
Results: The in vivo release of DSP or bevacizumab, can be
sustained in the vitreous chamber for more than four months by a
single intravitreal injection of a lipid-based formulation, with a
sufficient rate of drug release to maintain a vitreous concentration of
drugs equivalent to that of commercial formulation. No toxic effects
were found. In the efficacy study, free DSP did not have a significant
effect on the blockade of BRB breakdown beyond 9 days postadministration. In contrast, the DSP/BioSeizer continued to
significantly inhibit BRB breakdown 72 days after administration.
Conclusions: These findings suggest that lipid-based drug delivery
system can provide long-term and therapeutic concentration of drugs
in vitreous.
Commercial Relationships: Chi-Chun Lai, None; Ling Yeung,
None; Lan-Hsin Chuang, None; Yih-Shiou Hwang, None; ChihChiang Tsai, Taiwan Liposome Company (E); Po-Chun Chang,
Taiwan Liposome Company (E); Luke S.S. Guo, None; Yun-Long
Tseng, None; Sheue-Fang Shih, Taiwan Liposome Company (E);
Keelung Hong, Taiwan Liposome Company (E), TLC
Biopharmaceuticals, Inc. (E)
Support: NSC 99-2314-B-182-020-MY3 (Taiwan)
Noninvasive Dexamethasone Sodium Phosphate Ocular Drug
Delivery System for the Treatment of Intermediate and Posterior
Uveitis
Kongnara Papangkorn1, 2, Donald Mix1, Charlotte Butler1, John
Higuchi1, Balbir Brar1, William I. Higuchi2, 1. 1Aciont Inc, Salt Lake
City, UT; 2University of Utah, Salt Lake City, UT.
Purpose: To assess the efficacy and safety of dexamethasone sodium
phosphate Visulex system (DSP-V) with various dosing regimens in
treating intermediate and posterior uveitis in New Zealand white
(NZW) rabbit.
Methods: Fifteen NZW rabbits were assigned to 5 groups according
to DSP concentration, dosing, and Visulex application time (see
table). Uveitis was induced in NZW rabbits by subcutaneous
injections of complete Freund’s adjuvant and an IVT injection of H37
RA antigen as described by Cheng, et. al. After induction, DSP
treatment was given to the right eye of each rabbit by DSP-V except
the control group. Efficacy and safety of DSP-V were evaluated daily
by indirect ophthalmoscopy (IO) and at the end of the study by
histopathological examinations (HE) to evaluate the pathology and
inflammatory cell infiltration (ICI) into the eye tissues including both
anterior (conjunctiva, cornea, anterior chamber) and posterior
sections (vitreous, choroid, retina).
Results: The control group exhibited significant inflammation in the
vitreous, choroid, and retina; and slight or no inflammation in the
conjunctiva, cornea, anterior chamber, and iris. All treatments with
DSP-V showed effectiveness in the reduction of vitreous opacity
(VO) and ICI of the vitreous, choroid, and retina. The VO of the
control group indicate that uveitis occurred within 24 h after
induction and remained throughout the 29 day study. After Day 3,
there was a clear differentiation between the control and the 10 and
15 min treatment groups. By Day 10, the highest dosing regimen
(Group 2) showed complete resolution of the vitreal inflammation
and by Day 21, the lowest dosing regimen (Group 5) was also
completely clear. With the exception of the 8% single dose treatment,
there appears to be a DSP dose-efficacy relationship. As for the
safety of DSP-V, the differences in inflammation scores between the
treatment and control groups would imply DSP-V is a safe modality
for uveitis treatment. The HE also supports the safety aspect of DSPV treatment.
Conclusions: DSP-V containing 8% to 15% DSP administered for 5
to 15 min is well tolerated and highly efficacious in this chronic
intermediate and posterior uveitis model. Even a single dose of 8%
DSP can suppress intermediate and posterior uveitis for 29 days.
Conclusions: Administration site, compound properties and
formulation impact ocular and systemic pharmacokinetics. Our
results inform our understanding of R406 administration and the
design and formulation of ocular therapeutics in general.
Commercial Relationships: Kongnara Papangkorn, Aciont Inc
(E); Donald Mix, Aciont, Inc (E); Charlotte Butler, Aciont Inc (E);
John Higuchi, Aciont Inc. (E); Balbir Brar, Aciont Inc (C),
Atheronova Inc (C), Imprimis Pharma (C); William I. Higuchi,
Aciont Inc. (F)
Support: NEI SBIR Grant R44EY014772
Ocular Delivery of the Novel Spleen Tyrosine Kinase Inhibitor
R406 for Retinoblastoma
Eleanor M. Pritchard1, 2, Fangyi Zhu1, Lei Yang1, Cori Bradley2,
Elizabeth Stewart3, 2, Jiakun Zhang2, Burgess B. Freeman4, Michael
Dyer2, 5, R. Kip Guy1. 1Chemical Biology and Therapeutics, St. Jude
Children's Research Hospital, Memphis, TN; 2Developmental
Neurobiology, St. Jude Children's Research Hospital, Memphis, TN;
3
Hematology/Oncology, St. Jude Children's Research Hospital,
Memphis, TN; 4Pharmaceutical Sciences, St. Jude Children's
Research Hospital, Memphis, TN; 5Ophthalmology, University of
Tennessee Health Sciences Center, Memphis, TN.
Purpose: Drug delivery to the eye remains a major challenge due to
limited blood retinal barrier (BRB) penetration and systemic sideeffects. Local administration routes represent a promising potential
solution, but unfortunately relatively little is known about the
relationship between compound properties and in vivo ocular
pharmacokinetics. R406 is an inhibitor of a spleen tyrosine kinase
(SYK), which was recently identified as a novel therapeutic target for
retinoblastoma. Our objective was to investigate the effects of
formulation, administration site and physical-chemical compound
properties on ocular and systemic R406 pharmacokinetics.
Methods: Systemic and intraocular pharmacokinetics were evaluated
in mice for various formulations of three forms of R406 with varying
compound properties (R406 free base, R406 phenylsulfonate salt, and
the phosphate prodrug of R406, R788) after single dose
subconjunctival administration. PK of orally administered R788 was
also characterized.
Results: Tmax occurs at 0.5 hr for all formulations and routes tested,
indicating rapid absorption of R406 into the eye. Local
subconjunctival delivery of R788 increases vitreous exposure and
decreases systemic exposure compared with R788 administered
orally. At comparable doses, R788 in cosolvent solution achieves a
maximum vitreous concentration 5.7-fold higher than the water
insoluble R406 free base in emulsion, which suggests aqueous
solubility impacts ocular penetration. In contrast, R788 administered
in suspension exhibits a lower vitreous AUC and higher systemic
exposure than the less water soluble R406 salt administered in
DMSO, which suggests that at higher doses the more water soluble
prodrug diffuses more readily from the injection site into systemic
circulation.
Concentration versus time plots of plasma (top) and vitreous (bottom)
exposure following systemic (oral, squares) and local
(subconjunctival injection, circles) administration of R788
Formulation details and vitreous pharmacokinetic parameters for
R406 formulations investigated including AUC (area under the
concentration-time curve), Cmax (maximum concentration) and tmax
(time Cmax observed)
Commercial Relationships: Eleanor M. Pritchard, None; Fangyi
Zhu, None; Lei Yang, None; Cori Bradley, None; Elizabeth
Stewart, None; Jiakun Zhang, None; Burgess B. Freeman, None;
Michael Dyer, None; R. Kip Guy, None
Intravitreal long-lasting micelle formulation of
hexadecyloxypropyl-cidofovir (HDP-CDV) for cytomegalovirus
retinitis
Feiyan Ma1, Su-Na Lee1, Huiyuan Hou1, James Beadle2, William R.
Freeman1, Karl Hostetler2, Lingyun Cheng1. 1Ophthalmology, Shiley
Eye Center, UCSD, La Jolla, CA; 2Department of Medicine, San
Diego VA Healthcare System and UC San Diego, LA JOLLA, CA.
Purpose: We have previously shown that micelle formulation of
HDP-CDV had a long vitreous half-life and good ocular safety
profile. In the current study, we applied a pre-treatment strategy to
evaluate the sustained therapeutic effect of hexadecyloxypropylcidofovir (HDP-CDV) against a rabbit HSV retinitis model.
Methods: Twenty-seven New Zealand pigmented rabbits (27 eyes)
received either an intravitreal equal molar concentrations of HDPCDV (42μg/50μl), CDV (28 μg/50μl) or 5% dextrose (50μl) as
controls 5 weeks or 9 weeks before HSV intravitreal inoculation.
Intravitreal injection of a 5×10-5 dilution of 108TCID/mL HSV was
performed to induce HSV retinitis. After the virus injection, the
rabbit eyes were monitored by indirect ophthalmoscopy and the
retinitis was graded as 0.5, 1, 2, 3, and 4 as we described previously
on day 3, day 6, day 9, and day 14 on which rabbits were sacrificed
for pathological evaluation. The retinitis scores were compared
among the HDP-CDV, free CDV, and dextrose groups.
Results: HSV retinitis in rabbits is a rapidly spreading retinitis
model. It presents on day 4 to 6 after virus inoculation and shows
rapid progression between day 6 and day 9 before a complete retinitis
on day 14. For the 5-week pretreatment study, the retinitis scores on
day 6 and day 9 were pooled and treated as a repeated measurement
as ordinary scale to compare the retinitis severity among the three
groups. HDP-CDV showed significantly less retinitis than dextrose
controls (p=0.0047), though no significant difference from CDV
group (p=0.22). For the 9-week pretreatment study, HDP-CDV group
showed a significantly less retinitis than CDV group (p=0.0053) and
dextrose group (p=0.0097). At week 9, CDV had same severe
retinitis as the dextrose injected eyes (p=0.23). On day 14 after the
virus inoculation, there were 2 eyes that developed retinitis out of 7
eyes with HDP-CDV, 5 eyes out of 5 eyes with CDV, and 5 eyes out
of 5 eyes with dextrose (p=0.0062).
Conclusions: CDV can provide 5 weeks antiviral effect after
intravitreal injection. However, HDP-CDV can provide at least 9
weeks antiviral effect following a single intravitreal injection of 42
µg.
Commercial Relationships: Feiyan Ma, None; Su-Na Lee, None;
Huiyuan Hou, None; James Beadle, Chimerix Pharmaceuticals (I);
William R. Freeman, OD-OS, Inc. (C); Karl Hostetler, None;
Lingyun Cheng, Spinnaker Biosciences (C)
Characterization of the In Vitro Release Kinetics of Bevacizumab
from a Biocompatible Reverse Thermal Gel
Britta M. Rauck1, Carlos A. Medina-Mendez2, Thomas R. Friberg2,
Yadong Wang1. 1Bioengineering, University of Pittsburgh, Pittsburgh,
PA; 2Ophthalmology, University of Pittsburgh Medical Center,
Pittsburgh, PA.
Purpose: A biodegradable, biocompatible reverse thermal gel
demonstrates excellent promise as an intraocular drug delivery
system as it is minimally invasive and can sustain the release of drugs
substantially. In order to maximize the release profile, we loaded
several doses of bevacizumab (Avastin) using multiple gel
concentrations and studied the release behavior in vitro. Moreover,
we confirmed the ability of the release system to maintain the
bioactivity of the released drug.
Methods: We synthesized poly(ethylene glycol)-poly(serinol
hexamethylene urethane) (ESHU), an amphiphilic block copolymer
that, in solution, undergoes a sol-gel transition upon heating from
room temperature to body temperature. We performed in vitro release
studies by injecting 10, 15 or 20% (w/v) ESHU containing 0.625,
1.25, 2.5 or 5 mg of bevacizumab into a 1% solution of hyaluronic
acid at 37°C. At 1, 3, and 7 days and weekly thereafter, samples were
obtained from the supernatant, and an enzyme-linked immunosorbent
assay (ELISA) was used to determine the concentration of
bevacizumab released. We evaluated the bioactivity of the released
drug by quantifying the inhibition of tube formation using human
umbilical vein endothelial cells (HUVECs) cultured on Matrigel.
Results: The release profile of bevacizumab from ESHU is both
dose- and concentration-dependent. In all experimental groups ESHU
is capable of sustaining drug release in a near-linear fashion for
approximately 12 weeks. The burst release from the gel at day 1 is
less than 10% of the total drug loaded. Low gel concentration and/or
high drug loading results in a larger burst and ultimately a more rapid
release profile. The bevacizumab that is released from the gel is
capable of inhibiting HUVEC tube formation when samples are
mixed with culture media, suggesting that the released bevacizumab
is bioactive, and that ESHU does not interfere with its mechanism of
action.
Conclusions: By altering drug dose and gel concentration parameters
the release profile of ESHU can be controlled. Compared to a clinical
setting, in which bevacizumab must be injected monthly due to its
short half-life, ESHU is capable of sustaining release over
approximately 3 months in vitro, suggesting that it may ultimately be
used to reduce injection frequency, improve patient compliance and
enhance therapeutic efficacy.
Commercial Relationships: Britta M. Rauck, None; Carlos A.
Medina-Mendez, None; Thomas R. Friberg, 13/581,518 (P);
Yadong Wang, None
339 AMD: New Drugs, Delivery Systems, and Mechanisms of
Action
Tuesday, May 07, 2013 11:00 AM-12:45 PM
Rationale for Treating Wet AMD in Human Using an Oral Pill
Consisting of a VEGFR/PDGFR Inhibitor X-82
Chris Liang1, David M. Brown2, Nauman A. Chaudhry3, Michael J.
Elman4, Jeffrey S. Heier5. 1Xcovery, LLC, West Palm Beach, FL;
2
Retina Consultants of Houston, Houston, TX; 3New England Retina
Associates, New London, CT; 4Elman Retina Group, Baltimore, MD;
5
Ophthalmic Consultants of Boston, Boston, MA.
Purpose: To analyze the pre-clinical and clinical safety and biologic
activity of X-82, a VEGFR/PDGFR inhibitor.
Methods: X-82 was evaluated in vitro and via oral administration in
a rat CNV model, focusing on safety and biologic activity. The safety
and pharmacokinetic properties of X-82 in cancer patients were
analyzed to define the doses that might be safe and effective in AMD
patients.
Results: VEGFR and PDGFR inhibition occurred with <50 nM in
the HUVEC tube formation model. In the rat CNV model, 10 mg/kg
dosed orally once a day showed >80% inhibition of CNV. Oral
administration of X-82 in cancer patients was well tolerated with no
Grade 2 or higher toxicities at exposures of 5191 ng.hr/ml and Cmax
of 942 nM. At 50mg once a day, the mean AUC, Cmax and Ctrough
levels were 1282 ng.hr/ml, 268 nM and 41 nM, respectively.
Conclusions: Oral administration of X-82 at 50mg once a day
achieved exposures well above the concentration required to inhibit
new blood vessel formation; this level was < ¼ of the well tolerated
exposure noted in cancer patients, suggesting that the dose should be
well tolerated and could be efficacious in treating wet AMD patients.
A pilot clinical study of X-82 in patients with wet AMD has started.
Commercial Relationships: Chris Liang, Xcovery, LLC (E); David
M. Brown, Regeneron Pharmaceuticals, Inc. (F), Regeneron
Pharmaceuticals, Inc. (C), Regeneron Pharmaceuticals, Inc. (R),
Bayer HealthCare (F), Bayer HealthCare (C), Bayer HealthCare (R),
Genentech (C), Roche (C), Alimera (C), Alcon (C), Novartis (C),
Thrombogenics (C), Genentech (F), Roche (F), Thrombogenics (F),
GSK (F), Alimera (F), Alcon (F), Allergan (F), Eli Lilly (F);
Nauman A. Chaudhry, Genentech (F), Regeneron Pharmaceuticals
(F), Xcovery Vision (F), Lpath (F), DRCR network (F); Michael J.
Elman, Genentech (C), Ohr Pharmaceuticals (I), Novartis (F),
DRCRnet (F); Jeffrey S. Heier, Acucela (C), Aerpio (C), Alimera
(F), Allergan (C), Bayer (C), Forsight Labs (C), Fovea (F),
Genentech (C), Genzyme (C), Genentech (F), Genzyme (F),
Thrombogenics (C), Sequenom (C), Notal Vision (F), Novartis (F),
Ophthotech (F), Ophthotech (C), Oraya (C), Paloma (F), Regeneron
(F), Regeneron (C)
A Novel Endogenous Glycan Therapy For Retinal Diseases:
Safety, In vitro Stability, Ocular Pharmacokinetics and Biodistribution
Shankar Swaminathan1, Huiling Li1, Mallika Palamoor1, Dorababu
Madhura2, Bernd Meibohm2, Monica M. Jablonski1.
1
Ophthalmology, Hamilton Eye Inst, Univ TN HSC, Memphis, TN;
2
Pharmaceutical Sciences, The University of Tennessee Health
Science Center, Memphis, TN.
Purpose: Asialo tri-antennary oligosaccharide (NA3 glycan) is a
novel endogenous compound which supports the proper folding of
outer segment membranes; promotes normal ultrastructure and
maintains protein expression patterns of photoreceptors and Müller
cells in the absence of RPE support. Hence it is a potential new
therapeutic for atrophic AMD. Our purpose was to evaluate the
safety, in vitro stability, ocular pharmacokinetics and biodistribution
of NA3 in the eye.
Methods: NA3 was injected into the vitreous of NZW rabbits at two
concentrations viz. 1 nM (MEC) and 100 nM (100XMEC) at three
time points (1d-14d). Safety was evaluated using routine clinical and
laboratory tests. NA3 was tagged with tritium. Ocular
pharmacokinetics and biodistribution were estimated at
predetermined time points (2h-14d). Rabbits were sacrificed and
various parts of the eye, multiple peripheral organs and plasma were
isolated. NA3 was quantified using scintillation counting.
Pharmacokinetics was estimated by non-compartmental modeling
(WinNonlin 6.2). A 2-aminobenzamide labeling method and
subsequent HILIC chromatographic separation were used to assess
plasma and vitreous stability at simulated biological conditions.
Results: NA3 was well tolerated by the eye. The concentration of
NA3 in various eye tissues was in the following order:
vitreous>retina>sclera/choroid>aqueous humor>cornea>lens. AUC∞
was maximum in the vitreous thereby providing a positive
concentration gradient for the drug to reach the retina. Half-lives in
critical eye tissues were between 40-60 h. NA3 concentrations were
negligible in peripheral organs. Radioactivity from tritiated NA3 was
excreted via urine and feces. By using an optimized HILIC method
with fluorescence detection, NA3 was found to be stable at 37°C in
excised vitreous over a minimum of 6 days, while it degraded within
a few hours in plasma thereby providing a breakthrough targeted
therapy for atrophic AMD and other retinal diseases.
Conclusions: NA3 glycan is safe for intraocular applications,
degrades rapidly in plasma with minimal organ distribution, while
being stable in vitreous. Ocular pharmacokinetics confirms the
movement of NA3 from vitreous to retina, the site of action. With
novel formulation strategies, the residence time of NA3 would be
increased for therapeutic applications in chronic retinal diseases.
Commercial Relationships: Shankar Swaminathan, None;
Huiling Li, None; Mallika Palamoor, None; Dorababu Madhura,
None; Bernd Meibohm, None; Monica M. Jablonski, 8,092,825 (P)
Support: Research to Prevent Blindness, University of Tennessee
Research Foundation, Knights Templar Eye Foundation, Fight for
Sight, March of Dimes, International Retinal Research Foundation
A Single 700 μg Dexamethasone Intravitreal Implant (Ozurdex)
Effectively Treats Complex Post-Operative Cystoid Macular
Edema
Daniel F. Kiernan1, 2, Glenn Stoller1, Ken B. Carnevale1, Nina C.
Mondoc1, Eric Donnenfeld1. 1Ophthalmic Consultants of Long Island,
Rockville Centre, NY; 2Ophthalmology, Winthrop Hospital Medical
Center, Mineola, NY.
Purpose: Severe post-operative cystoid macular edema (CME)
associated with posterior uveitis may occur after anterior (Irvine-Gass
syndrome) or posterior segment intraocular surgery and may result in
visual loss. There is no consensus on the efficacy of various topical,
oral, peribulbar, retrobulbar or intravitreal therapeutic options
compared to natural history, but patients with this condition are often
referred to specialists for treatment consultation.
Methods: Consecutive, retrospective review of patients diagnosed
and treated with this condition in a multi-specialty practice. All
patients had optical coherence tomography (OCT) and fluorescein
angiography (FA) at baseline, one week, one month and on most
recent follow-up.
Results: Six pseudophakic patients with acute, post-operative (≤8
weeks post-surgery, 4 after anterior segment procedures and 2 after
posterior segment procedures with vitrectomy) CME associated with
posterior uveitis and decreased vision (average logMAR VA= .996,
range 20/60 - 20/400) and edema on OCT (avg central subfoveal
thickness [CST] = 640 μm, range 447 to >1000 μm) were treated with
one 700 μg intravitreal dose of Ozurdex (Allergan Inc., Irvine CA).
Within 1 week VA improved to an average of logMAR 0.619
(~20/80) and avg CST decreased to 389 μm. At 1 month avg
logMAR VA= 0.309, range 20/25 - 20/50 (p= 0.017 compared to
baseline), and avg CST = 291 μm, range 233-352 μm (p=0.016
compared with baseline). At the most recent visit (average total
follow-up of 18.4 weeks, range 12-21 weeks), logMAR VA and CST
were not statistically different than at the 1-month interval. No
patients developed serious adverse events, including vitreous
hemorrhage, ocular hypertension or endophthalmitis, or developed
recurrent edema. FA demonstrated a progressive diminution of late
leakage at all post-treatment time points compared to baseline.
Conclusions: A single intravitreal injection of Ozurdex may rapidly
and permanently treat severe post-operative CME associated with
posterior uveitis in in both vitrectomized and non-vitrectomized eyes.
This therapy may result in faster and more definitive visual
rehabilitation and macular edema resolution compared to observation
or other treatment options.
D407 cells. This inhibition of hypoxia pathway and downstream
genes may form the platform for indication of ritonavir in treatment
of various sight threatening diseases. The process of traditional drug
development could be fastened since ritonavir is clinically approved
for human use.
Commercial Relationships: Ramya Krishna Vadlapatla, None;
Aswani Dutt Vadlapudi, None; Dhananjay Pal, None; Mridul
Mukherji, None; Ashim K. Mitra, None
Support: NIH Grants R01 EY09171-16 and R01 EY010659-14
Example of a patient within this series who had maximal visual
recovery with macular edema and angiography leakage resolution
after a single Ozurdex injection on Day 0.
Commercial Relationships: Daniel F. Kiernan, Allergan (C),
Allergan (R); Glenn Stoller, Lpath (C), Regeneron (F), Lpath (P),
SKS (I); Ken B. Carnevale, None; Nina C. Mondoc, None; Eric
Donnenfeld, Allergan (C), Alcon (C), Abbott (C), Bausch & Lomb
(C), Glaukos (C), Aquysis (C), Katena (C), Sarcode (C), Wavetec (C)
Ritonavir Inhibits HIF-1α Mediated VEGF Expression in Retinal
Pigment Epithelial Cells
Ramya Krishna Vadlapatla, Aswani Dutt Vadlapudi, Dhananjay Pal,
Mridul Mukherji, Ashim K. Mitra. Department of Pharmaceutical
Sciences, University of Missouri Kansas City, Kansas City, MO.
Purpose: Retinal hypoxia (oxygen deficiency) leading to
angiogenesis is a major signaling mechanism underlying a number of
sight threatening diseases. One of the most important target genes of
hypoxia inducible factor (HIF) includes vascular endothelial growth
factor (VEGF). Intravitreal injections of VEGF antagonists resulted
in serious complications and systemic side effects. Inhibiting the
signaling mechanism underlying neovascularization (HIF-pathway)
with already approved therapeutic molecule may have promising
anti-angiogenic role with fewer side-effects. Hence, the primary
objective of this study is to examine the expression of HIF-1α and
VEGF in human retinal pigment epithelial (ARPE-19 and D407) cells
treated with ritonavir under hypoxic and normoxic conditions.
Methods: ARPE-19 and D407 cells were cultured in normoxic and
hypoxic conditions (3, 6 and 12 hours), alone or in the presence of
ritonavir (5, 10 and 20μM). Quantitative real time polymerase chain
reaction (qPCR), immunoblot analysis and sandwich ELISA were
performed to check the RNA and protein expression levels of HIF-1α
and VEGF. Further, in vitro anti-angiogenic activity of ritonavir was
also assessed by measuring proliferation of choroid-retinal
endothelial (RF/6A) cells.
Results: Hypoxic exposure to 12 hours demonstrated elevated RNA
expression levels of HIF-1α and VEGF in both ARPE-19 and D407
cells. Hence, this time point was chosen for subsequent studies.
Presence of ritonavir in culture medium strongly inhibited the
expression of HIF-1α and VEGF in a concentration-dependent
manner in hypoxic conditions. Immunoblot analysis demonstrated
that ritonavir inhibited the protein expression of HIF-1α. Further,
hypoxic exposure increased VEGF secretion while presence of
ritonavir reduced this secretion, as demonstrated by an ELISA.
Finally, ritonavir significantly inhibited the proliferation of RF/6A
cells, demonstrating potential anti-angiogenic activity.
Conclusions: This study demonstrates for the first time that ritonavir
can inhibit the expression of HIF-1α and VEGF in ARPE-19 and
Protection of RPE cells by sulindac against oxidative damage is
through ischemic preconditioning (IPC) and involves activation
of the peroxisome proliferator activated receptor alpha (PPAR α)
Arunodoy Sur1, Diane Baronas-Lowell2, Manas R. Biswal1, Howard
M. Prentice3, Herbert Weissbach2, Janet C. Blanks1. 1Complex
Systems, Florida Atlantic University, Boca Raton, FL; 2CMBB,
Florida Atlantic University, Jupiter, FL; 3College of Medicine,
Florida Atlantic University, Boca Raton, FL.
Purpose: Age related macular degeneration (AMD) is the leading
cause of blindness among the elderly population and oxidative stress
has been implicated as a major cause of this ocular disorder.
Previously we had reported that sulindac protects cultured RPE cells
(ARPE19) against reactive oxygen species (ROS) induced stress. The
purpose of this current study is to understand the protective
mechanism involved in protection by sulindac of RPE from oxidative
stress induced by either tert-butyl hydrogen peroxide (TBHP) or
ultraviolet light (UVB). Protection of RPE cells against ROS was
also observed with fenofibrate, a known agonist of PPARα. The
results support a mechanism in which the protection of RPE cells
against oxidative stress is through a preconditioning mechanism
involving activation of PPAR α.
Methods: RPE cells were incubated with sulindac or fenofibrate for
24 hours, following which the cells were either exposed to a range of
TBHP concentrations or 1200mj UVB light. Cell viability was
determined by the MTT assay and upregulation of preconditioning
markers was detected using western blotting. In a light damage
mouse model, sulindac was administered by intravitreal injection and
evaluation of the retinal tissues for photoreceptor damage was
performed.
Results: The results indicate that sulindac confers >30 % protection
of cultured ARPE19 cells against oxidative damage. The sulindac
protection in vitro could be replaced by fenofibrate and was inhibited
by an antagonist of PPARα, indicating a role of PPARα in the
sulindac effect. Other results indicated that the protective effect of
sulindac was through an IPC mechanism. In a preliminary in vivo
study, sulindac protected photoreceptors against photooxidative
damage in a light damage model in mice.
Conclusions: Our findings indicate that preconditioning pathways
and activation of PPARα play key roles in sulindac’s protective
mechanism. A more extensive in vivo study is planned to establish
therapeutic efficacy and other modes of administration. Implementing
pharmacological preconditioning to deter oxidative stress outlined
here represents a possible clinical approach to treat AMD and other
oxidative stress induced disorders.
Commercial Relationships: Arunodoy Sur, None; Diane BaronasLowell, None; Manas R. Biswal, None; Howard M. Prentice,
None; Herbert Weissbach, CHS Pharma (C), Florida Atlantic
University (P), CHS Pharma (S); Janet C. Blanks, None
Support: FAU Research Priority Grant (awarded to JB and HP)
NAT2 STUDY: OMEGA-3 LEVELS IN RED BLOOD CELLS
MEMBRANES CORRELATES THE PREVENTIVE EFFECT
Eric H. Souied, Cecile Delcourt, Giuseppe Querques, Benedicte
Merle, Theodore Smith, Pascale Benlian. Retina Creteil, University
Paris Est Creteil, Creteil, France.
Purpose: The NAT2 (Nutritional AMD treatment-2) study was a
randomized, placebo-controlled, double-blind, parallel, comparative
study. Our purpose was to evaluate the efficacy of docosahexaenoic
acid (DHA) enriched oral supplementation in preventing exudative
age-related macular degeneration (AMD). Here we correlated the red
blood cells membrane (RBCM) levels in omega-3 and the occurrence
of choroidal neovascularisation (CNV)
Methods: 263 patients with early lesions of age-related maculopathy
and visual acuity better than 0.4 LogMAR units in the study eye and
neovascular AMD in the fellow-eye. Patients were randomly
assigned to receive either 840 mg/day DHA and 270 mg/day
eicosapentaenoic acid (EPA) from fish oil capsules or the placebo
(olive oil capsules) for 3 years.
Results: Time to occurrence and incidence of CNV in the study eye
were not significantly different between the DHA (19.5 ± 10.9
months, 28.4%, respectively) and placebo groups (18.7 ± 10.6
months, 25.6%, respectively). EPA+DHA level significantly
increased in RBCM in the DHA group (+70%; p<0.001), suggesting
that DHA easily penetrated cells, but unexpectedly also in the
placebo group (+9%; p=0.007). We observed a wide range of
EPA+EPA levels at month 6 and year 3 in both placebo and DHA
groups. In the DHA-allocated group, patients steadily achieving the
highest tertile of EPA+DHA levels in RBCM had significantly lower
risk (-68%; p=0.047; HR=0.32 (0.10-0.99) of developing CNV over 3
years).
Conclusions: In patients with unilateral exudative AMD, 3 years of
oral DHA-enriched supplementation had the same effect on CNV
incidence in the second eye as did the placebo. However, the RBCM
measurements revealed that CNV incidence was significantly
reduced in DHA-supplemented patients showing steadily high
EPA+DHA index over 3 years.
Commercial Relationships: Eric H. Souied, BAUSCH + LOMB
(C), NOVARTIS (C), BAYER (C), THEA (C), ALLERGAN (C);
Cecile Delcourt, Laboratoires Théa (F), Novartis (C), Bausch+Lomb
(C); Giuseppe Querques, None; Benedicte Merle, Laboratoires
Théa (F); Theodore Smith, None; Pascale Benlian, Bausch & Lomb
(R)
Clinical Trial: ISRCTN98246501
Role of Oil Palm Phenolics in Angiogenesis and Inflammation
Souska Zandi1, 2, Shintaro Nakao1, 3, Dawei Sun1, Kenneth Hayes4,
Farhad Hafezi2, Ali Hafezi-Moghadam1. 1Radiology, Brigham and
Women’s Hospital, Boston, MA; 2Ophthalmology, Geneva
University Hospitals, Geneva, Switzerland; 3Ophthalmology, Kyushu
University, Fukuoka, Japan; 4Nutrition and Biology, Brandeis
University, Waltham, MA.
Purpose: To date the treatment options for Choroidal
neovascularization (CNV), the main cause of severe vision loss in
patients with age-related macular degeneration (AMD) are limited.
Minimally-invasive methods, such as repeated intravitreal injections
of VEGF-inhibitors are mainly performed.
We investigate the role of the nutritional supplement, oil palm
phenolics (OPP), in CNV formation.
Methods: To induce CNV, C57BL/6 mice were anesthetized and
pupils were dilated. Using a 532-nm laser, four spots (100mW,
50µm, 100ms) were placed in each eye. Development of a bubble
under laser confirmed the rupture of the Bruch’s membrane. One
group received drinking water, whereas the other group drank a
natural liquid containing oil palm phenolics ad libitum. 14 days after
laser injury, the size of the CNV lesions was measured in choroidal
flat mounts. The volume of the lesions was quantified, using confocal
microscopy.
On day 3 after laser injury 10μm frozen sections of the posterior
segment were prepared. The sections were incubated with a mouse
anti-F4/80 mAb (10μg/ml), and subsequently with the secondary
antibody. Photomicrographs of the CNV lesions were taken and the
number of F4/80 positive macrophages was counted.
Results: 14 days after laser injury, the mice fed with OPP showed a
significant decrease in CNV size, compared with vehicle fed animals
(n=4, 30 lesions per group, P<0.01).
OPP significantly reduced the number of accumulated macrophages
in CNV lesions (n=3, 24 lesions, P<0.01).
Conclusions: OPP effectively suppresses laser-induced CNV and
reduces macrophage recruitment to the lesions. Current results
suggest OPP as an attractive nutritional supplement in the prevention
and treatment of AMD.
Commercial Relationships: Souska Zandi, None; Shintaro Nakao,
None; Dawei Sun, None; Kenneth Hayes, Malaysian Palm Oil
Board (F), Brandeis University (P); Farhad Hafezi, Schwind (F),
Ziemer (F), PCT/CH 2012/000090 (P); Ali Hafezi-Moghadam,
None
Support: NIH grants AI050775, American Health Assistance
Foundation (AHAF), and MPOB
Bivalent ranibizumab is more active in vitro than bevacizumab
Hanieh Khalili1, 2, Steve Brocchini1, 2, Peng T. Khaw2. 1UCL School
of Pharmacy, London, United Kingdom; 2National Institute for
Health Research (NIHR) Biomedical Research Centre at Moorfield
Eye Hospital NHS Foundation Trust and UCL Institute of
Ophthalmology, London, United Kingdom.
Purpose: Ranibizumab is a monovalent Fab therapeutic. Its
complimentary binding region (CDR) has a higher apparent affinity
for VEGF than does the CDR in bevacizumab which, as a full IgG, is
bivalent. We have developed a practical method to make Fab-PEGFab conjugates that mimic IgG molecules. Conjugation of the Fab to
PEG occurs essentially in the same place where the Fab is linked
within a native IgG. We hypothesised that two ranibizumab (Fabrani)
molecules could be conjugated to give Fabrani-PEG-Fabrani which
would achieve equivalent or better binding than bevacizumab.
Methods: Ranibizumab was conjugated with a thiol-specific bisalkylation PEG to give Fab-PEG-Fab. Fabbeva was obtained from
papain digestion of bevacizumab. Purification gave pure Fab-PEGFab (silver stain). SPR binding analyses and in vitro angiogenesis
assay were performed on Fabrani-PEG20-Fabrani (N=5), Fabrani
(N=4) and bevacizumab (N=5).
Results: Fabrani-PEG20-Fabrani (Figure 1, A, lane 2) was obtained
from Fabrani (lane 1). Fabbeva-PEG20-Fabbeva was prepared for
comparison. SPR analysis indicates that Fabrani-PEG20-Fabrani
conjugates bind to VEGF. The dissociation rate constant (kd) of
Fabbeva-PEG20-Fabbeva was slower than bevacizumab and Fabbeva
(Figure 1, B). Fabrani-PEG-Fabrani inhibited angiogenesis in vitro to
a greater extent than bevacizumab as determined by the reduction in
tubule formation in a concentration-dependent manner. The average
number of junctions made in the wells treated with Fabrani-PEG20Fabrani was 6, where wells treated with VEGF only (positive
control), bevacizumab and Fabrani resulted in an average 830, 382
and 233 number of junctions respectively. The average number of
junctions calculated for the wells treated with medium (negative
control) was only 11.
Conclusions: Fabrani-PEG20-Fabrani inhibited angiogenesis in vitro
better than bevacizumab and ranibizumab. This work has the
potential to optimise new protein-based therapeutics, particularly
antibodies.
Figure 1. A; SDS-PAGE gels of the disulfide bridging PEGylation of
the Fab; Novex Bis-Tris 4-12% gel stained with colloidal blue for
protein (lanes M-2) and silver staining (lane 3) to detect impurity. M:
Standard protein markers. Lane 1: purified Fab. Lanes 2: 3: purified
Fabrani-PEG20-Fabrani. B; The dissociation rate profiles for
Fabbeva, Fabbeva-PEG20-Fabbeva and bevacizumab using CM3
chip with 53 RU VEGF at 25 0C.
Commercial Relationships: Hanieh Khalili, None; Steve
Brocchini, None; Peng T. Khaw, University College Moorfields (P)
Support: NIHR Moorfields Biomedical Research Centre
Results: Both OCT and CFM revealed the CNV size were
significantly reduced, and FA grading showed significantly less
leakage in the ω-3-fed-mice compared with the ω-6-fed mice. The
concentrations of the principal ω-3 LCPUFAs (EPA and DHA) were
significantly increased in serum of ω-3-fed mice. The serum levels of
AA-derived eicosanoids were significantly reduced, whereas those of
EPA-derived- and DHA-derived eicosanoids were significantly
increased in ω-3-fed mice. The mRNA expression and abundance of
protein of ICAM-1 and E-selectin in the retina or choroid after CNV
induction were significantly reduced in ω-3-fed mice. The rolling
velocity of leucocytes on the combination of P-selectin and ICAM-1
coated chamber was significantly greater in ω-3-fed mice. The
abundance of VEGF protein in both the retina and choroid was
significantly reduced in ω-3-fed mice.
Conclusions: We demonstrate that dietary supplementation of ω-3
LCPUFAs mediate choroidal neovessel regression. A clear
understanding of the mechanisms of these molecules in AMD could
bring a major shift in our approach to disease treatment. Currently
there is an urgent need for safe nutritional or pharmacological
interventions for the treatment and ideally the prevention of AMD.
Commercial Relationships: Ryoji Yanai, None; Lama Mulki,
None; Kimio Takeuchi, None; John H. Sweigard, None; Jun
Suzuki, None; Philipp Y. Gaissert, None; Demetrios Vavvas,
MEEI (P), Kala pharmaceuticals (C), Roche (C), Genentech (C);
Wolf-Hagen Schunck, None; Joan W. Miller, Massachusetts Eye
and Ear Infirmary (P), Novartis (I), Alcon (C), KalVista
Pharmaceuticals (C); Kip M. Connor, None
Support: the Massachusetts Lions Eye Research Fund, Inc., National
Institutes of Health (NIH) grants R01EY022084-01/S1 (KMC), and
P30EY014104 (Core support at the Massachusetts Eye and Ear
Infirmary), and Research to Prevent Blindness Unrestricted Grant and
an award from the Japan Eye Bank Association (RY).
Omega-3 long chain polyunsaturated fatty acids promote
choroidal neovessel regression
Ryoji Yanai1, Lama Mulki1, Kimio Takeuchi1, John H. Sweigard1, Jun
Suzuki1, Philipp Y. Gaissert1, Demetrios Vavvas1, Wolf-Hagen
Schunck2, Joan W. Miller1, Kip M. Connor1. 1Ophthalmology,
Massachusetts Eye & Ear Infirmary Harvard Medical School, Boston,
MA; 2Max Delbrück Center for Molecular Medicine, Berlin,
Germany.
Purpose: Age-related macular degeneration (AMD) is the primary
cause of blindness in elderly individuals of industrialized countries.
Prospective clinical studies have indicated that dietary intake of
omega (ω)-3 long-chain polyunsaturated fatty acids (LCPUFAs) may
have a protective effect against AMD. This study characterizes the
effects of dietary intake of ω-3 and ω-6 LCPUFAs in a mouse model
of AMD.
Methods: The present studies adhered to ARVO’s Statement for the
Use of Animals in Ophthalmic and Vision Research. C57BL/6 mice
were fed a diet enriched with either ω-3 or ω-6 LCPUFAs for 2
weeks prior to choroidal neovascularization (CNV) induction by
photocoagulation using a 532-nm laser. CNV development was
evaluated by Fluorescein Angiography (FA), Optical Coherence
Tomography (OCT), and choroidal flatmount (CFM). The primary
eicosanoids and their downstream metabolites were analyzed by LC
MS/MS lipidomics. Expression of ICAM-1, E-selectin, and VEGF in
the retina, choroid, or laser-captured CNV were evaluated by RTPCR
and immunoblot analysis. Macrophage invasion was evaluated in
Cx3cr1GFP/+ mice. Leukocyte rolling velocity was analyzed using
the autoperfused microflow chamber assay.
Preprogrammed Hematopoietic Stem Cells as a Systemic
Therapy for Dry AMD
Maria B. Grant1, Xiaoping Qi2, Yuanqing Yan1, Lynn C. Shaw1,
Alfred S. Lewin3, Michael E. Boulton2. 1Pharmacology and
Therapeutics, University of Florida, Gainesville, FL; 2Anatomy and
Cell Biology, University of Florida, Gainesville, FL; 3Molecular
Genetics and Microbiology, University of Florida, Gainesville, FL.
Purpose: Dry AMD represents 85% of AMD and is associated with
retinal pigment epithelial (RPE) dysfunction and cell death for which
there is no treatment. Previously we showed that systemic delivery of
genetically reprogrammed hematopoietic stem cells (HSC) with the
RPE differentiation marker, RPE 65, enhances repair of the RPE
layer and results in recovery of visual function (VF) in the sodium
iodate model of acute RPE cell loss (Sengupta et al 2009). In this
study, we tested the efficacy of programming HSC in the superoxide
dismutase 2 knockdown (SOD2 KD) model for dry AMD which
generates a defect predominantly in the RPE.
Methods: SOD2 KD was achieved by subretinal injection of AAV
containing an SOD2 ribozyme under the direction of an RPE specific
promoter (VMD2). Systemic administration of HSC infected with
lentivirus expressing human RPE 65 (LV hRPE65) or infected with
lentivirus expressing LacZ (LV LacZ) was undertaken at 1, 3 and 6
months post induction of the AMD phenotype with SOD2 KD. ERGs
and optomotor response tests were used to assess VF. Donor RPE65HSC integration was assessed by confocal immunofluorescence
microscopy. Histology and immunohistochemistry (IH) were used to
identify retinal histology and RPE repair.
Results: LV gave a 75.5±5.5% (p<0.01) efficient transfection of
HSC and RPE65 expression was confirmed by IH. Systemic
administration of LV hRPE65 programmed mouse HSCs maintained
VF, retained retinal thickness and prevented degeneration of the
retina of SOD2 KD mice. This preservation was not observed by null
or LV LacZ programmed HSC. At 1, 3 and 6 months following HSC
injections, photopic and scotopic ERGs were significantly higher
only in SOD2 KD mice administered LV hRPE65-mHSC but not
mice receiving LV LacZ-mHSC, untreated HSCs or untreated SOD2
KD mice. Using optomotor responses to further assess VF, at 1
month post SOD KD, mice injected with LV RPE65-mHSC retained
VF to a level similar to WT mice, VF declined slightly(<20%) at the
6-month time point.
Conclusions: Introduction of RPE-65 programmed HSC maintained
VF, retained retinal thickness and prevented degeneration. This
experimental paradigm offers a minimally invasive cellular therapy
that can be given systemically overcoming the need for invasive
ocular surgery and offering the potential for prevention, rather than
intervention in early AMD and other RPE based diseases.
Commercial Relationships: Maria B. Grant, None; Xiaoping Qi,
None; Yuanqing Yan, None; Lynn C. Shaw, None; Alfred S.
Lewin, University of Florida (P); Michael E. Boulton, None
Support: American Health Assistance Foundation M2011017 and
NEI grant R01 EY02025
Structure-Activity Relationship Studies of a Natural Product
Inhibitor of Choroidal Angiogenesis
Halesha D. Basavarajappa1, 2, Bit Lee3, Xiang Fei3, Carlos Magaña4,
Catherine Waller5, Neil R. Crouch6, 7, Dulcie A. Mulholland5, SeungYong Seo3, Timothy W. Corson1, 2. 1Biochemistry and Molecular
Biology, Indiana University School of Medicine, Indianapolis, IN;
2
Ophthalmology, Indiana University school of Medicine,
Indianapolis, IN; 3Pharmacy, Gachon University, Incheon, Republic
of Korea; 4Melvin and Bren Simon Cancer Center, Indiana University
School of Medicine, Indianapolis, IN; 5Chemistry, University of
Surrey, Guildford, United Kingdom; 6Chemistry and Physics,
University of KwaZulu-Natal, Durban, South Africa; 7Ethnobotany,
South African National Biodiversity Institute, Durban, South Africa.
Purpose: Preventing pathological angiogenesis in the eye is key to
treating retinopathy of prematurity (ROP), diabetic retinopathy and
age-related macular degeneration (AMD). At present there is no
small molecule drug on the market that specifically targets this
process. There is thus a need for developing novel small molecules
targeting angiogenesis. Cremastranone is a homoisoflavanone,
previously isolated from the bulb of a medicinal orchid, Cremastra
appendiculata. This compound has anti-angiogenic activity both in
vitro and in vivo. In mouse models of ROP and AMD,
homoisoflavanone inhibits pathogenic retinal and choroidal
neovascularization respectively. However the mechanism of action
and structure-activity relationship (SAR) of this compound are not
yet understood. We set out to describe the SAR of cremastranone and
show the anti-angiogenic activity of the synthetic compound in vitro.
Methods: We synthesized cremastranone for the first time. The antiproliferative effects of cremastranone along with more than 50
natural product and synthetic analogs were tested with the Alamar
blue cell proliferation assay, using human umbilical vein endothelial
cells (HUVECs) and human retinal microvascular endothelial cells
(HRMVECs). Retinoblastoma Y-79 and uveal melanoma 92-1 cells
were used as control cell lines to detect non-specific ocular cytotoxic
compounds. The anti-proliferative activity of the compounds was
confirmed by the EdU incorporation assay.
Results: Synthetic cremastranone had growth inhibitory properties
comparable to values reported for the natural-source compound. The
HRMVEC growth inhibitory (GI50) values of other active analogs
ranged from 150 nM to 95 µM. While some compounds showed
specific activity against HUVECs and HRMVECs and no toxicity
towards Y79 and 92-1, others had nearly equal cytotoxic effects on
all the cell lines tested. The fused ring-system of the chromanone
moiety is essential for activity, as is the benzyl group. Small
modifications on the aromatic rings of the compound are tolerated
and can increase the specificity to endothelial cells, providing an
opportunity to develop specific angiogenesis inhibitors.
Conclusions: This study offers the first evidence that synthetic
cremastranone has antiangiogenic activity, and might be developed as
a specific antiangiogenic drug to treat ROP, AMD and diabetic
retinopathy.
Commercial Relationships: Halesha D. Basavarajappa, Kemin
Industries, Inc (F); Bit Lee, None; Xiang Fei, None; Carlos
Magaña, None; Catherine Waller, None; Neil R. Crouch, None;
Dulcie A. Mulholland, None; Seung-Yong Seo, None; Timothy W.
Corson, None
Support: International Retinal Research Foundation, Carl Marshall
Reeves & Mildred Almen Reeves Foundation, Ausich Graduate
Student Research Scholarship
Intravitreal Tanibirumab, a Fully Human Monoclonal Antibody
against Vascular Endothelial Growth Factor Receptor 2,
Suppresses and Regresses Laser-induced Choroidal
Neovascularization in a Rat Model
Jaeryung Kim1, Sang Ryeol Shim2, Sung-Woo Kim2, Weon Sup Lee2,
Jin-San Yoo2, Sang Hoon Lee2, Sang Jin Kim1. 1Ophthalmology,
Samsung Medical Center, Seoul, Republic of Korea; 2PharmAbcine,
Inc., Daejeon, Republic of Korea.
Purpose: To investigate the effect of intravitreally administered
tanibirumab, a fully human monoclonal antibody against vascular
endothelial growth factor (VEGF) receptor 2, on a rat model of laserinduced choroidal neovascularization (CNV)
Methods: The anti-angiogenic effects of tanibirumab were validated
by rat aortic ring assay. CNV was induced by laser photocoagulation
on Day 0 in eyes of Brown Norway rats. Intravitreal injection of
tanibirumab or PBS was done on Day 0 (prevention arm) or Day 7
(treatment arm). Seven days after injection, eyes were enucleated and
retinal pigment epithelium-choroid-sclera flat mounts were prepared.
Areas of CNV were determined in flat mounts using intravenously
administered FITC-dextran and TRITC-BS isolectin labeling, and
quantified using an image analysis program.
Results: In the rat aortic ring assay, tanibirumab significantly
inhibited VEGF-mediated vessel sprouting. In the prevention arm, the
mean area of CNV was reduced by 28.2% and 53.9 % in
tanibirumab-treated (20ug and 60ug, respectively) eyes compared
with PBS-treated control eyes on Day 7 (P=0.028 and <0.001,
respectively). In the treatment arm, the mean area of CNV was
reduced by 28.7% and 46.0 % in tanibirumab-treated (20ug and 60ug,
respectively) eyes compared with PBS-treated control eyes on Day
14 (P=0.020 and <0.001, respectively).
Conclusions: Intravitreally administered tanibirumab resulted in the
suppression of formation of new, and regression of pre-formed laserinduced CNV in the rat model. Tanibirumab may be a feasible
treatment for CNV associated with age-related macular degeneration
or other causes.
Commercial Relationships: Jaeryung Kim, PharmAbcine, Inc. (F);
Sang Ryeol Shim, None; Sung-Woo Kim, PharmAbcine Inc. (F),
PharmAbcine Inc. (E), PharmAbcine Inc. (P); Weon Sup Lee, None;
Jin-San Yoo, None; Sang Hoon Lee, None; Sang Jin Kim, Daewoo
Pharm. (F), Pharmabcine (F)
Functional characterization of recombinant anti-VEGF variants
in vitro
Tobias Wimmer, Birgit Lorenz, Knut Stieger. Department of
Ophthalmology, Justus-Liebig University Giessen, Giessen,
Germany.
Purpose: Most retinal neovascular disorders are caused by an upregulation of the vascular endothelial growth factor (VEGF).
Especially disorders like age-related macular degeneration (AMD),
diabetic retinopathy (DR) and retinal vein occlusion (RVO) are
treated with repeated injections of anti-VEGF molecules like
Bevacizumab (Avastin®), Ranibizumab (Lucentis®), or Aflibercept
(Eyelea®). Repeated injections of anti-VEGF molecules can be
connected to severe side effects like endophthalmitis and can
represent a financial burden to the patients. The aim of this project is
to develop an alternative strategy i.e. controlled expression of antiVEGF molecules by the retina itself.
Methods: The DNA sequences of the light and the heavy chain of the
Ranibizumab F(ab)-fragment including secretory leader sequences
were cloned into pIREShrGFP1a separated by an internal ribosomal
entry site (IRES) (Ra01). This construct was PCR mutated to
generate a Ranibizumab variant (Ra02), which is expressed as just
one molecule containing a 6x glycine linker. HEK293 were
transfected with both constructs, presence in the culture medium
verified by western blot analysis and the concentrations measured
with a custom made Ranibizumab/Lucentis® ELISA. VEGF binding
affinities were determined in a cell free binding assay and the
biological activity evaluated in a HUVEC (human umbilical
endothelial cell) migration assay.
Results: Ra01 and Ra02 were detected in cell culture medium. The
concentrations were 374,1 ng/ml and 403,4 ng/ml for Ra01 and Ra02,
respectively. VEGF binding affinity was significantly lower for Ra01
and Ra02 compared to injectable Lucentis® for each tested
concentration. The inhibition of VEGF induced endothelial cell
migration by Ra01 and Ra02 was comparable with Lucentis® over
all tested concentrations, but the maximum inhibitory effect of Ra01
and Ra02 was reached at lower doses compared to Lucentis®.
Conclusions: Ra01 and Ra02 can be produced in eukaryotic cells in
vitro and display comparable biological activities as commercially
available Lucentis®. In vivo studies in human VEGF overexpressing
mice are ongoing. These results are the basis for a gene-based therapy
of human neovascular disorders.
Commercial Relationships: Tobias Wimmer, None; Birgit
Lorenz, Optos (F); Knut Stieger, None
Support: DFG Grant Sti 597/2-1
Long-term efficacy of ciliary muscle gene transfer of three sFlt-1
variants in a rat model of laser-induced choroidal
neovascularization
Mohamed El Sanharawi1, 2, Elodie Touchard3, Romain Benard3,
Pascal Bigey4, Virginie Escriou4, Chadi Mehanna1, 2, Marie-Chritine
Naud1, Jean-Claude P. Jeanny1, Marianne Berdugo Polak1, Francine
F. Behar-Cohen1, 2. 1INSERM UMRS 872, Paris, France; 2Université
Pierre et Marie-Curie, Paris, France; 3Eyevensys, Paris, France;
4
INSERM U640, Paris, France.
Purpose: Inhibition of vascular endothelial growth factor (VEGF)
has become the standard of care for patients presenting wet age-
related macular degeneration. However, monthly intravitreal
injections are required for optimal efficacy. We have previously
shown that electroporation enabled ciliary muscle gene transfer
results in sustained protein secretion into the vitreous for up to 9
months.
Methods: Here we evaluated the long-term efficacy of ciliary muscle
gene transfer of three soluble VEGF receptor-1 (sFlt-1) variants in a
rat model of laser-induced choroidal neovascularization (CNV).
Fluorescein angiography (FA) was performed to evaluate vascular
leakage. CNV growth was evaluated using retinal pigment epithelium
(RPE)/choroid flatmount and infracyanine angiography. Intra-ocular
VEGF levels were measured using ELISA. The mRNA expression of
VEGF was determined using RT-PCR in the RPE/choroid
complexes.
Results: All three sFlt-1 variants significantly diminished vascular
leakage and neovascularization as measured by FA and flatmount
choroid at 3 weeks. FA and infracyanine angiography demonstrated
that inhibition of CNV was maintained for up to 6 months after gene
transfer of the two shortest sFlt-1 variants. Throughout, clinical
efficacy was correlated with sustained VEGF neutralization in the
ocular media. Interestingly, treatment with sFlt-1 induced a 50%
down-regulation of VEGF mRNA levels in the retinal pigment
epithelium and the choroid.
Conclusions: We demonstrate for the first time that non-viral gene
transfer can achieve a long-term reduction of VEGF levels and
efficacy in the treatment of choroidal neovascularization.
Commercial Relationships: Mohamed El Sanharawi, None;
Elodie Touchard, None; Romain Benard, None; Pascal Bigey,
INSERM (P), Université Paris Descartes (P); Virginie Escriou,
None; Chadi Mehanna, Sisène (E); Marie-Chritine Naud, None;
Jean-Claude P. Jeanny, None; Marianne Berdugo Polak, None;
Francine F. Behar-Cohen, Inserm/Univesrité ParisDescartes (P)
Immune-Like Complexes of Bevacizumab Bind to Retinal
Pigment Epithelial Cells and Endothelial Cells in vitro
Yang Liu1, Terra B. Potocky2, Jingtai Cao1, Joel Martin2, Nicholas
Papadopoulos2, Stanley J. Wiegand1. 1Ophthalmology, Regeneron
Pharmaceuticals Inc, Tarrytown, NY; 2Molecular Biology, Bioassay,
& Protein Development, Regeneron Pharmaceuticals Inc, Tarrytown,
NY.
Purpose: The VEGF inhibitors ranibizumab (RAN), aflibercept
(AFL), and bevacizumab (BEV) bind VEGF with different binding
stoichiometries. AFL binds VEGF exclusively as a 1:1 complex
(MW: 155 kDa). In contrast, BEV:VEGF complexes are
heterogeneous in size (~400kDa to >2,000 kDa) (IOVS 2012, EAbstract 440), while one or two RAN molecules may bind to a single
VEGF dimer, forming 1:1 or 2:1 complexes. We compared the
binding patterns of BEV, RAN, and AFL to ARPE-19 cells and
HUVEC, and investigated the mechanisms underlying differential
binding of drug:VEGF complexes to cells.
Methods: ARPE-19 cells were treated with equimolar concentration
of BEV, RAN, and AFL. Cell surface bound VEGF blockers were
detected by immunofluorescence staining. Cells and culture media
were collected for TaqMan and ELISA of VEGF165 RNA and
protein levels. For acute binding assay, ARPE-19 cells were
incubated with serial dilutions of VEGF blocker alone, VEGF
blocker +VEGF121 or VEGF165 followed by immunostaining of cell
surface bound VEGF blockers. ARPE-19 cells were incubated with
serial dilutions of soluble neuropilin-1 (NRP-1) or heparin with
VEGF165 and BEV. Cells were then processed as above for signal
analysis. Binding studies were conducted on HUVEC using the same
protocols.
Results: BEV showed a time-dependent increase in binding to
ARPE-19 cells coincident with the up-regulation of VEGF165 in
cultures. In contrast, AFL showed no specific binding, while RAN
exhibited very light binding to RPE cells. Acute binding of BEV to
ARPE-19 cells was noted in the presence of exogenous VEGF165,
but not VEGF121. At the same concentrations, no staining was
observed with AFL or RAN + VEGF165 or VEGF121. Similar
patterns of cell surface binding of BEV were noted in cultured
HUVEC. Exogenous heparin blocked the binding of BEV:VEGF165
complexes to ARPE-19 cells, whereas, NRP-1 blocked the binding of
BEV:VEGF165 complexes to HUVEC.
Conclusions: These results show that BEV can bind to cultured
ARPE-19 cells and HUVEC. Acute binding of BEV:VEGF165
complexes occurs at molar ratios previously shown to favor the
formation of high molecular weight immune like complexes of
BEV:VEGF. Neither AFL nor RAN bind appreciably to RPE or
endothelial cells under identical conditions. Binding of
BEV:VEGF165 complexes to RPE or HUVEC appears to be
mediated via interactions of bound VEGF165 with lower affinity
binding partners, heparin or NRP-1.
Commercial Relationships: Yang Liu, Regeneron Pharmaceuticals,
INC (E); Terra B. Potocky, Regeneron Pharmaceuticals (E); Jingtai
Cao, Regeneron Pharmaceuticals, Inc. (E); Joel Martin, Regeneron
Pharmaceuticals (E); Nicholas Papadopoulos, Regeneron
Pharmaceuticals (E); Stanley J. Wiegand, Regeneron
Pharmaceuticals, Inc (E)
A Mechanism-Based Binding Model for the Pharmacokinetics
and Pharmacodynamics (PK-PD) of RN6G (PF-04382923), a
Humanized Monoclonal Antibody against Amyloid β Peptides, in
Subjects with Dry, Age-Related Macular Degeneration Including
Geographic Atrophy
Kai H. Liao1, Pamela D. Garzone2, Sangeetha S. Bollini2, Philip M.
Fanning2, Gilbert Wong2, Xu Meng1. 1Pfizer Inc, San Diego, CA;
2
Pfizer Inc, South San Francisco, CA.
Purpose: RN6G (PF-04382923) is a humanized IgG2Δa monoclonal
antibody that binds with high affinity to amyloid β (Aβ) peptides Aβ140 and Aβ1-42 at the free C-terminal region. It is under development as
treatment for patients with geographic atrophy (GA) related to dry
age-related macular degeneration (ARMD). Data from a single
ascending dose (SAD) and emerging data from a multiple ascending
dose (MAD) study in subjects with dry ARMD showed distinctive
RN6G PK profiles characterized by a dose-dependent distribution
phase, which cannot be fully explained by the observed PD (Aβ)
concentration profiles in plasma. The objective of this analysis was to
develop and evaluate a mechanism-based predictive population PKPD model that describes the data from these Phase I studies in
subjects with early and with advanced dry ARMD.
Methods: The database included 714 RN6G and 1178 Aβ1-x (n=864
from Gyros assay, n=314 from liquid chromatography-tandem mass
spectrometry) samples from the Phase I studies (B1181001 and
B1181002). These data were simultaneously analyzed via nonlinear
mixed-effects modeling with NONMEM, v 7.1.2.
Results: The time course of RN6G and its interplay with Aβ1-x were
delineated in the mechanism-based PK-PD model. By incorporating
an Aβ1-x-rich compartment, the model was able to adequately
describe the dose-dependent distribution phase of RN6G, as well as
the Aβ1-x profiles observed in plasma. The initial model from the
SAD study was used to select the dosage regimen for the MAD
study. The preliminary PK/PD data from the MAD study provided
verification of the predictions by the model, which was then used for
the dose regimen selection for the proof-of-concept study. Simulation
results suggest that the level of sequestration for free Aβ in plasma
might not be indicative of that in tissues with higher Aβ1-x
concentration such as choroid or vitreous humour.
Conclusions: A mechanism-based binding model was developed to
adequately describe the distinctive disposition profiles of RN6G and
the observed Aβ1-x concentration profiles in plasma. This modeling
work provided the rationale for dosage regimens for RN6G early
clinical trials.
Commercial Relationships: Kai H. Liao, Pfizer Inc (E); Pamela D.
Garzone, Pfizer (E); Sangeetha S. Bollini, Pfizer (E); Philip M.
Fanning, Pfizer (E); Gilbert Wong, Pfizer, Inc (E); Xu Meng, Pfizer
Inc (E)
Short Term Effect of Intravitreal Aflibercept Injection In
IntraOuclar Pressure
Kasra Attaran-Rezaei, Carmel Moazez, Clive H. Sell, Alan J.
Gordon, Rahul Reddy, Henry M. Kwong, Stephen De Souza, Matthew
C. Ziemianski, Belinda Shirkey, Shepard Bryan. Ophthalmology &
Visual Science, Associated Retina Consultants, Phoenix, AZ.
Purpose: Age-related macular degeneration (AMD) is the leading
cause of vision loss in patients older than 55 years, in the United
States. The most severe vision loss occurs in the neovascular form of
AMD. Intravitreal Aflibercept injection has been tried for the
treatment of Wet form of AMD. In this study we evaluate the shortterm effect of Intravitreal Aflibercept injections on intraocular
pressure.
Methods: 30 patients with Wet form of age related macular
degeneration, who have the inclusion criteria for intravitreal
Aflibercept injection were enrolled in the study. Aflibercept with
dose of 2 mg administered by intravitreal injection every 4 weeks, for
three months. The intraocular pressure was measured before each
injection with Tonopen (Reichert, NY, USA) in every visit.
Results: 30 patient enrolled in the study, 20 female and 10 male with
average age of 80 years. The IOP measurement were 17.35 mmhg at
the beginning of the study and 15.95, 16.00 and 14.85 mmhg in the
next three months visits. A one-way within-subjects ANOVA was
conducted with the factor being time (baseline, 1st month, 2nd month
and 3rd month and the dependent variable being intraocular pressure
(IOP). ( p = 0.07).
Conclusions: Aflibercept has been prescribed as a new and effective
treatment for wet AMD. In this study we show that intarvitreal
injections of Aflibercept does not increase the intraocular pressure in
a short term. We suggest that intarvitreal Aflibercept can be use
safely in patient with history of glaucoma.
Commercial Relationships: Kasra Attaran-Rezaei, None; Carmel
Moazez, None; Clive H. Sell, None; Alan J. Gordon, None; Rahul
Reddy, None; Henry M. Kwong, None; Stephen De Souza, None;
Matthew C. Ziemianski, None; Belinda Shirkey, None; Shepard
Bryan, None
Support: Associated retina consultant research support
In vivo imaging of fluorescent probes linked to antibodies against
human and rat vascular endothelial growth factor (VEGF)
Johanna Meyer1, Alexander Cunea1, Pia Welker2, Kai Licha2,
Dagmar Sonntag-Bensch1, Steffen Schmitz-Valckenberg1, Frank G.
Holz1. 1Department of Ophthalmology, University of Bonn, Bonn,
Germany; 2Mivenion GmbH, Berlin, Germany.
Purpose: To investigate fluorescent molecular probe linked to
antibodies against VEGF for in vivo imaging of VEGF.
Methods: Bevacizumab (a humanized monoclonal antibody, Roche),
antiRatVEGF (a polyclonal antibody against rat VEGF164, R&D
Systems) and B20-4.1.1 (a polyclonal antibody against human and rat
VEGF, Genentech) were covalently attached to indocyanine green
(ICG), a near-infrared dye, yielding soluble conjugates. Binding
properties were assessed by an in vitro proliferation assay. Using
confocal scanning laser ophthalmoscopy (cSLO), in vivo reflectance
and fluorescence imaging was performed in Dark Agouti rats that had
undergone argon laser photocoagulation to induce choroidal
neovascularisations (CNV). Retinal uptake and fluorescence were
recorded following intravenous and intravitreal injection of the dye
conjugates for up to 100 days.
Results: In vivo imaging before dye application showed ill-defined
retinal lesions at day 7. Immediately following intravenous and
intravitreal injection a strong fluorescence was visible. Twenty-four
hours following injection an accumulation of the antibody-conjugate
at the site of the roundish laser lesions for all antibody-conjugates
were observed. Furthermore, multiple fluorescent spots were visible
up to 35 days following intravenous injection of Bevacizumab-ICG
and for up to 60 days following intravitreal injection of
Bevacizumab-ICG, B20-4.1.1-ICG and antiRatVEGF-ICG. No
fluorescent spots occurred after intravenous injection of B20-4.1.1ICG or antiRatVEGF-ICG. Over time, a continuous decrease of the
fluorescence intensity was observed for all antibody-conjugates.
Conclusions: Pharmacokinetics of fluorescent-labeled bevacizumab,
antiRatVEGF and B20-4.1.1 can be investigated in vivo following
intravenous and intravitreal injection. Strong accumulations in the
site of the laser lesion were observed for all antibody-conjugates.
This novel molecular imaging approach of VEGF may be applicable
in patients for earlier diagnosis and more refined individualized antiVEGF therapies with the aim of optimizing functional outcomes.
Commercial Relationships: Johanna Meyer, Heidelberg
Engineering (F), Carl-Zeiss Meditec AG (F); Alexander Cunea, Carl
Zeiss Meditec (F), Heidelberg Engineering (F); Pia Welker,
mivenion (E); Kai Licha, mivenion GmbH (E); Dagmar SonntagBensch, Heidelberg Engineering (F), Carl-Zeiss Meditec AG (F);
Steffen Schmitz-Valckenberg, Heidelberg Engineering (F), Optos
(F), Carl Zeiss Meditec (F), Heidelberg Engineering (R), Genentech
(C), Novartis (C), Novartis (R), Roche (R), Novartis (F); Frank G.
Holz, Acucela (C), Allergan (C), Genentech (F), Heidelberg
Engineering (F), Zeiss (F), Novartis (F), Novartis (C), Optos (F),
Merz (C), Bayer (F), Bayer (C), Boehringer Ingelheim (C)
Support: German Ministry of Education and Research, BMBF FKZ
13N10285
Generation of Combination PDGF / VEGF-antagonist ECT
devices
Vincent Ling, Arne Nystuen, Susan A. Elliott, Lisa Orecchio, Brenda
Dean, Konrad Kauper, Weng Tao. Biological Sciences, Neurotech
USA, Cumberland, RI.
Purpose: PDGF and VEGF are potent pro-angiogenic molecules
implicated in neovascular diseases. Anti-VEGF therapies that inhibit
blood vessel growth have been successfully used in the treatment of
certain cases of wet AMD, but with only ~30% of treated patients
gain 3 lines of vision. It is hypothesized that regression of CNV may
be improved with the addition of anti-PDGF therapies that target the
pericyte cellular scaffolding surrounding blood vessels. To that end,
we generated PDGFR-Fc producing cell lines and ECT devices for
the potential treatment of wet AMD. We also explored the concept of
combination delivery of PDGFR-Fc and NT-503 VEGFR-Fc from an
ECT device system.
Methods: PDGFR-Fc cell lines were produced by standard
transfection into NTC-200 cells, followed by by screening for highest
recombinant protein productivity via ELISA. These new NT-506
PDGFR-Fc cell lines and Neurotech NT-503 VEGFR-Fc cell lines
were encapsulated by loading into 7.2 mm ECT devices either
separately, or in combination as a 50% mixture of each cell line.
Devices were implanted into rabbits for 1 month, after which device
explants were assessed for secretion of PDGFR-Fc and VEGFR-Fc
molecules.
Results: Stable PDGFR-Fc cell lines were produced that secreted ~
15 picogram per cell per day (pcd). PDGFR-Fc / PDGF binding was
established by direct binding and solution-phase ELISA assays on
purified PDGFR-Fc. Bioactivity of PDGFR-Fc was demonstrated by
inhibition of PDGF-based cellular migration assays. In-vivo PDGFRFc ECT implants exhibited sustained production of PDGFR-Fc
protein after one month, as based on explant device culture and
vitreal PDGFR-Fc accumulation. Mixed-cell line ECT implants were
also effective in the simultaneous production of PDGFR-Fc and
VEGFR-Fc, proportional to initial cell loading.
Conclusions: Here we show the ability of ECT to sustain production
of a PDGF antagonist which may have practical application in
conjunction with anti-VEGF therapy. As an initial proof-of-concept,
we show that a mixed-cell line ECT implant affords simultaneous
delivery of PDGF / VEGF antagonists and may represent a potent
future treatment modality for wet AMD.
Commercial Relationships: Vincent Ling, Neurotech
Pharmaceuticals (E); Arne Nystuen, NeurotechUSA, Inc (E); Susan
A. Elliott, Neurotech (E); Lisa Orecchio, Neurotech USA, Inc. (E);
Brenda Dean, Neurotech USA, Inc (E); Konrad Kauper, Neurotech
Pharmaceuticals (E); Weng Tao, Neurotech (E)
MORPHOLOGICAL STUDY OF THE GANGLION CELL
LAYER OF RABBITS AFTER INTRAVITREAL INJECTION
OF MYCOPHENOLIC ACID
Andre M. Liber1, Rafael M. Ferraro1, Dora F. Ventura1, Francisco
Max Damico2, 1, Christina Joselevitch1. 1Psychology Experimental,
Universidade de Sao Paulo, Sao Paulo, Brazil; 2medical school,
universidade de são Paulo, São Paulo, Brazil.
Purpose: To investigate the effects of different concentrations of
mycophenolic acid (MPA) injected in the vitreous of New Zealand
albino rabbits.
Methods: Rabbits were anesthetized with ketamine 10% and
xylazine 2%. Intravitreal injections were performed after
paracentesis. Right eyes were injected with 1 or 10 mg MPA, and left
eyes were injected with 0.1 mL of vehicle (polysorbate 80). After 30
days, animal were sacrificed by intravenous injection of sodium
pentobarbital (70 mg/kg). After enucleation, retinas were isolated and
fixed in 10% formalin for 48 h and kept in 4% formalin. Isolated
retinas were flattened on gelatinized slides and processed for Nissl
staining using cresyl violet as dye. Sample retinal fields were
photographed throughout the retina at 1 mm intervals with a digital
camera attached to a microscope. Neurons in the ganglion cell layer
(GCL) within each field were counted using NIH Scion Image 2.0
software. The average density of cells was estimated for each retina
along the nasotemporal and dorsoventral axes and the data from each
group were compared with those of the corresponding control.
Results: Cell density values along the dorsoventral and nasotemporal
axes in the eyes injected with MPA did not differ from those injected
with either salineor vehicle. Highest mean densities (±SD) in the
dorsoventral axis were 4,094±1,856 (saline), 4,396±1,899 (vehicle),
2,901+1,030 (MPA 1 mg) and 5,156 cells/mm2 (MPA 10 mg), and
those in the nasotemporal axis were 2,646±2,085 (saline),
3,612±2,044 (vehicle), 2,678±1,329 (MPA 1 mg) and 3,145±1,709
cells/mm2 in the nasotemporal axis. All values are very close to those
reported in literature for albino rabbits.
Conclusions: MPA at 1 and 10 mg does not cause significant
decrease in GCL density in New Zealand albino rabbits 30 days after
intravitreal injection. Our results suggest multiple interpretations: (a)
the doses of MPA used are safe to the retina, (b) the GCL recovers by
day 30 post intravitreal injection, (c) damage starts more than 30 days
after treatment, or (d) the method used is not sensitive enough to
measure retinal toxicity.
Commercial Relationships: Andre M. Liber, None; Rafael M.
Ferraro, None; Dora F. Ventura, None; Francisco Max Damico,
None; Christina Joselevitch, None
Development and Characterization of a 3-Dimensional Human
Corneal Full Thickness Tissue Model
Yulia Kaluzhny, Laurence d’Argembeau-Thornton, Helena
Kandarova, Pat Hayden, Mitch Klausner. MatTek Corporation,
Ashland, MA.
Purpose: The FDA and other regulatory agencies require ocular
irritation testing of ophthalmologic pharmaceuticals and many
consumer products. Although widely used, animal testing has
limitations due to interspecies differences, variability, high cost, and
animal welfare issues. A highly reproducible, cost effective, human
cell-based in vitro corneal tissue model would be of high utility for
ocular irritation testing and ophthalmic drug development.
Methods: Using primary culture and state of the art tissue
engineering techniques, we have developed a human corneal full
thickness (CFT) tissue model which includes epithelial, stromal, and
endothelial layers. This model has been characterized in terms of
histology and by testing a battery of test articles (TA, n=65) spanning
the range from ocular corrosives and severe irritants to non-irritants
(NI). In vivo irritancy levels were obtained from a published database
and in vitro effects were determined using the MTT tissue viability
assay and transepithelial electrical resistance (TEER) measurements.
Results: Using a tissue viability cutoff of 50%, the ocular CFT
(OCFT) model was able to detect irritants with 100.0% sensitivity
and non-irritants with 75.0% specificity (overall accuracy = 87.7%).
In addition, the OCFT model was used to assess tissue recovery
following exposure to increasing concentrations of surfactants and
TA spanning the range of irritancy. Significant recovery at 24 and 48
hours was observed for NI chemicals. For the same 65 TA, an ex
vivo assay, the Bovine Corneal Opacity and Permeability (BCOP),
had 87.9%, 68.8%, and 78.5% assay sensitivity, specificity, and
accuracy, respectively.
Conclusions: The OCFT model will address the needs of ophthalmic
and other formulators who need to screen their products for irritancy
and efficacy. Similar to other in vitro assays, high levels of
reproducibility at a low cost are anticipated. The OCFT model will
facilitate the development and testing of ophthalmic pharmaceuticals
and allow for basic studies related to corneal physiology, wound
repair, and pathologies.
Histological cross-section: Formalin fixed, paraffin embedded, H&E
stained cross-section of OCFT tissue model (4X). 1. Corneal
epithelium (10X); 2. Stroma with embedded keratocytes; 3.
Endothelial layer (20X); 4. Micorporous membrane; 5. Air-liquid
interface; 6. Culture medium fed through the membrane.
Commercial Relationships: Yulia Kaluzhny, MatTek Corporation
(E); Laurence d’Argembeau-Thornton, MatTek Corporation (E);
Helena Kandarova, MatTek Corporation (E); Pat Hayden, MatTek
Corporation (E); Mitch Klausner, MatTek Corporation (E)
Support: NIH SBIR Grant # 9R44ES020074-02
Nutritional supplementation for age related macular
degeneration in Italy
Ilaria Zucchiatti1, Maurizio B. Parodi1, maria vittoria cicinelli2,
Maria Lucia Cascavilla1, Francesco Fasce1, Matteo Prati1,
Francesco Bandello1. 1Department of Ophthalmology, University
Vita-Salute, Scientific Institute San Raffaele, Milan, Milano, Italy;
2
University Vita Salute San Raffaele, Milan, Italy.
Purpose: To evaluate the rate of adherence to nutritional
supplementation prescription in Italian patients affected by advanced
form of age-related macular degeneration (AMD).
Methods: All Italian patients referred to the Department of
Ophthalmology of Scientific Institute San Raffaele Hospital, in Milan
from August to November 2012 were prospectively evaluated.
Dilated funduscopic examinations were performed to classify patients
according to AREDS categories. Inclusion criteria were the presence
of AMD in AREDS category 3 and 4. Exclusion criteria were the
presence of other ocular disorders. Primary outcome measure was the
rate of adherence to the supplementation prescription.
Results: 100 patients were included in the study, meeting the
AREDS criteria for taking nutritional supplementation (95 patients
with AREDS category 4, and 5 with category 3). Thirty-six patients
(36%) were taking oral supplementation, 33 of them (94%) assuming
is 1 tablet/day.
Oral supplementation was recommended by the personal
ophthalmologist in all patients assuming it (100%). Fifty-eight
patients (58%) declared to have had no information from their
ophthalmologist, whereas fifteen (15%) declared to have partial
knowledge about the effects of supplementation in AMD.
Conclusions: Our data reveal that Italian patients with AREDS
category 3 and 4 have a low adherence to nutritional
supplementation. More than 70% of patients have not been
adequately informed by their ophthalmologist regarding the potential
benefits of oral supplementation for AMD. Italian ophthalmologists
should stress the importance of nutritional recommendation.
Commercial Relationships: Ilaria Zucchiatti, None; Maurizio B.
Parodi, None; maria vittoria cicinelli, None; Maria Lucia
Cascavilla, None; Francesco Fasce, None; Matteo Prati, None;
Francesco Bandello, ALLERGAN Inc. (S), NOVARTIS
PHARMACEUTICALS CORPORATION (S), FARMILA-THEA
(S), BAYER SCHERING PHARMA (S), PFIZER Inc. (S), ALCON
Inc. (S), BAUSCH AND LOMB (S), GENENTECH Inc. (S),
ALIMERA SCIENCES Inc. (S), SANOFI AVENTIS (S),
THROMBOGENICS (S)
Feasibility of Cryopreservation of the Encapsulated Cell
Technology Devices
Cahil McGovern1, John D. Duggan3, Crystal Cortellessa1, Sandy
Sherman1, Melissa Stiles2, Konrad Kauper4, Allyse Mazzarelli6, Weng
Tao5. 1Manufacturing, Neurotech, Cumberland, RI; 2Quality Control,
Neurotech, Cumberland, RI; 3Quality Assurance, Neurotech,
Cumberland, RI; 4Engineering, Neurotech, Cumberland, RI;
5
Research and Development, Neurotech, Cumberland, RI; 6Histology,
Neurotech, Cumberland, RI.
Purpose: To investigate the feasibility of cryopreservation as a
potential means of Encapsulated Cell Technology (ECT) device
storage after manufacturing.
Methods: An NTC-200 cell line secreting an antiangiogenic factor
was used for the encapsulation. Cells were expanded in a growth
media, harvested, and formulated at a specific density in a serum free
media, either with or without various concentrations of a
cryopreservative. Prepared cells were loaded into Neurotech
ophthalmic ECT devices. Control devices were kept at 37C while
cryopreserved devices were frozen at a rate of 1C/min and stored in
liquid nitrogen (vapor phase) for 2 weeks. Cryopreserved devices
were then removed from liquid nitrogen, rapidly thawed, washed in a
balanced salt solution, and incubated in a serum free media for 6
days. The devices were then cultured in fresh serum free media for 24
hours. The condition media samples were collected and assayed for
the antiangiogenic factor by ELISA. The devices were subsequently
analyzed for metabolic activity using the CCK-8 assay (Dojindo) and
then subjected to either total DNA using the Hoefer DyNA Quant
200 fluorometer (Pharmacia) or histological analysis using standard
hematoxylin and eosin staining techniques.
Results: During the pre-encapsulation culture period, the cells
maintained a typical and consistent cuboidal morphology. Cells
encapsulated in devices in the fresh media group and cryopreserved
group both maintained a normal morphology with a high density of
healthy cells distributed throughout the device during the testing
period. No deterioration of the device capsule was observed by visual
inspection. Factor secretion levels, cell metabolic activity, and the
overall device cell load between fresh and cryopreserved devices
were also comparable. Furthermore, secreted proteins with expected
molecular weights were detected in the culture media from both
device groups. No differences in protein size or degradation between
the fresh and cryopreserved device groups were detected by Western
blot.
Conclusions: These data suggested that the cryopreservation is a
feasible method of extending the shelf life of ECT products. Longer
term and in vivo studies of cryopreserved devices are currently under
investigation. Optimizing cryopreservation techniques in the
manufacturing process will allow for the long term storage of ECT
product and may greatly enhance accessibility of ECT products
worldwide.
Commercial Relationships: Cahil McGovern, Neurotech (E); John
D. Duggan, Patent U.S.S.N. 61/653,191 (P), Neurotech USA, Inc
(E); Crystal Cortellessa, U.S.S.N. 61/653,191 (P), Neurotech USA
Inc. (E); Sandy Sherman, Neurotech USA Inc (E); Melissa Stiles,
None; Konrad Kauper, Neurotech Pharmaceuticals (E); Allyse
Mazzarelli, Neurotech USA (E); Weng Tao, Neurotech (E)
Continuous Intraocular Drug Delivery over 5 ½ Years: Ciliary
Neurotrophic Factor (CNTF)Production by Encapsulated Cell
Technology Implants Treating Patients with Retinitis Pigmentosa
and Geographic Atrophy
Konrad Kauper, Cahil McGovern, Paul Stabila, Sandy Sherman,
Pam Heatherton, Brenda Dean, Crystal Cortellessa, Alice Lee, Weng
Tao. Core Technology Development, Neurotech USA, Cumberland,
RI.
Purpose: Continuous intraocular delivery of biotherapeutics for
treatment of chronic retinal diseases remains a major hurdle for drug
development. We have previously reported the pharmacokinetics of
CNTF delivered over 2 years by an intraocular encapsulated cell
technology (ECT) implant in patients with retinitis pigmentosa (RP)
and geographic atrophy (GA). This is a follow-up evaluation of
patients implanted up to a 5.5 - year period.
Methods: All study patients received an ECT-CNTF implant,
designated NT-501, in one eye. For the phase 1 RP (CNTF1) study,
the protocol mandated explant of all patients at 6 months. For the
phase 2 studies, including phase 2 GA (CNTF2), and phase 2 late and
early stage RP (CNTF3, and CNTF4), explants were optional and
occurred at 12, 18 and 24 months. Several additional patients from
the CNTF4 study chose to be explanted at 30 (n=6), 44 (n=1), 54
(n=1) and 66 (n=1) months post implant. The rate of CNTF secretion
from the explants and the corresponding vitreous CNTF levels, if
available, were evaluated at each time point. Serum samples from
these patients were evaluated for CNTF, anti-CNTF antibodies and
antibodies to the encapsulated cells.
Results: Cumulatively, the data demonstrates NT-501 implants
produce CNTF continuously over a 5.5 year period. The range of
explant CNTF production rate at each time point was statistically
equivalent between the 0.5 year and 5.5 year implant period. The
mean rates of CNTF production over this period varied between
approximately 1 ng/day to 2 ng/day, a rate shown to be effective in
protecting cone photoreceptors in RP patients (Talcott et el. IOVS,
2011). Encapsulated cells, subjectively evaluated following H&E
staining of explant capsules, were viable and remained at a high
population density, similar to the pre-implant condition. CNTF, antiCNTF antibodies and antibodies to the encapsulated cells were not
detected in the serum of patients.
Conclusions: This follow-up study demonstrates that the intraocular
ECT implant continues to maintain a favorable pharmacokinetic
profile for the treatment of chronic retinal degenerative diseases
without systemic exposure for over a half decade.
Commercial Relationships: Konrad Kauper, Neurotech
Pharmaceuticals (E); Cahil McGovern, Neurotech (E); Paul Stabila,
Neurotech Pharmaceuticals (E); Sandy Sherman, Neurotech USA
Inc (E); Pam Heatherton, None; Brenda Dean, Neurotech USA, Inc
(E); Crystal Cortellessa, U.S.S.N. 61/653,191 (P), Neurotech USA
Inc. (E); Alice Lee, Neurotech USA, Inc. (E); Weng Tao, Neurotech
(E)
Effect of Apolipoprotein A1-mimetic peptides on cell viability of
cultured retinal pigment epithelial cells
Yoko Miura1, 2, Ingrid Fritz1, Salvatore Grisanti1, Martin Rudolf1.
1
Department of Ophthalmology, University of Luebeck, Luebeck,
Germany; 2Institute of Biomedical Optics, University of Luebeck,
Luebeck, Germany.
Purpose: Previous studies of our research group have shown that
ApoA-I mimetic peptides like D-4F and L-4F are able to reduce
pathological lipid deposits in and on top of Bruch’s membrane
(Rudolf et al. ARVO 2011, 2013). Since the function of the retinal
pigment epithelium (RPE) plays a central role in photoreceptor health
and function we evaluate the effect of these peptides on RPE cell
viability in vitro.
Methods: Third passage porcine RPE cell cultures were treated with
different concentrations of L-4F or its stereoisomer D-4F (0; 0.1; 10;
100; 500; 1000; 2000; 4000 µg/ml) for 48 hrs. Nonfunctional
scrambled L-4F (sL-4F) served as a control. MTT assays
(Dimethylthiazol diphenyltetrazolium bromide) were performed to
analyse the peptides’ cytotoxicity on RPE cells. RPE cell
proliferation activity was assessed by measuring the optical density at
577 nm using a photometric multiplate reader. Further test runs were
performed in smaller concentration steps to detect the maximum
tolerated dose of these peptides.
Results: D-4F and L-4F showed no significant effect in MTT assays
with concentration ≤ 10 µg/ml. With higher concentrations, L-4F ≤
375 µg/ml and ≤ 125 µg/ml for D-4F induced a significant increase
of optical density (p<0.01). L-4F concentrations ≥ 500 µg/ml reduced
significantly RPE cell proliferation (p<0.01). The same effect was
detected for D-4F at concentrations ≥ 250 µg/ml. The control peptide
sL-4F did not show any significant difference on RPE cell
proliferation activity.
Conclusions: Our results demonstrated cytotoxic effects in RPE cell
culture if exposed to concentrations of L-4F ≥500 µg/ml and ≥ 250
µg/ml of D-4F. In RPE cell cultures concentrations up to 10 µg/ml
can be considered safe for both peptides. Additional tests are needed
to explain sufficiently the increase of optical density for
concentrations between 10 µg/ml and the mentioned toxicity levels
above.
Commercial Relationships: Yoko Miura, None; Ingrid Fritz,
None; Salvatore Grisanti, Novartis (C), Allergan (C), Bayer (C),
Pfizer (C), Thrombogenics (C); Martin Rudolf, UAB Research
Foundation (P)
Beta cyclodextrins bind and remove Lipofuscin Bisretinoids from
RPE
Marcelo M. Nociari. Ophthalmology, Weill Cornell Medical College,
New York, NY.
Purpose: Lipofuscin bisretinoids (LBs) accumulate irreversibly in
the lysosomes of Retinal Pigment Epithelium (RPE) cells with age
and in some genetic diseases, such as Stargardt and Best diseases.
Excessive LB accumulation is toxic and causes retinal degeneration.
The goal of this study is to develop a small drug to reduce the levels
of LBs from RPE.
Methods: Using a novel solvatochromic fluorescence shift screening
assay, we just developed, we searched for small molecules with
ability to bind the most abundant LB, A2E.
Results: We have identified beta cyclodextrins (β-CDs), cyclic rings
formed by 7 glucose residues, as small drugs that bind A2E. We
show that treatment with beta-CDs stabilizes A2E by preventing its
oxidation, and reduces bisretinoid content from lipofuscin granules in
RPE. Molecular modeling of the complex indicate strategies to
improve the LB binding of CDs. Furthermore, we tested the removal
properties of CDs in eyes of ABCA4/RDH8 DKO animals, a mouse
model for LB-driven retinal degeneration, and observed a reduction
in bisretinoid levels larger than 60%.
Conclusions: These results provide important clues to develop a
novel small drug therapy to preserve and/or improve retinal function
in individuals with LB driven retinal degeneration.
Commercial Relationships: Marcelo M. Nociari, None
Safety of implantation of the NT-503 device in patients with
Choroidal Neovascularization secondary to Age-related Macular
Degeneration
Jose Luis Guerrero-Naranjo1, Hugo Quiroz-Mercado3, Gustavo
Sanchez-Bermudez1, Fernando Schoonewolff1, Samantha Salinas
Longoria1, Rafael Romero Vera1, Weng Tao2, Richard L. Beckman2,
Virgilio Morales-Canton1. 1Retina, Asoc Para Evitar la Ceguera en
Mexico, Mexico City, Mexico; 2Neurotech, Inc., Cumberland, RI;
3
Denver Health Medical Center, Denver, CO.
Purpose: To determine the safety and incidence of complications in
the surgical implantation of a low dose NT-503 device (Encapsulated
Cellular Delivery System producing soluble VEGF receptor Neurotech, USA) in patients with choroidal neovascularization
(CNV) secondary to age related macular degeneration (ARMD).
Methods: Single-arm, multi-center, open-label Phase I study. 13
patients with a diagnosis of CNV secondary to ARMD were enrolled
in the study at our center from March 2010 to April 2011. Each
patient provided informed consent and was screened for eligibility by
means of complete eye examination, fluorescein angiography and
optical coherence tomography. Implantation was performed on
eligible patients under retrobulbar anesthesia. An inferior temporal
quadrant peritomy was performed followed by a 2mm sclerotomy at
3.75mm from the limbus, and the device was implanted and fixated
to the sclera with 9-0 Prolene. The wound was closed with
interrupted 9-0 Nylon sutures, and the peritomy was repaired with
interrupted 7-0 vicryl sutures. Topical steroids and antibiotics were
administered after the surgery. Follow up was at 1 day, 1 week, 1
month, then every 3 months out to 1 year and included visual acuity
(VA), intraocular pressure (IOP), biomicroscopy, indirect
ophthalmoscopy, fundus photography, optical coherence tomography
(OCT), and fluorescein angiography. Case Report Forms (CRF) were
used to report all data and adverse events.
Results: A total of 13 patients, 6 males and 7 females, with a mean
age of 71.9 years, were included. All patients were implanted
satisfactorily with no intraoperative complications. The most
commonly reported post operative complications were retinal
hemorrhage (N=4), ocular hyperemia (N=4), ecchymosis (N=2), and
conjunctival hemorrhage (N=1). There was one case of uveitis that
responded well to topical steroid treatment and the only SAE reported
was a death not related to treatment that occurred in one patient with
previous diagnosis of prostate cancer.
Conclusions: The present study shows that the NT-503 device can be
implanted safely in humans with choroiodal neovascularization
secondary to AMD and that the Encapsulated Cellular Delivery
System for soluble VEGF receptor warrants further investigation at
higher dose levels and in a larger cohort of patients.
Commercial Relationships: Jose Luis Guerrero-Naranjo,
Neurotech (F); Hugo Quiroz-Mercado, Allegro Pharmaceutical (C);
Gustavo Sanchez-Bermudez, None; Fernando Schoonewolff,
None; Samantha Salinas Longoria, None; Rafael Romero Vera,
None; Weng Tao, Neurotech (E); Richard L. Beckman, Neurotech
(E); Virgilio Morales-Canton, Clearside Biomedical (F)
Suprachoroidal Microinjection of Bevacizumab is Well Tolerated
in Human Patients
Virgilio Morales-Canton1, Jans J. Fromow-Guerra1, Samantha
Salinas Longoria1, Rafael Romero Vera1, Michelle Widmann2,
Samirkumar R. Patel2, Benjamin Yerxa1. 1The Asociación Para Evitar
La Ceguera (APEC), Mexico City, Mexico; 2Clearside Biomedical,
Alpharetta, GA.
Purpose: To evaluate the safety and tolerability of a single
microneedle injection of bevacizumab into the suprachoroidal space
(SCS) using a Clearside Biomedical proprietary microneedle
Methods: Four adult patients with choroidal neovascularization
(CNV), secondary to wet age-related macular degeneration (AMD),
were enrolled in a phase 1, single-center, open-label study. Each
patient provided informed consent and was screened for eligibility.
Following application of topical anesthesia, the patient was
administered a single unilateral injection of 100 µL bevacizumab
(Avastin®) into the SCS using an 850 µm 33 gauge Clearside
Biomedical microneedle. The microneedle was inserted into the
sclera approximately 8-12 mm posterior to the limbus in the superior
temporal quadrant. Treated patients remained in the clinic for 4 hours
for observation and then returned multiple times for follow-up during
a 2 month period. Major safety examinations included intraocular
pressure (IOP), angiograms, biomicroscopy, indirect
ophthalmoscopy, fundus photography, optical coherence tomography
(OCT), visual acuity (VA), and assessment of pain
Results: Preliminary data shows that four patients were successfully
dosed into the SCS which was confirmed via ophthalmoscope
immediately following injection. A moderate level of pain was
recorded for the administration. There were no unexpected or serious
adverse events related to bevacizumab or method of administration
on ophthalmic examinations. No negative effect on IOP was noted in
any patient. No patients required rescue therapy or reinjection during
the two months following treatment
Conclusions: Preliminary data suggests that the SCS can be
successfully and safely dosed via the Clearside Biomedical
proprietary microneedle using only topical anesthesia. These data
show that 100 µL bevacizumab can be delivered into the SCS without
unexpected or serious adverse events
Commercial Relationships: Virgilio Morales-Canton, Clearside
Biomedical (F); Jans J. Fromow-Guerra, None; Samantha Salinas
Longoria, None; Rafael Romero Vera, None; Michelle Widmann,
Clearside Biomedical, Inc. (E); Samirkumar R. Patel, Clearside
Biomedical (E), Clearside Biomedical (I), Clearside Biomedical (P);
Benjamin Yerxa, Clearside Biomedical (I)
369 Pharmacological Targets for Eye Disease: Present and
Future
Tuesday, May 07, 2013 2:45 PM-4:30 PM
Localization and Activity of Histone Deacetylases in the Retina
Oday Alsarraf, Jie Fan, C. James Chou, Craig E. Crosson.
Ophthalmology - Storm Eye Institute, Medical University of South
Purpose: The increase in histone deacetylase (HDAC) activity and
resulting dysregulation of protein acetylation is an integral event in
retinal degenerations associated with ischemia and ocular
hypertension. The purpose of the current study is to determine
activity profiles and distribution of individual HDACs in the retina,
and if the selective modulation of individual isoforms can limit
retinal injury.
Methods: Isolated mouse retinas were evaluated for class-I and II
HDAC activity using a class-specific fluorometric enzymatic assay.
The activities of individual HDAC isoforms were determined by the
addition of selective isoform inhibitors and gene deletion. Retinal
localization and relative abundance of individual HDAC isoforms
was determined by immunohistochemistry and Western blotting. In
selected knockout and wild type mice, electroretinogram (ERG)
analysis was used to evaluate responses to 45-minutes of ischemic
retinal injury at 7 days post-injury as compared to baseline values.
Results: Although class-II HDACs were detected by Western blot
analysis in retinas from control mice, class-I HDACs accounted for
all the measureable activity. Analysis of individual class-I isoforms
demonstrated that HDAC1 and 2 accounted for 34 ± 3.5% of total
activity, and HDAC3 for the remaining 66 ± 5.0% activity.
Immunolocalization demonstrated that HDAC1, 2 and 3 were
primarily located in the cell bodies of inner nuclear and retinal
ganglion cell layers. However, HDAC1 and 2 also exhibited limited
expression in the outer nuclear layer. In wild-type mice, ERG a- and
b-wave amplitudes from ischemic eyes were significantly reduced by
53.3 ± 6.3% and 59.8 ± 6.9%, respectively, as compared to preischemia baseline values. In HDAC2 heterozygote knockout mice,
baseline ERGs were not altered; however, ERG a- and b-wave
amplitudes from ischemic eyes were significantly greater by 66.4 ±
4.8% and 60.0 ± 7.6%, respectively, as compared to waveforms from
ischemic eyes in wild-type mice.
Conclusions: Although HDACs are detected in most retinal cell
bodies, these studies provide evidence that HDAC1, 2 and 3 isoforms
account for almost all activity and are predominantly expressed in
inner retinal layers. The selective reduction in HDAC2 expression is
sufficient to significantly reduce ischemic retinal injury. These data
support the idea that isoform selective HDAC inhibitors may provide
an efficacious strategy to protect the retina from injury.
Commercial Relationships: Oday Alsarraf, None; Jie Fan, None;
C. James Chou, None; Craig E. Crosson, Alimera Sciences (C),
Lexicon Pharmaceuticals, Inc (R)
Support: NH Grant EY021368-02
Sigma-1 Receptor Stimulation Protects Purified RGCs from
Ischemic Insult through the Phosphorylation of Extracellular
Signal Regulated Kinase 1/2
Brett H. Mueller1, 2, Yong H. Park1, 2, Hai-Ying Ma1, 2, Thomas Yorio1,
2 1
. Pharmacology & Neuroscience, Univ of North Texas Hlth Sci Ctr,
Fort Worth, TX; 2North Texas Eye Research Institute, University of
North Texas Health Science Center, Fort Worth, TX.
Purpose: Sigma-1 receptor activation and mitogen-activated protein
kinases (MAPKs) have been shown to have neuroprotective roles in
protecting retinal ganglion cells (RGCs) from cell death. The purpose
of this study was to determine if sigma-1 receptor stimulation with
pentazocine could promote neuroprotection under conditions of
ischemia through the phosphorylation of extracellular signal
regulated kinase (pERK)1/2.
Methods: Primary RGCs were isolated from P3-P7 Sprague-Dawley
rats and purified by sequential immunopanning using a Thy 1.1
antibody. RGCs were cultured for 7 days before subjecting the cells
to an ischemic insult (0.5% oxygen in glucose-free medium) for 6
hours. During the ischemic insult, RGCs were treated with
pentazocine (sigma-1 receptor agonist) with or without BD1047
(sigma-1 receptor antagonist). In other experiments primary RGCs
were treated with pentazocine, in the presence or absence of
PD98059 (MEK1 inhibitor). Cell survival/death was assessed by
staining with the calcein-AM/ethidium homodimer reagent. Levels of
pERK1/2, total ERK1/2, and beta tubulin expression were determined
with immunoblotting and immunofluorescence.
Results: RGCs subjected to an ischemic insult demonstrated more
than a 40% increase in cell death, compared to untreated controls.
RGCs maintained under ischemia also showed a 50% decrease in
expression of pERK1/2 (p<0.05). Cell death was attenuated when
RGCs were treated with pentazocine under ischemic conditions and
levels of pERK1/2 were increased more than 60% (p<0.05),
compared to untreated RGCs subjected to ischemia. Treatment with
BD1047 abrogated the pentazocine neuroprotection effects, and also
attenuated the increase in levels of pERK1/2 (p<0.05). Finally,
treatment with PD98059 also reversed the pentazocine mediated
neuroprotective effects on RGCs, and abolished the expression of
pERK1/2 (p<0.05).
Conclusions: These results establish a direct relationship between
sigma-1 receptor stimulation and neuroprotective effects under
ischemia through the involvement of the MAPK/Erk1/2 pathway in
purified RGCs. These findings support a role for sigma receptor
agonists as potential neuroprotective agents.
Commercial Relationships: Brett H. Mueller, None; Yong H.
Park, None; Hai-Ying Ma, None; Thomas Yorio, None
Support: Department of Defense: W81XH-10-2-0003
NO Activates Src Family Kinase and Inhibits Na,K-ATPase
Activity in Nonpigmented Ciliary Epithelium
Mohammad Shahidullah, Amritlal Mandal, Guojun Wei, Nicholas A.
Delamere. Physiology, Univ of Arizona, College of Medicine,
Tucson, AZ.
Purpose: We reported earlier that the nitric oxide (NO) donor SNP
reduces aqueous humor secretion in isolated porcine eyes and inhibits
Na,K-ATPase activity in nonpigmented ciliary epithelium (NPE)
through a cGMP/PKG-mediated pathway. Here we examine the
mechanism that links SNP-induced PKG activation to inhibition of
Na,K-ATPase activity.
Methods: Porcine NPE was established in primary culture. Na,KATPase activity was measured as ouabain-sensitive ATP hydrolysis
in NPE lysates. Na,K-ATPase-mediated transport was measured as
ouabain-sensitive potassium (86Rb) uptake by intact NPE
monolayers grown on permeable supports. Protein phosphorylation
was studied by western blot analysis.
Results: SNP and 8-Br-cGMP both inhibited Na,K-ATPase activity
and 86Rb uptake and the Src family kinase (SFK) inhibitor PP2
blocked the responses. SNP and 8-Br-cGMP caused activation of
SFK, ERK1/2 and p-38 MAPK as indicated by increased
phosphorylation. Although it is selective for SFK, PP2 prevented
SNP-mediated activation of ERK1/2 and p-38 MAPK as well as
SFK. The selective ERK1/2 inhibitor U0126 prevented SNP-induced
ERK1/2 and p-38 MAPK activation but not SNP-induced SFK
phosphorylation or Na,K-ATPase inhibition. SNP did not detectably
change Na,K-ATPase α1 abundance on the plasma membrane or
Ser16 phosphorylation of Na,K-ATPase α1. On the other hand, SNPtreated NPE displayed significant phosphorylation of Na,K-ATPase
α1 at Tyr10.
Conclusions: NO elicits phosphorylation of SFK through a
cGMP/PKG pathway and Na,K-ATPase activity reduction appears to
be SFK-mediated. The Na,K-ATPase response is associated with
Tyr10 phosphorylation of Na,K-ATPase α1 protein. NO-elicited
activation of ERK1/2 and p-38 MAPK does not play a role in the
regulation of Na,K-ATPase activity.
Commercial Relationships: Mohammad Shahidullah, None;
Amritlal Mandal, None; Guojun Wei, None; Nicholas A.
Delamere, None
Mechanisms of retinal cell swelling, volume sensing and
mechanical excitotoxicity
Daniel A. Ryskamp1, 2, Andrew O. Jo2, Amber Frye-Gordon2, Shiwani
Chauhan2, Tünde Molnár2, David Krizaj1, 2. 1Interdepartmental
Program in Neuroscience, University of Utah School of Medicine,
Salt Lake City, UT; 2Department of Ophthalmology and Visual
Sciences, Moran Eye Institute, University of Utah School of
Medicine, Salt Lake City, UT.
Purpose: We sought to identify the molecular determinants of retinal
cell volume regulation, volume sensing and cell survival by
characterizing the biophysical, electrophysiological and cellular
properties of osmoregulation in retinal neurons and glia. We
identified the osmosensor, TRPV4, and characterized the signaling
cascades through which osmotic stress differentially regulates the
amplitude and kinetics of calcium signals in retinal ganglion cells
(RGCs) and Müller glia.
Methods: Retinal cells from C57 mice were bathed in isotonic (300
mOsm) and hypotonic saline. Volume and Ca2+ changes were
measured in cells loaded with calcein and fura-2. Apoptosis and cell
survival were measured with TUNEL and Live/Dead assays. Force
transduction was examined with a high-resolution mechanoclamp
technique.
Results: Antibody labeling localized the cation channel TRPV4 to
RGCs and Müller glia. During agonist or hypotonic stimulation
(HTS), TRPV4 response properties in RGCs and Müller glia
exhibited contrasting kinetics and Ca2+ dynamics. Acute HTS dosedependently elevated Ca2+ in both cell types, whereas prolonged
swelling triggered apoptosis. BAPTA-AM (Ca2+ chelator) and
HC067047 (selective TRPV4 antagonist) suppressed apoptosis (e.g.
90% and 62% suppression in RGCs and glia by HC067047) and
swelling. [Ca2+]i levels in Müller glia were selectively elevated by
5’6’-EET, an eicosatrienoic acid derivative of arachidonic acid,
consistent with an indirect activation of glial TRPV4. Inhibition of
phospholipase A2 (PLA2) reduced HTS-induced swelling and Ca2+
elevations in Müller glia but had no effect on volume regulation in
RGCs. In contrast, mechanotransduction in RGCs was intrinsic and
2nd-messenger independent, as shown by high-speed pressure clamp
recordings of stretch-activated cation currents from outside-out RGC
patches (fast, pressure-dependent inward currents at 10-50 mmHg).
Conclusions: Although both Müller glia and RGCs transduce
mechanical stimuli into a TRPV4-dependent cation influx, glial
TRPV4 activation relies on a slow, indirect, PLA2-dependent force
transduction pathway, whereas the neuronal TRPV4 channel is
directly yet transiently activated by lipid bilayer deformation.
Inhibition of TRPV4 was strongly neuroprotective in a model of
retinal edema. This data has implications for our understanding of
retinal volume regulation as well as the molecular events that mediate
cytotoxic edema, ischemic injury and glaucoma.
Commercial Relationships: Daniel A. Ryskamp, None; Andrew
O. Jo, None; Amber Frye-Gordon, None; Shiwani Chauhan,
None; Tünde Molnár, None; David Krizaj, None
Support: Supported by the NIH, DOD, FFB, University of Utah and
an unrestricted award from RPB to the Moran Eye Institute.
Comprehensive Analysis of Prostanoid Receptor Antagonists
Effects on Intraocular Pressure in Ocular Hypertensive Monkeys
Carol B. Toris1, Tara L. Rudebush1, Stacey Wenthur1, David F.
Woodward2. 1Ophthalmology, Univ of Nebraska Medical Ctr,
Omaha, NE; 2Biological Sciences, Allergan, Inc., Irvine, CA.
Purpose: Most of what is known about prostanoid receptor mediated
control of IOP has been learned from studies of prostanoid agonists.
Prostanoid antagonist effects have been studied largely from the
perspective of confirming the pharmacological identity of prostanoid
agonists in producing their effects. This study examines the IOP
effects of 8 prostanoid receptor antagonists in monkeys with
unilateral laser-induced glaucoma to better understand a possible role
of endogenous prostaglandins in the regulation of IOP.
Methods: Eight trained conscious monkeys were studied. Baseline
IOPs were taken by pneumatonometry of both eyes at 9AM. The
study compound was applied topically to the glaucomatous eye at
9:05. IOP measurements were repeated at 10AM, 12PM, 2PM, and
4PM and daily at 9AM for the next three days. After 6 days of
washout, another compound was applied. IOPs were repeated as at
baseline. Randomized dosing and IOP measurements continued until
all compounds were tested. Antagonists dosed at 1% were SC-51322
(EP1), PF-04418948 (EP2), L-826266 (EP3), GW-627368 (EP4),
BW-A868C (DP1), AGN-211336 (prostamide), AS- 604872 (FP),
RO-3244019 (IP). The vehicle was 1% Polysorbate 80.
Results: When compared with vehicle treatment, significant (p<0.05)
findings were higher IOPs with the EP1 and EP2 antagonists at 3
hours, and IP and EP4 antagonists at 7 hours after dosing. The
increases were small and transient. No other antagonist changed IOP
compared to vehicle.
Conclusions: The implication of these findings is that endogenous
prostanoids, predominantly EPs, are involved in decreasing IOP.
Apparently exogenously administered prostanoid antagonists
interfere only with those receptors under endogenous prostanoid
control which are anatomically located such that they maintain an
IOP within a healthy range.
Commercial Relationships: Carol B. Toris, Alcon (F), Allergan
(F); Tara L. Rudebush, None; Stacey Wenthur, None; David F.
Woodward, Allergan Inc. (E)
Support: Research to Prevent Blindness, Allergan
Endothelium Independence of ROCK-Mediated Retinal
Arteriolar Constriction
Luke B. Potts1, Yi Ren2, Lih Kuo1, 2, Travis W. Hein2. 1SBTM, Texas
A&M Health Science Ctr, Temple, TX; 2Scott and White Eye
Institute, Temple, TX.
Purpose: The interplay of vascular endothelium and smooth muscle
is essential for retinal blood flow regulation by changing arteriolar
diameter. Since many retinal diseases are associated with endothelial
dysfunction along with protein kinase C (PKC) activation and
elevated levels of the vasoconstrictor peptide endothelin-1 (ET-1) in
the retina, it is important to understand the role of endothelium in
modulating vascular diameter under these pathophysiological
conditions. We previously reported the importance of Rho kinase
(ROCK) in mediating retinal arteriolar constrictions to PKC
activation and ET-1 and we herein examined the role of endothelium
in these ROCK-mediated vasomotor responses.
Methods: To determine whether endothelium has a role in
modulating retinal arteriolar constriction to endogenous and
exogenous ET-1 and to PKC activation, porcine retinal arterioles
were isolated, cannulated and pressurized for functional study.
Vasoconstrictions to ET-1 precursor big ET-1 (0.1 µM), ET-1 (0.1
nM), and PKC activator phorbol-12,13-dibutyrate (PDBu, 0.1 µM)
with and without the endothelium were examined. Denudation was
achieved by intraluminal injection of air and was confirmed
immunohistochemically, as well as via demonstration of the absence
of dilation to the endothelium-dependent vasodilator bradykinin (10
nM).
Results: Pressurized vessels developed the same degree of basal tone
(43±2% of max diameter), which was reversed by ROCK inhibitor
H-1152 (1 μM), with or without the endothelium. While the dilation
of denuded vessels to endothelium-independent vasodilator sodium
nitroprusside remained unaltered, vasoconstriction to endogenous
ET-1 via big ET-1 was reduced by 50% and reversed by H-1152.
Although the vasoconstrictions to ET-1 and PDBu were not altered
by denudation, their vasoconstrictor responses were sensitive to H1152.
Conclusions: It appears that the development of basal tone in porcine
retinal arterioles requires sustained ROCK activation in smooth
muscle cells. The endothelium does not modulate basal tone and has
little influence on the vasomotor responses to ET-1 and PKC
activation under resting conditions. Nonetheless, the endothelium is
responsible for a significant portion of overall retinal arteriolar
conversion of big ET-1 to ET-1 for vasoconstriction. Moreover,
ROCK is a major signaling molecule responsible for
vasoconstrictions to ET-1 and PKC activation independent of the
endothelium.
Commercial Relationships: Luke B. Potts, None; Yi Ren, None;
Lih Kuo, None; Travis W. Hein, None
Support: Retina Research Foundation, NIH EY018420, Scott and
White Research Foundation, Kruse Family Endowment Fund
Fenofibric Acid Protects Rod Precursor and Müller Cells from
Oxidative Stress and Hypoxia in a PPAR-alpha-Dependent
Mechanism
Elizabeth P. Moran1, 2, Lexi Ding1, 3, Ying Chen1, 3, Yang Hu1, 3,
Yusuke Takahashi1, 3, Jian-Xing Ma1, 3. 1Endocrinology, University of
Oklahoma Health Sciences Ctr, Oklahoma City, OK; 2Cll Biology,
University of Oklahoma Health Sciences Center, Oklahoma City,
OK; 3Physiology, University of Oklahoma Health Sciences Ctr,
Oklahoma City, OK.
Purpose: PPARα is a master regulator of glucose and lipid
homeostasis, and is thought to play a protective role in diabetic
retinopathy (DR) and other neurodegenerative disorders. Clinical
trials indicated that the PPARα agonist fenofibrate significantly
reduced diabetic retinopathy in human patients, but its physiological
and molecular mechanisms of action remain unknown. The purpose
of this study is to investigate the mechanism for fenofibrate and
PPARα’s neuroprotective effects.
Methods: The bioactive fenofibrate metabolite fenofibric acid (FA)
was used in vitro to determine its protective effect and molecular
mechanisms of action. Oxidative stress and hypoxia were induced
using 4-Hydroxynonenal and cobalt chloride, respectively, in rat rod
precursor and human Müller cells. FA’s protective effect was
evaluated using trypan blue dye exclusion. FA’s dependence upon
PPARα was determined using primary cells isolated from PPARα
knockout and wild-type mice. FA’s effect upon Ik-Bα and NOX4
signaling was assessed using western blotting and RT-qPCR, and
adenovirus expressing PPARα was utilized to determine whether
these effects were a consequence of PPARα activation.
Results: Our results showed that FA protected rod precursor and
Müller cells from oxidative stress and hypoxia. Furthermore, we
identified that FA did not protect primary cells isolated from PPARα
knockout mice, indicating that its cytoprotective effect is PPARαdependent. Moreover, both FA and PPARα over-expression upregulated IκBα, which is known to repress NFκβ signaling and thus
down-regulate NOX4, a primary source of ROS in both hypoxia and
oxidative stress. Further, both FA and PPARα over-expression downregulated NOX4 in hypoxia, suggesting that the effect is relevant in a
hypoxic environment, and is a direct consequence of PPARα
activation.
Conclusions: Taken together, our data have demonstrated for the
first time that FA/PPARα have a neuroprotective effect in the retina,
and moreover established that the protective effect is PPARαdependent. Further, because both FA and PPARα over-expression
activate the IκBα/NFκβ axis and subsequently down-regulate NOX4,
this event may also be PPARα-specific. We have also identified that
FA and PPARα affect IκBα down-regulation of NOX4, and we
hypothesize that this may be responsible, at least in part, for its
protective effect.
Commercial Relationships: Elizabeth P. Moran, None; Lexi Ding,
None; Ying Chen, None; Yang Hu, None; Yusuke Takahashi,
None; Jian-Xing Ma, None
Support: This study was supported by NIH grants EY018659,
EY012231, EY019309 and P20GM104934
409 Purine Signaling in the Eye: Role in Health and Disease Minisymposium
Wednesday, May 08, 2013 8:30 AM-10:15 AM
618-620 Minisymposium
Contributing Section(s): Cornea, Retinal Cell Biology
Introduction to Purine Signaling
Julie Sanderson. School of Pharmacy, University of East Anglia,
Norwich, United Kingdom.
Commercial Relationships: Julie Sanderson, None
Purine Signaling in the Trabecular Meshwork: A Regulator of
Intraocular Pressure
Mortimer M. Civan. Physiology-Richards Bldg, Univ of
Pennsylvania Sch of Med, Philadelphia, PA.
Commercial Relationships: Mortimer M. Civan, None
Purinergic Receptors in Lacrimal Gland Health and Dry Eye
Disease
Darlene A. Dartt. Schepens Eye Research Institute, Boston, MA.
Commercial Relationships: Darlene A. Dartt, None
TRPV4 and Hemichannels Cooperate to Play a Critical Role in
Purine Signaling in the Lens
Nicholas A. Delamere. Physiology, University of Arizona, Tucson,
AZ.
Commercial Relationships: Nicholas A. Delamere, None
Purine Signaling in Müller Cells During Development and in the
Adult Retina in Health and Disease
Antje Grosche. Pathophysiology of Neuroglia, University of Leipzig,
Leipzig, Germany.
Commercial Relationships: Antje Grosche, None
Purinergic Signaling in Retinal Processing
Thomas E. Salt. Visual Neuroscience, UCL Institute of
Ophthalmology, London, United Kingdom.
Commercial Relationships: Thomas E. Salt, Lundbeck (F), Merck
(C), Phytopharm (C), Neurexpert (I)
Purines and Low Grade Inflammation at Either End of the
Retina; ATP and Cytokines in Both RGCs and RPE
Claire H. Mitchell. Anatomy and Cell Biology, University of
Pennsylvania Library, Philadelphia, PA.
Commercial Relationships: Claire H. Mitchell, University of
Pennsylvania (P)
418 Antibiotics and Corneal Disease
Wednesday, May 08, 2013 8:30 AM-10:15 AM
Contributing Section(s): Cornea
The use of predatory prokaryotes to control human ocular
pathogens
Robert M. Shanks1, Kevin To2, Nicholas A. Stella1, Kimberly M.
Brothers1, Regis P. Kowalski1, Eric G. Romanowski1, Daniel E.
Kadouri2. 1Ophthalmology, University of Pittsburgh, Pittsburgh, PA;
2
Oral Biology, UMDNJ, Newark, NJ.
Purpose: Antibiotic resistant, disease-causing microorganisms are an
increasing cause for concern in ocular infections. In an attempt to
find innovative approaches to eradicate bacteria that cause ocular
infections, we measured whether predatory bacteria could
successfully kill clinical keratitis isolates of Pseudomonas
aeruginosa and Serratia marcescens in vitro. The cytotoxic impact of
predatory bacteria on a human corneal cell line was also assessed.
Methods: The predatory bacteria used in this study were
Bdellovibrio bacteriovorus, strains HD100 and 109J, and Micavibrio
aeruginosavorus ARL-13. These are non-pathogenic, soil-derived,
Gram-negative, obligatory parasites that feed on other Gram-negative
bacteria. 108 CFU of fluoroquinolone-resistant and susceptible ocular
isolates of P. aeruginosa and multidrug resistant strains of S.
marcescens were mixed with 108 CFU of predatory bacteria. After 24
and 48 hours, the numbers of surviving bacteria were enumerated.
Human corneal limbal epithelial cells (HCLE) were challenged with
~108 CFU of predatory bacteria or 5 x107 CFU of P. aeruginosa;
HCLE viability was measured at 4 and 24 hours using alamar blue.
Results: B. bacteriovorus predatory bacteria reduced viable counts of
the S. marcescens isolates examined (n=9) by 2-4 logs. B.
bacteriovorus and M. aeruginosavorus predatory bacteria reduced
fluoroquinolone-resistant P. aeruginosa isolates (n=9) viable counts
by up to 5 logs, and had mixed results with fluoroquinolone-
susceptible isolates, ranging from no effect to a 4-log reduction.
Although predatory bacteria were lethal to ocular pathogens, little
cytotoxic effect was observed when high doses of the predators were
exposed to a human corneal cell line.
Conclusions: This work highlights the potential use of predatory
bacteria as biological based agent for eradicating antibiotic-resistant
ocular infections.
Commercial Relationships: Robert M. Shanks, Bausch and Lomb
(C); Kevin To, None; Nicholas A. Stella, None; Kimberly M.
Brothers, None; Regis P. Kowalski, Rempex (F); Eric G.
Romanowski, Rempex (F), Allergan (F), Alcon/Novartis (F), 3-V
Biosciences (F); Daniel E. Kadouri, None
Support: NIH Grant AI085570, Research to Prevent Blindness
Career Development Award, NIH Grant EY08098
Susceptibility of Stenotrophomonasmaltophilia to Antibiotics and
Contact Lens Multipurpose Solutions
Keizo Watanabe1, 2, Hua Zhu2, Rani Bandara2, Shiro Higaki1,
Masahiko Fukuda1, Yoshikazu Shimomura1, Mark D. Willcox2, Brien
A. Holden1. 1Ophthalmology, Kinki University Faculty of Medicine,
Osakasayama, Japan; 2Brien Holden Vision Institute, Sydney, NSW,
Australia.
Purpose: To determine the susceptibility of Australian ocular isolates
of S. maltophilia to antibiotics, and to various contact-lensmultipurpose solutions (MPS).
Methods: A total of 40 strains of S. maltophilia isolated from either
contact lenses or lens storage cases of contact lens wearers were
tested in the study. Bacterial susceptibilities to antibiotics were
determined using a disc diffusion test. The antibiotics tested included
trimethoprim-sulfamethozole, tigecycline, fluoroquinolones,
aminoglycosides, β-lactams, chloramphenicol, and polymyxin B.
Susceptibility of test strains to seven commonly used MPS was
determined by using a broth microdilution method.
Results: The resistant rates of isolates were 93% to imipenem, 15%
to aztreonam, 13% to chloramphenicol, and 8% to cefepime. None of
the isolates was resistant to trimethoprim-sulfamethozole, tigecycline,
ceftazidime, gatifloxacin, levofloxacin, or moxifloxacin. The
susceptibility of test strains to the test MPS varied with MIC levels
ranging from 3% to 100% (full strength) MPS. The test strains were
less susceptible to the MPSs containing single disinfectant (PHMB)
and the MPS containing PQ-1 and Aldox than to the three recently
available MPS containing dual disinfectants (p < 0.05).
Conclusions: The Australian ocular isolates of S. maltophilia remain
susceptible to trimethoprim-sulfamethozole, tigecycline, and most
fluoroquinolones, but are less susceptible to MPS containing single or
certain dual disinfectant/s.
Commercial Relationships: Keizo Watanabe, None; Hua Zhu,
Brien Holden Vision Institute (E); Rani Bandara, None; Shiro
Higaki, None; Masahiko Fukuda, None; Yoshikazu Shimomura,
None; Mark D. Willcox, Allergan Inc (C), Allergan Inc (R), Brien
Holden Vision Institute (P), Bausch + Lomb (C), Basuch + Lomb
(R); Brien A. Holden, Allergan (F), AMO (I)
A Comparison of Prophylactic Antibacterial Efficacy of
Besifloxacin 0.6% versus Moxifloacin 0.5% at 1 hour and 3 days
Prior to Phacoemulsification
Frank A. Bucci. Bucci Laser Vision Institute, Wilkes-Barre, PA.
Purpose: To compare antibacterial efficacy of besifloxacin (BESI)
versus moxifloxacin (MOXI) on ocular surface tissues after 1 hour
and 3 days of treatment respectively.
Methods: 60 pts were randomized in a single center study to receive
either BESI or MOXI for 3 days (QID) prior to cataract surgery in
their surgical eye, and 1 hour prior to their final cultures in their nonsurgical eye, on the day of surgery. Lid and conjunctival cultures
were obtained prior to treatment in all eyes.
Results: The lid cultures for the surgical eyes (3 day tx) revealed a
significant decrease in colony forming units (CFU) for both BESI
(4,925+/-1,830 to 315+/-8 CFU) (p=0.01) and MOXI (1,124+/- 337
to 207+/- 20 CFU)(p=0.01). However the percent of NO GROWTH
lid cultures in these surgical eyes (3 day tx) for BESI increased from
13 % to 44 % pre-tx to post tx, but only from 16% to 20 % for MOXI
eyes. In addition, the CFUs of the lid cultures for the non-surgical
eyes (1hr tx) decreased significantly in the BESI eyes (3,217+/-1,022
to 558+/-377 CFU) (p=0.02) but INCREASED for the MOXI eyes
(1,781+/-325 to 2,268+/-897 CFU)(p=0.55). The percent of NO
GROWTH lid cultures in these non-surgical eyes (3 day tx) for BESI
increased significantly from 10% to 52% pre-tx to post tx, but
decreased in the MOXI eyes from 10% to 0%. The conjunctival
cultures also revealed similar results with greater decreases in CFUs
favoring BESI vs. MOXI in the non surgical (1 hr tx) eyes, but
statistical significance was not reached due to smaller CFUs and large
statistical variances. The mean CFU in MRSE BESI surgical eyes (3
day tx)(n=8)(7,020 to 15)(p=0.01) and non surgical eyes (1 hr tx)
(n=6) (7,961 to 633)(p=0.03) decreased significantly. The mean CFU
in MRSE MOXI surgical eyes (3 day tx)(n=8)(219 to 139) showed no
significant decrease (p=.051) and the non surgical eyes (1hr tx)(n=9)
showed a substantial INCREASE in CFU (1,051 to 3,290).
Conclusions: : Both BESI and MOXI revealed significant reductions
in lid CFUs after 3 days of treatment. In contrast, only BESI revealed
significant reductions in lid CFU after 1 hour of treatment. BESI also
revealed more rapid and significant killing of MRSE. These results
suggest that BESI has a significantly more rapid kill rate and is more
efficacious in MRSE eyes versus MOXI in a typical population of pts
undergoing cataract surgery.
Commercial Relationships: Frank A. Bucci, Bausch & Lomb (F)
Support: Physician initiated independent research Bausch Lomb
Cytotoxicities of various ophthalmic antimicrobial solutions in
SV40-immortalized human corneal epithelial cells
Jae Lim Chung1, Sang Wroul Song1, Byung Yeop Kim1, Joon H. Lee2,
Kyoung Yul Seo3. 1Konyang University Kim's Eye Hospital, Seoul,
Republic of Korea; 2Myunggok Eye Research Institute, Konyang
University, Seoul, Republic of Korea; 3Shinchon Severance Hospital,
Yonsei University, Seoul, Republic of Korea.
Purpose: Currently high concentration ophthalmic antibiotics were
introduced to increase the pharmacokinetic properties. However
toxicity is a concern in using these agents. This study evaluated the
cytotoxicities of various antibiotic eyedrops on SV40-immortalized
human corneal epithelial cells by using 3-(4,5-dimethylthiazol-2-yl)5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium
(MTS) assay.
Methods: Tested drugs were Cravit (levofloxacin 0.5%), Cravit 1.5
(levofloxacin 1.5%), Quixin (levofloxacin 0.5%, Benzalkonium
chloride (BAK) 0.005%), Iquix (levofloxacin 1.5%), Vigamox
(moxifloxacin 0.5%), Moxeza (moxifloxacin 0.5%, xanthan gum),
Gatiflo (gatifloxacin 0.3%), Zymaxid (gatifloxacin 0.5%, BAK
0.005%), Besivance (besifloxacin, BAK 0.01%, durasite), Azasite
(azithromycin 1%, BAK 0.003%, durasite), Tobrex (tobramycin
0.3%, BAK 0.01%) and standard powders of each drug. MTS assay
was performed after 5~120 minutes of exposure to each solution.
Results: After the exposure to undiluted solutions cell viabilities
were decreased to 9~73% from 5 minutes. At 30 minutes tobrex,
zymaxid and quixin showed marked toxicity, Cravit 1.5, Iqux and
Vigamox showed moderate toxicity and Cravit and Gatiflo showed
mild toxicity. When exposed to 10 times diluted solutions, Tobrex,
Besivance, Zymaxid, Quixin and Azasite, BAK containing products,
showed moderate toxicity. After the exposure to standard powder,
0.6% besifloxacin, 0.5% gatifloxacin, 0.5% moxifloxacin, 1.5%
levofloxacin and 0.5% levofloxacin showed toxicity results with
decreased order. However 1% azithromycin and 0.3% tobramycin
showed almost negative cytotoxicity.
Conclusions: High concentration drugs didn’t show increased
cytotoxicity. The most important factor for the determination of
cytotoxicity of antibiotic solutions was the concentration of BAK.
Commercial Relationships: Jae Lim Chung, None; Sang Wroul
Song, None; Byung Yeop Kim, None; Joon H. Lee, None; Kyoung
Yul Seo, None
The In Vitro Activity of Tigecycline for Clinically Relevant
Ocular Pathogens
Regis P. Kowalski, Tyler Kowalski, Eric G. Romanowski, Robert M.
Shanks, Leela Raju. Ophthalmology/Microbiology, Univ of
Pittsburgh, Pittsburgh, PA.
Purpose: Tigecycline is a glycylcycline antibiotic that is FDA
approved for the treatment of skin and soft tissue infections,
community-acquired pneumonia, and intra-abdominal infections.
Previous studies of clinical isolates from these body sites
demonstrated that tigecycline is active in vitro against isolates of a
number of Gram-positive and Gram-negative pathogens, including
methicillin-resistant Staphylococcus aureus (MRSA), but lacks
significant potency against Pseudomonas aeruginosa to be useful for
treatment of systemic infections A topical ophthalmic formulation
(RPX-978) is in development. The goal of the current study was to
determine the in vitro activity of tigecycline against multiple
clinically relevant ocular pathogens.
Methods: Minimum Inhibitory Concentrations (MIC) using CLSI
reference methods for broth dilution were determined for 110 clinical
conjunctivitis isolates based on incidence at the Campbell Lab:
Staphylococcus aureus (SA) (n=36), coagulase-negative
Staphylococcus (CNS) (14), Streptococcus pneumoniae (SP) (22),
other Gram-positive bacteria (GP) (8) (2 Streptococcus viridans
group and 6 beta-hemolytic Streptococcus species), Haemophilus
species (HS) (20), and other Gram-negative bacteria (10) (2 Serratia
marcescens, 2 Proteus mirablis, 3 Pseudomonas aeruginosa, 1
Enterobacter aerogenes, 1 Pseudomonas fluorescens, and 1 Klebsiella
species). In addition, MICs were performed on 26 keratitis isolates of
Pseudomonas aeruginosa (PA) and 10 endophthalmitis isolates each
of MRSA, MSSA, MRCNS, and MSCNS. A total of 176 isolates
were tested.
Results: : Data is expressed as (MIC50, MIC90 and Range of MICs)
in μg/ml respectively. Conjunctivitis: SA (0.25, 0.5, 0.125-0.5); CNS
(0.25, 0.5, 0.05-1.0); SP (<0.03, 0.125, <0.03-0.25); GP (0.125, 0.5,
<0.03-1.0); HS (2.0, 4.0, 2.0-4.0); GN (1.0, 4.0, 0.5-8.0). Keratitis:
PA (4.0, 8.0, 0.5-8.0). Endophthalmitis: MRSA (0.5, 0.5, 0.25-0.5);
MSSA (0.125, 0.5, 0.125-8.0); MRCNS (0.25, 0.25, 0.125-0.25);
MSCNS (0.25, 0.5, 0.125-0.5).
Conclusions: Tigecycline demonstrated broad spectrum in vitro
activity against clinically relevant ocular pathogens. Potent activity
was demonstrated against MRSA isolates, and lower than expected
MICs were demonstrated for PA. Further development of topical
tigecycline to treat eye infections is warranted.
Commercial Relationships: Regis P. Kowalski, Rempex (F); Tyler
Kowalski, None; Eric G. Romanowski, Rempex (F), Allergan (F),
Alcon/Novartis (F), 3-V Biosciences (F); Robert M. Shanks, Bausch
and Lomb (C); Leela Raju, None
Support: REMPEX
TEAR OSMOLARITY IN PEDIATRIC PATIENTS WITH
CYSTIC FIBROSIS
Livio Giulio Marco Franco1, Vittorio De Grande1, Santo Stella1,
Michele Reibaldi1, Elena Lionetti2, Chiara Franzonello2, Salvatore
Leonardi2, Caterina Gagliano1, Andrea Russo1, Mario La Rosa2.
1
Institute of Ophthalmology, University of Catania, Catania, Italy;
2
Department of Pediatrics, University of Catania, Catania, Italy.
Purpose: To evaluate the tear osmolarity in pediatric patients with
cistyc fibrosis compaired with healthy control, and to correlate the
osmolarity values with the genetic profiles.
Methods: 30 eyes of 30 patients (20 males; mean age: 11 years,
range: 8-16), with diagnosis of cistyc fibrosis and 30 eyes of 30
healthy patients age and sex matched, were enrolled. Only one eye of
each patient was randomized to the analysis. The tear osmolarity was
measured with the TearLab System (TearLab Corporation, San
Diego, CA). Tear samples were collected from the lateral meniscus of
the eye of each patient. All measurements were performed by the
same investigator under similar testing conditions. The genetic
mutations in patients with cistyc fibrosis were classified in severe
(Class I-II-III) and mild (Class IV-V).
Results: In the cistyc fibrosis group, the mean osmolarity value was
315.6 ± 30.5 mOsmol/L; in the control group the mean osmolarity
value was 298.2 ± 16.4 mOsmol/L; the difference was statistically
significant (t-Test; P < 0.001). The genetic mutation classes were
significantly correlated with the osmolarity values (r= 0.64).
Conclusions: Tear osmolarity is increased in children with cystic
fibrosis, especially when a severe mutation is present. Screening for
dry eye disease maybe indicated in patients with cistyc fibrosis.
Commercial Relationships: Livio Giulio Marco Franco, None;
Vittorio De Grande, None; Santo Stella, None; Michele Reibaldi,
None; Elena Lionetti, None; Chiara Franzonello, None; Salvatore
Leonardi, None; Caterina Gagliano, None; Andrea Russo, None;
Mario La Rosa, None
The Pharmacokinetics and Aqueous Humor Penetration of
Besifloxacin 0.6% and Moxifloxacin 0.5% in Patients Undergoing
Cataract Surgery
Ruth Evans, Frank A. Bucci. Bucci Laser Vision Institute, Wilkes
Barre, PA.
Purpose: To compare the aqueous penetration and pharmacokinetics
of besifloxacin 0.6% and moxifloxacin 0.5% in patients undergoing
phacoemulsification.
Methods: 120 patients participated in a randomized, single center,
single-masked, active comparator, parallel group absorption study.
Patients were randomized in a 1:1 ratio to receive either BESI (n=60)
or MOXI (n=60) to 1 of 4 subgroups. Each pt. was instructed to take
1 drop QID for 3 days prior to their surgery. On the day of surgery,
pts. were instructed to use their drops at 1 of 4 prearranged times.
Aqueous samples were collected at 1,2,4, or 6 hours following their
last drop.
Results: Aqueous concentrations were higher for MOXI when
compared to BESI at all time periods. The mean aqueous penetration
levels at ONE hour were BESI 49 / MOXI 489 ng/ml (p=0.001). The
two hour levels were BESI 55 / MOXI 623 ng/ml (p=0.001). At
FOUR hours the levels were BESI 74 / MOXI 331 ng/ml (p=0.01).
At SIX hours following the last study drops the levels were BESI 47 /
MOXI 329 ng/ml (p=0.005). The calculated mean AUC 1-6 was
significantly greater for MOXI (2170.1 +/- 171.1) versus BESI
(302.8 +/- 28.3) (p<0.0001). Penetration levels for MOXI peaked at 2
hours, and the 4 & 6 hour levels fell below that achieved at the initial
1 hour time period. Penetration levels for BESI peaked at the 4 hour
time period, and the 6 hour level was essentially equal to the initial 1
hour time period.
Conclusions: MOXI 0.5% demonstrated significantly greater
aqueous penetration vs. BESI 0.6% at all 4 time intervals studied.
The AUC 1-6 for MOXI 0.5% was significantly greater than BESI
0.6%. Data for both penetration AND potency are required to
evaluate the efficacy of a topical antibiotic. This study provides
valuable information regarding the penetration to the aqueous of
BESI vs. MOXI when used topically over time.
Commercial Relationships: Ruth Evans, None; Frank A. Bucci,
Bausch & Lomb (F)
Support: Physician initiated independent research Bausch & Lomb
Ocular Pharmacokinetics, Safety and Efficacy of Intracameral
Moxifloxacin 0.5% Solution in a Rabbit Model
Yonca Akova1, 2, Leyla Asena2, Mustafa T. Göktas5, Atilla Bozkurt5,
Umit Yasar5, Gulten Karabay4, Ebru Demiralay3. 1Ophthalmology,
Bayindir Kavaklidere Hospital, Ankara, Turkey; 2Ophthalmology,
Baskent University Faculty of Medicine, Ankara, Turkey;
3
Pathology, Baskent University Faculty of Medicine, Ankara,
Turkey; 4Histology, Baskent University Faculty of Medicine, Ankara,
Turkey; 5Pharmacology, Hacettepe University Faculty of Medicine,
Ankara, Turkey.
Purpose: This study was carried out to determine the ocular
pharmacokinetics, efficacy and potential endothelial toxicity of
moxifloxacin (MF) after a single intracameral bolus injection of
500µg/0.1ml in a rabbit model.
Methods: Forty-eight eyes of 24 New Zealand White Rabbits were
separated into 6 groups, each including 4 rabbits. 0.1 ml of 0.5%
intracameral moxifloxacin (500µg) injection was performed to the
right eyes and 0.1 ml of balanced salt solution to the left eyes
(control). Aqueous humor (AH) and vitreous samples were collected
at the 0.5th, 1st, 3rd, 6th, 12th and 24th hours from boths eyes of
group 1, 2, 3, 4, 5 and 6 respectively. MF concentrations were
determined by High Performance Liquid Chromatography. These
were compared with the minimum inhibitory concentrations (MIC)
and mutant prevention concentrations (MPC) for frequent
endophthalmitis pathogens. Electron and light microscopical
evaluation of the corneas were performed.
Results: Moxifloxacin reaches higher concentrations than the MIC of
all common endophthalmitis pathogens in the aqueous humor and
exceeds the mutant prevention concentration levels for Streptococcus
pneumonia, Streptococcus viridans, flouroquinolone susceptible
Coagulase-negative staphylococcus and flouroquinolone susceptible
Staphylococcus aureus for 6 hours. The half-life of moxifloxacin in
the AH was 2.2 hours. Electron and light microscopic evaluation
revealed no noticeable sign of toxicity.
Conclusions: Peroperative intracameral moxifloxacin injection for
endophthalmitis prophylaxis is a safe and effective method in
uncomplicated phacoemulsification surgery.
Commercial Relationships: Yonca Akova, None; Leyla Asena,
None; Mustafa T. Göktas, None; Atilla Bozkurt, None; Umit
Yasar, None; Gulten Karabay, None; Ebru Demiralay, None
High affinity LPS-binding branched peptides display synergistic
activity with other antibiotics
Rajamani Lakshminarayanan1, 2, Shouping Liu1, 2, Roger W.
Beuerman1, 2. 1SINGAPORE EYE RESEARCH INSTITUTE,
Singapore, Singapore; 2SRP Iin Neuroscience and Behavioral
Disorders, Duke-NUS Graduate Medical School, Singapore,
Singapore.
Purpose: The purpose of this study is to understand the relationship
between outermembrane permeability and synergism in two branched
peptides, B2088 and B2088_99 which carry two copies of putative
sequence RGRKVVRR and RGRKGGRR, respectively.
Methods: The outermembrane permeability of the peptides were
analyzed by N-1-phenyl naphthalene (NPN) fluorescence assay. We
used various biophysical techniques such as Circular Dichroism
spectropolarimetry, fluorescence spectrometry, isothermal titration
calorimetry and Surface plasmon resonance to probe the interactions
between lipopolysaccharide and peptides.
Results: Both the peptides displayed potent antimicrobial activity
against Gram-negative pathogens (2.7 μM). NPN assay suggested
that both the peptides permeabilized the outer membrane of P.
aeruginosa. B2088 permeabilized more effectively compared to
B2088_99. E. coli mutants which lack O-antigen, outer core and
inner core polysaccharides were hypersensitive to peptides.
BODIPY-cadaverine assay and TEM studies indicated that the
peptides bind to Lipid A and disrupt the supramolecular structure of
LPS. ITC studies indicated that both the peptides bind to LPS with
moderate affinity. Both the peptides were non-haemolytic (~3%
haemolysis) up to ~8.5 mM (20 mg/mL). To probe if the permeability
of peptides allows other antibiotics to enhance the antibacterial
action, we performed checker board assay and estimated the
fractional inhibitory concentration index (FICI) against multi-drug
resistant P. aeruginosa. Both the peptides displayed
synergism/additive action against 8 different commercially available
antibiotics.
Conclusions: The results showed that moderate affinity and
disruption of LPS supramolecular structure are important for rapid
bactericidal action and synergism with other antibiotics.
Commercial Relationships: Rajamani Lakshminarayanan, None;
Shouping Liu, None; Roger W. Beuerman, Allergan (F), SERI (P),
Santen (R)
Support: Supported by TCR Grant R618/41/2008 and NIGSERI/2009-R754/38/2010
The Comparison of Topical RPX-978 (an Ophthalmic
Formulation of Tigecycline) to Topical Vancomycin in a MRSA
Rabbit Keratitis Model
Eric G. Romanowski, Kathleen A. Yates, Katherine E. O'Connor,
Francis S. Mah, Leela Raju, Robert M. Shanks, Regis P. Kowalski.
The Charles T. Campbell Laboratory, UPMC Eye Center, University
of Pittsburgh, Pittsburgh, PA.
Purpose: Tigecycline is a glycylcycline antibiotic indicated for the
IV treatment of systemic infections. RPX-978 (RPX) is a topical
ophthalmic formulation of tigecycline in development for the
treatment of ocular infections. The efficacy of RPX was compared to
topical vancomycin (VAN) using a methicillin-resistant
Staphylococcus aureus (MRSA) keratitis model.
Methods: In 32 NZW rabbits, corneal epithelial defects were created
in the left eyes using an Amoils epithelial scrubber (abraded corneas),
while the epithelia of the right corneas remained intact (intact
corneas) to determine if corneal abrasion affected drug efficacy by
enhancing penetration. The corneas were intrastromally injected with
1000 CFU of MRSA. Rabbits were separated into 4 groups (n=8): A)
RPX (0.5% tigecycline), B) VAN 5%, C) normal saline (SAL), and
D) no treatment (euthanized before treatment for baseline CFU). 4h
after MRSA challenge, the topical treatment regimen of one drop
every 15 minutes for 5h was initiated. After treatment, all eyes were
slit lamp examined for presentations of infection. The eyes were
graded using a 0-3 severity scale. After examination and 1h after
treatment, the animals were euthanized and the corneas were
harvested for CFU determination. The data were non-parametrically
analyzed.
Results: The total clinical scores for RPX treatment were less than
SAL which was less than VAN (p<0.05, Kruskal-Wallis) in both
abraded and intact corneas. This indicated that clinical pathology
from infection and drug toxicity was less for RPX. VAN and RPX
produced similar reductions in CFU, and both were less than SAL
(P<0.05, K-W) in both abraded and intact corneas. Both treatments
demonstrated a 99.9% reduction in comparison to baseline CFU
(P<0.05, K-W) in both intact and abraded corneas. The comparable
reduction in CFU of abraded and intact corneas (P>0.05, Mann
Whitney) by RPX suggest high penetration through the corneal
epithelium. For RPX and VAN, the CFU of abraded and intact
corneas were statistically equivalent suggesting that RPX penetrated
through the corneal epithelium as well as VAN.
Conclusions: RPX was equally efficacious as fortified VAN in this
MRSA keratitis model. Additional studies are warranted to determine
the potential of RPX in the treatment of ocular infections due to other
bacteria.
Commercial Relationships: Eric G. Romanowski, Rempex (F),
Allergan (F), Alcon/Novartis (F), 3-V Biosciences (F); Kathleen A.
Yates, Rempex Pharmaceuticals (F); Katherine E. O'Connor,
Rempex Pharmaceuticals (F); Francis S. Mah, Alcon (C), Allergan
(C), B&L (C), ForeSight (C); Leela Raju, None; Robert M. Shanks,
Bausch and Lomb (C); Regis P. Kowalski, Rempex (F)
Support: Rempex Pharmaceuticals, NIH Core Grant P30 EY008098,
RPB, Eye & Ear Foundation
The Monthly Eye Drop: Development of a Long-term,
Noninvasive Glaucoma Treatment System
Morgan V. Fedorchak1, 2, Anthony Cugini1, Joel S. Schuman1, 3,
Steven R. Little1, 2. 1University of Pittsburgh, Pittsburgh, PA;
2
McGowan Institute for Regenerative Medicine, Pittsburgh, PA;
3
UPMC Eye Center, Pittsburgh, PA.
Purpose: IOP reduction in patients with glaucoma is typically
accomplished through the administration of medicated eye drops
several times daily, the difficult and frequent nature of which
contributes to low patient compliance rates. Newer drug delivery
methods for glaucoma require clinician administration of invasive
injections or implants. The purpose of this study was to develop and
test a hydrogel/microparticle formulation that provides one month of
brimonidine tartrate (BT) from a noninvasive, patient-administered
drop.
Methods: BT-loaded poly(lactic-co-glycolic) acid (PLGA)
microparticles and poly-(N-isopropylacrylamide)/poly(ethylene
glycol) (pNIPAAm/PEG) hydrogels were combined following
polymerization of the gel. The gel/microparticle formulation was
characterized for degradation and BT release for over one month.
Additionally, a single drop was administered to the inferior fornix of
New Zealand white rabbits. IOP was measured periodically using
tonometry and histology was used to assess biocompatibility of the
gel/microparticle system.
Results: Microparticles were confirmed to have a diameter of
7.5±2.9 μm with a primarily poreless morphology. The pNIPAAm
gel demonstrated a lower critical solution temperature (LCST) of
approximately 34°C. Degradation of the gel was negligible. Drug
release was within the calculated therapeutic range of topical BT
drops (Figure 2). Cytotoxicity testing demonstrated no significant
effect on conjunctival cell viability. In vivo testing demonstrated that
the gel/microparticle drop could be easily administered and form a
stable, opaque gel (Figure 1). The gel eye drop was easily removed,
leaving no evidence of gel or microparticles.
Conclusions: This controlled-release BT delivery system represents
a simple and novel patient-administered formulation that we believe
will provide adequate IOP reduction and biocompatibility without the
need for intraocular injections or frequent drop administration.
Figure 1: In vivo testing of thermo-gelling eye drop demonstrating A)
administration of liquid drop, B) rapid gelling in the lower fornix,
and C) simple removal with tweezers.
Figure 2: In vitro release of brimonidine tartrate from PLGA
microparticles alone and combined with pNIPAAm gels (n=3). Also
shown are the theoretical maximum and minimum amounts of BT
absorbed from traditional eye drops, based on 2 drops per day of
0.15% BT solution and 1-7% absorption.
Commercial Relationships: Morgan V. Fedorchak, None;
Anthony Cugini, None; Joel S. Schuman, Carl Zeiss Meditec, Inc.
(P); Steven R. Little, None
Chronic alcohol consumption and corneal pathologies: the role of
aldehyde dehydrogenases
Naseem Ansari1, 2, Min Zhang1, Cheng Wang1, John
Papaconstantinou1, Vasilis Vasiliou3, Bhupendra Kaphalia4.
1
Biochemistry and Molecular Biology, University of Texas Medical
Branch, Galveston, TX; 2Ophthalmology & Visual Science,
University of Texas Medical Branch, Galveston, TX;
3
Pharmaceutical Sciences, University of Colorado, Aurora, CO;
Pathology, University of Texas Medical Branch, Galveston, TX.
Purpose: Chronic alcoholism has been associated with various
pathologies involving oxidative damage, and may be an important
and underrated risk factor for eye disease, especially since the ocular
tissues are constantly exposed to oxidants. Corneal thickness and
structural abnormality has been reported in chronic alcoholics,
however this subject has not received due attention. Aldehyde
dehydrogenases (ALDHs) play a crucial role in the detoxification of
reactive and toxic lipid aldehydes, such as 4-hydroxynonenal (HNE)
and thus protecting this tissue and the rest of the eye against UVlight-induced oxidative damage. We have therefore investigated the
status of oxidative damage, inflammation and ALDH isozymes in
corneal pathology developed in mice upon chronic alcohol
consumption.
Methods: To have blood alcohol levels similar to those observed in
chronic alcoholics, deer mice (deficient in hepatic alcohol
dehydrogenase) were fed 3.5% ethanol via Lieber-DeCarli diet for 6
months while control mice were fed the same diet with equivalent
calories replaced by maltose-dextrin. Mice were euthanized and eyes
excised and fixed for morphological studies (hematoxylin and eosin
staining) and immunohistochemical studies (for ALDH1A1, ALDH2,
ALDH3A1, protein-acetylated lysine adducts, protein-HNE adducts,
and nitric oxide synthase staining). Stained images of corneal
sections were used to measure the thickness of the middle and side
portions of the cornea.
Results: Chronic ethanol treatment resulted in a dramatic increase of
the corneal thickness of both the stroma and epithelial layer.
Maximum change in the corneal thickness (in microns) occurred in
the middle portion (Control group: stroma, 59 ±7; epithelial layer, 28
±3 and Alcohol-treated group: stroma, 110±2; epithelial layer, 41 ±
2). Alcohol treatment led to a decrease in the expression of most of
the ALDH isozymes with a concomitant increase in the oxidative and
inflammatory markers and protein-acetylated lysine adducts, a
potential marker of ethanol consumption.
Conclusions: Our results suggest that impairment in detoxification of
lipid aldehydes and acetaldehyde under chronic alcohol exposure
may be central to alcohol-induced changes in corneal structure and
function.. Further studies using the ALDH knockout mice would lay
the foundation for a novel therapeutic strategy of preserving corneal
pathology in chronic alcoholics.
Commercial Relationships: Naseem Ansari, None; Min Zhang,
None; Cheng Wang, None; John Papaconstantinou, None; Vasilis
Vasiliou, None; Bhupendra Kaphalia, None
4
Small non-hydrophobic, cationic peptide with in vitro and in vivo
efficacy against Gram negative bacteria
Roger W. Beuerman1, Shouping Liu2, Rajamani Lakshminarayanan3,
Jianguo Li4, Bai Yang5. 1Singapore Eye Research Inst, Singapore,
Singapore; 2Singapore Eye Research Institute, Singapore, Singapore;
3
Singapore Eye Research Institute, Singapore, Singapore; 4Singapore
Eye Research Institute, Singapore, Singapore; 5Singapore Eye
Research Institute, Singapore, Singapore.
Purpose: This study has tested the concept that antimicrobial
peptides must include hydrophobic residues in order to show a high
level of bacterial killing both in vitro and in vivo.
Methods: Two short 8 AA peptides were designed and synthesized
in two analogues one with two alanines (088) and one with two
glycines (099), these linear peptides were covalentluy linked through
the C-terminal lysine. MICs were determined for resistant and
sensitive strains of Pseudomonas along with time kill and lab
simulations for resistance, membrane interactions with bacteria
modeled by molecular dynamics, NMR and finally a mouse model of
infection with Pseudomonas ATCC 9027.
Results: MICs using MHB dilution methods showed that the MICs
for Pseudomonas, E. coli, and K. pneumonia were 1.5μM for the 099
compound and 2.73 μM for the 088 compound, but the MICs for
Staphalococcus aureus were 5.9 and 4.7μM respectively. MICs were
determined for 10 clinical isolates of Pseudomonas of which 2 strains
were DR and 1 strain MDR (gatifloxicin MICs >22-125μg/ml) while
MICS for these as well as 3 clinical E. coli were between 1.55.9μM/ml. Laboratory simulations of resistance using Pseudomonas
ATCC 9027 with norfloxicin and gentamicin used as comparison
showed that resistance was not induced, but was induced in the other
antibiotics, MICs increased by 30-140 fold. Both compounds tested
on a rabbit model of corneal wound healing of a 5mm dia abrasion
showed no differences compared to PBS control. In a mouse model
of corneal infection with Pseudomonas with 106 CFU, with
gatifloxicin (3mg/ml) used for comparison there was no difference in
infection control with B2088/99 at either 1 or 3mg/ml all applications
at 5/day.
Conclusions: This new family of non-natural peptides has important
characteristics which should be useful for the rapdily emerging
problem of antibiotic resistant strains of Gram negative bacteria,
especially Pseudomonas
Commercial Relationships: Roger W. Beuerman, Allergan (F),
SERI (P), Santen (R); Shouping Liu, None; Rajamani
Lakshminarayanan, None; Jianguo Li, None; Bai Yang, None
Support: TCR, Exploit
Molecular Design of Novel Membrane Targeting Antimicrobials
with Improved Membrane Selectivity Using Natural Compound
as a Scaffold
Shouping Liu1, 2, Hanxun Zou1, Jun-Jie Koh1, Jianguo Li3, Rajamani
Lakshminarayanan1, 2, Roger W. Beuerman1, 2. 1Singapore Eye
Research Institute, Singapore, Singapore; 2Duke-NUS Medical
School, SRP Neuroscience and Behavioural Disorders, Singapore
169857, Singapore, Singapore; 3Bioinformatics Institute, Singapore
138671, Singapore, Singapore.
Purpose: To set up a new platform for design and prediction of
membrane targeting small organic molecules based antimicrobials
with improved membrane selectivity
Methods: This work describes how to tune the amphiphilic
conformation of α-mangostin, a natural compound with a
hydrophobic xanthone scaffold, to improve the antimicrobial activity
and selectivity toward Gram positive bacteria. A series of xanthone
derivatives were obtained by cationic modification of free hydroxyl
groups of α-mangostin at C3 and C6 positions with amines groups of
different pKa values.
Results: The results show that the antimicrobial activities of the
cationic xanthone derivatives can be generally predicted based on the
pKa values of the corresponding amines. We have identified AM0016 (3b) as the most potent compound in the series with potent
antimicrobial activity with MIC values of 0.095-0.39 (µg/mL) against
Gram-positive bacteria including MRSA, improved selectivity up to
200, rapid time-kill in 10-30mins, avoidance of antibiotic resistance
and good biocompatibity. Biophysical studies and molecular dynamic
simulations also revealed that 3b disrupted the bacterial membrane by
forming an amphiphilic conformation with the cationic groups
located at the hydrophobic-water interface. In contrast, conjugation
moieties with low pKa value to the xanthone scaffold diminished the
antimicrobial activities.
Conclusions: A series of novel antimicrobials have been designed
and prepared by cationic modifications of α-mangostin, a natural
xanthone with a planar hydrophobic core, to yield an amphiphilic
structure which improves selectivity for bacterial membranes through
the hydrophobic-water interface perturbation. This design strategy is
important as we provide a new approach to modify natural
compounds to yield excellent antimicrobial properties, high
selectivity and safety. This strategy could improve “hits” in the
development of new antibiotics for drug-resistant pathogens.
Commercial Relationships: Shouping Liu, None; Hanxun Zou,
None; Jun-Jie Koh, None; Jianguo Li, None; Rajamani
Lakshminarayanan, None; Roger W. Beuerman, Allergan (F),
SERI (P), Santen (R)
Support: NMRC/NIG/1010/2010, Singapore
In vitro analysis of the effect of steroid in combination with
antimicrobial on co-cultures of bacteria and fungi isolated from
keratitis
Herlinda Mejia-Lopez1, Lucero Y. Martínez-López2, Alejandro
Clinent-Flores1, Aida V. Rodríguez-Tovar2, Luis A. BautistaHernández1, Victo M. Bautista de Lucio1, María A. Martínez-Rivera2.
1
Research Unit, Inst of Ophthal "Conde de Valenciana", Mexico City,
Mexico; 2Laboratory of Medical Mycology, Department of
Microbiology, National School of Biological Sciences, Instituto
Politécnico Nacional, México, Mexico.
Purpose: Some of major diseases affecting the cornea are keratitis
and may be of various origins including microbial, Currently, as a
result of various risk factors such as frequent use of broad spectrum
antibiotics and topical steroids, the self-medication, use of contact
lenses, chronic systemic diseases, immunosuppression, among others,
increased frequency of keratitis. Clinical manifestations depend on
the causative agent; however, misdiagnosis generates chronicity with
possibility of multiple treatments. The aim of this study was to
analyze, in vitro, the effect of steroids in combination with
antimicrobial agents on the growth of bacteria and fungi isolated
from keratitis, as well as their co-cultures.
Methods: Aspergillus fumigatus, Fusarium solani, Staphylococcus
aureus and Staphylococcus epidermidis isolated from patients with
keratitis whom received multiple treatments, were studied. Minimum
inhibitory concentration of amphotericin B and itraconazole and
moxifloxacin and gatifloxacin were determinated. The effect of the
dexamethasone and prednisolone on fungal or bacterial growth,
individually and in co-cultures fungus-bacteria was studied also.
Experiments were performed six times and analyzed by the ANOVA
test to calculate the significance with respect to the untreated
controls.
Results: The growth of Fusarium solani was inhibited by the
presence of prednisolone at 312.5 mg/mL (p ≤ 0.005). On the
contrary, the growth of Aspergillus fumigatus was increased in
presence of dexamethasone at 2.22 ug/ml and 570.0 mg/mL
concentrations (p ≤ 0.05). There was no effect of corticosteroids on
growth of bacteria. Moxifloxacin reduced the growth of F. solani
dose-dependent manner. The antifungal combination with
corticosteroids or quinolones favors the growth of fungi. S. aureus in
coculture with A. fumigatus and prednisolone favored the growth of
this fungus.
Conclusions: The cortiesteroids has dose-dependent inhibition effect
on the growth of F. solani and A. fumigatus; similarly moxifloxacin
on F. oxysporum. Combination of corticosteroids-antifungal as well
as corticosteroids-quinolones favors growth of both fungi. In co-
culture of S. aureus with A. fumigatus treated with prednisolone, the
bacterium favored the growth of the fungus.
Commercial Relationships: Herlinda Mejia-Lopez, None; Lucero
Y. Martínez-López, None; Alejandro Clinent-Flores, None; Aida
V. Rodríguez-Tovar, None; Luis A. Bautista-Hernández, None;
Victo M. Bautista de Lucio, None; María A. Martínez-Rivera,
None
Support: Private Assistance Foundation "Conde de Valenciana"
Iontophoresis transcorneal delivery technique for transepithelial
corneal collagen crosslinking with riboflavin in a rabbit model
Vincent J. Soler1, 2, Myriam Cassagne1, 2, Camille Laurent3, Anne
Galinier4, Pierre R. Fournie1, 2, Stéphane Galiacy1, Pierre Roy5,
Francois J. Malecaze1, 2. 1UMRS 563, CPTP, Universite Paul
Sabatier, Toulouse, France; 2Ophthalmology, Toulouse Purpan
Hospital, Toulouse, France; 3Laboratory of Pathology, Toulouse
Purpan Hospital, Toulouse, France; 4Department of Biochemistry,
Toulouse Rangueil Hospital, Toulouse, France; 5Hexamed, Paris,
France.
Purpose: To evaluate a new iontophoresis transcorneal riboflavin
delivery technique for transepithelial corneal collagen crosslinking
(CXL).
Methods: ANIMALS: A total of 108 eyes from New Zealand white
albino rabbits were included in this study. Corneas after riboflavin
application by iontophoresis (n=25) were compared with corneas
treated by conventional riboflavin application after deepithelialization (n=16). Then I-CXL (n=18) was compared with CCXL (n=9) 14 days after treatment. All these groups were compared
with specific controls (n=40).
METHODS: Iontophoresis involved the administration of a new
formulation of charged riboflavin (Ricrolin® +) into cornea by
applying a current of 1mA for 5 min. C-CXL was performed
according to the Dresden protocol.
MAIN OUTCOMES: Riboflavin diffusion in the eyes was
investigated by two-photon microscopy in corneas and by highperformance liquid chromatography (HPLC) in corneas and aqueous
humors. Stromal collagen structure modifications were analyzed
using second harmonic generation imaging (SHG).
Riboflavin diffusion in corneas was analyzed by measuring riboflavin
emission at 500-550 nm with a two-photon microscope on corneal
sections and riboflavin concentration in corneas and aqueous humors
was measured by HPLC.
Corneal stromal modifications were evaluated by analyzing the
collagen fiber organization through SHG using a two-photon
microscope.
Results: Images from two-photon microscope showed that after
iontophoresis, riboflavin fluorescence and its diffusion throughout
cornea were similar to that observed in a conventional application. In
parallel, iontophoresis showed no more toxicity in the corneal
epithelium than conventional application of riboflavin.
Using HPLC, the corneal concentration of riboflavin was two-fold
less after iontophoresis than after conventional application (936.2 ±
312.5 ng/ml and 1708 ± 908.3 ng/ml, respectively, p<0.05).
Z-stack imaging using SHG showed that collagen fibers were more
strongly interlinked, with a higher, lamellar organization in the
anterior stroma 14 days after I-CXL or C-CXL as compared to
controls.
Conclusions: This experimental study suggests that I-CXL is a
promising alternative methodology for riboflavin delivery in
crosslinking treatment, preserving the epithelium.
Commercial Relationships: Vincent J. Soler, None; Myriam
Cassagne, None; Camille Laurent, None; Anne Galinier, None;
Pierre R. Fournie, None; Stéphane Galiacy, None; Pierre Roy,
Hexamed (P); Francois J. Malecaze, None
Comparative Analysis of In Vitro Biocompatibility Assays of
Polyhexamethylene Biguanide Disinfecting Contact Lens MultiPurpose Solutions
Mercedes Salvador-Silva1, Ling C. Huang1, James Cook2. 1Biology
R&D, Abbott Medical Optics (AMO), Santa Ana, CA; 2Corneal
Product Development R&D, Abbott Medical Optics (AMO), Santa
Ana, CA.
Purpose: This study evaluated the effects of polyhexamethylene
biguanide (PHMB) contact lens multi-purpose (MPS) solution on cell
cytotoxicity, metabolic activity, membrane integrity, and
biocompatibility.
Methods: Five MPS were used - MPS-1: polyhexamethylene
biguanide (PHMB) + poloxamer (PLX), MPS-2: PHMB + PLX,
MPS-3: PHMB + poloxamine (PLA), MPS-4: PHMB +
polyquaternium (PQ1) + PLA, and MPS-5: PHMB + PLX. In vitro
biocompatibility was assessed according to ISO 10993. MPS effect
on colony formation rate was evaluated in V79 Chinese Hamster
Lung Fibroblast for 7 days. MPS were evaluated at 1.25%, 2.5%, 5%
and 10% as diluted in cultured medium. Cytotoxicity and metabolic
activity were determined using alamarBlue dye. Corneal epithelial
barrier function was assessed by Zonula Occludens (ZO-1)
immunohistochemistry (IHC) and trans-epithelial electrical resistance
(TEER) in Simian virus transformed human corneal epithelial cell
line (SV40 HCEC).
Results: MPS-1 formulation was comparable to MPS-2 at 1.25% and
2.5% and MPS-3, MPS-4, and MPS-5 at 5% and 10% in cell
cytotoxicity. Treatment with 1ppm PHMB alone did not induce a
statistically significant inhibition on V79 colony formation (n=3,
p>0.05). MPS-1 at 50% and 75% was comparable to MPS-2, MPS-3,
MPS-4, and better than MPS-5 at 100% in corneal barrier integrity as
evaluated by ZO-1 IHC. Exposure to MPS-1 for 120 mins at 50%
concentration was superior to MPS-3, MPS-4, and MPS-5 and similar
to MPS-2 on maintaining SV40 HCEC viability as assessed by
alamarBlue. TEER of SV40 HCEC showed that exposure to MPS-1
for 120 mins at 50% concentration was better than MPS-3 but equal
to MPS-2, MPS-4, and MPS-5. MPS-1 for 120 mins at 75%
concentration was similar to MPS-5 and superior to MPS-3 and MPS4 to maintain corneal cell membrane integrity (n=3-4, p<0.05).
Conclusions: PHMB effects on in vitro cell cytotoxicity are best
demonstrated by correlating results from multiple assays. Results of
membrane integrity assays (ZO-1 IHC and TEER) showed the
greatest differences among PHMB-formulated MPS solutions,
followed by metabolic activity (alamarBlue) and colony forming
assays. These in vitro results demonstrated that MPS-1 (PHMB +
PLX) to be better than or equal to MPS-3, MPS-4, and MPS-5 in
maintaining corneal barrier integrity and cell viability.
Commercial Relationships: Mercedes Salvador-Silva, Abbott
Medical Optics (E); Ling C. Huang, Abbott Medical Optics, Inc.
(E); James Cook, Abbott Medical Optics (E)
Benzalkonium Chloride Stimulation of THP-1 Differentiated
Macrophages in Vitro
Sylvain Michee1, 2, Francoise Brignole-Baudouin2, Luisa Riancho2,
William H. Rostene2, Christophe Baudouin1, 2, Antoine Labbe1, 2.
1
department 3, CHNO des 15-20, Paris, France; 2UMRS 968, vision
institute, PARIS, France.
Purpose: To characterize the phenotype, function and cytokine
production of THP-1 derived macrophages when exposed to the most
common preservative in eye drops, benzalkonium chloride (BAK).
Methods: Macrophages, obtained after differentiation of THP-1
cells, a human monocytic leukemia cell line, with phorbol myristate
acetate (PMA) were exposed for 24 hours to 10-5% benzalkonium
chloride (BAK) or phosphate buffered saline (PBS) as control. The
expressions of CD11b, CD11c, CD33 and CD54/ICAM-1 were
evaluated using immunohistochemistry and flow cytometry (FCM).
Phagocytosis function was analyzed using carboxylate-modified
fluorescent microspheres and FCM. Cytokine production was
quantified in culture supernatants using a human cytokine array.
Results: Stimulation of macrophages with BAK increased CD11b
and CD11c expressions and decreased CD33 as well as they
increased their phagocytosis function and their secretion of cytokines
in supernatants especially CCL1, CCL4/MIP-1β, TNF-α, soluble
CD54/ICAM-1 and IL-1β.
Conclusions: In vitro, a low concentration of BAK induced a direct
stimulating effect on macrophages, increasing phagocytosis, cytokine
release and expressions of CD11b and CD11c. Long-term exposure
to low concentrations of BAK should be considered as a stimulating
factor responsible for inflammation through macrophage activation.
Commercial Relationships: Sylvain Michee, None; Francoise
Brignole-Baudouin, None; Luisa Riancho, None; William H.
Rostene, None; Christophe Baudouin, None; Antoine Labbe,
None
Delayed corneal wound healing resulting from the administration
of fluoroquinolone antibacterial ophthalmic solutions and its
causes
Masamichi Fukuda, Yusuke Seki, Yusuke Kurihara, Shino Enta,
Naoko Shibata, Kenta Hagiwara, Hiromi Osada, Sinsuke Shibata, Eri
Kubo, Hiroshi Sasaki. Ophthalmology, Kanazawa Medical
University, Uchinada, Japan.
Purpose: The purpose of this study was to examine delayed corneal
wound healing resulting from the administration of fluoroquinolone
antibacterial ophthalmic solutions and the factors affecting this delay.
Methods: Corneal epithelial detachment was induced in mature
domestic white rabbits (male, weight: 2.5-3.0kg) with 1-heptanol and
these rabbits were subsequently used in the experiment. Three types
of antibacterial ophthalmic solutions, VEGAMOXTM 0.5%
ophthalmic solution (MFLX), Cravit® 1.5% ophthalmic solution
(LVFX), and Gatifloxacin® 0.3% ophthalmic solution (GFLX), were
used in this experiment where rabbit models were divided into
antibacterial agent instillation groups and a saline solution instillation
group (control) and the impact on corneal wound healing was
assessed using a corneal electrical resistance measurement method
(corneal resistance device: CRD method). Seven eye drops were
instilled every 30 minutes following corneal epithelial abrasion and
corneal resistance CR(%) was measured from 1-96 hours following
the completion of instillation. The degree of corneal disorder was
assessed with fluorescein staining. The effect of each antibacterial
agent on cell proliferation was also assessed using rabbit-derived
corneal cell lines (SIRC).
Results: CR(%) as measured by the CRD method increased along
with the reduction in the fluorescein staining area. There was
significant reduction in the CR(%) of the three antibacterial
ophthalmic solution groups as compared to the control group
(P<0.05) and corneal wound healing was delayed. It is possible that
the CRD method can detect disorder that cannot be evaluated by
fluorescein staining. There was a significant reduction in the cell
proliferation rate (%) in the LVFX and MFLX groups as compared
with the control group (P<0.05), however there was no significant
reduction in the GFLX group.
Conclusions: Delayed corneal wound healing resulting from the
administration of three antibacterial ophthalmic solutions could be
confirmed. The cause of this is believed to be the suppression of
corneal cell proliferation as a consequence of the DNA synthesis
inhibitory effect of the antibacterial agent along with the increase in
concentration of the ophthalmic solutions.
Commercial Relationships: Masamichi Fukuda, None; Yusuke
Seki, None; Yusuke Kurihara, None; Shino Enta, None; Naoko
Shibata, None; Kenta Hagiwara, None; Hiromi Osada, None;
Sinsuke Shibata, None; Eri Kubo, None; Hiroshi Sasaki, None
Penetration of Eye Drop Solutions of Fluoroquinolones into the
Cornea and Anterior Chamber of Human Enucleated Eyes
Sidney J. Sousa, Gleilton C. Mendonça. Ophthalmology, Univ of Sao
Paulo-Sch of Med, Ribeirao Preto, Brazil.
Purpose: Many Eye-banks decontaminate the donated eyeballs by
fully immersing them, for ten minutes, into commercial eye-drop
solutions of antibiotics. The purpose of this study was to compare the
penetration of ciprofloxacin 0.3%, ofloxacin 0.3%, and moxifloxacin
0.5% into the cornea and aqueous humor of enucleated human eyes
immersed into these solutions.
Methods: Three groups of 30 enucleated human eyes, inappropriate
for corneal transplantation, were immersed, for ten minutes, into
commercial eye drop solutions of ciprofloxacin 0.3%, ofloxacin 0.3%
and moxifloxacin 0.5%, respectively. Samples of the cornea and the
aqueous humor were stocked under -70 C in liquid nitrogen until the
time of analysis with high performance chromatography (HPLC).
Results: The 95% CI of the average concentration within the corneal
stroma ranged from 2.50 to 4.78 µg/g for ciprofloxacin 0,3%, from
5.42 to 7.87 µg/g for ofloxacin 0,3% and from 10.81 to 19.16 µg/g
for moxifloxacin 0.5%. The differences among the three antibiotics
were statistically significant. The 95% CI of the average
concentration in the aqueous humor ranged from 0.80 to 1.30 µg/g
for ciprofloxacin 0.3% , from 1.20 to 1.59 µg/g for ofloxacin 0.3%
and from 3.38 to 5.75 µg/g for moxifloxacin 0.5%. The advantage of
the moxifloxacin over the other antibiotics was statistically
significant. However, it was impossible to demonstrate a significant
difference in the aqueous concentration of ofloxacin and
ciprofloxacin.
Conclusions: The corneal concentration expected for moxifloxacin is
almost twice as much as for ofloxacin and five times greater than for
ciprofloxacin, under the conditions of the experiment. The
concentration expected for moxifloxacin in the aqueous humor is
about 3.5 times bigger than for ciprofloxacin or for ofloxacin. In
summary the superiority of the moxifloxacin penetration into the eye
seems to be remarkable, albeit not so intense to rise concerns with the
endothelial toxicity. Ofloxacin seems to penetrate better than
ciprofloxacin into the cornea. However, in the aqueous humor we
could not tell if the difference found in favor of the former was real
our fortuitous. Since most of the intraocular penetration of drugs is
due to passive diffusion (Healy et al. 2004) this method seems to be a
promising tool for comparing relative drug penetration into the
cornea and anterior chamber.
Commercial Relationships: Sidney J. Sousa, None; Gleilton C.
Mendonça, None
Support: FAEPA
To evaluate the efficacy of corneal collagen crosslinking in fungal
corneal ulcers
shah nawaz, Prafulla K. Maharana, Namrata Sharma, Rasik B.
Vajpayee. Opthalmology, R.P.Centre, AIIMS, New Delhi, India.
Purpose: To study the efficacy of corneal collagen
crosslinking(CXL) in treatment of moderate fungal corneal ulcers.
Methods: Thirty-seven cases of micro-biologically proven moderate
fungal corneal ulcers were divided in two groups.Group 1 received
medical therapy with CXL on day 1 of presentation. Group 2
received medical therapy alone.Medical therapy included topical
Natamycin 5% drops .In cases of deep and large ulcers additional
systemic therapy was given. CXL was done using riboflavin 1% with
UVA 370nm. The parameters analyzed included healing time, final
BCVA and perforation rate.
Results: Mean age in group 1 was 43.5 ±17.01 years and 39.6±19.7
years in group 2, statistically insignificant(p value 0.604). Average
ulcer size in-group 1 was 25.79±16.7 mm2 and 25.4±12.4 mm2 ingroup 2, statistically insignificant (p value 0.915). BCVA at
presentation in group 1 was 1.48 ±0.46 Log MAR and 1.56±0.435
Log MAR in group 2, statistically insignificant (p value 0.6618). 13
out of 15 cases (86.6%) in group 1 and 11/12 (91.67%) cases in
group 2 healed (p value 0.56), statistically not significant .The
average healing time of ulcer in group 1 was 43.08 days ±26.6 and
39.2± 19.7 days in group 2, statistically insignificant (p value 0.673).
Final BCVA in group 1 was 1.3522 ±0.46 Log MAR and 1.26± 0.43
Log MAR in group 2, statistically insignificant (p value 0.434).
Conclusions: CXL offers no advantage as an adjunctive treatment in
the treatment of moderate fungal corneal ulcers.
Commercial Relationships: shah nawaz, None; Prafulla K.
Maharana, None; Namrata Sharma, None; Rasik B. Vajpayee,
None
Improvement of post-cataract dry eye by 0.05%
cyanocobalamine plus 0.5% taurine and 0.5% long-chained
hyaluronic acid
Federico Solignani1, Monica Zurria2, Maurizio Rolando1, 3.
1
Ophthalmology Dept - Ocular surface disease research center,
University of Genoa, Genoa, Italy; 2Medical Dept, Alfa Intes Ind Ter
Spl srl, Casoria, Italy; 3Is.Pre Oftalmica, Genoa, Italy.
Purpose: To study the effects of 0.05% cyanocobalamine in
combination with 0.5% taurine and 0.5% long-chained hyaluronic
acid (IALUVIT® ophthalmic solution, Alfa Intes, Italy) and its
impact on functional recovery related to patient comfort, in postcataract dry eye (PCDE).
Methods: Records from 20 patients (mean age 75.35+7.07 years, 8
males and 12 females) undergone phacoemulsification (sutureless,
temporal incision) were selected. In addition to routine post-operative
protocol, 10 patients had received IALUVIT® for 3 months; 10
patients were considered as control. Break-up time (BUT),
conjunctival and corneal staining, temporal, central and nasal corneal
sensitivity (Cochet-Bonnet aesthesiometer), were considered before
surgery and 1, 7, 30 and 90 days after surgery. Frequency and extent
of symptoms (foreign body sensation, stinging, burning, discomfort
at blinking) had been registered using a VARS before and 7, 30 and
90 days after surgery.
Results: BUT, staining scores, and corneal sensitivity were affected
by surgery in all patients. BUT increased during the follow-up and it
was significantly better in treated patients than controls at day 30 and
90 (respectively, 13.3+0.94 vs 10.8+1.31 at day30 and 13.7+1.05 vs
11.1+1.52 at day90, p<0.0001). Staining scores recovered within 1
month in both groups, with a statistically significant effect of the
treatment both at day30 and day90 (p<0.0001). Corneal sensitivity
returned to baseline levels in all patients at day90, however the time
course in treated eyes was significantly different within each
measurement (p<0.0001). Foreign body sensation, stinging and
discomfort at blinking were significantly different in treated patients,
both as frequency and extent of symptom perception (p<0.0001). No
significant change in burning was observed. Finally, there was a
strong correlation between symptoms and corneal sensitivity values,
at each considered point.
Conclusions: Despite successful surgery, patients always complain
of PCDE related to different causes, including the disruption of
normal cornea nerves and sensitivity. The present data show that
adding to post-surgery therapy an ophthalmic solution whose
ingredients (especially cyanocobalamine and taurine) have an impact
on nerves could improve cornea health thus reducing patient
dissatisfaction, when the adjunctive therapy is extended from 1 to 3
months after surgery.
Commercial Relationships: Federico Solignani, None; Monica
Zurria, Alfa Intes Ind Ter Spl srl (E); Maurizio Rolando, Bausch &
Lomb (F), Allergan (R), Alcon (R), Santen (R), Merck (R), Thea (C)
Title: Susceptibility Profiles in Infectious Keratitis as an Aid in
Antibiotic Selection
Mark L. Hill, Duncan A. Friedman, Elizabeth Cooper, John Parker.
Ophthalmology, UAB, Homewood, AL.
Purpose: Previous studies have pointed out the need for broad
antibiotic coverage in infectious keratitis. With increasing rates of
antibiotic resistance, the method of empiric treatment of infectious
keratitis should be revisited frequently through retrospective analysis
of organisms isolated from these infections.
Methods: A retrospective chart review of all cases of infectious
keratitis was performed for all patients presenting to a Level 1 Eye
Trauma hospital between 2010 and 2012. Data included patient
demographics, source of culture, bacterial or fungal organism
cultured, and susceptibility profiles for positive bacterial cultures. An
antibiogram was constructed from all positive cultures.
Results: A total of 289 culture positive cases of infectious keratitis
occurred over the three year period observed. Of the positive cultures,
41 (14%) grew multiple microbes. Thirty-five (10.6%) of the cultures
were fungal isolates. Two hundred nineteen (66%) of the cultures
were gram-positive bacteria, and all Gram positive organisms were
susceptible to vancomycin. The antibiotics with the highest
sensitivities were vancomycin (100%), ceftriaxone or cefotaxime
(100%), tobramycin (100%), linezolid (100%), cefepime (100%),
rifampin (98.6%), meropenem (97.6%), gentamicin (96.0%),
tetracycline (90.16%), and trimethoprim-sulfamethoxazole (87.80%).
Rates of fluoroquinolone sensitivity ranged from 67.47% (ofloxacin)
to 84.26% (levofloxacin).
Conclusions: Infectious keratitis is a serious cause of visual loss, and
treatment is often initiated prior to culture and sensitivity results.
Susceptibility testing reveals that most organisms are susceptible to
vancomycin, tobramycin, and third-generation cephalosporins with
no resistant strains found. Growing rates of resistance to
commercially available antibiotics should drive the development of
better empiric treatments for bacterial keratitis.
Commercial Relationships: Mark L. Hill, None; Duncan A.
Friedman, None; Elizabeth Cooper, None; John Parker, None
Photodynamic antimicrobial chemotherapy for methicillinresistant Staphylococcus aureus -in vitro, biofilm, and ex vivo
bovine keratitis model
Hsiao-sang Chu1, Fung-Rong Hu1, Chin-Tin Chen2, 3. 1Department of
Ophthlamology, National Taiwan University Hospital, Taipei,
Taiwan; 2Center for Optoelectronic Biomedicine, College of
Medicine, National Taiwan University, Taipei, Taiwan; 3Institute of
Microbiology and Biochemistry, College of Life Science, National
Taiwan University, Taipei, Taiwan.
Purpose: To evaluate the efficacy of micelle encapsulated chlorin e6
(mCe6) photodynamic antimicrobial therapy (PACT) on methicillinresistant Staphylococcus aureus (MRSA) infection.
Methods: Chlorin e6 was encapsulated into micelles by the reversedphase evaporation method. 405nm purple laser with adjustable
fluence rate was used as the light source. MRSA suspension
(2×108/ml) was incubated with various concentration of mCe6,
followed by exposure of light. Colony forming units (CFUs) of
diluted suspensions were counted for survival assay after treatment.
For biofilm growth, stainless steel mircodisc was incubated with
MRSA suspensions (108/ml) over night. The microdiscs were washed
and incubated with different concentration of mCe6, followed by
exposure of light. The biofilm on the microdisc was removed by
mechanical oscillation for survival assay after PACT. For keratitis
model, fresh bovine eyes were inoculated with MRSA
(100CFU/10μL) in superficial corneal stroma and incubated in moist
chambers. Biomicroscopic examinations were performed 12 and 16
hours after infection. Twelve hours after infection, bovine eyes were
divided into four groups: no treatment, treated with hourly topical
vancomycin (50mg/ml)(V), treated with 10μL mCe6 (100mM)
topically every 5 minutes for 30minutes, followed by exposure of
light at a fluence rate of 35mW/cm2 for 10 minutes(P), combined
mCe6 mediated PACT with hourly topical vancomycin
(50mg/ml)(P+V). Four hours after treatments, the corneal buttons
were trephined for tissue homogenization and bacterial survival
assay.
Results: mCe6 mediated PACT showed a dose-dependent inhibition
on MRSA suspension and biofilm. Under the light dose of 10J/cm2,
at a concentration of 1μM, mCe6 was able to decrease 5 log bacteria
in suspension. Under the light dose of 20J/cm2, at a concentration of
100μM, mCe6 was able to decrease 2 to 3 log bacteria in biofilm. For
ex vivo bovine keratitis model, compared with the control, the
vancomycin treated)(V), and the mCe6-PACT treated(P) groups,
significantly fewer CFUs were noted in the combined treatment
group)(P+V).
Conclusions: This study demonstrated the effectiveness of mCe6PACT against MRSA. mCe6 mediated PACT could be a potential
alternative treatment for MRSA keratitis.
Commercial Relationships: Hsiao-sang Chu, None; Fung-Rong
Hu, None; Chin-Tin Chen, None
Support: National Taiwna University Research Grant NTUH10S1596
438 Retinal Flow and VEGF
Wednesday, May 08, 2013 11:00 AM-12:45 PM
Program #/Board # Range: 4628-4673/B0092-B0137
Contributing Section(s): Retinal Cell Biology
Program Number: 4628 Poster Board Number: B0092
Subfoveal Choroidal blood flow in senescence
John V. Lovasik, Helene Kergoat, Mireille Parent. School of
Optometry, University of Montreal, Montreal, QC, Canada.
Purpose: The sub-foveal choroid is the sole blood supply for the
cone photoreceptors within the foveal avascular zone (FAZ). A
reduction in the sub-foveal choroidal blood flow (ChBF) has been
reported before the onset of age-related macular degeneration,
suggesting a cause-effect relationship. However, a reduction in ChBF
can also be the consequence of a sub-clinical retinopathy in the FAZ,
or increasing age per se. Thus, the interdependence among the ChBF
and the structural integrity and function of the retina in the FAZ
remains poorly defined. In the present study we quantified the effects
of senescence on choroidal hemodynamics and its relation to central
visual function.
Methods: 20 healthy volunteers for each decade of life between 20
and 80 years of age participated in the study. The best corrected
Snellen visual acuity (VA) was recorded during a complete eye exam
for each subject. A confocal near infrared (780 nm) Laser Doppler
Flowmeter was used to record ChBF (AU), velocity (ChBVel-kHz),
and volume (ChBVol-AU) in the choriocapillaris within the FAZ at
25 Hz. Each subject was directed to fixate the center of the laser
probe that appeared as a small dim red spot of light. Foveation of the
laser spot was continued until a 10-20 sec interval with a constant DC
output (indicating steady fixation) was obtained. The corresponding
records of ChBF, ChBVel, ChBVol were cleaned of blink artifacts
for each subject and then averaged over time. The mean values for all
subjects were graphed as a function of subject age and a linear
regression line drawn through each parameter to glean the effects of
age on choroidal hemodynamics. An alpha value of 0.05 was used for
statistical significance.
Results: ChBVel decreased (r= -0.223, p= 0.02) while ChBVol
increased (r= 0.222, p= 0.02) with age. In contrast, the ChBF
remained constant with age (r= 0.005, p> 0.05). The group average
ChBF across all age groups, Mean= 0.957 ± SD= 0.405, was
associated with a central VA of 0.9 minarc ± SD= 0.14 across all
subjects.
Conclusions: An acuity of 20/20 or better can be maintained in
septuagenarians. The ChBF in the FAZ does not change between 20
to 80 years of age in healthy individuals. Because the ChBF varied by
almost 100% within the 6 age groups studied, singular measurements
of the ChBF cannot distinguish patients differing by just one or two
lines from the standard 20/20.
Commercial Relationships: John V. Lovasik, None; Helene
Kergoat, None; Mireille Parent, None
Support: CFI, NSERC, CIHR
Time-dependent intracellular pattern of Bevacizumab in RPE
cells
Shereen Hassan M. Aboul Naga1, 2, Michaela Dithmer1, Johann
Roider1, Alexa K. Klettner1. 1Ophthalmology, University of Kiel,
University Medical Centre, Kiel, Germany; 2Ophthalmology, Kasr Al
Aini, Cairo University Hospitals, Cairo, Egypt.
Purpose: Bevacizumab is taken up into RPE cells and is
intracellularly found for at least seven days. In this study, we
investigate Bevacizumab uptake and intracellular localisation of
Bevacizumab at different time points in RPE cells.
Methods: For this study, RPE cell line Arpe19 and primary porcine
RPE cells, passage 2, were used. Cells were treated once with
Bevacizumab and intracellular Bevacizumab was investigated after
various time periods (1 h - 7 d). For the detection of intracellular
Bevacizumab, Western blot and immunofluorescence was utilized.
For intracellular localization, antibodies against Rab5 (early
endosome), Rab7 (late endosome), Lamp2 (lysosome) and
microtubuli were used. Actin was stained using Phalloidin.
Results: Bevacizumab is abundantly found in porcine RPE cells and
Arpe19 cells as detected in immunofluorescence and Western blot.
After 1 h and 4 h of stimulation, Bevacizumab is primarily located
close to the cell membrane and can partly be found in close proximity
to or colocalizing with Rab 5, indicating that sections of
Bevacizumab are taken up into early endosomes. During 1 day to 5
days after Bevacizumab challenge, intracellular Bevacizumab
displays a net-like pattern. After 7 d of Bevacizumab challenge, the
pattern intracellular Bevacizumab displays becomes more diffuse.
Colocalization with microtubule can hardly be seen, while
Bevacizumab is found in close proximity to or colocalizing with actin
filaments, suggesting an actin-mediated intracellular transport.
Colocalization with Lamp2 is rarely found, suggesting that
Bevacizumab is not degraded in lysosomes.
Conclusions: Bevacizumab displays a distinct, time dependent
localization in RPE cells. The pattern suggests a controlled
intracellular transport via actin filaments. Taken-up Bevacizumab
does not seem to be transported into the lysosomal pathway and does
not seem to be intracellularly degraded.
Commercial Relationships: Shereen Hassan M. Aboul Naga,
None; Michaela Dithmer, None; Johann Roider, Novartis (F),
Bayer (F); Alexa K. Klettner, Novartis (F), Novartis (C), Novartis
(R), Santen (R)
Support: DFG KL 2425/2-1, DAAD (German Academic Exchange
Service)
Antagonism of PDGFRβ Inhibits Pericyte Recruitment in a
Mouse Model of Corneal Neovascularization
Amy Jensen1, Rosemarie Cepeda1, Michael Maker1, Chad E.
Bigelow1, Joy Ghosh1, Guochun Li1, Patricia A. D'Amore2, Gunther
Spohn1, Bruce D. Jaffee1, Sassan Azarian1. 1Ophthalmology,
Novartis, Cambridge, MA; 2Ophthalmology, Schepens Eye Research
Institute, Boston, MA.
Purpose: To explore the pathobiology of pericytes in ocular diseases
such as diabetic retinopathy, in which pericyte loss is a hallmark.
Pericytes are abluminally associated with the capillary endothelium
and contribute to vessel formation, stabilization, and maturation. To
examine the role of PDGF in these functions, we inhibited the PDGF
pathway in a mouse model of corneal neovascularization (CoNV).
Methods: CoNV was induced in anesthetized C57BL/6 mice by
mechanical abrasion of corneas. Animals were treated every other
day, with control antibodies or with PDGFRβ and VEGF neutralizing
antibodies administered IP. To quantify pericytes and
neovascularization (NV), corneal flat mounts were stained with αNG2 and α-PECAM1 antibodies, to detect pericytes and endothelial
cells respectively, and imaged by fluorescence microscopy. Pixel area
was measured using a program written in MatLab, and pericyte
coverage (%) was calculated as NG2-labeled pixels/PECAM1labeled pixels*100. NV area was measured using AxioVision
software. Vessel morphology was assessed using AngioTool.
Results: In unabraded mice, pericytes were associated only with
perilimbal vessels as there were no corneal vessels. In the new
vessels observed in the abraded mice there was an increase in
pericyte area (6.2-fold), vessel area (3.6-fold), and pericyte coverage
(1.6-fold) relative to normal limbal vessels in unabraded controls.
Neutralization of PDGFRβ dramatically reduced pericyte coverage
but had no effect on vessel area or morphology. α-VEGF treatment
resulted in reduced vessel area and altered vessel morphology.
Neutralizing both PDGFRβ and VEGF did not elicit any additional
effects on pericyte area, vessel area or morphology compared to
neutralizing VEGF alone.
Conclusions: Corneal abrasion resulted in NV which could be
inhibited with an antibody against VEGF. The new vessels were
accompanied by an increase in pericyte area and coverage, which
represents recruitment of new pericytes and can be blocked with
systemic α-PDGFRβ treatment. This model allows visualization of
both existing and newly recruited pericytes and appears to be a
suitable model for studying pericytes.
Commercial Relationships: Amy Jensen, Novartis (E), Novartis
(F); Rosemarie Cepeda, Novartis Institute for Biomedical Research
Inc. (F), Novartis Institute for Biomedical Research Inc. (E); Michael
Maker, Novartis Institutes for Biomedical Research (E); Chad E.
Bigelow, Novartis (E); Joy Ghosh, Novartis (E); Guochun Li,
Novartis (E); Patricia A. D'Amore, Valeant (C); Gunther Spohn,
Novartis (E); Bruce D. Jaffee, Novartis (E); Sassan Azarian,
Novartis (E)
Dynamic retinal venous oscillations are changed in diabetes
mellitus type 1
Konstantin E. Kotliar1, 2, Ines M. Lanzl2, Thorsten Siegmund4, Arno
Schmidt-Trucksaess5, 3. 1Biomedical Engineering, Aachen University
of Applied Sciences, Juelich, Germany; 2Ophthalmology, Munich
University of Technology, Munich, Germany; 3Preventive Sports
Medicine, Munich University of Technology, Munich, Germany;
4
Endocrinology and Diabetes, Bogenhausen Hospital, Munich,
Germany; 5Division of Sports Medicine, University of Basel, Basel,
Switzerland.
Purpose: We demonstrated previously that non-stimulated temporal
retinal arterial and venous oscillations (pulsations and vasomotions)
are changed in healthy volunteers with age and in patients with
primary open angle glaucoma. Whether this dynamic retinal vessel
behavior is altered in diabetes mellitus type 1 (DM1) is investigated.
Methods: 33 untreated patients with DM1 (age 51.7±8.3 years) with
no or non-proliferative retinopathy and 33 age and sex matched
medically healthy volunteers were examined by Dynamic Vessel
Analyzer (DVA, IMEDOS, Jena, Germany). Temporal changes of
vessel diameters of retinal arterial and venous vessel segments were
examined with DVA in all subjects during 40 seconds. Oscillatory
temporal changes of vessel diameter were divided into high-frequent
(period < 1,5 s) and low-frequent (period ≥ 1,5 s) and were evaluated
using methods of mathematical signal analysis.
Results: Quantitative parameters characterizing dynamic retinal
vessel behavior did not show any differences in retinal arterial
oscillations between DM1 patients and healthy control subjects. In
veins there was a significant difference in the rate of periodicity of
high-frequency (control: 0.08(0.06; 0.17), DM1: 0.12(0.08; 0.23)
[median (1.quartile; 3. quartile)], p < 0.05) as well as of lowfrequency oscillations (control: 0.16(0.10; 0.24), DM1: 0.62(0.49;
1.23), p < 0.001) between the groups.
Conclusions: Functional and morphological alterations in the retinal
veins in DM1 with no or mild non-proliferative retinopathy are
shown using a non-invasive in-vivo methodology. Changed retinal
venous behavior in the initial stages of diabetes mellitus might be an
indication for alterations in the vascular endothelium and blood flow
regulation in this disease.
Commercial Relationships: Konstantin E. Kotliar, None; Ines M.
Lanzl, allergan (C), alcon (R), pfizer (R), santen (R), novartis (R);
Thorsten Siegmund, None; Arno Schmidt-Trucksaess, None
Parameters related to Choroidal thickness
Yasuki Ito1, Kazuhiro Oiwa1, Eiji Iwata1, Akiko Takahashi1,
Tetsuhiro Yasuma1, Kenichi Kawano1, Nobuyuki Hamajima2, Hiroko
Terasaki1. 1Ophthalmology, Nagoya University Graduate School of
Medicine,, Nagoya, Japan; 2Department of Preventive Medicine,
Nagoya University Graduate School of Medicine,, Nagoya, Japan.
Purpose: The choroidal thickness has been reported to be
significantly correlated with age and axial length. In addition, the
choroid in eyes with central serous chorioretinopathy (CSC) has been
reported to be thicker. Because smoking is a risk factor of CSC, we
tested the hypothesis that the choroid will be thicker in smokers. We
also examined whether the choroidal thickness was affected by body
weight.
Methods: A total of 495 subjects (197 men, 298 women) who were
≥40-years-of-age and were in the Comprehensive Health
Examination Program (Yakumo Study) in 2011 were studied. All
were examined by optical coherence tomography, fundus
photography, and axial length measurements. The choroidal thickness
was measured in the OCT images, and the relationship with the
ocular parameters, bodyweight, sex, and smoking were determined.
Results: The average choroidal thickness was 222.3 ± 90.7 µm. The
choroidal thickness was significantly correlated with age (P
<0.0001), axial length (P <0.0001), and body weight (P <0.05). The
choroidal thickness was thicker in smokers than in non-smokers
(258.0 ± 110.2 µm vs 217.5 ± 87.1 µm, respectively; P <0.05).
Regression analysis also showed that the choroidal thickness was
significantly correlated negatively with age (P <0.0001), negatively
with axial length (P <0.0001), positively with body weight (P <0.001)
and with smoking (P <0.05). Sex was not significantly associated
with choroidal thickness (P >0.05).
Conclusions: The choroidal thickness is associated with not only
axial length and age, but also bodyweight and smoking. Smoking
may disturb choroidal circulation and cause the thickening of choroid
and may eventually increase the risk of CSC. In addition, body
weight should also be considered to evaluate the choroidal thickness.
Commercial Relationships: Yasuki Ito, None; Kazuhiro Oiwa,
None; Eiji Iwata, None; Akiko Takahashi, None; Tetsuhiro
Yasuma, None; Kenichi Kawano, None; Nobuyuki Hamajima,
None; Hiroko Terasaki, None
Blockade of PGF/VEGFR1 signaling significantly prevents BRB
breakdown, apoptosis and inflammation in diabetic retinopathy
Hu Huang, Da'Kuawn Johnson, Dorothy Kim, Gerard A. Lutty.
Ophthalmology, Johns Hopkins Wilmer Eye Inst, Baltimore, MD.
Purpose: The goal of this study is to investigate the effects of
blocking placental growth factor/vascular endothelia growth factor
receptor 1 (PGF/VEGFR1) signaling on adverse complications in
diabetic retinopathy (DR).
Methods: Placental growth factor knockout (PGFKO) mouse was
crossed with the Ins2Akita (Akita) diabetic mouse to create the new
Akita.PGFKO double mutant mouse strain. VEGFR1 activity in DR
was blocked via neutralizing antibody (MF1). The integrity of bloodretinal barrier (BRB) was assessed by measuring vascular leakage
into the retina using [3H]Mannitol as a tracer. Perfusion of retinal
blood vessels was assessed by comparing the areas of perfuseddextran with GSA-lectin staining. Retinal capillary drop-out assays
were performed on the isolated retinal microvasculature after elastase
digestion. Cell apoptosis was determined by the activated Caspase-3
or TUNEL staining. Inflammation was investigated by counting the
number of leukocyte adherent to the blood vessels (or retinal
leukostasis). The expression of inflammatory and BRB makers was
determined by real-time quantitative PCR and/or immunofluorescent
staining.
Results: Vascular leakage was significantly increased in Akita
diabetic mice but was not in the Akita.PGFKO diabetic mice
compared to their respective non-diabetic littermates. Retinal
capillary degeneration was significantly increased in the Akita
diabetic mice as compared with the wild type (WT) non-diabetic
mice or the Akita.PGFKO diabetic mice. Apoptotic cell death was
significantly increased in the Akita diabetic mice as compared with
the Akita.PGFKO diabetic mice. Similarly, blockade of VEGFR1 by
MF1 significantly inhibited diabetes-caused BRB breakdown, cell
apoptosis and retinal leukostasis. Furthermore, in diabetic conditions,
the expression of inflammatory markers, such as ICAM-1 and IL-1α
were significantly suppressed by blockade of PGF/VEGFR1
signaling. In contrast, the expression of BRB markers, such as Ecadherin and ZO-1, were significantly elevated due to its blockade.
Conclusions: The results suggested PGF/VEGFR1 signaling is
involved in the adverse complications during DR and is a potential
target in the treatment of diabetes complications, such as diabetic
macular edema.
Commercial Relationships: Hu Huang, None; Da'Kuawn
Johnson, None; Dorothy Kim, None; Gerard A. Lutty, None
Computational Model of Oxygen Transport in Retina and Optic
Nerve
David Bragason1, Einar Stefánsson1, 2. 1Ophthalmology, Landspitali
University Hospital, Reykjavik, Iceland; 2Medicine, University of
Iceland, Reykjavik, Iceland.
Purpose: In order to increase our understanding of oxygen saturation
patterns observed in retinal vessels, we propose a computational
model to describe oxygen transport and its dependence on local,
systemic and extrinsic factors, such as vessel width, blood flow,
illumination and oxygen supplementation.
Methods: A mathematical model of arteriole-venule pairs and
surrounding tissue was developed. Coupled non-linear partial
differential equations describing convection, diffusion and interaction
of oxygen with hemoglobin and oxygen-consuming tissue are solved
numerically. Taking into account non-uniform blood-flow and
hematocrit profiles, longitudinal and radial oxygen saturation and
partial pressure gradients are calculated. The results are compared
with measurements obtained with the Oxymap retinal oximeter
(Oxymap ehf., Reykjavik, Iceland), as well as with data previously
published by researchers using other methods.
Results: Oxygen saturation gradients along major retinal vessels
reflect oxygen consumption of perivascular tissue, with a smaller
component due to countercurrent exchange between closely spaced
vessels. Our model predicts longitudinal saturation gradients
consistent with those measured in retinal oximetry. Oxygen
penetrates by diffusion into a perivascular tissue layer comparable in
thickness to the capillary-free zone. A gradient of 1 - 4 % in
saturation is predicted in central retinal vessels along the optic nerve.
The model predicts reduced oxygen saturation with decreased blood
flow. It helps explain a distribution width of 5 - 10 % in saturation in
short segments of retinal vessels and the variability in saturation
observed between normal eyes. According to the model, the 3 %
increase in retinal arteriolar saturation observed in darkness can be
accounted for by increased blood flow in the dark. Diffusion currents
of oxygen in vitreous close to major retinal arterioles are predicted to
be approx. 10-6 ml O2/cm2/sec, compatible with previously published
results, obtained with polarographic methods.
Conclusions: Our model predicts retinal vessel oxygen saturation
patterns that are consistent with those observed in retinal oximetry
and helps us gain a quantitative understanding of some aspects of
oxygen transport in the retina and optic nerve.
Saturation profile in central retinal vessels along 10 mm in optic
nerve
Commercial Relationships: David Bragason, None; Einar
Stefánsson, Oxymap ehf (P), Oxymap ehf (I), Oculis ehf (P), Oculis
ehf (I), Risk ehf (I), Acta Ophthalmologica (E)
Support: Helga Jonsdottir and Sigurlidi Kristjansson Memorial Fund
Increased Retinal Vascular Tortuosity in Obstructive Sleep
Apnea
Amir Mohsenin1, Vahid Mohsenin2, Ron A. Adelman1. 1Department of
Ophthalmology, Yale University School of Med, New Haven, CT;
2
Yale Center for Sleep Medicine, Yale University School of
Medicine, New Haven, CT.
Purpose: Obstructive sleep apnea (OSA) is a highly prevalent
disorder with significant vascular morbidity and mortality. Affected
patients are subjected to intermittent hypoxemia, hypercapnia, arterial
blood pressure surges and increased intracranial pressure during
sleep. We sought to examine the effects of OSA on retinal
vasculature tortuosity.
Methods: A pilot retrospective chart review was conducted
identifying patients with and without OSA who had undergone
fundus photography. 7 control subjects and 9 subjects with OSA were
analyzed. Measurements were taken of the superior and inferior
temporal retinal artery and vein starting at the optic disc rim to the
crossing point of 2 circles centered on the optic disc with diameters
of 5 disc diameters (DD) and 10DD. Retinal vascular tortuosity (tau)
was assessed by calculating the arc length/chord length of each vessel
segment as per previously published methods.
Results: Patients with OSA have significant increases in arterial and
venous tortuosity when measured at the 10DD length. At 5DD, there
was no statistically sig difference in arterial tortuosity between the
two groups. Venular tortuosity was significantly increased at the 5DD
mark in patients with OSA.
Conclusions: These findings demonstrate an association between
obstructive sleep apnea and increased retinal vessel tortuosity. A
larger study will be necessary to examine the prevalence and
significance of this vascular abnormality in OSA.
Commercial Relationships: Amir Mohsenin, None; Vahid
Mohsenin, None; Ron A. Adelman, None
Support: Leir Foundation. Newman's Own Foundation.
Oxygen saturation in retinal hemorrhages
Olafur Palsson1, 2, Sveinn H. Hardarson1, 2, Thorunn S. Eliasdottir2,
Einar Stefánsson1, 2. 1Department of Ophthalmology, Landspítali
University Hospital, Reykjavik, Iceland; 2University of Iceland,
Reykjavik, Iceland.
Purpose: To develop and test a method to measure oxygen saturation
of extravasated hemoglobin in retinal tissue in disorders such as
central retinal vein occlusion (CRVO).
Methods: The spectrophotometric retinal oximeter (Oxymap ehf,
Reykjavik, Iceland) simultaneously delivers two monochromatic
images of the same area of the fundus, one which is is sensitive to
oxygen saturation (600nm) and one which is not (570nm). The light
absorbance of retinal hemorrhages was measured at these two
wavelengths in 7 CRVO patients. The light absorbance was used to
estimate oxygen saturation in the hemorrhages. Variability between
measurements of areas within the same retinal hemorrhage was
determined. The mean oxygen saturation in retinal vessels was
measured with automated software.
Results: The mean saturation in the retinal hemorrhages was
58±18% (mean±SD, n=7). The mean saturation in retinal venules in
the same patients was 33±14% (p=0.03). Standard deviation between
repeated measurements on the same hemorrhage in the same image
was 14.6% (saturation percentage).
Conclusions: The results suggest that measurement of oxygen
saturation of extravascular hemoglobin in retinal tissues is possible.
Saturation values in hemorrhages are between saturation values in
arterioles and venules. The oxygen saturation can be correlated to
PO2 and therefore might be an indicator of the oxygenation of the
tissue.
Commercial Relationships: Olafur Palsson, None; Sveinn H.
Hardarson, Oxymap ehf. (F), Oxymap ehf. (I), Oxymap ehf. (C),
Oxymap ehf. (R), Patent no. 7774036 (P), Patent application
13/377,749 (P), Optos plc. (F); Thorunn S. Eliasdottir, None; Einar
Stefánsson, Oxymap ehf (P), Oxymap ehf (I), Oculis ehf (P), Oculis
ehf (I), Risk ehf (I), Acta Ophthalmologica (E)
Support: The Icelandic Center for Research (Rannís), The
University of Iceland Research Fund, The Landspítali University
Hospital Research Fund
Assessment of Total Retinal Blood Flow under Systemic
Hypercapnia and Hypocapnia
Ayda M. Shahidi1, Sunni R. Patel1, John G. Flanagan1, 3, Ou Tan2,
David Huang2, Christopher Hudson1, 3. 1Ophthalmology & Vision
Science, University Health Network, Toronto, ON, Canada;
2
Ophthalmology, Oregon Health and Science University, Portland,
OR; 3Optometry & Vision Science, University of Waterloo,
Waterloo, ON, Canada.
Purpose: To investigate the effect of change in systemic partial
pressure of CO2 on total retinal blood flow (TRBF) as measured by
Doppler Fourier-domain optical coherence tomography (OCT)
Methods: TRBF scans were captured in nine healthy individuals
(mean age ± standard deviation: 27±4, 6 males) using the RTVue™
OCT double ring blood flow protocol. Measurements were captured
during homeostatic PETCO2 levels, hypercapnia (+5/+10/+15 mmHg
PETCO2), back to baseline and hypocapnia (-5/-10/-15 mmHg
PETCO2) using a custom-designed computer controlled gas blender
(RespirAct™) with a sequential gas delivery rebreathing system. The
order for hyper- and hypo-capnia conditions was randomized.
Repeated measure analysis of variance (reANOVA) and Tukey’s
post-hoc analysis were used to compare Doppler OCT measurements
amongst breathing conditions. The effect of end-tidal CO2 on the
outcomes was investigated using regression models.
Results: TRBF (45.9 ± 10.9, vs 60.9 ± 12.1 µl/min), superior RBF
(24.0 ± 9.3 vs 33.8 ± 9.5 µl/min) , arterial (14.1 ± 2.7 vs 19.9 ± 4.5
mm/s) and venous (12.5 ± 1.7 vs 15.6 ± 1.3 mm/s) velocities were
significantly different between baseline measurements and extreme
hypercapnia (p<0.0001). There were general trends towards reduced
retinal blood flow parameters during hypocapnia but the differences
with the baseline, except for the superior arterial vessel area
(p=0.006), were not statistically significant. Increased PETCO2 had a
significant effect on the outcomes (p<0.002 for all states).
Conclusions: In healthy individuals, a 15% increase in end tidal CO2
significantly raised TRBF and vessel velocity, while, hypocapnia
significantly reduced retinal hemodynamics only in the superior
arterial area.
Commercial Relationships: Ayda M. Shahidi, None; Sunni R.
Patel, None; John G. Flanagan, Heidelberg Engineering (C),
Heidelberg Engineering (R), Heidleberg Engineering (F), Carl Zeiss
Meditec (C), Carl Zeiss Meditiec (R), Carl Zeiss Meditiec (R), Alcon
Pharmaceuticals (R), Alcon Pharmaceuticals (R), Optovue Inc (F),
Optovue Inc (F), Photon etc (F), Photon etc (F); Ou Tan, Optovue
(F), Optovue (P), Carl Zeiss Meditec (P); David Huang, Optovue
(F), Optovue (I), Optovue (P), Optovue (R), Carl Zeiss Meditec (P);
Christopher Hudson, Opovue Inc (F)
Support: Ontario Research Fund - Research Excellence
One-year results of Central Retinal Sensitivity in eyes treated
with Pegaptanib for Proliferative Diabetic Retinopathy with
extended dosing
Blanca C. Flores, Victor H. Gonzalez, Roberto Diaz-Rohena. Valley
Retina Institute, McAllen, TX.
Purpose: To evaluate retinal sensitivity in the macular area in
patientes treated for proliferative diabetic retinopathy (PDR) using a
modified pharmacologic approach versus drug induction combined
with selective laser.
Methods: Prospective longitudinal 54 week follow-up of patients
enrolled in an open label clinical trial. Twenty eyes (20 patients) with
high-risk PDR were enrolled. Ten eyes (Group A) received 3
intravitreal injections of Pegaptanib at 6 week intervals, then 3
additional injections at 12 week intervals. Ten eyes (Group B)
received selective laser photocoagulation after 3 intravitreal
injections of Pegaptanib at 6 week intervals. The fellow eye of each
patient was treated with standard panretinal photocoagulation serving
as control. Retinal sensitivity in the central 8 degrees was evaluated
by MP-1 microperimeter at baseline and at 1 year after treatment.
Results: At 1 year, mean retinal sensitivity within the central 8
degrees field of the study eye in Group A improved from 14.5 ± 5.5
to 15 ± 4.9 (P = 0.823) and the fellow eye improved from 10.5 ± 7.5
to 12.8 ± 6.6 (P = 0.135). In Group B, the central retinal sensitivity of
the study eye improved from 9.4 ± 6.1 to 11.4 ± 5.8 (P = 0.237) and
the fellow eye improved from 9.4 ± 5.3 to 10.8 ± 4.7 (P = 0.405).
Mean change in best corrected visual acuity at 1 year from baseline in
the study eye of Group A was +7.9 letters (P = 0.016) on the Early
Treatment Diabetic Retinopathy Study (ETDRS) chart and -2.7 (P =
0.351) in the study eye of Group B.
Conclusions: Results show a tendency of improvement in central
retinal sensitivity in all groups after 1 year of treatment. The
administration of Pegaptanib alone resulted in higher gain of letters
on ETDRS chart as opposed to a combined treatment with selective
laser photocoagulation. A larger sample and further follow-up are
needed.
Commercial Relationships: Blanca C. Flores, None; Victor H.
Gonzalez, Genetech (C), Regeneron (C), Pfizer (C), Valiant (C),
Alimera (C); Roberto Diaz-Rohena, None
Persistent hyaloid vessels counteract insufficient retinal perfusion
in the mouse eye
Christina Seide, Marina Garcia Garrido, Vithiyanjali Sothilingam,
Naoyuki Tanimoto, Susanne C. Beck, Mathias W. Seeliger. Div of
Ocular Neurodegeneration, Ctr for Ophthal Inst for Ophth Rsrch,
Tuebingen, Germany.
Purpose: Persistence of embryonic vitreal vasculature beyond the
time of its physiologic regression during ocular development was
observed in a number of mouse models where retinal vascularization
is impaired. Here, we evaluate the nature and distribution of
persistent hyaloid vessels in relation to the topography of retinal
perfusion defects
Methods: In vivo imaging was performed with a Heidelberg
Engineering HRA I using native confocal scanning-laser
ophthalmoscopy (cSLO) as well as fluorescein (FA) and indocyanine
green angiography (ICGA). Mouse models with insufficiencies in
retinal vascularization already starting during development were
selected for this study. The setting of different confocal planes
allowed a selective analysis of the distribution of hyaloid and retinal
surface vasculature. The topography of vascular alterations of retinal
vessels was then correlated with the distribution of vitreal vascular
persistence.
Results: The combination of FA and ICGA revealed a detailed
overview of the distribution of retinal vessels and their state of
perfusion. Retinal regions with a reduced or lacking circulation could
often be identified via a leakage of fluorescein indicating enhanced
vascular ultrafiltration due to VEGF upregulation in dependent
tissues. In all cases studied, areas of insufficient retinal perfusion
colocalized with persistent hyaloid vasculature. The strongest
inhibition of hyaloid vessel regression was present in mice with a
total lack of parts of the retinal vasculature.
Conclusions: The physiology of the regression of vitreal vessels
during development is not entirely understood. Some aspects suggest
a more or less fixed time window for this process, in particular with
respect to the apoptotic death of vascular cells and their removal from
the vitreous. On the other hand, we show here that insufficient retinal
perfusion, presumably via factors like VEGF, clearly leads to a local
persistence of hyaloid vasculature, which in turn improves the
compromised perfusion at least for some time. This finding may help
to develop symptomatic therapeutic concepts in respective diseases
like ROP.
Commercial Relationships: Christina Seide, None; Marina
Garcia Garrido, None; Vithiyanjali Sothilingam, None; Naoyuki
Tanimoto, None; Susanne C. Beck, None; Mathias W. Seeliger,
None
Support: DFG Se837/6-2; GIF 1127-155.2/2010
Retinal Vessel Diameter at High Altitude
Gabriel Willmann1, Andreas Schatz1, M Dominik Fischer1, 3, Kai
Schommer2, Eberhart Zrenner1, Karl-Ulrich Bartz-Schmidt1, Florian
Gekeler1. 1Centre for Ophthalmology, University of Tübingen,
Tübingen, Germany; 2Department of Sports Medicine, University
Hospital Heidelberg, Heidelberg, Germany; 3Nuffield Laboratory of
Ophthalmology, University of Oxford, Oxford, United Kingdom.
Purpose: This study aimed to quantify the impact of acute high
altitude exposure on retinal vessel diameter and to assess possible
correlations to symptoms of acute mountain sickness (AMS) and high
altitude headache (HAH). This work is related to the Tuebingen High
Altitude Ophthalmology (THAO) study.
Methods: VesselMap 1 analyzer (Imedos Systems, Germany) was
used to quantify changes of retinal vessel diameter within one diopter
distance of the papilla in 18 healthy subjects during acute high
altitude exposure to 4559 m compared to baseline recordings (341 m)
using infrared fundus images obtained from a Spectralis® device
(Heidelberg Engineering, Germany). Intra-individual differences
were calculated using ANOVA with a significance level of p < 0.05.
Pearson’s correlation was used to assess a possible linkage between
retinal vessel diameter and scores of AMS and HAH.
Results: Analysis of intra-individual differences revealed a
significant (p < 0.05) increase of mean arterial (MAD; increased
MADaltitude = 13.6 μm) and venous diameter (MVD; increased
MVDaltitude = 26.7 μm) at high altitude in healthy subjects
compared to baseline recordings. Average arterial and vein diameters
at baseline and high altitude were: MADbaseline = 122.72±14.78 μm
vs. MADaltitude = 136.36±19.84 μm; MVDbaseline = 148.02±15.32
μm vs. MVDaltitude = 171.74±22.09 μm; mean±sd) Changes were
completely reversible upon descend. Pearson’s coefficient showed
neither a correlation between increased retinal vessel diameter and
AMS (MAD vs. AMS-c score: r = 0.02, p = 0.95; MVD vs. AMS-c
score: r = -0.17, p = 0.51) nor with HAH (MAD vs. headacheAMS-c
score: r = -0.17, p = 0.50; MVD vs. headacheAMS-c score: r = -0.10,
p = 0.71).
Conclusions: A significant increase in central retinal vessels for both
arteries and veins occurs in response to acute exposure to high
altitude in healthy subjects. This may be attributed to the physiologic
response to the effects of hypoxia during acute high altitude exposure
in non-acclimatized subjects. The missing correlation of retinal vessel
diameter and symptoms of AMS or HAH is of special interest as
restricted cerebral and retinal venous outflow is currently debated to
be associated with a greater headache burden in response to high
altitude hypoxia. Our findings may provide a novel basis for this
debate.
Commercial Relationships: Gabriel Willmann, None; Andreas
Schatz, None; M Dominik Fischer, None; Kai Schommer, None;
Eberhart Zrenner, Retina Implant AG (F), Retina Implant AG (I),
Retina Implant AG (C), Retina Implant AG (P), QLT Inc (C),
Servier, Paris (C), Steinbeis GmbH&CoKG, Stuttgart (I), Steinbeis
GmbH&CoKG, Stuttgart (C), Neurotech, USA (C), Pfizer, USA (C);
Karl-Ulrich Bartz-Schmidt, Retina Implant (P); Florian Gekeler,
Retina Implant AG (F), Okuvision GmbH (F), Retina Implant AG
(C), Retina Implant AG (P)
Retinal and Cerebral Oxygenation Response to Graded Hypoxia
Sunni R. Patel1, Christopher Hudson2, 3, Ayda M. Shahidi1, Susith
Kulasekara2, Joseph Fisher4, John G. Flanagan2, 3, W Alan Mutch5.
1
Ophthalmology and Vision Sciences, Toronto Western Hospital,
Toronto, ON, Canada; 2Department of Ophthalmology and Vision
Science, University of Toronto, Toronto, ON, Canada; 3School of
Optometry and Vision Science, University of Waterloo, Toronto, ON,
Canada; 4School of Physiology, University of Toronto, Toronto, ON,
Canada; 5Perioperative Medicine and Anaesthesiology, University of
Manitoba, Winnepeg, MB, Canada.
Purpose: To determine whether major retinal vessel oxygen
saturation (SO2) and frontal cortex tissue oxygenation (StO2)
changes with controlled manipulation of inhaled oxygen (ETO2)
occur alongside one another.
Methods: Non-invasive hyperspectral retinal imaging (Photon HRC,
Photon etc, QC, Canada) and cortical near-infrared spectroscopy
(FORE-SIGHT™ Cerebral Oximeter, CasMed, CT, USA)
measurements were acquired in 11 healthy volunteers (29±4 yrs,
8M). Continuous readings were acquired during normoxia and graded
hypoxic conditions (i.e., end-tidal [ET] O2 80mmHg, 60mmHg and
50mmHg), whilst end-tidal CO2 tensions were stabilized at
homeostatic baseline values, using a computer-controlled sequential
gas blender system (RespirAct™, Thornhill Research, ON, Canada).
Results: There was a marked decrease in retinal artery SO2
(Friedman analysis; p=0.006) and cerebral StO2 (p=0.001) during
hypoxia which was most pronounced during ETO2 50mmHg (95±8%
vs. 77±10% and 70±3% vs. 61±3%).
Conclusions: This novel preliminary study demonstrated a
significant decrease in SO2 and StO2 of healthy individuals that
occur concurrently during controlled isocapnic hypoxia.
Commercial Relationships: Sunni R. Patel, None; Christopher
Hudson, Opovue Inc (F); Ayda M. Shahidi, None; Susith
Kulasekara, None; Joseph Fisher, Thornhill Research Inc. (I),
thornhill Research Inc (E); John G. Flanagan, Heidelberg
Engineering (C), Heidelberg Engineering (R), Heidleberg
Engineering (F), Carl Zeiss Meditec (C), Carl Zeiss Meditiec (R),
Carl Zeiss Meditiec (R), Alcon Pharmaceuticals (R), Alcon
Pharmaceuticals (R), Optovue Inc (F), Optovue Inc (F), Photon etc
(F), Photon etc (F); W Alan Mutch, None
Support: ORF - RE
3-D Computer-Automated Threshold Amsler Grid to Quantify
Retinal Deficits Before and After Standard Treatment of Wet
Age-related Macular Degeneration
Kristie Lin1, Wolfgang Fink2, 3, Sami Kamjoo1, Michael Davis1, Tom
Chang1. 1Retina Institute of California, Arcadia, CA; 2Physics,
Mathematics and Astronomy, California Institute of Technology,
Pasadena, CA; 3Biomedical Engineering, University of Arizona,
Tuscon, AZ.
Purpose: To characterize and quantify disease activity and response
to treatment of wet Age-related Macular Degeneration (AMD) with
standard intravitreal anti-VEGF agents using 3-D ComputerAutomated Threshold Amsler Grid (3D-CTAG) as a functional
assessment of macular function over time.
Methods: 10 eyes in 10 patients with wet AMD underwent
quantification of central field abnormalities with 3D-CTAG (Fink &
Sadun, JBO 2004) before and one week after treatment with standard
intravitreal anti-VEGF agents including bevacizumab (Avastin,
Genentech), ranibizumab (Lucentis, Genentech), and aflibercept
(Eyelea, Regeneron). Quantitative analysis of 3D visual field
abnormalities included: Lost Area Grade (LAG = scotoma area ratio
as a function of contrast sensitivity), Preserved Area Grade (PAG =
intact visual field area ratio as a function of contrast sensitivity), and
Hill-of-Vision Volume Loss (HVL = ratio of not seen vs. total
number of Amsler grid points).
Results: Wet AMD patients exhibited a central defect at low vs. a
larger central defect at higher contrast sensitivity levels. We
demonstrated the quantification and improvement of macular
structure and function using 3D-CTAG before and after standard
treatment of wet AMD: LAG, PAG, and HVL indices showed
marked improvement following treatment, which correlated with
regression of the choroidal neovascular membrane complex after use
of standard anti-VEGF agents. Overall, 3D-CTAG provided easy
clinical access to measuring and assessing the functional health of the
retina in vivo to quantify change over time.
Conclusions: 3D-CTAG is a direct, non-invasive, and easy-to-use
functional retinal imaging technology. It allows characterization and
quantification of longitudinal changes in macular function before and
after treatment of patients with wet AMD. 3D-CTAG may prove
useful as a screening tool in wet AMD and other central macular
diseases like geographic atrophy due to dry AMD. As newer
therapeutics are developed, the distinctive and quantitative
measurements offered by 3D-CTAG may become an increasingly
important marker to characterize AMD, monitor disease severity, and
provide quantitative outcome measures of therapy. Objective indices
(e.g., LAG, PAG, HVL) delivered by 3D-CTAG may serve as useful
adjunctive measures in clinical trials guided by functional outcomes.
Commercial Relationships: Kristie Lin, Janssen Pharmaceutical
Companies (C), Thrombogenics (C), Sequenom (C); Wolfgang Fink,
University of Arizona (P), California Institute of Technology (P);
Sami Kamjoo, None; Michael Davis, Sequenom (C), Johnson and
Johnson Research and Development (C), Synergetics (C), Allergan
(C), Citi Financial Group (C); Tom Chang, None
Clinical Trial: PRO00007677
Treatment of retinal capillary hemangioblastoma using inhibitors
of the HIF pathway
Mridul Mukherji, Divya Teja Vavilala, Prakash Swami, VK
Chaithanya Ponnaluri. Pharmacy - Pharmaceutical Sci, Univ of
Missouri - Kansas City, Kansas City, MO.
Purpose: Retinal capillary hemangioblastoma (RCH, aka blood
vessel tumor) is observed in patients with mutations in the VHL
tumor suppressor gene. This disease is a multisystem tumor
syndrome where the mutated VHL protein (pVHL) does not bind and
degrade the regulatory α-subunits of the hypoxia-inducible factor
even under normoxic conditions. This results in overexpression of
HIF-dependent pro-angiogenic factors. As a result,
hemangioblastomas of retina, CNS, and kidney are highly vascular in
nature. All current therapies for RCH have significant limitations and
side-effects; e.g., photocoagulation and cryotherapies cause tissue
destruction and inflammation, while anti-VEGF therapies had
minimal detectable beneficial effects. This lack of efficacy of antiVEGF therapies is possibly due to overexpression of other proangiogenic factors (e.g. EPO, PDGF, etc.) in RCH. Thus, there
remains a critical need for the development of an effective agent for
the treatment of RCH. Since inhibition of the HIF pathway is
sufficient to suppress tumor growth by mutated VHL cells in mouse
xenografts, it offers an attractive target to treat RCH.
Methods: Four renal cell carcinoma derived cell lines (RCC4, T314,
PRC3 and WT8) and two human retinal pigment epithelial cells
(D407 and ARPE19) were used to evaluate the expression of cancer
stem cell (CSM) and angiogenic markers. Honokiol, digoxin and
doxorubicin were used as HIF inhibitors in cell culture model.
Inhibition of CSM and angiogenic marker expression by these small
molecules was evaluated using qPCR, western blot analysis and
ELISA.
Results: RCC4 and PRC3 cells which lack VHL showed
upregulation of HIF pathway, thus induction of CSM and proangiogenic genes as compared to T314 and WT8 (both have
functional pVHL), respectively. In D407 and ARPE19 cells HIF is
stabilized under hypoxic conditions, thereby mimicking VHL disease
phenotype, showing induction of CSM and pro-angiogenic genes.
Honokiol, digoxin and doxorubicin significantly inhibited the HIF
pathway, thus lowering the induction of CSM and pro-angiogenic
genes in these cell lines.
Conclusions: Loss of VHL in RCH leads to activation of HIF
pathway and thus induces expression of CSM and pro-angiogenic
genes. Small molecule inhibitors of HIF pathway like honokiol,
digoxin and doxorubicin lowered expression of CSM and proangiogenic genes, and thus presents a novel therapeutic strategy for
RCH treatment.
Commercial Relationships: Mridul Mukherji, None; Divya Teja
Vavilala, None; Prakash Swami, None; VK Chaithanya
Ponnaluri, None
Short-term Effects of Cocoa on Retinal Reactivity in Individuals
with Prediabetes and Type 2 Diabetes
Mary E. Lott1, Alicia Johns1, Joshua Oman1, Julia E. Slocomb2,
Michael Herr1, Robert A. Gabbay3, David A. Quillen4, Kerstin
Bettermann2. 1Heart and Vascular Institute, Penn State Milton S
Hershey Med Ctr, Hershey, PA; 2Neurology, Penn State Milton S
Hershey Med Ctr, Hershey, PA; 3Endocrinology, Penn State Milton S
Hershey Med Ctr, Hershey, PA; 4Opthalmology, Penn State Milton S
Hershey Med Ctr, Hershey, PA.
Purpose: To examine the effects of short term cocoa flavanol
ingestion (i.e. seven days) on retinal blood vessel endothelial function
in individuals at different disease stages of diabetes. We hypothesize
that short term ingestion of high flavanol cocoa compared to a
placebo will be associated with enhanced vascular reactivity. We also
hypothesize that the degree of vascular response to cocoa will likely
be greater in groups without structural vessel damage (i.e. controls
and prediabetics).
Methods: Individuals with prediabetes (n=5), type 2 diabetes (n=5)
and healthy controls (n=5) were recruited. Subjects’ ages ranged from
40 to 71 years. Using a double blinded, randomized crossover design,
this pilot study had subjects consume a high flavanol cocoa (13
grams of pure cocoa) vs. placebo (0 grams of pure cocoa) drink for
seven days with a one week washout period. Retinal blood vessel
vasoreactivity was measured using a flickering light stimulus
(Dynamic Vessel Analyzer, Imedos, Jena, Germany). Fasting glucose
and insulin were also measured.
Results: High flavanol cocoa ingestion led to an increase in peak
retinal vein vasodilation to flickering light in prediabetic individuals
(pre 2.32% ± 0.59% vs. post 4.52% ± 0.75%, P=0.04). Similar effects
were shown in the retinal artery but did not reach significance (pre
1.71% ± 0.82% vs. post 3.18% ± 0.80%, P=0.24). There was a trend
for a small improvement the retinal vein with the placebo drink (pre
2.34% ± 0.60% vs. post 3.08% ± 0.33%, P=0.08) without any change
in the artery diameter (pre 1.45% ± 0.72% vs. post 1.59% ± 0.29%,
P=0.82). There were no significant diameter changes in the control or
type 2 diabetic groups to high flavanol cocoa or placebo. Although
the drinks did not alter fasting glucose and insulin in the prediabetic
or control groups, fasting glucose slightly increased in individuals
with type 2 diabetes (135 to 154 mg/dl; P=0.01).
Conclusions: Short term high flavanol cocoa appears to improve
retinal endothelial function in individuals with prediabetes within one
week. However, a larger sample size is needed to confirm these
findings and a longer duration to potentially see improvements in
type 2 diabetic individuals.
Commercial Relationships: Mary E. Lott, None; Alicia Johns,
None; Joshua Oman, None; Julia E. Slocomb, None; Michael
Herr, None; Robert A. Gabbay, None; David A. Quillen, None;
Kerstin Bettermann, None
Support: Funded by Barsumian Trust Grant; Product supplied by
The Hershey Company
Altered Vascular Microenvironment by Bevacizumab in Diabetic
Fibrovascular Membrane
Shintaro Nakao1, Keijiro Ishikawa1, Shigeo Yoshida1, Ri-ichiro
Kohno1, Masanori Miyazaki1, Hiroshi Enaida1, Toshihiro Kono2,
Tatsuro Ishibashi1. 1Ophthalmology, Kyushu University, Fukuoka,
Japan; 2Ophthalmology, Fukuoka University, Fukuoka, Japan.
Purpose: The purpose of this study was to evaluate the impact of
intravitreal bevacizumab (IVB) on 3 cellular components (vascular
endothelial cells, pericytes, and myofibroblasts) of the vascular
microenvironment in fibrovascular membranes (FVMs) of
proliferative diabetic retinopathy (PDR) patients.
Methods: Immunohistological studies with Abs of CD34, α-SMA,
and TGF-β were performed on 20 surgical specimens obtained during
a pars plana vitrectomy from 8 IVB-treated eyes, while 12 remained
untreated. Four different indexes of vascular phenotype (vascular
area, vascular major axis, CD34(+) endothelial area, and blood vessel
density), and α-SMA expression in vascular and stromal components
were quantitatively-analyzed.
Results: The intraluminal area of blood vessels, CD34(+) endothelial
area, and the blood vessel density in IVB-treated FVMs were
significantly less than in untreated FVMs. The number of CD34(+)
blood vessels in IVB-treated FVMs was similar to in untreated
FVMs. IVB could not affect vascular as well as stromal αSMA(+)
area significantly. However, the ratio of vascular αSMA(+)
area/CD34(+) area was significantly higher in IVB-treated FVMs
than in untreated FVMs. TGF-β expression could be observed in the
IVB-treated FVM.
Conclusions: IVB might primarily affect blood vessels, and the
effects on pericytes and myofibroblasts might be secondary. IVB
treatment regulates vascular microenvironment by the contraction of
blood vessels, the increasing pericyte ratio, and TGF-β expression in
FVMs of PDR patients.
Commercial Relationships: Shintaro Nakao, None; Keijiro
Ishikawa, None; Shigeo Yoshida, None; Ri-ichiro Kohno, None;
Masanori Miyazaki, None; Hiroshi Enaida, None; Toshihiro
Kono, None; Tatsuro Ishibashi, None
Retinal Oxygenation During Intravitreal Treatment for Central
Retinal Vein Occlusion
Sindri Traustason, Morten D. de La Cour, Michael Larsen. Dept of
Ophthalmology,Glostrup Univ Hosp, Copenhagen University,
Glostrup, Denmark.
Purpose: Intravitreal ranibizumab has in recent years become
standard of care treatment for macular edema in central retinal vein
occlusion. While the treatment generally reduces macular edema and
improves visual acuity, cases of increased retinal ischemia have been
reported. The purpose of this study was to investigate the effect of
intravitreal anti-VEGF on retinal oxygenation in patients with central
retinal vein occlusion.
Methods: Patients with central retinal vein occlusion in one eye were
included in a prospective single-group observational study. Included
patients received treatment according to standard clinical procedure
and were followed over a period of six month. All patients received
three initial monthly injections of intravitreal ranibizumab (Lucentis,
Novartis), followed by a three month follow-up period, during which
injections were given as needed.
At each visit, patients underwent examinations which included best
corrected visual acuity, central macular thickness with SD-OCT and
retinal oximetry.
Results: At baseline, retinal venous oxygen saturation was reduced in
the affected eye, compared to fellow eye (35 ± 14% and to 57 ± 9%,
respectively, mean ± SD, p = 3.1 x 10-5, paired t-test).
Retinal venous oxygen saturation and BCVA increased and macular
thickness decreases during ranibizumab treatment. No significant
changes were found in retinal arterial saturation (Fig. 1).
Conclusions: Our results indicate that retinal venous saturation is
increased during treatment with intravitreal ranibizumab and do not
point towards increased retinal ischemia.
Changes in retinal oximetry, best corrected visual acuity (BCVA) on
the ETDRS scale and central retinal thickness (OCT) from baseline to
month 4 and 7. P-values indicate results from one sample t-tests
(μ=0).
Commercial Relationships: Sindri Traustason, Novartis (F),
Oxymap (R); Morten D. de La Cour, Alcon (C), Novartis (C);
Michael Larsen, None
Support: Novartis, Th. Elmquist’s Fund for Eye Research, Velux
Foundation, Carl and Nicoline Larsen's Fund, The Danish Eye Health
Society, Synoptik Foundation
Rationale for Bimonthly Ranibizumab and Quarterly
Aflibercept. A Drug and Disease Assessment Model in Wet Agerelated Macular Degeneration
Daniele Veritti1, Gianluca Gorni2, Laura Perissin3, Paolo Lanzetta1.
1
Department of Ophthalmology, University of Udine, Udine, Italy;
2
Department of Mathematics and Computer Science, University of
Udine, Udine, Italy; 3Department of Biomedical Sciences and
Technologies, University of Udine, Udine, Italy.
Purpose: A drug and disease assessment model was used to evaluate
the impact of different treatment regimens on intraocular ranibizumab
and aflibercept concentration and free vascular endothelial growth
factor (VEGF) proportion.
Methods: A time-dependent mathematical model using Wolfram
Mathematica software was developed on the 12-month data from
PIER, MONT BLANC, CATT, VIEW. Pharmacokinetic and affinity
data for ranibizumab and aflibercept were obtained from published
reports. Two alternative regimens with bimonthly ranibizumab and
quarterly aflibercept were simulated.
Results: The mathematical model showed good correlation between
clinical trial data and intraocular VEGF proportion. In the fixed
monthly 0.5 mg ranibizumab regimen that has been evaluated in
MARINA, ANCHOR and CATT trials highest free VEGF levels stay
below 1/100,000 of total VEGF. Following the quarterly
administration of 0.5 mg ranibizumab (PIER study), highest free
VEGF levels reach the 60% of total VEGF. In the individualized
regimen studied in the MONT BLANC trial free VEGF proportion
reaches 100% during the maintenance phase. The aggressive
individualized regimen with 0.5 mg ranibizumab studied in the
CATT trial with a simulated even distribution of injections maintains
free VEGF levels constantly below 0.5% of total VEGF. Simulations
of the two alternative regimens suggest that such treatment
approaches maintain the free VEGF proportion under threshold
levels. Free VEGF proportion remains stably below 0.5% and 0.1%
with bimonthly ranibizumab and quarterly aflibercept respectively.
Conclusions: Fixed bimonthly ranibizumab or quarterly aflibercept
regimens may result in visual acuity improvement with reduced
burden over treatment strategies applied in clinical trials.
Commercial Relationships: Daniele Veritti, None; Gianluca
Gorni, None; Laura Perissin, None; Paolo Lanzetta, Alimera (C),
Allergan (C), Bayer (C), Novartis (C), Novartis (R), Roche (C),
Iridex (P)
Retinal Vein Occlusions in Young Patients: Visual Outcomes and
Associated Systemic Risk Factors
Noureen Khan1, 2, Michael M. Lai1, 2. 1Dept. of Ophthalmology,
Georgetown University/Washington Hospital Center, Washington,
DC; 2The Retina Group of Washington, Chevy Chase, MD.
Purpose: To assess the visual outcomes in patients forty years of age
and younger with retinal vein occlusion and to identify associated
systemic risk factors.
Methods: This is a retrospective, comparative, consecutive case
series. Fifty eyes from 50 patients were diagnosed with branched
retinal vein occlusion (BRVO), hemi-central retinal vein occlusion
(HRVO) or central retinal vein occlusion (CRVO) that presented in
patients 40 years or younger from 1997 to 2002. Risk factors
(hypertension, hyperlipidemia, diabetes, obesity, glaucoma, smoking,
OCP use, hypercoagulable lab abnormalities, and final visual acuity
(VA) were recorded and compared.
Results: Final visual acuity (VA) found that 46% had improved
visual outcomes. Seventy eight percent ended up with at least 20/60
vision or better. Patients with good baseline vision (20/60 or better)
were found to have statistically significant better final VA compared
to patients with baseline vision of less than 20/60 (P= <0.0001).
Additionally, patients with retinal ischemia on FA are also found to
have a statistically significant worse outcome on visual prognosis (P=
0.0017).
Forty eight percent of patients were found to have at least one
systemic risk factor, 30% two or more risk factors, and 24% had
abnormal hypercoagulable work up. The most common systemic risk
factors include HTN (24%) and abnormal hypercoagulable work up
(24%). The most common abnormal lab work up was
Methylenetetrahydrofolate reductase mutation, which was found in
five patients.
Conclusions: There is a strong association between vein occlusion in
young patients and at least one systemic risk factor (78%). Most
patients presented with good VA (mean 20/50) and non-ischemic
disease (86%). Many young RVO patients (24%) had an abnormal
hypercoagulable work-up. Most patients improved or maintained
vision during follow-up (78%) and obtained good final VA (mean
20/40). Poor baseline VA and retinal ischemia were associated with a
worse VA outcome.
Given such findings, we recommend that any young patient
presenting with retinal vein occlusions undergo extensive evaluation
to identify systemic risk factors of thrombosis, including prothrombotic medication and undiagnosed hypercoagulable states.
Initial evaluation should emphasize baseline VA as well as retinal
perfusion status, given that they are strong predictors of final visual
outcomes.
Commercial Relationships: Noureen Khan, None; Michael M.
Lai, None
Effects of Prostacyclin on Isolated Porcine Retinal Arterioles:
Cross-Talk between Nitric Oxide and Prostacyclin
Shinji Ono, Taiji Nagaoka, Tsuneaki Omae, Takayuki Kamiya,
Akitoshi Yoshida. Ophthalmology, Asahikawa Med University,
Asahikawa, Japan.
Purpose: To investigate the vasomotor activity of prostacyclin
(PGI2) on porcine retinal arterioles.
Methods: Porcine retinal arterioles were isolated, cannulated, and
pressurized without flow in vitro. Changes in diameter were recorded
using videomicroscopic techniques. To confirm the role of the
endothelium, we compared the responses before and after endothelial
removal. To examine the involvement of endothelium-derived
relaxing factor nitric oxide (NO), we assessed the responses in the
presence of NO synthase inhibitor NG-nitro-L-arginine methyl ester
(L-NAME).
Results: The retinal arterioles dilated in a concentration-dependent
manner in response to PGI2. This vasodilation decreased
significantly after the endothelium was removed. L-NAME
significantly inhibited PGI2-induced vasodilation comparable to
denudation.
Conclusions: PGI2 elicits vasodilation of the retinal arterioles
mediated not only by the vascular smooth muscle but also the
endothelium, suggesting that an endothelium-dependent component
of PGI2-induced vasodilation may be involved because of production
of NO. We speculated that cross-talk between NO and PGI2 may
exist in the retinal circulation.
Commercial Relationships: Shinji Ono, Kaken Pharmaceutical
Co.,Ltd. (F); Taiji Nagaoka, None; Tsuneaki Omae, None;
Takayuki Kamiya, None; Akitoshi Yoshida, None
Perifoveal Microvasculature in Human Eyes with Vascular
Comorbidities
Geoffrey Chan1, 2, Chandra Bala1, 2, Paula Yu1, 2, William Morgan1,
Ian L. McAllister1, Stephen J. Cringle1, 2, Yu Dao-Yi1, 2. 1Centre for
Ophthalmology and Visual Science, Perth, WA, Australia;
2
Australian Research Council Centre of Excellence in Vision
Science, Canberra, ACT, Australia.
Purpose: The microvasculature change which precedes the
development of clinically manifest ocular disease has not been
clarified. This study delineates the morphological characteristics of
the perifoveal microvasculature in patients with vascular
comorbidities.
Methods: Comparisons were made between 11 human eyes from
patients with vascular comorbidities and 17 healthy control eyes. All
eyes were absent of clinically evident ocular disease.
Microcannulation and targeted perfusion techniques were used to
label the retinal microvasculature. Retinae were then flat mounted
and the peripapillary region 2mm nasal to the fovea imaged using
confocal laser scanning microscopy. Two- and three-dimensional
image reconstructions were used to perform quantitative
measurements of individual capillary networks within the perifovea.
Parameters measured included capillary diameter, capillary loop area,
capillary density and capillary surface area. Comparisons were made
between normal eyes we previously studied and those from patients
with vascular comorbidities.
Results: Capillary diameter was increased in all layers of the retina
in patients with vascular comorbidities with the exception of the
nerve fibre layer capillary network. Capillary loop area was reduced
in all layers of the retina in patients with vascular comorbidities
except the deep capillary network of the inner nuclear layer.
Capillary density was reduced within the nerve fibre layer of eyes
with vascular comorbidities. There was no difference in the relative
occupied capillary surface area between healthy and diseased eyes.
Conclusions: Morphometric differences in retinal capillary networks
are present between healthy patients and those with vascular
comorbidities. Understanding the capillary changes that precede
clinical ocular disease may be useful for defining the pathogenesis of
retinal vascular disease.
Commercial Relationships: Geoffrey Chan, None; Chandra Bala,
None; Paula Yu, None; William Morgan, National Health and
Medical Research Council (F); Ian L. McAllister, None; Stephen J.
Cringle, None; Yu Dao-Yi, None
Flavonoid-rich dark chocolate improves endothelial function in
retinal vessels
Naim Terai, Alexandra Gedenk, Eberhard Spoerl, Richard P.
Stodtmeister. Ophthalmology, University of Dresden, Dresden,
Germany.
Purpose: As a sign of autoregulation retinal arterioles and venoles
dilate in response to flicker stimulation. This response can be
attributed to vascular endothelial function. It has been shown that in
coronary arteries the endothelial-dependent vasomotion improves
significantly after flavonoid-rich dark chocolate. Aim of the present
study was to investigate whether dark chocolate leads to an
improvement of retinal endothelial function in glaucoma patients and
age-matched healthy subjects using the Dynamic Vessel Analyzer
(DVA,Imedos, Jena, Germany).
Methods: Subjects: Patients with primary open-angle glaucoma (GL)
n=18; m/f=5/13, control subjects (CS) n=18 m/f=7/1; Age GL: 65±6
(arithmet mean±s, years); CS: 64±7 (p=0.72). RR prior DVA:
GL141/82±18/9 mmHg; CS: 133/79±14/11 (p=0.17/0.22). Visual
field: GL: Mean defect: -17.9,-2.1,2.7 db (Minimum, median,
maximum); PSD: 1.1, 2.4, 15.6.
The patients and controls were assigned to dark or white chocolate
(C) by randomisation with forced equal distribution. The number in
each of the four groups was 9. Examination: Following pupil dilation
measurement of blood pressure, intraocular pressure, blood glucose
was done followed by DVA. This instrument consists of a fundus
camera, video equipment and computer-aided process control.
Recording procedure: Online measurement of the diameter of a
retinal arteriolar and a venolar vessel segment, 50s baseline, three
periods of 20s flicker stimulation and 80s follow- up registration.
Three registrations were averaged.
The change of the vessel diameter following flicker stimulation
before and 2 hours after chocolate ingestion was measured. Statistics:
Eight paired Wilcoxon tests with Bonferroni-Holm correction. H0:
Vessel dilation (DIL)before=DILafter. H1:DILafter>DILbefore. One
sided 5% sigificance level p=0.1.
Results: GL: DIL of arterioles before and after comsuption of darkC
or whiteC didn't differ. Lowest p=0.41. CS: DIL of the arterioles
didn't differ significantly. Lowest p=0.56. DIL of venules before
darkC =1.9, 3.1, 4.4; after darkC=1.9, 5.2, 6.9; p=0.08. DIL of veins
before whiteC=2.4, 2.8, 5.6; after whiteC=1.4, 3.0, 5.8; p=0.95.
Conclusions: In GL patients retinal vessel autoregulation remained
unchanged by darkC or whiteC. In our control subjects, however, the
extent of the autoregulatory response to flicker stimulation improved
statistically significantly by darkC but not by whiteC which contains
no flavonoids.
Hyoung Kim, None; Young S. Yu, None; Jin Sook Kim, None;
Jeong Hun Kim, None
Support: the Bio-Signal Analysis Technology Innovation Program
of MEST/NRF, Republic of Korea (2011-0027723), Mid-Career
Researcher Program, Republic of Korea (2011-0017910) of
MEST/NRF, grant from the Korea Institute of Oriental Medicine
(KIOM) (K11040)
Commercial Relationships: Naim Terai, None; Alexandra
Gedenk, None; Eberhard Spoerl, None; Richard P. Stodtmeister,
Novartis (F)
Inhibition of retinal neovascularization by luteolin via
suppression of VEGF expression and VEGFR signaling pathway
Sung Wook Park1, 2, Chang Sik Cho1, Hyoung Oh Jun1, Nam Hee
Ryu3, Jin Hyoung Kim1, Young S. Yu1, Jin Sook Kim3, Jeong Hun
Kim1, 2. 1FARB, Department of Ophthalmology, Seoul National
University Hospital, Seoul, Republic of Korea; 2Biomedical Sciences,
Seoul National University College of Medicine, Seoul, Republic of
Korea; 3Diabetic Complications Research Center, Division of
Traditional Korean Medicine Integrated Research, Korea Institute of
Oriental Medicine, Daejeon, Republic of Korea.
Purpose: This study was to investigate the anti-angiogenic effect of
luteolin against reactive oxygen species (ROS) induced retinal
neovascularization
Methods: The toxicity of luteolin was evaluated through modified 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay in
human retinal microvascular endothelial cells (HRMECs) as well as
terminal deoxynucleotidyl transferase dUTP nick-end labeling
staining in the retina of C57BL/6J. After intravitreal injection of
luteolin in the mouse model of ROP, retinal neovascularization was
examined by fluorescence angiography and vessel counting. Antiangiogenic activity of luteolin was evaluated by VEGF-induced
migration and tube formation assay. The effect of luteolin on t-BHinduced ROS production was measured with 2’7’-dichlorofluorescein
diacetate. The effect of luteolin on t-BH induced and hypoxiainduced VEGF transcription and expression were evaluated by RTPCR and Western blot, respectively.
Results: Luteolin never affected the viability of HRMECs up to 10
μM, where luteolin never induce any structural change in all retinal
layers. Luteolin inhibited retinal neovascularization in the mouse
model of ROP. Moreover, VEGF-induced migration and tube
formation were significantly decreased by co-treatment of luteolin.
Luteolin attenuated VEGF transcription via blockade of t-BH induced
ROS production. Luteolin suppressed hypoxia-induced VEGF
expression via attenuating hypoxia inducible factor 1 α expression.
Conclusions: Our results suggest that luteolin could be a potent antiangiogenic agent for retinal neovascularization, which is related to
anti-oxidative activity to block ROS production and to subsequently
suppress VEGF expression and the pro-angiogenic effect of VEGF
Commercial Relationships: Sung Wook Park, None; Chang Sik
Cho, None; Hyoung Oh Jun, None; Nam Hee Ryu, None; Jin
Plasma VEGF-levels are variable in patients with persistent renal
dysfunction and diabetes: possible implications and risks for
ophthalmic anti-VEGF therapy
Markus van der Giet, Mirjam Schuchardt, Nicole Pruefer, Jasmin
Pruefer. Med. Klinik - SP Nephrology, Charite - Universitätsmedizin
Berlin, Berlin, Germany.
Purpose: Ophthalmic anti-VEGF therapies are used commonly
without monitoring of systemic risks and their intraocular application
might cause unexpected effects to systemic VEGF-dependent
regulatory mechanisms. Our study aims at quantifying systemic
VEGF-levels in situations commonly found in the target population
of ocular anti-VEGFs
Methods: Blood was drawn form 30 healthy controls (age 28-78
years), 30 patients with type I or II diabetes without renal dysfunction
(24-84 years, DM), 30 patients with chronic kidney disease (CKD)
stage III-V (incl. hemodialysis patients) and 30 patients with type I/II
diabetes and renal dysfunction (CKD III-V, CKD/DM). VEGF A-D
were quantified using 96-well ELISA (Cusabio, Wuhan, China)
according to manufacturer’s protocol. VEGF levels were quantified
by comparison to standard concentration curves; all values are given
as means.
Results: Mean plasma VEGF A levels were 85.8 pg/ml in healthy
controls and 77.6 pg/ml in DM patients. These levels were
moderately elevated in CKD (129.92pg/ml) and CKD/DM (111.9
pg/ml) patients (p<0.05). Levels of VEGF-B were significantly
elevated in DM (169.3 pg/ml), CKD (271.4 p/ml) and CKD/DM
patients (697.1 pg/ml) in comparison to controls (105.6 pg/ml).
Changes of VEGF C levels were massive and significant in CKD
(1187.4 pg/ml), DM (214.7 pg/ml), and CKD/DM (3939.1pg/ml)
patients compared to controls (162.2pg/ml). VEGF D levels were
very similar to VEGF A, with no significant differences between
controls (89,3 pg/ml) and DM patients (61.0 pg/ml) while there was a
significant increase in CKD (209,4 pg/ml) and CKD/DM
(208.2pg/ml).
Conclusions: Our data show that the plasma VEGF levels in elderly
patients (target population for ophthalmic anti-VEGF treatment) are
affected by disease status as seen in Diabetes Mellitus and even more
in Chronic Kidney Disease, especially for the VEGF B and C
isoforms. The physiopathology and relevance of these changes
remain unclear but caution should be taken in risk patients when
injecting ophthalmic anti-VEGFs with a long plasma half-life,
implying a high systemic exposure.
Commercial Relationships: Markus van der Giet, Novartis (F),
Roche (F), Novartis (C), Takeda (F), Bayer (C); Mirjam
Schuchardt, Deutsche Hochdruckliga (F), Peter und Traudl
Engelhorn Stiftung (F), Sonnenfeld Stiftung (F); Nicole Pruefer,
None; Jasmin Pruefer, None
Support: Novartis Pharma Gmbh; Nuremberg, Germany
Pathological retinal neovascularization is exacerbated by
selective pharmacological blockade of VEGFR2, but ameliorated
by combination with Placental Growth Factor (PlGF) inhibition
in the Oxygen induced Retinopathy (OIR) model in mice
Eunice Cheung, Ivan B. Lobov, George D. Yancopoulos, Stanley J.
Wiegand. Ophthalmology, Regeneron Pharmaceuticals, Inc.,
Tarrytown, NY.
Purpose: Vascular endothelial growth factor A (VEGF-A) signaling
through VEGF receptor 1 (VEGFR1) and VEGFR2 regulates both
physiological and pathological angiogenesis [Luttun et al., Nature
Med. 2002, 8:831-840]. We have reported that genetic deletion or
inhibition of the VEGFR1 ligand, PlGF, reduced vasoobliteration and
neovascularization in OIR [IOVS 2009; 50:E-Abstract 2943; IOVS
2010; 51:E-Abstract 4486]. We also demonstrated that inhibition of
either PlGF or VEGFR1 could improve normal blood vessel regrowth
and also lessen neovascularizations [IOVS 2011; 52:E-Abstract
6064]. In this study, we administered DC101, a VEGFR2 blocking
antibody, alone or in combination with a PlGF blocking antibody
after vessel loss was complete, to evaluate the effects of selective
blockade of activity of VEGFR2 alone, or in combination with
inhibition of the VEGFR1 ligand, PlGF, in the murine model of OIR.
Methods: Mice were place in a hyperoxic environment (75% O2) at
postnatal day 6 (P6) and returned to room air at P11. Pups were
injected systemically with 25mg/kg DC101 or a control protein (Fc)
at P12. Left eyes of DC101 treated pups were injected intravitreally
(IVT) with 5ug of a polyclonal Goat anti-Mouse PlGF-2 or a control
IgG (Normal Goat IgG) at P13. Retinal flatmounts were collected at
P16 and endothelial cells stained with FITC-labeled Griffornia
simplicifolia lectin I and anti-NG2, a pericyte marker.
Results: At P16, pathological neovascularizations worsened in mice
treated with DC101 relative to controls. In contrast, IVT
administration of PlGF antibody in mice treated with DC101
produced significant improvement of pathological neovascularization
relative to animals treated with DC101 and control IgG. No
appreciable differences were noted among groups in the extent of the
residual avascular area on P16.
Conclusions: This study demonstrates that selective pharmacological
neutralization of VEGFR2 is ineffective in ameliorating pathological
neovascularization in OIR, while co-incident neutralization of the
VEGFR1 ligand, PlGF results in significant amelioration of
pathological neovascularization.Thus, this study further supports the
hypothesis that inhibition of VEGFR1 ligand, such as PlGF, can play
an important role in the inhibition of pathological angiogenesis.
Commercial Relationships: Eunice Cheung, Regeneron
Pharmaceuticals (E); Ivan B. Lobov, Regeneron Pharmaceuticals,
Inc. (E); George D. Yancopoulos, Regeneron Pharmaceuticals (E),
Regeneron Pharmaceuticals (I), Regeneron Pharmaceuticals (P);
Stanley J. Wiegand, Regeneron Pharmaceuticals, Inc (E)
Support: None in the Support field
Treatment of proliferative idiopathic macular telangiectasia Type
2 with intravitreal bevacizumab
Ranjit Sandhu, Robin D. Hamilton. Medical Retina, Moorfields Eye
Hospital, London, United Kingdom.
Purpose: Idiopathic macular telangiectasia Type 2 is bilateral and
characterised by retinal opacification, vascular telangiectasia, rightangled venules, intraretinal crystalline deposits, foveal thinning,
retinal pigment epithelial hypertrophy and rarely, choroidal
neovascularisation (proliferative form).
The purpose of this study was to determine the effect of treatment
with intravitreal bevacizumab on retinal thickness and visual acuity
in the proliferative form of MacTel Type 2 in a small case series.
Methods: A retrospective review of clinical notes of patients with
idiopathic macular telangiectasia Type 2 complicated by choroidal
neovascularisation based on fundus fluorescein angiography who
received treatment with intravitreal bevacizumab injections was
carried out. All patients had Snellen visual acuity testing, fundus
fluorescein angiography and optical coherence tomography (OCT) at
baseline. The patients were treated until the choroidal neovascular
membrane (CNV) showed no signs of activity based on the OCT
findings. Visual acuity and central macular thickness were recorded
at final follow-up visit.
Results: Ten eyes of seven patients, of which two were males and
eight females, were included. The average age was 60 years ± 5 years
and the average follow-up was 14 months ± 12 months (range, 2-34
months). The visual acuity was 0.6 ± 0.5 logMAR at baseline and 0.6
logMAR ± 0.4 logMAR at final follow-up. The central macular
thickness was 223µ ± 41µ at baseline and 229µ ± 53µ at final followup. The average number of bevacizumab injections administered
were 3 (range, 1-8). There was no improvement in the visual acuity
or central macular thickness. The CNV resolved in all ten eyes of
seven patients. One patient did not require bevacizumab as the CNV
regressed spontaneously.
Conclusions: Although the use of intravitreal bevacizumab in the
proliferative form of idiopathic macular telangiectasia Type 2 did not
improve the visual acuity or reduce the central macular thickness, it
resulted in the regression of the CNV. As the probability of
progression from absence to presence of an IS/OS PR break has been
recently reported to be as high as 72% after 5 years by the MacTel
Study group and CNV complicating MacTel posing a further threat to
central visual loss, treatment with intravitreal bevacizumab prevents
further visual loss in this potentially sight threatening condition.
Commercial Relationships: Ranjit Sandhu, None; Robin D.
Hamilton, Novartis (R), Bayer (R), Ellex (R), Valon (R)
Computational model of blood flow through the choriocapillaris
highlights marked heterogeneity of blood flow
Moussa A. Zouache1, 2, Philip J. Luthert1, 2, Ian Eames3. 1Institute of
Ophthalmology, University College London, London, United
Kingdom; 2NIHR Biomedical Research Centre in Ophthalmology,
London, United Kingdom; 3Department of Mechanical Engineering,
University College London, London, United Kingdom.
Purpose: The choroid is a pigmented vascular layer located between
the retina and the sclera. It supplies the outer retina with oxygen and
nutrients through a dense, one-layered network of capillaries called
the choriocapillaris. The blood flow through the choriocapillaris is
remarkably high. A discrete lobular segmentation of the plexus is
observed in the macula and the posterior fundus. The aim of this
study was to investigate, theoretically, the heterogeneity of blood
renewal within the choriocapillary plexus over the macular region
and posterior pole.
Methods: A computational model informed by the angioarchitecture
of the choriocapillaris as described in the literature and as revealed in
whole mounts stained for CD31, a vascular marker, was built in order
to simulate blood flow within choriocapillary lobules. Using a simple
geometry, analytical and numerical solutions for the flow were
obtained.
Results: The computer model included the spacing and distribution
of arterioles and venules connecting with the plexus and derived
parameters including the pressure distribution and velocity fields.
The model revealed significant heterogeneities in blood renewal over
the vascular network. The number of regions of minimal blood
renewal was shown to equate to the number of arterioles and venules
supplying each choriocapillary lobule.
Conclusions: The choriocapillary plexus displays significant
heterogeneities in blood renewal resulting from the intrinsic
anatomical and functional organization of the plexus. This implies
that there may be areas of critical perfusion even given the very high
blood flow rates within the choriocapillaris as a whole. Observations
may have relevance for the pathogenesis of degenerative diseases
such as macular degeneration. Furthermore, this model provides the
basis for a more comprehensive model of the pathophysiology of the
back of the eye.
Commercial Relationships: Moussa A. Zouache, None; Philip J.
Luthert, None; Ian Eames, None
Support: Special Trustees of Moorfields Eye Hospital and
University College London Impact Studentship Scheme
Periocular injection of a broad spectrum antiangiogenic therapy
regresses choroidal neovascularization
Ofra Benny, Zai-Long Chi, Lauren Bazinet, Robert J. D'Amato.
Boston Children's Hospital, Harvard Medical School, Boston, MA.
Purpose: Current therapies with intravitreal injections of antivascular endothelial growth factor (VEGF) agents for intraocular
neovascularization suppress neovascularization and vessel leakage.
However, nearly half of AMD patients do not respond to this therapy,
and those that do require injections repeatedly and chronically,
poising them for clinical ocular complications. The purpose of our
study is to develop a less invasive, local treatment that can safely be
administered chronically, as a means of simultaneously improving
the potency of treatment and reducing the burden for patients with
AMD.
Methods: Lodamin is a polyethylenglycol-poly lactic acid (PEGPLA) conjugate of the antiangiogenic drug TNP-470, a Methionine
aminopeptidase 2 (MetAp2) inhibitor and selective cytostatic agent of
endothelial cells. A laser-induced CNV model was used in C57/Bl
mice, with 6 lesions inflicted per retina. Average CNV size was
measured following isolectin IB4 staining of retinal vessels.
Results: Lodamin was administered as a single retrobulbar or
peribulbar injection (100ug per eye) on the day of laser treatment. By
day 7, 42% inhibition and 34% inhibition in CNV size was recorded,
respectively. Additionally, experiments were performed with
established CNV lesions where treatment was given on Day 7 postlaser. In these cases, Lodamin was able to regress CNV by 45% as
measured on day 14 post CNV induction.
Conclusions: We have previously demonstrated Lodamin to be a
potent broad-spectrum antiangiogenic drug in multiple angiogenic
models. Lodamin was effective in treating CNV when administered
orally. However in the interest of reducing systemic exposure, local
administration (intravitreal) was explored, yielding positive results.
We now show that Lodamin is also effective when given by
periocular administration, thus minimizing the risks clinically
associated with intravitreal injection. Lodamin’s ability to regress
preexisting neovascularization together with its improved delivery
may change the paradigm of patient therapy and improve current
disease treatment.
Commercial Relationships: Ofra Benny, None; Zai-Long Chi,
None; Lauren Bazinet, None; Robert J. D'Amato, Boston
Childrens Hospital (P)
Efficacy and safety of subconjunctival bevacizumab for recurrent
pterygium
Larissa S. Stival1, Anelise M. Lago1, Marisa N. Figueiredo1, Ricardo
G. Bittar2, Marcia L. Machado1, Joao J. Nassaralla2. 1Cornea and
Refractive Surgery, Instituto de Olhos de Goiania, Goiania, Brazil;
2
Retina and Vitreous, IOG, Goiania, Brazil.
Purpose: To evaluate clinical outcomes and complications of
subconjunctival bevacizumab (Avastin) in patients with recurrent
pterygium.
Methods: This prospective clinical trial included 36 patients (36
eyes), who underwent pterygium surgery and who were diagnosed
with recurrent pterygium. All pacientes received subconjunctival
application of bevacizumab (0,5ml). Pterygium vascularity and
thickness was graded according Tan's rating. The size of the
pterygium (measured in mm) was recorded from baseline to 8 weeks
after injection. Treatment-related complications and adverse events
were reported. The main outcome of measurements was the change in
size, vascularity and thickness.
Results: There were 18 males (50%) and 18 females (50%) of 36
patients with a mean age of 58,75 years (SD 10,98 years). 30.6% of
pacients had recurrent pterygium in both eyes, being 47.2% in the left
eye and 22.2% in the right eye. 44.4% of patients were from Goiânia
(Brazil) and 55.6% from other cities. More than half of patients
(58.3%) had a family history of pterygium. There was a significant
difference in the size of pterygium at different intervals (P < 0.05)
and the mean surface area was reduced. 66.7% of patients presented
hyposphagma on the 2nd day after the subconjunctival application,
decreasing to 30.6% by day 7 and finally no patient after 1 month.
Most patients (69.4%) had improvement of irritative symptoms
within 2 days, 88.9% after 7 days and 97.2% after 1 month.
Conclusions: Subconjuctival bevacizumab injection is useful in
management of patients with recurrent pterygium without significant
local or systemic adverse effects.
Commercial Relationships: Larissa S. Stival, None; Anelise M.
Lago, None; Marisa N. Figueiredo, None; Ricardo G. Bittar,
None; Marcia L. Machado, None; Joao J. Nassaralla, None
A Treat and Extend Regimen Using Anti-VEGF Therapies for
Central Retinal Vein Occlusion
Christopher J. Brady, Rayan Alshareef, Andre J. Witkin, Carl D.
Regillo. Wills Eye Institute, Jefferson Univ School of Medicine,
Philadelphia, PA.
Purpose: With the FDA approval of ranibizumab and aflibercept for
macular edema due to central retinal vein occlusion (CRVO), a new
standard of care has been adopted for this condition. The initial
clinical trials were designed using a monthly treatment approach
followed by as-needed injections. A treat and extend (T&E) approach
has been described in the treatment of neovascular age-related
macular degeneration (ARMD), and is being increasingly adopted.
Such an approach has the potential to reduce the burden of treatment
on patients and physicians while maintaining effectiveness in the
treatment of CRVO.
Methods: Nine physicians who routinely adopt a T&E extend
approach were identified. A retrospective analysis of their patients
with CRVO treated with either bevacizumab or ranibizumab for at
least six months was undertaken. Change in Snellen visual acuity and
central macular thickness (CMT) were analyzed. Adherence to a
T&E protocol was also analyzed.
Results: Sixty-six patients were identified, 25 of whom were
treatment-naïve, and thus included in the analysis. The mean age was
73.6 years, and 63% were female. Sixty-seven percent reported
hypertension and 14.8% reported diabetes mellitus. Mean duration of
symptoms prior to therapy was 131 days. After 6 months follow-up,
mean visual acuity improved to 20/80 from 20/200+1. Mean CMT
improved 229.4 microns (from 652 to 422 microns).
Conclusions: There are several theoretical advantages to a T&E
regimen for CRVO compared with monthly and as needed strategies
including fewer office visits and fewer injections, and therefore lower
risk of side effects including endophthalmitis. Compared with
ARMD, T&E for CRVO appears to be less successful. This may be
due to differences in pathophysiology, or perhaps demographics that
hinder adherence to a strict treatment protocol. Given the logistical
benefits and reduced risk profile, an initial attempt at T&E with a low
threshold to maintain therapy at fixed interval is reasonable.
Commercial Relationships: Christopher J. Brady, None; Rayan
Alshareef, None; Andre J. Witkin, None; Carl D. Regillo,
Genentech (C), Regeneron (C), Alcon (C), Thrombogenics (F), GSK
(F), ACT (F)
In vitro studies on the antiangiogenic effects of Pigment
Epithelium Derived Factor and Somatostatin
Anna Salas Torras, Andrea R. Carvalho, Ibane Abasolo, Miguel A.
Zapata, Laura N. Distefano, Simo Schwartz, Jose Garcia-Arumi.
Ophthalmology, Vall d'Hebron Research Inst, Barcelona, Spain.
Purpose: Pigment Epithelium Derived Factor (PEDF) and
Somatostatin (SST) have been demonstrated to be two of the
principal natural antiangiogenic and anti-VEGF factors present in the
retina. Both peptides are promising tools to be used in different
strategies for a variety of retinal disorders affecting the endothelial
compartment such as PDR and wet AMD.
The aim of this study is to evaluate the antiangiogenic and
antiproliferative properties of PEDF and SST in several in vitro
models, as a preliminary proof-of-concept assay before testing their
efficacy in vivo.
Methods: To test the antiangiogenic properties of PEDF and SST in
vitro, two cell types were used: the ARPE-19 cell line derived from
the retinal pigment epithelium, and a primary culture of human
umbilical vein endothelial cells (HUVECs), as a model of vascular
endothelium. Both are the main cell types affected in proliferative
eye diseases.
First we determined the expression of SST receptors (SSTR) in
HUVECs and ARPE-19 by RT-PCR. PEDF receptors could not be
verified, as they are still not identified. Antiproliferative action of
PEDF and SST were tested in HUVEC and ARPE-19 cells by MTT
assay after 24 and 48 h of incubation. In addition, HUVECs were
used as an endothelial model to perform migration and tubule
formation assays under VEGF treatment, in order to test the
antiangiogenic activity of PEDF and SST.
Results: Using RT-PCR, ARPE-19 cells were found to express
SSTR-1, -2, -3 and -5, while HUVEC cells mainly express SSTR-1
and -2. In proliferation assays, SST was found to significantly inhibit
ARPE-19 proliferation up to a 40% at 10-4M, (p<0,001). PEDF only
inhibited proliferation of ARPE-19 cells in a 13% (10-8M, p<0,001).
Both PEDF and SST showed a similar suppression of endothelial
migration (35% and 39% respectively, p<0,05) in HUVEC cells
treated with 25 ng/ml of VEGF. Finally, tube formation assay showed
that PEDF was able to reduce the tubule length as well as the number
of tubules and connections (22%, 30% and 57% respectively,
p<0,05). On the other hand, SST was able to reduce the number of
tubules and connections but not the tubule length (51% and 68%
respectively, p<0,05).
Conclusions: Our data demonstrate that PEDF and SST have both
antiproliferative and antiangiogenic effects in retinal pigment
epithelium and endothelial cells and can be good candidates for the
treatment of retino-epithelial disorders in vivo.
Commercial Relationships: Anna Salas Torras, None; Andrea R.
Carvalho, None; Ibane Abasolo, None; Miguel A. Zapata, None;
Laura N. Distefano, None; Simo Schwartz, None; Jose GarciaArumi, None
Support: ISCIII grant PI11/00706
Our experience with Sequential treatment of macular oedema
from retinal vascular occlusions with anti-VEGF agents and
Ozurdex
Evangelia Papavasileiou, Andre Grixti, Vineeth Kumar, Som Prasad.
Eye Treatment Centre, Arrowe Park University Hospital, London,
United Kingdom.
Purpose: To report our experience with sequential treatment with
ant-VEGF agents and dexamethasone 0.7mg sustained-release
intravitreal implant (Ozurdex) in patients with macular oedema due
to central or branch retinal vein occlusion.
Methods: An interventional case series of 31 patients treated with
intravitreal anti-VEGF initially and then with Ozurdex was
undertaken in our hospital. Included were demographics, underlying
disease, BCVA, number of anti-VEGF injections required, IOP
measurements, other prior therapies, examination, and adverse
events. Patients underwent a comprehensive examination including
optical coherence tomography, fluorescein angiography, and fundus
photography. Ozurdex was administered in accordance with the
manufacturer’s instructions using the 22-gauge applicator device.
Patients were re-treated with anti-VEGF agents post Ozurdex
injection only if OCT central field thickness increased by 50 microns
or vision decreased by 5 letters.
Results: 31 eyes of 31 patients were included. 23 patients had
macular oedema due to branch retinal vein occlusion and 8 due to
central retinal vein occlusion. 4 patients had one previous
triamcinolone injection and one had pan retinal photocoagulation.
Mean number of intravitreal Anti-VEGF injections pre-Ozurdex was
2.32 and mean number post-Ozurdex dropped to 0.13. Mean OCT
central field thickness decreased by -73μm with combined treatment
whereas increased by +97.74μm on Avastin monotherapy, prior to
treatment with Ozurdex. For BRVO patients the treatment outcomes
appear more favourable than CRVO patients. Characteristics of nonresponders were the presence of subretinal fluid and epiretinal
membrane formation. No serious systemic or ocular treatment-related
events occurred.
Conclusions: Our series confirms that sequential treatment with antiVEGF agents and Ozurdex is effective for treating refractory macular
oedema due to retinal vascular occlusions, and is synergistic,
providing sustained increases in visual acuity, as well as reduction in
central field thickness. In addition, this therapy can provide
predictable intervals for re-treatment.
This series is, to the best of our knowledge, one of first to report
clinical experience with sequential use of anti-VEGF agents and
Ozurdex. Further prospective, controlled studies need to be
performed to better determine efficacy, duration of effect, and side
effects.
Commercial Relationships: Evangelia Papavasileiou, None;
Andre Grixti, None; Vineeth Kumar, None; Som Prasad,
Thrombogenics (C), Bausch & Lomb (C), Alcon (R), Novartis (R),
Bayer (C), Nidek (C), Allergan (R)
Short Term Ocular Response of retinal blood flow to intravitreal
injection of bevacizumab (Avastin○R) treatment in eyes with
central retinal vein occlusion
Seung W. Lee. Ophthalmology, Dongguk Unv, Gyeongju Hosp,
Gyeongju, Republic of Korea.
Purpose: To study the effect of intravitreal bevacizumab (1.25mg)
injection (IVBI) on the retinal blood flow and the retinal vessel
caliber in patients with central retinal vein occlusion (CRVO).
Methods: We conducted a retrospective, uncontrolled study of 26
patients. Twelve eyes underwent IVBI and were considered to be the
study group. The control group composed of 14 eyes, which were
observed at least 3 months before retinal photocoagulation.
Depending on the clinical requirements, all eyes in the study group
had at least one IVBI. Retinal arteriovenous transit time (RAVTT)
and visual acuity were used to monitor the evolution of CRVO, and
follow up. The diameter of the retinal vessel diameter was measured
with using the software available on the IMAGEnet program before
the first IVT injection and 30 days after the third injection.
Results: Initially, the mean RAVTT was 23.0 ± 6.1 seconds in the
study group and 28.2 ± 5.4 seconds in the control group. Three
months later, RAVTT improved to 17.1 ± 2.9 seconds in the study
group, but did not change to 28.4 ± 6.5 second in the control group.
The change in RAVTT was statistically significant, only in the study
group (p= 0.003). In the study group, the significant vasoconstriction
was observed in the retinal vein (P<0.001) after IVBI.
Conclusions: These results suggest that IVBI may improve retinal
blood flow and reduce retinal vein diameter in patients with CRVO.
Further studies, evaluating larger sample sizes, are needed to confirm
these results and potential adverse effects on the retinal circulation in
patients with CRVO.
Commercial Relationships: Seung W. Lee, None
Support: None in the Support field
The Effect of Diluted Sterile Penetration Enhancer Solution in
Nebulized Mist and Liquid Drop Forms on Ocular Circulation in
Patients with Diabetic Retinopathy
Lina Krepste1, Alon Harris2, Ingrida Januleviciene1, Vilma J.
Balciuniene1, Lina Siaudvytyte1, Ruta Barsauskaite1, Austin L.
Gerber2, Brent Siesky2. 1Eye Clinic, Lithuanian University of Health
Sciences, Kaunas, Lithuania; 2Eugene and Marilyn Glick Eye
Institute, Indiana University School of Medicine, Indianapolis, IN.
Purpose: To compare the retrobulbar blood flow (RBF) effects and
subjective discomfort of diluted penetration enhancer solution
(DPES) in nebulized mist as opposed to liquid drop in patients with
diabetic retinopathy (DR).
Methods: A prospective, nonrandomized clinical trial collected data
from 20 patients (40 eyes) with mild-to-moderate nonproliferative
DR. Color Doppler imaging (CDI) was used to assess peak systolic
(PSV) and end diastolic (EDV) velocities, and calculated resistance
index (RI) in the ophthalmic (OA) and central retinal arteries (CRA).
Sterile DPES was made in 4:1 proportions of sterile distilled water
and saponin liquid (consisted of water, sodium laureth sulfate,
cocamidopropyl betaine, glycerin, ammonium xylenesulfonate,
disodium phosphate, citric acid). A saline nebulizer, DPES nebulizer
or DPES drop was individually applied to one eye, and RBF was
analyzed at baseline, 1 minute and 5 minutes post-treatment. Patients
were asked about discomfort upon every application. Statistical data
analysis was performed using the computer program SPSS 17.0 for
Windows. Student’s t Test, Mann-Whitney U test and Paired Samples
t test were used.
Results: Saline mist application significantly increased OA velocities
at 5 minutes (PSV from 40.3±6.5 to 43.1±7.6 cm/s, EDV from
6.7±2.7 from 7.9±3.2 cm/s). The DPES mist significantly increased
OA velocities at 5 minutes (PSV from 35.3±8.5 to 38.8±9.2 cm/s,
EDV from 6.4±3.7 to 7.4±3.2 cm/s in the left eye during the first
stage of the study; PSV from 40.7±6.8 to 44.4±5.9 cm/s, EDV from
7.0±2.6 cm/s to 9.2±2.5 cm in the right eye during the second stage),
and increased CRA PSV at 1 minute (from 10.6±1.8 to 11.4±1.8 cm/s
in the left eye during the first stage; from 10.6±2.3 to 12.0±3.1 cm/s
in the right eye during the second stage) and at 5 minutes (to
12.0±2.1 cm/s and to 11.5±2.1 cm/s, respectively). DPES drop
application increased OA PSV from 36.6±9.6 to 39.8±10.9 cm/s at 1
minute and to 40.5±10.1 cm/s at 5 minutes and CRA PSV from
10.6±2.3 to 11.2±2.3 cm/s at 1 minute and to 11.7±2.0 cm/s at 5
minutes. Irritation was noted by 20 patients after DPES drip and by
none after application of mists.
Conclusions: Application of a DPES mist in patients with DR does
not cause ocular irritation as compared to liquid drop application
while showing similar increase in RBF and decrease in RI.
Commercial Relationships: Lina Krepste, None; Alon Harris,
MSD (R), Alcon (R), Merck (C), Pharmalight (C), ONO (C),
Sucampo (C), Adom (I); Ingrida Januleviciene, Pharmalight (F),
Alcon (C), MSD (C), Santen (C); Vilma J. Balciuniene, None; Lina
Siaudvytyte, None; Ruta Barsauskaite, None; Austin L. Gerber,
None; Brent Siesky, None
Clinical Trial: P1-21/2010
Measurement of Retinal Blood Flow Velocity Using a Newly
Developed Doppler Fourier-Domain Optical Coherence
Tomography Instruments in Humans
Akitoshi Yoshida, Taiji Nagaoka, Tomofumi Tani, Eiichi Sato,
Takafumi Yoshioka, Kenji Sogawa, Seigo Nakabayashi.
Ophthalmology, Asahikawa Medical University, Asahikawa, Japan.
Purpose: To report the reproducibility of retinal blood flow (RBF)
measurements using a newly developed Doppler Fourier-domain
optical coherence tomography (FD-OCT) instrument in humans.
Methods: We measured the RBF using the FD-OCT instrument and
bidirectional laser Doppler Velocimetry instrument (LDV) (Canon
Laser Blood Flowmeter, Canon Co., Japan) in 6 healthy volunteers.
The reproducibility of the RBF measurements was assessed by
calculating the coefficients of variation (CVs) for repeated
measurements (5 times) of the retinal blood velocity (V) in the
temporal retinal arterioles and venules.
Results: The measurements of the V in the temporal superior
arterioles and venules were 40.0 ± 6.2 mm/second (mean ± standard
deviation) and 23.5 ± 2.7 mm/second with FD-OCT and 35.5 ± 6.4
mm/second and 21.6 ± 2.5 mm/second with the bidirectional LDV
apparatus, respectively. The blood velocity results obtained using the
two methods did not differ significantly (arterioles, p = 0.80; venules,
p = 0.23). The CVs for V were 12.1 ± 4.9% in the arterioles and 14.5
± 6.6% in the venules with FD-OCT and 11.9 ± 5.6% in the arterioles
and 11.7 ± 5.7% in the venules with the bidirectional LDV apparatus.
There were no significant differences in CVs obtained by two
methods (arterioles, p = 0.55; venules, p = 0.49).
Conclusions: The current study showed that the newly developed
Doppler FD-OCT enables accurate and reproducible measurements
of the blood velocity in the retinal arterioles and venules in humans.
Commercial Relationships: Akitoshi Yoshida, None; Taiji
Nagaoka, None; Tomofumi Tani, None; Eiichi Sato, None;
Takafumi Yoshioka, None; Kenji Sogawa, None; Seigo
Nakabayashi, None
Mechanisms of retinal venous pulsation inferred from diameter
waveforms analysis
Fabrice Moret1, Wolf Lagreze2, Charlotte M. Poloschek1, 2, Michael
Bach1. 1Sect. Visual Function and Electrophys, Eye Hospital,
University of Freiburg, Freiburg, Germany; 2Sect.
Neuroophthalmology, Eye Hospital, University of Freiburg, Freiburg,
Germany.
Purpose: The retinal venous outflow resistance is a candidate
element in the etiology of glaucoma. The cardiac pulsation,
especially its pressure waveform, can characterize the nature of
resistive elements in vascular segments. Expanding on recent data
collected to investigate the spontaneous venous pulsation's time of
collapse, we examine the pulsation waveform in young healthy
subjects and test explanatory hypotheses.
Methods: We analyzed fundus movies collected in 12 subjects.
Arterial lateral displacement and venous diameter waveforms were
measured near the disk on sequences with and without image
processing improvement. We then reviewed the various mechanisms
proposed so far and assessed each of them with our data. Proposed
mechanisms include: the "classical view", a pulse transfer through the
microcirculation, a spontaneous pulsation, a Venturi effect, a
common adventitial sheath segment, the "constant inflow/variable
outflow" hypothesis, and an intracranial pressure interaction.
Results: Venous pulsations show different amplitudes and
waveforms, suggesting more than one mechanism at play. None of
the previous mechanisms alone fully explains the measured
waveforms. We propose two mechanisms: 1. For waveforms showing
a damped-like version of the arterial pressure: initially (and wellestablished), the pulsation of the choroidal volume governed by a
Windkessel effect leads to an intraocular pressure pulsation of similar
waveform, then this pulsation is transferred to the retinal circulation
upstream of the measurement sites. 2. For venous diameter
waveforms matching the arterial pressure waveforms: the pressure
pulsation of the central retinal artery is transferred to the central
retinal vein in a non-extensible common adventitial sheath segment
in the optic nerve.
Conclusions: The waveform of the retinal venous pulsation obtained
from fundus movies informs on the nature of the venous outflow
resistance in ways not matched by an analysis of the amplitude alone.
It would now be of interest as of whether quantitative changes are
observed in glaucoma.
Commercial Relationships: Fabrice Moret, None; Wolf Lagreze,
Merz (C), Allergan (C); Charlotte M. Poloschek, None; Michael
Bach, None
Support: Deutsche Forschungsgemeinschaft BA877/19-2
Effect of Nitric Oxide on Increased Retinal Blood Flow in
Response to Flicker Stimuli in Cats
Takafumi Yoshioka, Taiji Nagaoka, Akitoshi Yoshida.
Purpose: To investigate the role of nitric oxide (NO) in the increase
in retinal blood flow (RBF) during flicker stimuli.
Methods: Twelve adult cats were used in this study. After anesthesia
was induced with sevoflurane in each animal, laser Doppler
velocimetry (LDV) was used to measure the vessel diameter (D) and
blood velocity (V) simultaneously and calculate the RBF in the
second-order retinal arterioles during flicker stimulation. In the first
series of studies, the flicker frequencies and light intensities ranged
from 2 to 64 Hz and 300 to 3000 lux, respectively. The durations of
dark adaptation and flicker stimulation ranged from 0 to 30 minutes
and 60 to 300 seconds, respectively. In the second series of studies,
we injected phosphate-buffered saline (PBS) or NG-nitro-L-argininemethyl ester (L-NAME) into the vitreous and measured the D, V, and
RBF during flicker stimuli 2 hours later.
Results: Flicker stimulations from 2 to 32 Hz increased the RBF in
the retinal arterioles. In contrast, flicker stimulation with 64 Hz did
not affect the RBF. At 16 Hz, the increase in RBF was enhanced in
response to light intensities of 300~3,000 lux, dark adaptation times
of 0~30 minutes, and flicker stimulation times of 60~180 seconds. At
16 Hz, 3,000 lux, 30 minutes of dark adaptation, and 180 seconds of
stimulation time, flicker stimulation resulted in a 51% increase in
RBF over baseline in the PBS group (n=6) and an 11% increase in
the L-NAME group (n=6), a difference that was significant
(P<0.0001).
Conclusions: We confirmed that the LDV system allows evaluation
of the flicker-induced increase in RBF in the retinal arteries in
anesthetized cats. Our results suggested that NO plays an important
role in the increase in RBF during flicker stimuli.
Commercial Relationships: Takafumi Yoshioka, None; Taiji
Nagaoka, None; Akitoshi Yoshida, None
Measurement Of Retinal Blood Flow and Retinal Oxygen
Tension During Acute Decreasing Systemic Blood Pressure In
Cats
Tomofumi Tani, Taiji Nagaoka, Takafumi Yoshioka, Akitoshi
Yoshida. Ophthalmology, Asahikawa Medical University,
Asahikawa, Japan.
Purpose: To investigate retinal arteriolar blood flow (RBF) and
retinal oxygen tension (PO(2)) during decreases in systemic blood
pressure (BP) in cats.
Methods: The effect of acute decreased systemic blood pressure on
RBF and PO(2) on near the arteriolar were assessed with laser
Doppler velocimetry (CLBF model 100, Canon) and oxygen
microelectrodes (POE-10N, BRC). The systemic BP was decreased
over 5 minutes with continuous withdrawal of blood from the
descending aorta at 24% of total blood volume using a syringe pomp
(Model 11 Plus Advanced, Harvard Apparatus). Ocular perfusion
pressure (OPP) was calculated as OPP = mean arterial BP (MABP) IOP.
Results: The RBF was maintained until 70mmHg of OPP and started
to decrease (P<0.01) below 60 mmHg of OPP. The PO(2) was also
maintained until 70mmHg of OPP and started to decrease (P<0.01)
below 60 mmHg of OPP.
Conclusions: These results suggest that RBF may be maintained
more than 70 mmHg of OPP. The PO(2) seems to reflect RBF
changes during acute decreasing systemic BP in cats.
Commercial Relationships: Tomofumi Tani, None; Taiji
Nagaoka, None; Takafumi Yoshioka, None; Akitoshi Yoshida,
None
Measurement of Retinal Blood Flow Using a Newly Developed
Doppler Fourier-Domain Optical Coherence Tomography
Instruments in Cats
Taiji Nagaoka, Tomofumi Tani, Eiichi Sato, Takafumi Yoshioka,
Kenji Sogawa, Seigo Nakabayashi, Akitoshi Yoshida.
Purpose: To report the reproducibility of retinal blood flow
measurements by using a newly developed Doppler Fourier-domain
optical coherence tomography (FD-OCT) instrument in anesthetized
cats.
Methods: Four cats (2.5-5.0 kg) were anesthetized using a mixture of
sevoflurane and room air. We measured retinal blood flow by using
both the FD-OCT and the bidirectional LDV (Canon Laser Blood
Flowmeter, Canon Co., Japan) which was customized for cats. The
reproducibility of retinal blood flow measurements was assessed by
calculating the coefficients of variation (CV) for repeated
measurements (5X) of retinal vessel diameter, time-average
centerline blood velocity, and blood flow (RBF) at temporal retinal
arterioles and venules. In addition, we examined the changes in
retinal blood flow in arterioles in response to systemic inhalation of
100% pure oxygen (hyperoxia) by using both instruments.
Results: The averaged values of CV (mean ± SD) for RBF were
9.7% ± 3.8% and 8.2% ± 3.5% in arterioles and 10.3% ± 0.8% and
7.4% ± 0.9 % in venules with LDV and FD-OCT, respectively (p =
0.34 and p = 0.18). After 10 minutes of hyperoxia, RBF decreased by
-43.8 ± 11.2% and -43.9 ± 7.4% with LDV and FD-OCT,
respectively. There was no significant change in RBF in response to
hyperoxia between LDV and FD-OCT (p = 0.87).
Conclusions: The current study revealed that newly developed FDOCT enable the accurate and reproducible measurements of blood
velocity in retinal arterioles and venules in in vivo cats models.
Commercial Relationships: Taiji Nagaoka, None; Tomofumi
Tani, None; Eiichi Sato, None; Takafumi Yoshioka, None; Kenji
Sogawa, None; Seigo Nakabayashi, None; Akitoshi Yoshida, None
Development of a prototype of laser Doppler flowmeter for the
measurement of blood flow of retinal vessels and optic nerve head
tissue in small animals (rats)
Marielle Mentek1, Christophe Chiquet3, 1, Frederic Truffer2, Mario
Bernabei2, Benjamin Mottet3, Jean-Paul Romanet3, Diane GodinRibuot1, Martial Geiser2. 1INSERM U1042, Grenoble, France;
2
Institute of system engineering, University of Applied Sciences of
Western Switzerland, Sion, Switzerland; 3Ophthalmology
department, CHU Grenoble, Joseph Fourier University, Grenoble,
France.
Purpose: The aim of the present study was to validate a new laser
Doppler flowmeter (LDF) device, specifically designed to measure
retinal and optic nerve head blood flow in the rat. We investigated the
reproducibility of blood flow parameters measurements on optic
nerve head and retinal arteries in Wistar rats along with the effects of
hyperoxia.
Methods: Rats were immobilized on a modified stereotactic table,
which allows free rotation of the instrument around the eye, in order
to aim the probing beam on the fundus. The fundus is illuminated
with a green light and an image is captured with a CCD for easy
targeting (measurement diameter about 60 μm). In vivo validation
consisted of: (1) Study of linearity of the instrument with a rotating
diffusing wheel. (2) Measurement reproducibility during a single
experimental session (n = 5 rats, 1 hour, 4 measurements of 5-min
duration) by calculation of intra-individual Variation Coefficients
(VC) and Intra-class Correlation Coefficient (ICC). (3) Blood Flow
(BF), velocity (Vel) and volume (Vol) (mean ± SD) were evaluated
in response to 100% oxygen breathing (n = 8 rats).
Results: Vel varied linearly with the rotating speed (R2=0.9806)
while Vol did not change (standard deviation/mean = 2.9%). Shortterm reproducibility of retinal arterial Vel shows strong agreement
(ICC=0.9, IC 95%=0.7-0.99), associated with low intra-individual
VC (7.8 ± 6.6 %). After 2 minutes of 100% oxygen breathing, retinal
arterial Vel was significant decreased by 17.0 ± 13.7 % (p=0.012),
and optic nerve head BF by 11.0 ± 9.7 % (p=0.018).
Conclusions: This new LDF device allows repeatable measurements
of ocular blood flow in small animals. Rat optic nerve head and
retinal blood flow measurements are of crucial interest in
physiological and pathophysiological studies. This prototype will be
used to investigate various animal models (diabetes mellitus,
glaucoma, vascular disorders) and for therapeutic evaluation.
Optical scheme of the laser Doppler flowmeter prototype (LDF),
based on the Schlieren arrangement of the laser delivery and
detection system, and rat fundus image.
Typical rat posture for ocular LDF recording. LDF is held on a
horizontal movable stereotatic table with a special mechanical device
permitting three-dimensional movements (θ,φ,z) and micrometric
positioning of the probing beam on the rat's fundus.
Commercial Relationships: Marielle Mentek, None; Christophe
Chiquet, None; Frederic Truffer, None; Mario Bernabei, None;
Benjamin Mottet, None; Jean-Paul Romanet, None; Diane GodinRibuot, None; Martial Geiser, None
Support: Germaine de Staël
Retinal blood velocity significantly decreased in rhegmatogenous
retinal detachment and recovered after surgical treatment in
both artery and vein
Hirofumi Kinoshita, Kiyoshi Suzuma, Masafumi Uematsu, Ryotaro
Ueki, Takashi Kitaoka. Nagasaki Univ School of Medicine,
Nagasaki, Japan.
Purpose: To evaluate retinal blood velocity by laser speckle
flowgraphy (LSFG) in rhegmatogenous retinal detachment (RRD)
patients before and after vitrectomy surgery.
Methods: Subjects included 36 patients (36 eyes) who had
undergone successful vitrectomy surgery for unilateral RRD from
May 2010 to March 2011. Preoperative, and 1- and 3-month
postoperative retinal blood velocities were measured. We determined
mean retinal blood velocity via LSFG measurements of the mean blur
rate (MBR) of the major vessels in the optic disc, retinal artery and
retinal vein beside the optic disc corresponds to detachment area.
When we investigated correlation between the preoperative MBR and
the postoperative MBR in this study, we calculated the values as the
percent of the preoperative MBR.
Results: 18 males and 18 females patients (mean age 56.3 ± 10.6
years, ranging from 32 to 74) with RRD were included in this study.
All of the patients underwent 23-gauge pars plana vitrectomy, which
included SF6 gas tamponade. The average preoperative visual acuity,
which was expressed as the LogMAR, was 0.62 ± 0.76. Significant
improvement of the LogMAR was noted at 3 months after the
surgery (0.10 ± 0.28; P < 0.0001). After surgery, average MBR of the
major vessels in the optic disc of the affected eyes gradually
improved, and the average 3-month postoperative MBR (130.43 ±
36.18%; P < 0.01) significantly increased as compared to the average
preoperative MBR. Moreover, 1-month and 3-month postoperative
MBR of retinal artery in detachment area were 141.1 ± 32.7% and
144.1 ± 30.2%, respectively as compared to preoperative MBR of
retinal artery in detachment area. 1-month and 3-month postoperative
MBR of retinal vein in detachment area were also increased to 117.5
± 18.8% and 125.4 ± 20.4%, respectively as compared to
preoperative MBR of retinal vein in detachment area.
Conclusions: LSFG results showed that the mean retinal blood
velocity in the optic disc, both retinal artery and vein in detachment
area recovered after successful vitrectomy surgery for RRD patients.
Commercial Relationships: Hirofumi Kinoshita, None; Kiyoshi
Suzuma, None; Masafumi Uematsu, None; Ryotaro Ueki, None;
Takashi Kitaoka, None
Ocular rigidity as estimated based on measurement of pulse
amplitude using pneumotonometry and fundus pulse is
dependent on intraocular pressure
Anton B. Hommer1, 2, Gerhard Garhofer2, Leopold Schmetterer2.
1
Krankenhaus Hera Vienna, Vienna, Austria; 2Dep.of. Clinical
Pharmacology, University Clinic, Vienna, Austria.
Purpose: Evidence has accumulated that the biomechanical
properties of the optic nerve head and the sclera play a role in the
pathophysiology of glaucoma. In this study, the hypothesis was tested
that abnormal ocular structural stiffness as estimated based on
measurements of intraocular pressure amplitude and ocular fundus
pulsation amplitude is dependent on intraocular pressure.
Methods: Onehundredsixtyeight healthy subjects aged between 18
and 45 years were included in this study. The ocular pulse amplitude
and pulsatile ocular blood flow were assessed with
pneumotonometry. The fundus pulsation amplitude was measured by
using laser interferometry. Based on the Friedenwald equation, a
coefficient of ocular rigidity (E1) was calculated relating pulse
amplitude to fundus pulsation amplitude
Results: The ocular fundus pulsation amplitude was 4.21 ± 0.99 μm,
the ocular pulse amplitude was 3.07 ± 0.61 mmHg. This resulted in a
calculated factor E1 of 0.047 ± 0.007 AU. Multiple regression
analysis revealed that fundus pulsation amplitude and pulse
amplitude were dependent on pulse pressure amplitude (p < 0.001)
whereas E1 was dependent on intraocular pressure (p < 0.001).
Conclusions: The present study indicates that ocular rigidity is
dependent on intraocular pressure.
Commercial Relationships: Anton B. Hommer, Allergan (R),
Allergan (C), Alcon (R), Santen (R), Merck (R); Gerhard Garhofer,
None; Leopold Schmetterer, None
Sphingosine 1-phosphate elicits constriction of isolated porcine
retinal arterioles
Takayuki Kamiya, Taiji Nagaoka, Tsuneaki Omae, Shinji Ono,
Akitoshi Yoshida. Asahikawa Medical University, Asahikawa, Japan.
Purpose: Sphingosine 1-phosphate (S1P), a member of a large
family of lipid metabolites called sphingolipids, induces a wide
variety of biologic responses, i.e., immune responses, inflammatory
processes, organ perfusion, and regulation of vascular tone in
different organs through various high-affinity G-protein-coupled
receptors (S1PR) 1-5. However, few reports have addressed the effect
of S1P on the retinal circulation. We examined the effect of S1P to
determine the signaling mechanisms involved in the retinal
microvasculature.
Methods: Porcine retinal arterioles (internal diameter, 70-100 µm)
were isolated, cannulated, and pressurized (55 cmH2O) without flow
in this in vitro study. Videomicroscopic techniques were used to
record the changes in diameter in response to S1P.
Results: The retinal arterioles were constricted in a dose-dependent
manner (1 nM-10 µM) in response to S1P. This vasoconstrictive
response to S1P was not attenuated after removal of the endothelium.
Blockade of S1PR2 by the S1PR2 antagonist JTE-013 abolished the
vasoconstrictive response to S1P, whereas the S1PR1 antagonist
(compound 5) and the S1PR3 antagonist (suramin) did not affect the
vasoconstrictive response. The PKC inhibitor chelerythrine and storeoperated calcium channels (SOCE) blocker (2-APB) significantly
(p<0.0001) inhibited the effect of S1P-induced vasoconstriction.
Conclusions: S1P elicits vasoconstriction of the retinal arterioles via
S1PR2. This vasoconstriction may be mediated by Ca2+-dependent
and Ca2+-independent pathway.
Commercial Relationships: Takayuki Kamiya, None; Taiji
Nagaoka, None; Tsuneaki Omae, None; Shinji Ono, Kaken
Pharmaceutical Co.,Ltd. (F); Akitoshi Yoshida, None
Support: do not use
Ocular Pulse Amplitude Waveform Reflects Ventricular
Bigeminy and Aortic Insufficiency
Jean B. Kassem1, 2, Steven E. Katz1, 2, Cynthia J. Roberts2, 3, Ashraf
M. Mahmoud2, 3, Robert H. Small4, 3, Subha V. Raman5, 3. 1NeuroOrbit-Oculoplastics, Havener Eye Institute, Columbus, OH;
2
Ophthalmology, The Ohio State University, Columbus, OH;
3
Biomedical Engineering, The Ohio State University, Columbus, OH;
4
Anesthesiology, The Ohio State University, Columbus, OH;
5
Cardiovascular Medicine, The Ohio State University, Columbus,
OH.
Purpose: Intraocular pressure (IOP) is pulsatile in nature due to the
filling of the ocular blood vessels with each ventricular contraction.
Ocular pulse amplitude (OPA) is defined as the difference between
maximum and minimum deflections in this signal. Average values of
OPA in healthy subjects range from 1-4mmHg. Normal appearance
of the waveform includes regular cycles corresponding to the
heartbeat. The purpose of the current investigation was to determine
the source of an irregular waveform of extreme values.
Methods: Ocular pressure waveforms were digitally recorded from
both eyes of a subject being examined during training to use the
PASCAL Dynamic Contour Tonometer (DCT), with custom software
provided by the manufacturer (Ziemer, Port, Switzerland). It was
noted that the waveform had an unusual shape consistent with an
early ventricular contraction every other beat, as seen in Figure 1. It
was also noted that ocular pulse amplitude (OPA) was >9mmHg,
which is extraordinarily high. The subject presented for a thorough
examination by a cardiologist.
Results: The subject was determined to be in ventricular bigeminy,
as seen in Figure 2, which was the source of the irregular appearance
of the OPA waveform. In addition, it was determined that he had
aortic insufficiency which explained the extreme value for OPA that
corresponded to bounding carotid pulses via palpation. After
replacement of the aortic valve, the bigeminy resolved and the ocular
pulse waveform became regular in appearance with an OPA of 1.62.0mmHg
Conclusions: The ocular pulse waveform is a direct reflection of
hemodynamics. Evaluating this waveform may provide an additional
opportunity for screening subjects for cardiovascular abnormalities.
Wednesday, May 08, 2013 2:45 PM-4:30 PM
Program #/Board # Range: 5043-5075/B0001-B0033
A minimally invasive and localised intra-scleral delivery of drugloaded thermoresponsive polymeric implants to treat posterior
ocular diseases
Hannah L. McMillan, Steven J. Fallows, Thakur Raghu R. Singh,
David S. Jones. School of Pharmacy, Queen's University Belfast,
Belfast, United Kingdom.
Purpose: This study evaluates minimally invasive hollow
microneedles (HMNs) to deliver thermoresponsive-based intrascleral
implants for sustained drug release.
Methods: Gel formulations containing thermo-responsive polymer
i.e. poloxamer, loaded with fluorescein sodium (FNa), were evaluated
for rheological analysis, syringeability through HMNs & penetration
forces of HMNs into rabbit sclera. Visualisation of gel-based implant
formation in sclera and scleral recovery were also determined, using
optical coherence tomography (OCT). Ex vivo FNa release following
intrascleral delivery of gels was tested in Franz-diffusion cells.
HMNs were fabricated from different hypodermic needles (i.e. 26G,
29G & 30G) that were adjusted to different heights (i.e., 400, 500 or
600 µm).
Results: Gelation temperatures of poloxomer formulations ranged
from 20.8 - 30.7°C, which showed Newtonian behaviour at 20°C and
pseudoplastic (shear-thinning) behaviour at 37°C. The maximum
force and work required in expelling gels from HMNs increased with
poloxamer concentration, with volume of gel expelled (30, 50 or 100
µL) and with decrease in needle aperture (26G to 30G). Nevertheless
low force (i.e., 0.123 - 2.021 N) & work (0.139 - 6.000 Ns) was
required across all variables tested. Forces required to penetrate the
scleral tissue correlated well with increasing needle size and depth of
penetration (equator; 0.549 - 1.159 N, anterior; 0.710 - 1.346 N,
posterior; 0.905 - 1.589 N). OCT studies revealed that HMNs has
successfully able to penetrate to the required depth and allowed
intrascleral injection of 50 µL of gel. Recovery of the sclera
following injection correlated well with penetration depth. FNa
release was 80.88- 87.56%, 70.33 - 75.02% and 52.86 - 60.63%,
respectively after 24 h, when 50 µL of FNa-poloxamer gels were
injected at a depth of 400 µm, 500 µm and 600 µm, intrasclerally.
Conclusions: This study has indicated the potential for sustained
delivery when using thermo-responsive polymers injected
intrasclerally via HMNs and also scleral recovery and implant
visualization. Results suggest that this method may offer a minimally
invasive means of treating posterior segment diseases.
Commercial Relationships: Jean B. Kassem, None; Steven E.
Katz, None; Cynthia J. Roberts, Oculus Optikgerate GmbH (C),
Ziemer Ophthalmic Systems AG (C), Sooft Italia (R), Carl Zeiss
Meditec (F); Ashraf M. Mahmoud, None; Robert H. Small, Ziemer
(F); Subha V. Raman, None
468 Drug Delivery III
OCT images of 50 µL (red) injected to a depth of 400 µm at 0 h (a), 1
h (b) and 2 h (c). Dashed yellow shows empty space in sclera created
via injection and its subsequent closure.
Commercial Relationships: Hannah L. McMillan, None; Steven J.
Fallows, Wellcome Trust (F); Thakur Raghu R. Singh, None;
David S. Jones, None
Support: Wellcome Trust Biomedical Vacation Scholarship 2012
Hindered convective transport of nanoparticles and
macromolecules in the vitreous humor
Anita N. Penkova1, 2, Komsan Rattanakijsuntorn1, Yang Tang2, Rex
Moats2, 3, Michael R. Robinson4, 2, Susan S. Lee4, 2, Satwindar S.
Sadhal1, 2. 1Aerospace & Mechanical Engineering, University of
Southern California, Los Angeles, CA; 2SAIRC, Saban Research
Center, Children’s Hospital Los Angeles, Los Angeles, CA;
3
Biomedical Engineering, University of Southern California, Los
Angeles, CA; 4Global Pharmaceutical Sciences, Allergan, Inc, Irvine,
CA.
Purpose: 1. To investigate the extent of transport enhancement of
macromolecules and nanoparticles due to convective flow of water in
the vitreous humor.
2. To establish deviation from convective transport predictions by
forced-flow experiments and comparison with conventional theory
calculations.
3. To propose corrections to convective transport to allow for largeparticle hindrance in the vitreous humor.
Methods: Fresh ex vivo whole bovine eyes were individually
injected with two gadolinium-based contrast agents [Gd-Albumin,
and 30 nm gadolinium-based particles (Gado CELLTrackTM,
Biopal, Worcester, MA)]. After imaging the eye, the contrast-agent
bolus was subjected to a high rate of water flow with a syringe pump.
The water (0.9% saline solution) was injected at the top of the bovine
eye at 100 μl/min, and was allowed to drain through small slits cut at
the bottom of the eyeball. After one hour of pumping, the eye was
imaged again and the contrast-agent distribution was obtained. For
theoretical comparison the convective transport was also evaluated by
numerical modeling based on the conventional convection-diffusion
equation.
Results: The experimentally measured convective transport of GdAlbumin and 30 nm nanoparticles was found to be somewhat slower
than the carrier (0.9% saline solution). The conventional convectiondiffusion equation with the u.▽c term for convective transport seems
to over-predict the convection rate, indicating some degree of
hindrance to convection for nanoparticles and Gd-Albumin.
Conclusions: It has been claimed in several theoretically-based
investigations that convection significantly enhances the transport of
large particles and macromolecules in the vitreous humor. However,
current investigations have raised questions about the validity of
using the unhindered convective term u.▽c which does not
discriminate between the different solutes. We have hypothesized
that large molecules, when subjected to high rate of water flow in the
vitreous humor, will experience resistance, depending on the
respective permeabilities of the injected surrogate solute. We are
proposing that the usual convection term be adjusted to allow for the
filtration effect on the larger particles in the form (1-σ)u.▽c with
important implications for computational modeling.
Commercial Relationships: Anita N. Penkova, Allergan, Inc (F);
Komsan Rattanakijsuntorn, None; Yang Tang, None; Rex Moats,
Allergan (F); Michael R. Robinson, Allergan (I); Susan S. Lee,
Allergan, Inc. (E); Satwindar S. Sadhal, Allergan, Inc. (F)
Support: This work has been supported with a Research Grant by
Allergan, Inc.
Slow-release intraocular drug delivery by injectable PEA
microfibrils (DSM, NL)
Gabriele Thumann1, George Mihov2, Jens Thies2, Anja Kemp2,
Katharina Morawa1, Martina Kropp1. 1Department of
Ophthalmology, University of Geneva, Geneva, Switzerland; 2DSM,
Geleen, Netherlands.
Purpose: Ophthalmic drug therapy of the posterior segment requires
that effective concentrations of drug reach the target tissue, a goal
complicated by limited penetration, short half-life of many drugs and
difficult access to the posterior segment. Generally drug delivery to
the posterior segment of the eye is accomplished by intravitreal (ivt)
injection, often requiring frequent injections, e.g. AMD treatment
with ranibizumab or aflibercept. Continuous drug delivery systems
would be useful to avoid frequent administration and to deliver the
drug at a more physiological concentration. Here we report properties
of biodegradable amino acid polyester amide (PEA) polymers (DSM,
NL) that can deliver a spectrum of drugs in a sustained, zero-order
behavior and degrade via an enzyme-mediated pathway.
Methods: Deformability and in vitro degradation of the PEA fibrils
(120-300 µm in diameter) was analyzed by incubation at 37o C in
PBS, in α-chymotrypsin, in human, rabbit and bovine vitreous. In
vivo biocompatibility and degradation was analyzed by
subconjunctival (sc) and ivt implantation in normal and VEGFtreated rabbits that simulate blood-retinal barrier breakdown. Drug
release by single PEA fibrils loaded with 10% dexamethasone was
analyzed in vitro.
Results: During the first 2 hours the fibrils decreased in length 57%
in PBS, 62% in rabbit vitreous and 59% in human vitreous; the
decrease in length was accompanied by an increase in diameter.
Degradation by chymotrypsin depended on the amino acid
composition of the PEA. Degradation in normal vitreous was slight
but increased in vitreous from VEGF-treated rabbits. Sc and ivt PEA
fibrils were well tolerated with no evidence of inflammation, retinal
damage or changes in IOP. After an initial peak dexamethasone was
released at a constant rate of 0.05-1.0 µg/ml for 140 days, whereas
PLGA 75/25 did not release significant amounts of dexamethasone
during the first 120 days, followed by 90% release from day 120 to
day 140.
Conclusions: PEA polymers (DSM, NL) are well tolerated in the
ocular environment, degrade slowly intravitreally and can be loaded
with drugs that are released with zero-order kinetics over a period of
months. Since PEAs are well tolerated in the ocular environment and
can be manufactured to be loaded with different classes of drugs,
these materials would be ideal for sustained delivery of drugs to the
posterior segment.
Commercial Relationships: Gabriele Thumann, DSM (F); George
Mihov, DSM (E); Jens Thies, None; Anja Kemp, DSM (E);
Katharina Morawa, DSM Biomedical (F); Martina Kropp, DSM
(F)
Support: DSM
Fucoidan but not Mannan inhibits Bevacizumab uptake
independently of phagocytosis and reduces VEGF expression in
the RPE
Michaela Dithmer1, Tim Meyer2, 3, Elisabeth Richert1, Johann
Roider1, Alexa K. Klettner1. 1Ophthalmology, University of Kiel,
University Medical Center, Kiel, Germany; 2Immunology, University
of Kiel, University Medical Center, Kiel, Germany; 3Medizinische
Klinik I, Charite-Campus Benjamin Franklin, Berlin, Germany.
Purpose: We have previously shown that Bevacizumab is taken up in
RPE cells and stored for at least seven days. In this study, we further
investigate the mechanisms of uptake, focussing on the influence of
sugar moieties.
Methods: For the experiments, Arpe19 cells and primary porcine
RPE cells of passage 2 and 3 were used. Cells were treated with 250
µg/ml Bevacizumab and evaluated after different time intervals (1 h 7 d). Fucoidan or Mannan, respectively, was applied at a
concentration of 100 µg/ml. Bevacizumab uptake was evaluated with
immunofluorescence. Phagocytosis was measured in a phagocytosis
assay using opsonized FITC latex beads. VEGF expression was
determined in Western blot.
Results: Fucoidan, but not Mannan, strongly reduces Bevacizumab
uptake in RPE cells. Phagocytotic ability was not compromised by
either Fucoidan or Mannan. Fucoidan, but not Mannan, significantly
reduces VEGF165 and VEGF121 expression in Western blot.
Conclusions: Fucoidan inhibits Bevacizumab uptake, which is not
mediated via phagocytosis but may be connected to the reduction of
VEGF expression in RPE cells by Fucoidan. This may hint towards
uptake mechanism that involves immunocomplex binding.
Commercial Relationships: Michaela Dithmer, None; Tim Meyer,
None; Elisabeth Richert, None; Johann Roider, Novartis (F),
Bayer (F); Alexa K. Klettner, Novartis (F), Novartis (C), Novartis
(R), Santen (R)
Support: DFG KL2425/2-1
In Vivo Pharmacokinetics of Injectable, Intravitreal, SustainedRelease Latanoprost Formulations
William S. White, Mae Hu, Glenn Huang, Tan Pham, Faina
Karasina, Aaron Lee, Maria Gorlina, Vernon Wong. Icon
Bioscience, Inc, Sunnyvale, CA.
Purpose: To study the in vivo pharmacokinetics of injectable,
intravitreal, sustained-release latanoprost formulations
Methods: In vivo anterior chamber drug levels were measured in
New Zealand White rabbits receiving single doses of one of two
formulations of sustained-release latanoprost administered by
intravitreal injection. Aqueous humor samples were obtained weekly
and analyzed for latanoprost acid concentrations using highperformance liquid chromatography with mass spectrometry.
Results: Sustained levels of latanoprost acid in the aqueous humor
were demonstrated for 3 to 7 months in animals injected with single
doses of sustained-release latanoprost formulations. The sustainedrelease formulation containing 559 µg of latanoprost demonstrated
sustained levels of anterior chamber latanoprost acid which decreased
to an average latanoprost acid aqueous humor level of 17.5 pg/mL on
day 84. The sustained-release formulation containing 1118 µg of
latanoprost demonstrated sustained anterior chamber levels of
latanoprost acid which decreased to an average latanoprost acid
aqueous humor level of 19.1 pg/mL on day 231.
Conclusions: In vivo anterior chamber levels of latanoprost acid
were sustained for up to 7 months and were well-tolerated in rabbits
receiving sustained-release latanoprost formulations injected
intravitreally.
Commercial Relationships: William S. White, Icon Bioscience (E);
Mae Hu, Icon Bioscience, Inc (E), Icon Bioscience, Inc (I); Glenn
Huang, Icon Bioscience, Inc. (P), Icon Bioscience, Inc. (E), Icon
Bioscience, Inc. (I); Tan Pham, Icon Bioscience Inc (E), Icon
Bioscience Inc (P); Faina Karasina, Icon Bioscience Inc (I), Icon
Bioscience Inc (E); Aaron Lee, Icon Bioscience Inc (C), Icon
Bioscience Inc (R); Maria Gorlina, None; Vernon Wong, Icon
Bioscience Inc (E)
Hystem, a bio-absorbable protein delivery polymer: safety,
tolerability and efficacy in a rabbit corneal debridement model
MaryJane Rafii1, Barbara M. Wirostko1, 2, Liliana Werner2, Nick
Mamalis2, Thomas Zarembinski4, Stacy Pritt3, Glenwood G. Gum3.
1
Ophthalmics, Jade Therapeutics, Salt Lake City, UT;
2
Ophthalmology, University of Utah, SLC, UT; 3Absorption Systems,
SD, CA; 4BioTime, Almeda, CA.
Purpose: HyStemTM (BioTime, CA), a biodegradable hyaluronic
acid (HA)-based polymer shown to promote wound healing, can be
used for local, ocular sustained delivery of proteins. Safety,
tolerability, and efficacy of this polymer, alone and in combination
with recombinant human growth hormone (rhGH) to accelerate
wound healing, was evaluated in a rabbit corneal debridement model
(CDM). rhGH was selected based on its ability to activate growth
factors, e.g., Insulin-like Growth Factor and Epidermal Growth
Factor, that have been shown to be involved in corneal reepithelialization.
Methods: To assess the tolerability of HyStem cross-linked with
glutathione (GSSG); non-cross-linked HyStem, HyStem/GSSG, and
Ringers lactate (RL) (control) were applied four times a day (QID)
for 4 days topically in a CDM using New Zealand rabbits (NZR)(
N=3, 2 eyes/arm). Twice daily, slit lamp exams with photos were
employed to evaluate healing. Tissues were harvested on day 5 and
histopathology was performed. To evaluate the efficacy of
HyStem/GSSG/rhGH in accelerating wound healing, a validated
NZR CDM model (N=11) was used. All 22 eyes received topical
dexamethasone (dex) QID for 7 days, alone (control; n=8 eyes), with
HyStem/GSSG BID (n=4 eyes), or with HyStem/GSSG/rhGH BID (4
μg/50 μL drop; n=10 eyes). Healing was assessed via daily slit lamp
photos. Tissues were harvested on day 7 and histopathology was
performed. Time to complete healing and daily % healing were
compared across groups.
Results: Tolerability study revealed that HyStem and HyStem/GSSG
were well tolerated, with a trend for faster return to normal histology
vs. RL. In the efficacy study, HyStem/GSSG and
HyStem/GSSG/rhGH yielded excellent safety and tolerability:
histopathology was normal, with no inflammation or angiogenesis. In
spite of the small sample size, an efficacy trend was seen with a faster
rate to complete defect closure in the HyStem/GSSG/rhGH group as
early as Day 5. By Day 6, 80% of eyes with HyStem/GSSG/rhGH
were completely healed vs. 50% of HyStem/GSSG vs. 38% of dex
alone.
Conclusions: HyStem can deliver, rhGH, such that an efficacy signal
for faster corneal wound healing is demonstrated. HyStem and
Hystem/GSSG/rhGH were well tolerated in vivo, with normal
histopathology. HyStem is a viable polymer to deliver proteins, e.g.,
rhGH, for corneal defects that have impaired healing.
Commercial Relationships: MaryJane Rafii, Jade Therapeutics
(P), Jade Therapeutics (S), Jade Therapeutics (I), Pfizer Inc. (C),
Regenron (C); Barbara M. Wirostko, Jade Therapeutics (P), Jade
Therapeutics (I), Jade Therapeutics (E), Altheos Inc. (C), Merck (C),
SKS Ocular (C), USTAR (F); Liliana Werner, Aaren Scientific (F),
Abbott Medical Optics (F), Advanced Vision Science (F), Alcon
Laboratories (F), Anew Optics (F), Bausch & Lomb Surgical (F),
Calhoun Vision (F), Innovia (F), MRI Research (C), Powervision
(C), Rayner Intraocular Lenses (F), Visiogen (C); Nick Mamalis,
Abbott Medical Optics (F), Alcon Laboratories, Inc (F), Allergan (F),
Anew (C), Bausch & Lomb (F), Calhoun Vision, Inc (F), NuViiew,
Inc (F), OpticaMedica (C), Powervision (F); Thomas Zarembinski,
BioTime, Inc. (E); Stacy Pritt, Jade Therapeutics (E), Absorption
Systems (E), Amakem (E); Glenwood G. Gum, Jade Therapeutics
(F)
Support: The Utah Science Technology and Research initiative at
the University of Utah unrestricted TCIP grant
Delivery of Human Growth Hormone via DSM’s Poly(ester
amide)
Julien Bérard1, John Zupancich1, Audrey Hecka1, George Mihov1,
Sarah Reiver1, Jens Thies1, Kenneth A. Messier1, Barbara M.
Wirostko2, MaryJane Rafii2. 1DSM, Geleen, Netherlands; 2Jade
Therapeutics, LLC, Salt Lake City, UT.
Purpose: Fibrillar, degradable polymer-based constructs for the
local, sustained delivery of recombinant human growth hormone
(rhGH) were generated. The safety, tolerability and possible efficacy
of subconjunctivally placed devices was evaluated in a rabbit
debridement model. rhGH was selected based on an ability to upregulate and modulate various growth factors (i.e., insulin growth
factor, epidermal growth factor) that have been shown to be involved
in corneal re-epithelialization.
Methods: rhGH-loaded, multi-layer constructs were prepared via
encapsulation of solid protein within poly(ester amide) (PEA)
matrices through use of an innovative film formation and assembly
process. Constructs were subsequently shaped into fibers with
dimensions that facilitate passage through a 27 gauge needle.
Construct in vitro release performance was evaluated over a period of
30 days and the bioactivity of released rhGH confirmed via a
proliferative cell assay. In vivo experiments compared PEA/rhGH
(~12ug/device) and PEA (i.e., blank) constructs placed
subconjunctival, perilimbal to BSS and topical rhGH (100ug/ml)
delivered four times daily in a standardized rabbit corneal epithelial
debridement model (N=9, 18 eyes). Safety, tolerability, and efficacy
were assessed daily and histopathology was performed on Day 7.
Time to complete healing and daily percent healing was compared
across arms over 7 days.
Results: In vitro results demonstrate that over 90% of rhGH
encapsulated within early generation PEA fibers is released over the
course of 3 days. Advances in multi-layer construct design reveal the
potential to sustain the release of rhGH for multiple weeks. In vivo
results showed PEA/rhGH and PEA constructs were well tolerated,
with histopathology on all eyes revealing normal healing with no
inflammation or angiogenesis. An efficacy signal was difficult to
ascertain due to the small number of animals utilized in the current
study, and the rapid healing that occurs in normal healthy rabbits.
Conclusions: Subconjunctivally placed rhGH-loaded PEA fibers
were well tolerated in vivo and no histopathologic concerns were
identified. In order to test efficacy, a preclinical model of impaired
wound healing, such as an alkali burn, should be utilized. Study
results indicate that PEA could be a viable polymer for the sustained
delivery of proteins to the eye.
Commercial Relationships: Julien Bérard, DSM (E); John
Zupancich, DSM (E); Audrey Hecka, DSM (E); George Mihov,
DSM (E); Sarah Reiver, DSM (E); Jens Thies, None; Kenneth A.
Messier, DSM Biomedical (E); Barbara M. Wirostko, Jade
Therapeutics (P), Jade Therapeutics (I), Jade Therapeutics (E),
Altheos Inc. (C), Merck (C), SKS Ocular (C), USTAR (F);
MaryJane Rafii, Jade Therapeutics (P), Jade Therapeutics (S), Jade
Therapeutics (I), Pfizer Inc. (C), Regenron (C)
Design of a Non-Invasive Core-shell Nanoparticulate Drug
Delivery System for Posterior Part of the Eye
Binapani Mahaling, Dhirendra S. Katti. Biological Sciences and
Bioengineering, Indian Institute of Technology Kanpur, Kanpur,
India.
Purpose: Providing drug molecules to the posterior part of the eye in
therapeutic concentration and for adequate durations is difficult
because of barrier properties of different layers of the eye. The
differential permeability towards hydrophilic or hydrophobic
molecules and surface charge of different layers of the eye further
complicates the matter. Hence, the purpose of this study was to
develop a noninvasive core-shell nanoparticle-based drug delivery
system for targeting the posterior part of the eye. It was hypothesized
that a nanoparticulate system with a hydrophobic core and a
hydrophilic shell can potentially overcome the barriers due to the
preferential permeability of hydrophilic molecules in the outer and
hydrophobic molecules in the inner layers of the eye.
Methods: Nanoparticle cores of poly(lactic acid) (PLA) were coated
with a chitosan (CHI) shell. CHI was chosen due to hydrophilic
nature and its potential to overcome the tear fluid barrier because of
its positive charge, bioadhesive and permeability enhancing
properties. PLA and coumarin loaded PLA nanoparticles were
synthesized by emulsion solvent evaporation method and modified
with chitosan by adsorption followed by chemical crosslinking.
Coumarin loaded PLA and PLA-CHI formulations were administered
as eye drops onto the eye of C57BL/6J mice with PBS, PLA and
PLA-CHI nanoparticles as controls. At predetermined time points
mice were euthanized. The eyes were then enucleated, fixed,
sectioned and analyzed under a fluorescence microscope.
Results: Fluorescence was detected in coumarin loaded PLA and
PLA-CHI particles in retina indicating the penetration of
nanoparticles across the layers up to the retina, whereas, the control
groups showed no fluorescence in retina. The levels of fluorescence
observed for PLA-CHI core-shell nanoparticles was higher than that
of PLA nanoparticles at the retina indicating the possible role of
PLA-CHI in enhancing penetration.
Conclusions: These results suggest that PLA-CHI core-shell
nanoparticles show potential to be developed as a non-invasive drug
delivery system for posterior part of the eye.
Commercial Relationships: Binapani Mahaling, None; Dhirendra
S. Katti, None
Support: Indian Council of Medical Research
Development and characterization of silicone pressure sensitive
adhesive episcleral implant
He Wen, S Kevin Li. University of Cincinnati, Cincinnati, OH.
Purpose: Ocular implants provide prolonged delivery of therapeutic
agents to the eye, minimize systemic drug exposure, and reduce the
need of multiple intraocular drug injections. Episcleral implants are
ocular drug delivery systems that prolong drug delivery through the
transscleral route. Materials currently available for constructing
ocular implants are limited. Silicone pressure sensitive adhesives
(PSA) are non-toxic and highly compressible, have high cohesive
strength, and offer good adhesion to biological substrates. This
material can form a polymer-drug matrix that improves drug
compactibility and allows prolonged drug release at low polymer
content. The objectives of this study were to develop and characterize
a silicone PSA episcleral implant system for transscleral delivery of
small molecules and macromolecules.
Methods: Dexamethasone, atenolol, and bovine serum albumin
(BSA) were selected as the model drugs. Silicone PSA 7-4302 was
the polymer used to construct the episcleral implant. The implant had
an average diameter of 3.5 mm and thickness of 0.7 mm. In vitro
drug release experiments were conducted with implants of varying
polymer to drug mass ratios in a well-stirred vial system.
Results: The silicone PSA episcleral implants provided sustained
drug release that could last for over one month in the present study in
vitro. Drug release from the implants followed a dissolution
controlled release mechanism with the higher water solubility drug
showing faster release rate than the low solubility drug. Increasing
polymer content in the implant led to a significant decrease in the
drug release rate.
Conclusions: Drug release from the silicone PSA episcleral implant
is influenced by the solubility of the drug and the polymer content in
the implant. The episcleral implant demonstrates the ability to
provide drug release for an extended period of time, and therefore has
the potential to offer safe and effective treatment to chronic ocular
diseases via the transscleral route.
Commercial Relationships: He Wen, None; S Kevin Li, Aciont Inc
(C)
Ocular injection characteristics and in vitro release profiles of
lipophilic dye using thermogel PLGA-PE-PLGA as a sustainedrelease drug delivery device
Eva Abarca, Page M. Potter, Jacklyn H. Salmon, Brian C. Gilger.
North Carolina State University, Raleigh, NC.
Purpose: Thermosensitive biodegradable gels may be useful for
sustained delivery of drugs
intravitreally (IVT) or into the suprachoroidal space (SCS). The
purpose of this study is to determine if a thermogel polymer can
improve release kinetics of lipophilic carbocyanine dye (DiI) and to
evaluate if these polymers can be effectively injected into the eye.
Methods: Release kinetics of DiI from poly-(d,l-lactic acid-coglycolic acid)-polyethylene glycol (PLGA-PEG-PLGA) was
determined in vitro. Liquid 20% PLGA-PEG-PLGA (200 µl at 2°C)
was added to glass vials, either alone or mixed with 0.1 mg DiI
crystals, 0.1 mg DiI dissolved in ethanol, or 0.1 mg DiI dissolved in
ethanol with 2% hyaluronic acid (HA). A 5th group had no PLGAPEG-PLGA, just 0.1 mg DiI crystals. The vials were then maintained
at 37°C allowing gels to form,1 ml of PBS was added and daily
samples were collected for analysis by spectrofluoroscopy for 21
days. To characterize ocular injection and gel formation, 12
normothermic ex-vivo pig eyes were injected with liquid thermogel
(2°C) with 0.1 mg DiI. Six eyes were injected into the SCS using a
33 gauge 750 depth microneedle, whereas 6 eyes were injected IVT.
High-frequency (50Mhz) or 20 Mhz ultrasound was used to image
and to evaluate the effect and distension in the SCS or vitreous,
respectively.
Results: Release of DiI from thermogel was significantly higher than
DiI crystals over the first 7 days. Area under the curve (AUC) was
significantly higher (P<0.001) in PLGA-PEG-PLGA-HA gels
compared to the other gels, and the PLGA-PEG-PLGA-HA had
highest Cmax at day one (Tmax) showing an initial burst release.
Following SCS injection, a well demarcated hypoechoic structure
formed in the anterior SCS (1.9 +/- 0.4 mm by 7.1 +/-1.3 mm). After
IVT injection, a hyperechoic nearly spherical structure formed (3.9
+/- 0.8 mm by 5.3 +/-1.08 mm). Macroscopic exam of the sectioned
eye showed a well delimited gel-like structure in the SCS and IVT in
each eye.
Conclusions: PLGA-PE-PLGA thermogel resulted in sustained DiI
release for 7 days. The more hydrophilic PLGA-PG-PLGA with HA
exhibited a higher and more rapid release. SCS injections of liquid
(2°C) PLGA-PE-PLGA are feasible using microneedles resulting in a
well-defined gel in the SCS and IVT. The thermosensitive show
promise as a sustained delivery system targeting the choroid and
retina.
Commercial Relationships: Eva Abarca, Clearside (F); Page M.
Potter, None; Jacklyn H. Salmon, Clearside (F); Brian C. Gilger,
Clearside (F), Allergan (F)
Development of Aqueous Nanomicellar Formulation for Topical
Delivery of Biotinylated Lipid Prodrug of Acyclovir to Treat
Herpetic Keratitis
Aswani Dutt Vadlapudi, Kishore Cholkar, Ramya Krishna
Vadlapatla, Ashim K. Mitra. Division of Pharmaceutical Sciences,
University of Missouri-Kansas City, Kansas City, MO.
Purpose: The objective of this study was to develop a clear, aqueous
nanomicellar formulation and evaluate its in vitro ocular
biocompatibility as a novel carrier for topical delivery of biotinylated
lipid prodrug to the eye for treatment of herpetic keratitis (HK).
Methods: Biotin-12Hydroxystearic acid-acyclovir (B-12HS-ACV)
was synthesized and a micellar formulation was prepared by solvent
evaporation/film hydration method using two non-ionic surfactants vitamin E TPGS and octoxynol-40. The optimized formulation was
characterized for various parameters including in vitro prodrug
release. Human corneal epithelial cells (HCEC) were employed for
studying the cytotoxicity of the formulation. Further, the mRNA
expression levels of various cytokines were also studied with
quantitative real-time PCR (qPCR).
Results: B-12HS-ACV was successfully synthesized and confirmed
by LC-MS/MS and NMR. The micelle average size was 10.46 ± 0.05
nm with a PDI of 0.086 for blank micelles, and 10.78 ± 0.09 nm with
a PDI of 0.075 for prodrug loaded micelles. Both unloaded and
prodrug loaded micelles had negative zeta potential. The prodrug
encapsulation efficiency of mixed micelles was calculated to be ≈
90%. TEM analysis showed that the micelles were spherical,
homogenous and devoid of aggregates. B-12HS-ACV release from
micelles was slow and without a significant burst effect. Results
showed a sustained release of the prodrug from the micelles over a
period of 4 days. Neither the blank formulation nor prodrug loaded
micellar formulation demonstrated any cytotoxic effects. Further,
incubation of HCEC cells with blank and prodrug loaded micelles
groups did not significantly alter the expression levels of IL-1β, IL-6,
IL-8, IL-17, TNF-α and IFN-γ.
Conclusions: In summary, a topical aqueous nanomicellar
formulation comprised of vitamin E TPGS and octoxynol-40 loaded
with 0.1% B-12HS-ACV was successfully developed for the
treatment of HK. B-12HS-ACV loaded nanomicelles are relatively
small in size, spherical and homogenous, and devoid of aggregates.
The micelle formulations were perfectly transparent comparable to
water. Ocular biocompatibility studies indicated that mixed
nanomicelles were non-toxic and non-inflammatory to corneal
epithelial cells. Therefore, nanomicellar technology represents a
promising strategy for delivery of biotinylated lipid prodrugs of ACV
to treat HK.
Commercial Relationships: Aswani Dutt Vadlapudi, None;
Kishore Cholkar, None; Ramya Krishna Vadlapatla, None;
Ashim K. Mitra, None
Support: NIH grants R01EY09171-16 and R01EY010659-14
Dexamethasone Sustained Delivery From Polyesteramide
Microspheres For Intraocular Administration. Influence Of
Sterilization
Vanessa Andres-Guerrero1, Mengmeng Zong2, George Mihov2,
Aylvin A. Dias2, Rocio Herrero-Vanrell1. 1Pharmaceutical
Technology, Faculty of Pharmacy/Complutense University, Madrid,
Spain; 2DSM, Geleen, Netherlands.
Purpose: Amino acid based polyesteramides (PEAs) are a new
family of biodegradable polymeric materials based on α-amino acids,
aliphatic dicarboxylic acids and aliphatic α-ω diols. The aim of the
current study was to determine the effect of a sterilization dose of γirradiation (25kGy) on the release profile of dexamethasone (DX)loaded PEA microspheres (Ms), to evaluate the use of these systems
as carriers for controlled intraocular drug delivery.
Methods: DX-loaded Ms were prepared following an emulsionsolvent evaporation technique. Ms were characterized by particle size
(dynamic light scattering), morphology (scanning electron
microscopy) and DX-encapsulation efficiency. In vitro release studies
were carried out for 24 days by suspending the Ms (5mg) in 1.5ml of
PBS (pH 7.4, 37C) isotonized with NaCl. At pre-set times (1h, 24h
and twice a week for 24 days) the release medium was collected and
DX levels were quantified by HPLC. Freeze-dried DX-loaded Ms
were sterilized with a 60Co radiation source (25 kGy). Sterilized Ms
were characterized following the same procedures described above.
Results: PEA Ms were spherical and had smooth surface with sizes
ranging between 20-40 µm in all cases. The encapsulation efficiency
was 85.14±0.99% for non-sterilized DX- Ms and 85.28±0.81% for
sterilized DX-Ms (180.99±2.39 µg DX/mg Ms and 181.57±2.76 µg
DX/mg Ms, respectively). The DX release profile of non-sterilized
Ms had an initial burst of 17.69±0.58 µg DX/mg Ms released in the
first 24 h, similar to the one obtained for sterilized Ms (17.28±0.44
µg DX/mg Ms). A progressive sustained release of DX was observed
for the following days. At day 24, the cumulative release of DX was
80.86±2.40 µg DX/mg Ms for non-sterilized Ms and 79.45±4.74 µg
DX/mg Ms for sterilized Ms (45.27±1.11% and 43.80±3.28% of the
encapsulated DX, respectively).
Conclusions: PEA Ms developed are capable of providing a
sustained release of DX for at least 24 days. Non significant
differences were found between sterilized and non-sterilized MS.
Gamma-radiation had no significant effect on the release profile of
the microspheres. Biodegradable PEA Ms are potentially useful to
develop new controlled drug delivery systems for treating ophthalmic
diseases affecting the back of the eye.
Commercial Relationships: Vanessa Andres-Guerrero, None;
Mengmeng Zong, DSM (E); George Mihov, DSM (E); Aylvin A.
Dias, DSM Biomedical (E); Rocio Herrero-Vanrell, None
Support: PANOPTES (project number 246180) under the 7th
Research Framework Program of the European Union.
Pharmacodynamic study of Intravitreal liposomal doxorubicin
by matrix-assisted laser desorption/ionization-mass spectrometry
(MALDI/MS)
Hsi-Kung Kuo, Yi-Hao Chen, Pei-Chang Wu. Ophthalmology,
Kaohsiung Chang Gung Memorial Hospital and Chang Gung
University College of Medicine, Kaohsiung, Taiwan.
Purpose: to investigate the pharmacodynamics of doxorubicin and
liposomal doxorubicin (Lipo-Dox) after intravitreal injection.
Methods: Pigmented rabbits were used. One eye accepted
intravitreal injection of doxorubicin or Lipo-Dox (10 μg/ml). Another
eye was injected with 0.1 ml BSS as the control. To evaluate the
pharmacodynamic change of the drugs, the animals were sacrificed
on the day 1, 3, 5, 7 and 14 after the injection. The vitreous contents
and retina extracts were prepared for LC/MS/MS and MALDI/MS
study. On the matrix-assisted laser desorption/ionization-imaging
mass spectrometry (MALDI/IMS) group, the full eye sections are
prepared from cryo-section.
Results: Pharmacodynamic study by MALDI/MS showed liposomal
doxorubicin existed in the vitreous for at least 14 days with the
highest density on day 7. The result is compatible with the slowly
released property of liposomal drugs. To study the drug distribution
with MALDI/IMS was failed due to background interference.
Conclusions: Intravitreal injection of liposomal doxorubicin can
obtain a sustained drug concentration for at least 14 days with the
peak on day7.
Commercial Relationships: Hsi-Kung Kuo, None; Yi-Hao Chen,
None; Pei-Chang Wu, None
Support: CMRPG891281 from Chang-Gung Memorial Hospital,
Taiwan
Cavernous Sinus as Ocular Pharmacokinetic Compartment for
Better Understanding of Posterior Segment and Contralateral
Eye Drug Availability
Muhammad Abdulrazik. Ophthal/Innovative Interventions, East
Jerusalem Biomedical Institute, East Jerusalem, Occupied Palestinian
Territory.
Purpose: To study the possibility that the drainage of drug loaded
ocular blood to the cavernous sinus is coupled with returned drug
transfer in the opposite direction, not through the systemic
circulation.
Methods: Texas-Red labeled dextran (40-kDa) solution (2.5 mg/ml)
was injected into the sub-conjunctiva of the rat eye. Eyes were
enucleated and intracranial optic nerves, up to the chiasm, were
harvested. Tissues were snap-frozen and processed for visualization
by fluorescence microscopy. In an extension of previous brimonidine
ocular pharmacokinetic study, whole-tissue brimonidine in
contralateral eye tissues was quantified at 5 minutes after single
topical instillation of 50µl of 3H-radiolabeled brimonidine solution
(0.2%) to the right eye of albino rabbits.
Results: In the area of the optic nerve head, dextran was heavily
loaded in episcleral and periocular veins but absent in the posterior
ciliary artery. In the sampled intracranial segment of the optic nerve,
just before the junction with the optic-chiasm, nerve axons were free
of dextran while pial and sheath veins were draining dextran towards
the cavernous sinus. Whole-tissue brimonidine calculation was based
on the evidenced drug concentration per gram tissue and whole-tissue
weight. The sum of brimonidine accumulation at 5 min. post dosing
for all tissues of the contralateral eye was 151.08±39.21 ng. The
estimated amount of brimonidine that could have entered the eye via
blood circulation in first 5 minutes post dosing (67.3±26.8 ng) was
calculated by using previously reported value of blood flow per
minute into the rabbit eye and the evidenced brimonidine
concentration in the systemic blood in current study.
Conclusions: The dextran study has shown the accumulation of
dextran in the cavernous sinus area of the rat by non-systemically
mediated vascular drainage. The evidenced brimonidine
accumulation in the contralateral rabbit eye (151.08±39.21 ng) was
significantly higher than highest possible amount of brimonidine
(67.3±26.8 ng) that could be extracted from the systemic blood by
contralateral eye tissues, suggesting that contralateral eye
brimonidine accumulation represents in part returned drug transfer
from the cavernous sinus toward the eye via non-systemic vascular
connections. Further studies are needed to support the integration of
the cavernous sinus in ocular pharmacokinetic modeling.
Commercial Relationships: Muhammad Abdulrazik, None
Influence of Physicochemical Properties on Drug Delivery across
Sclera into Choroid-Retina
Ayumi Yoshimatsu1, Chiho Yabuta1, Akira Ohtori1, 2, Mitsuyoshi
Azuma1. 1Senju Laboratory of Ocular Sciences, Senju Pharmaceutical
Co., Ltd., Kobe, Japan; 2Laboratory of Ocular Drug Delivery System,
Kyushu Institute of Technology, Fukuoka, Japan.
Purpose: Intravitreal injections are currently used to deliver drugs to
the retina and other tissues in the back of eye. Topical instillation of
ophthalmic drugs would be more comfortable for patients and lower
the risk of infection. Reports indicate that topical instillation of some
compounds indeed diffuse from the posterior periocular tissues across
sclera and reach the posterior choroid-retina. The specific
physicochemical properties of such drugs would be important for
their permeability, diffusivity and partitioning into ocular tissues, but
such data are limited. Thus, the purpose of the present study was to
determine the influence of physicochemical properties of selected
drugs on their passage across sclera into choroid-retina.
Methods: Model compounds were selected to range in molecular
weight (MW) from 300 to 10,000 and included a wide range of
lipophilicity. Sclera and sclera-choroid-retina were excised from
rabbit globes, and were mounted side-by-side in Ussing diffusion
chambers. Model compounds in buffer were added to the donor
chamber adjacent to sclera, while buffer solution alone was present in
the receptor chamber. Permeation rates, time lags, and permeability
across sclera or sclera-choroid-retina preparations were determined
by pharmacokinetics modeling. Diffusion and partition coefficients
were then calculated based on a bilayer membrane model.
Results: Cumulative transport of drug across sclera and sclerachoroid-retina increased as drug concentration in donor solution
increased except for compound of MW 10,000. Permeability was
negatively correlated with MW in sclera and sclera-choroid-retina.
Drug diffusivity in sclera and choroid-retina decreased as MW
increased, and diffusion coefficients in choroid-retina were 3 to 10
times less than that in sclera. Partition coefficients into choroid-retina
increased as lipophilicity increased. This relationship was not
observed for sclera.
Conclusions: Optimal modification of ocular drugs used for topical
instillation needs to decrease MW and to balance solubility with
lipophilicity.
Commercial Relationships: Ayumi Yoshimatsu, Senju
Pharmaceutical Co., Ltd. (E); Chiho Yabuta, Senju Pharmaceutical
Co., Ltd. (E); Akira Ohtori, SENJU PHARMACEUTICAL CO.,
LTD. (E); Mitsuyoshi Azuma, Senju Pharmaceutical Co., Ltd. (E)
allogeneic rat corneal grafts after penetrating keratoplasty (PK).
Methods: A total of 48 PK were performed using Fisher rats
(allogeneic groups) and Lewis rats (syngeneic group) as donors and
Lewis rats as recipients: group 1 (n=12), syngeneic control; group 2
(n=12), allogeneic control without treatment; group 3 (n=12),
allogeneic grafts with subconjunctivally-implanted PA-loaded
microfilm treatment; group 4 (n=12): allogeneic grafts with 1% PA
eye drops treatment. All grafts were evaluated for 28 days by a
scoring rejection index (RI), assessing graft opacity, edema and
neovascularization by slit lamp biomicroscopy and anterior segment
optical coherence tomography (ASOCT). Time to rejection was
analyzed with Kaplan-Meier survival analysis. PA concentrations in
the aqueous humor were measured using high-performance liquid
chromatography. Histopathological and immunohistochemical
staining were also performed.
Results: The PA-loaded microfilms achieved a sustained and steady
release at a rate of 7.0ug/day, with a consistent drug concentration of
207-209 ng/ml in the aqueous. The mean survival was >28 days in
group 1, 9.9±0.8 days in group 2, 26.8±2.7 days in group 3 (P =
0.023 as compared with group 2), and 26.4±3.4 days in group 4 (P =
0.027 as compared with group 2). Statistically significant decrease in
CD4+, CD8+, CD163+, CD11c+, CD 25+ and CD54+ cell
infiltration in group 3 as compared with group 2 was observed on
immunohistochemistry (P<0.001). There was no significant
difference between group 3 and group 4 in the mean survival and
immunohistochemical analysis.
Conclusions: The sustained PA-loaded microfilm DDS effectively
prolongs corneal allograft survival in the rat model. It is as effective
as conventional PA eye drops, providing a promising clinically
applicable alternative for patients undergoing corneal transplantation.
Figure 1: Representative slit lamp biomicroscopy and ASOCT photos
showing the status of the corneal grafts 2 and 4 weeks after
penetrating keratoplasty
Sustained Prednisolone Acetate-loaded Microfilm Drug Delivery
System Effectively Prolongs corneal allograft survival in the rat
keratoplasty model
Yu-Chi Liu1, 2, Yan Peng3, Nyein Chan Lwin1, Subbu S Venkatraman3,
Tina Wong1, 2, Jodhbir S. Mehta1, 2. 1Singapore Eye Research
Institute, Singapore, Singapore; 2Singapore National Eye Centre,
Singapore, Singapore; 3School of Materials Science and Engineering,
Nanyang Technological University, Singapore, Singapore.
Purpose: To investigate the efficacy of a sustained prednisolone
acetate (PA)-loaded poly (d,l-lactide-co-ε-caprolactone) (PLC)
microfilm drug delivery system (DDS) for promoting the survival of
with 0.2mg SCS TA and 0.2 and 2.0mg IVT TA. Furthermore, 2.0mg
SCS TA mean cell counts and protein concentrations were not
significantly different than negative control eyes.
Conclusions: Results from this study suggest that delivery of TA to
the SCS provides effective control of acute posterior uveitis in a
model that is similar in anatomy, size, and retinal vascular pattern to
the human eye. There were no adverse effects, increased IOP, or
evidence of procedural or drug toxicity following injection of TA into
the SCS using microneedles in porcine eyes. This study supports the
further evaluation of the SCS as a site of drug delivery to ocular
posterior segment.
Commercial Relationships: Brian C. Gilger, Clearside (F),
Allergan (F); Eva M. Abarca, Clearside (F); Jacklyn H. Salmon,
Clearside (F); Samirkumar R. Patel, Clearside Biomedical (E),
Clearside Biomedical (I), Clearside Biomedical (P)
Support: Georgia Research Alliance
Figure 2: Kaplan-Meier analysis of the rejection-free graft survival
Commercial Relationships: Yu-Chi Liu, None; Yan Peng, None;
Nyein Chan Lwin, None; Subbu S Venkatraman, None; Tina
Wong, 61, 250,006 (P); Jodhbir S. Mehta, None
Support: Translational and Clinical Research (TCR) Programme
(NMRC/TCR/002-SERI/2008)
Treatment of Acute Posterior Uveitis by Injection of
Triamcinolone Acetonide into the Suprachoroidal Space Using
Microneedles
Brian C. Gilger1, Eva M. Abarca1, Jacklyn H. Salmon1, Samirkumar
R. Patel2. 1Clinical Sciences, North Carolina State University,
Raleigh, NC; 2Clearside Biomedical, Alpharetta, GA.
Purpose: To compare effects of microneedle injection of
triamcinolone acetonide (TA) into the suprachoroidal space (SCS)
versus intravitreal (IVT) TA injection in a model of acute posterior
uveitis.
Methods: Twenty weanling pigs had BSS or lipopolysaccharide
(LPS) IVT injection followed 24 hours later with SCS or IVT
injection of 0.2 mg or 2.0 mg TA. SCS injections were made using
purpose-designed microneedles and 27G needles were used for IVT
injections. Clinical ocular inflammatory scores (CIS) and intraocular
pressure measurements (IOP) were collected daily, while
electroretinography, optical coherence tomography (OCT), and widefield ocular fundus photography were performed on -1, 0, and 3 days
after treatment. Pigs were then euthanized, aqueous and vitreous
humor collected for cell counts and protein levels, and the eyes were
processed for histopathology.
Results: SCS TA injection using microneedles was simple, effective,
and not associated with adverse effects, elevated IOP, or toxicity.
Cumulative mean CIS of eyes receiving 0.2 and 2.0mg SCS TA were
not significantly different than either control (non-uveitis induced)
eyes or eyes receiving 2.0mg IVT TA at all examination times.
Cumulative mean CIS, however, of 0.2mg IVT TA was not
significantly less than LPS injected, vehicle treated eyes. OCT vitreal
cellular infiltrate was significantly lower after treatment with both 0.2
and 2.0 mg SCS TA than eyes treated with vehicle but were not
significantly different than eyes treated with IVT TA. Histologic
inflammatory suppression after 0.2 and 2.0mg SCS TA was similar to
eyes treated with 2.0mg IVT TA. Mean vitreal cell counts and protein
concentrations were not significantly different between eyes injected
Ocular and Systemic Pharmacokinetics of a PDE4 Inhibitor
Following Topical Administration (Eyedrop) in Male DutchBelted Rabbits
David C. Gale1, Caroline J. Sychterz2, Ciara Rodgers2, Sherry
Wang2, Tom Wilde2, Anu Shilpa Krishnatry2, Harma Ellens2.
1
Ophthalmology DPU, GlaxoSmithKline, King of Prussia, PA; 2PTS
DMPK-UM, GlaxoSmithKline, King of Prussia, PA.
Purpose: To evaluate the ocular and systemic pharmacokinetics of a
phosphodiesterase 4 (PDE4) inhibitor following topical eyedrop
administration in male Dutch-Belted rabbits.
Methods: All studies were conducted according to the GSK Policy
on the Care, Welfare and Treatment of Laboratory Animals after
review by the GSK Institutional Animal Care and Use Committee
and in compliance with the ARVO Statement on the Use of Animals
in Ophthalmic and Visual Research. Male Dutch-Belted rabbits were
topically dosed with a potent PDE4 inhibitor (GSK907188, IC50
<100pM) with either a 0.07 mg/mL or 7.6 mg/mL solution
formulation. Plasma samples were collected to measure the systemic
exposure of GSK907188. Ocular tissues were collected from postmortem enucleated eyes. GSK907188 levels were quantitated in
plasma and ocular tissue samples using an HPLC/MS/MS method.
The predicted human systemic exposure was estimated from rabbit
systemic exposure, clearance values and GastroPlusTM modeling.
Results: GSK907188 levels in rabbit plasma, following topical
administration of the 7.6 mg/mL formulation, were quantifiable out
to 6hrs (6hr plasma levels = 0.40 ± 0.16 ng/mL). The rabbit plasma
AUC(0-6hr) was 14.7 ± 3.50 ng*h/mL. The predicted human
systemic exposure is 140-fold lower than the systemic exposure
observed in rabbit. Ocular tissue levels were measured after topical
administration of the 0.07mg/mL formulation. The highest
GSK907188 levels were observed in the conjunctiva, where levels
decreased from a Cmax of 50.5 ng/g tissue at 1hr to 0.48 ng/g at
24hr. GSK907188 concentrations in the cornea were low compared to
the levels observed in the conjunctiva and remained relatively
consistent throughout the study. Levels of GSK907188 in the
lacrimal gland were low (<0.5 ng/g) but measureable out to 6 hr.
Aqueous humor levels were below the limit of quantitation (0.1
ng/mL) for all samples, except the 1hr time point (0.12 ng/mL).
Conclusions: PDE4 inhibitors may be an important new class of
molecules for inflammatory eye diseases. Preclinical
pharmacokinetic studies, in-vitro studies and modeling can be
utilized to provide an estimate of human systemic exposure, which is
an important consideration with PDE4 inhibitors. For GSK907188, a
formulation strength of >1 mg/mL may be required to generate
efficacious concentrations in the cornea, conjunctiva and lacrimal
gland.
Commercial Relationships: David C. Gale, GlaxoSmithKline (E);
Caroline J. Sychterz, GlaxoSmithKline (E); Ciara Rodgers,
GlaxoSmithKline (E); Sherry Wang, GlaxoSmithKline (E); Tom
Wilde, GlaxoSmithKline (E); Anu Shilpa Krishnatry,
GlaxoSmithkline (E); Harma Ellens, GlaxoSmit5hKline (E)
Aqueous humor concentration of Bromfenac 0.09% (Bromday)
compared with Bromfenac in DuraSite 0.075% (Bromsite) in
cataract patients undergoing phacoemulsification after 3 days
dosing
Kamran Hosseini1, Judith Hutcheson1, Lyle M. Bowman2. 1Clinical,
InSite Vision Inc, Alameda, CA; 2Development, InSite Vision,
Alameda, CA.
Purpose: To compare the aqueous humor penetration of two
different formulations of Bromfenac, namely commercial Bromday
(0.09%) with Bromfenac in Durasite (0.075%).
Methods: A multi center, double-masked study randomized 60
patients requiring cataract extraction. Patients were to receive one of
two treatment arms. Once a day dosing of Bromday and Bromfenac
in Durasite was instructed for 2 day prior to surgery. Patients were
instructed to instill the last (third) dose in the morning of surgery.
After completion of the paracentesis site, aqueous humor was
collected through the peripheral clear cornea with a 30-gauge needle.
Following collection, aqueous samples were frozen and stored prior
to analysis. Drug concentrations were analyzed by a validated liquid
chromatography tandem mass spectrometry method from extracted
tissue samples using an internal standard. The comparison of means
between groups was performed using an F-test.
Results: The average aqueous humor concentration of Bromfenac in
Durasite 0.075% was 2 times the concentration of Bromday 0.09%.
Bromfenac in Durasite achieved a mean peak aqueous humor
concentration of 49.33 compared with 23.64 ng/ml for Bromday.
Conclusions: Bromfenac in Durasite 0.075% achieved significantly
greater aqueous humor concentrations when compared to Bromfenac
0.09% in patients undergoing phacoemulsification.
Commercial Relationships: Kamran Hosseini, InSite Vision Inc.
(E); Judith Hutcheson, InSite Vision (E); Lyle M. Bowman, InSite
Vision (E)
Comparison of the Total Amount of Triamcinolone Acetonide
Delivered Via Suprachoroidal or Intravitreal Administration
Brian Burke, Rozemarijn S. Verhoeven, Samirkumar R. Patel.
Clearside Biomedical, Raleigh, NC.
Purpose: The purpose of this study was to compare the total amount
of triamcinolone acetonide (TA) delivered into a pig eye when
injected into the suprachoroidal space using a Clearside Biomedical
proprietary microneedle or into the vitreous using a standard 30
gauge needle.
Methods: Whole pig cadaver eyes (Sioux-Preme Packing)
enucleated within 24 hours after death were used for all injections.
Intravitreal and suprachoroidal injections of TA were performed
using Triesence® (Alcon Labs). Intravitreal injections were
performed using a 30 g needle (Becton-Dickinson) and
suprachoroidal injections were performed using a Clearside
Biomedical proprietary microneedle. 1 mL syringes (BectonDickinson) were loaded with the required amount of Triesence® at
each of the three volumes assessed: 50, 100, and 150 µL (2, 4, and 6
mg, respectively). The residual amount of TA present in the
syringe/needle assembly after injection was determined by RPHPLC. The total amount of TA delivered to the eye for each dose
volume was determined as the difference in the total amount loaded
into a syringe before injection into the pig eye versus the residual
amount of TA recovered from the syringe/needle assembly after
injection.
Results: Average total dose administered following 50, 100 and 150
µL TA injected into the suprachoroidal space ranged from 86-92% of
the target dose level, while average total dose administered following
50 and 100 µL TA injected into the vitreous ranged from 88-89%.
Virtually no difference was observed between the two routes of
administration and needles for each volume.
Conclusions: The target dose level of TA can be consistently
delivered into the SCS using a microneedle or into the vitreous using
a 30 g needle. Total amount of TA delivered was similar between the
two administration routes.
Commercial Relationships: Brian Burke, Clearside Biomedical
(E); Rozemarijn S. Verhoeven, Clearside Biomedical (E);
Samirkumar R. Patel, Clearside Biomedical (E), Clearside
Biomedical (I), Clearside Biomedical (P)
Suprachoroidal Microinjection Delivers Triamcinolone
Acetonide to Therapeutically-Relevant Posterior Ocular
Structures and Limits Exposure in the Anterior Segment
Henry F. Edelhauser1, 2, Samirkumar R. Patel2, Carol Meschter3,
Robin Dean3, Kendall Powell4, Rozemarijn S. Verhoeven2.
1
Ophthalmology, Emory Univ Eye Center, Atlanta, GA; 2Clearside
Biomedical, Alpharetta, GA; 3Comparative Biosciences, Sunnyvale,
CA; 4Tandem Labs, Durham, NC.
Purpose: To evaluate the ocular and systemic PK of triamcinolone
acetonide (TA) in the New Zealand White rabbit following
intravitreal (IVT) injection or administration into the suprachoroidal
space (SCS) using a Clearside Biomedical proprietary microneedle.
Methods: On Day 0, male rabbits (5/group) received a single
bilateral administration of 4 mg TA (100 µL Triesence®) via SCS
injection using a 33g 750µm microneedle or IVT injection using a
standard 30g needle. Clinical observations, body weights, and
intraocular pressure (IOP) were assessed up to 13 weeks post-dose.
Plasma and ocular matrixes (aqueous humor (AH), lens, iris/ciliary
body (ICB), vitreous humor (VH), sclera/choroid (SC), and retina)
were sampled on Days 1, 14, 28, 56, and 91. Plasma (LLOQ 0.5
ng/mL) and ocular matrixes (LLOQ 2 - 15 ng/mL) were analyzed
using LC-MS/MS, and resulting data were assessed for
noncompartmental PK parameters.
Results: Preliminary data shows that there were no observed adverse
effects related to treatment or method of administration. TA in
plasma peaked on Day 1 at 4 ng/mL in both groups, and TA was
quantifiable in all ocular matrixes through Day 91. Following SCS
TA, TA was observed (in decreasing order) in SC>retina
>VH>ICB>lens>AH. SCS TA Cmax and AUC (area under the
concentration curve) was increased in SC (Cmax: 11-fold, AUC: 11fold) compared with IVT TA. SCS and IVT TA retina Cmax and
AUC were roughly equivalent, but SCS TA peaked more quickly
(Day 1) compared with IVT TA (Day 14). Following IVT TA, TA
was observed in VH>ICB>retina>lens>SC>AH. IVT TA Cmax and
AUC was increased in AH (Cmax: 755-fold, AUC: 715-fold), lens
(Cmax: 290-fold, AUC: 682-fold), ICB (Cmax: 24-fold, AUC: 44fold) and VH (Cmax: 4-fold, AUC: 52-fold) compared with SCS TA.
Conclusions: Preliminary data suggest that both IVT and SCS TA
were well tolerated in the albino rabbit and systemic exposure was
minimal by either route. These data show that SCS TA is absorbed at
much greater proportions into the clear/choroid and retina, while IVT
TA distributes throughout the eye, indicating that SCS administration
using a microneedle is a targeted approach for delivering TA to
therapeutically-relevant ocular structures of posterior segment disease
and limiting anterior segment exposure.
Commercial Relationships: Henry F. Edelhauser, Clearside
Biomedical (P), Clearside Biomedical (I), Clearside Biomedical (C);
Samirkumar R. Patel, Clearside Biomedical (E), Clearside
Biomedical (I), Clearside Biomedical (P); Carol Meschter, None;
Robin Dean, None; Kendall Powell, None; Rozemarijn S.
Verhoeven, Clearside Biomedical (E)
Protective Effects of Transscleral Drug Delivery Device Against
Photoreceptor Cell Death in S334ter Rhodopsin Mutant Rats
Nobuhiro Nagai1, Hirokazu Kaji2, Hideyuki Onami1, Takuya
Yamada2, Yuki Katsukura1, Yumi Ishikawa1, Matsuhiko Nishizawa2,
Yukihiko Mashima3, Toshiaki Abe1. 1Graduate School of Medicine,
Tohoku University, Sendai, Japan; 2Graduate School of Engineering,
Tohoku University, Sendai, Japan; 3R-Tech Ueno, Tokyo, Japan.
Purpose: To evaluate the protective effects of a transscleral drug
delivery device that can release unoprostone isopropyl (UNO) in a
controlled release manner against photoreceptor cell death in S334ter
rhodopsin mutant rats.
Methods: The device consists of a reservoir, controlled-release
cover, and drug formulations, which were made of photopolymeized
poly(ethyleneglycol) dimethacrylate that partially contains
tri(ethyleneglycol) dimethacrylate. These parts were fabricated via a
microfabrication technique that used an AutoCAD design. UNO, a
prostanoid for antiglaucoma eyedrops marketed in Japan, was loaded
in the device. High-performance liquid chromatography was used to
evaluate the release amount of UNO. After the devices were placed
onto the sclera of eyes in 2 weeks old S334ter rats, flash
electroretinograms were recorded. Histological examinations were
perfomred to evaluate the thickness of the outer nuclear layer.
Results: UNO was released with zero-ordered kinetics from the
device. Electroretinographic amplitudes of the a- and b-waves
increased significantly in rats treated with UNO-loaded devices
compared with saline-loaded devices. The outer nuclear layer
thickness was thinned in the group treated with saline-loaded devices,
but the group treated with UNO-loaded devices suppressed the retinal
degeneration.
Conclusions: Transscleral UNO delivery device protected against
photoreceptor cell death in S334ter rhodopsin mutant rats. The device
may offer a less-invasive method of drug delivery to achieve
sustained release of medications for intravitreal drug delivery and the
treatment of various retinal diseases.
Commercial Relationships: Nobuhiro Nagai, R-Tech Ueno (F);
Hirokazu Kaji, None; Hideyuki Onami, None; Takuya Yamada,
None; Yuki Katsukura, None; Yumi Ishikawa, None; Matsuhiko
Nishizawa, None; Yukihiko Mashima, R-Tech Ueno (E); Toshiaki
Abe, R-TECH UENO, LTD. (F)
Support: Grant-in-Aid for Young Scientists (A) from the MEXT,
Japan, Health Labour Sciences Research Grant from the MHLW,
Japan
In vitro cytotoxicity screening and pharmacokinetic modeling: a
tool in the development of ocular drug delivery systems
Eva Tuominen1, George Mihov2, Mengmeng Zong2, Sanjay Sarkhel1,
Aylvin A. Dias2, Arto Urtti1. 1Centre for Drug Research, Faculty of
Pharmacy, University of Helsinki, Helsinki, Finland; 2DSM, Geleen,
Netherlands.
Purpose: The aim was to combine in vitro cytotoxicity screening
assays and ocular pharmacokinetic modeling in order to aid the
development of safe ocular drug delivery systems. Polyesteramide
(PEA) is a potential polymer platform for controlled release systems
of ocular drugs. Cytotoxicity of hydrolytic degradation products of
biodegradable polyesteramide were tested using this combination
method.
Methods: A cell based cytotoxicity screening platform has been
designed for testing of polymers, associated particles and degradation
products of polymers. Human retinal pigment epithelial cell line
(ARPE-19) was used as the test cell line. A broad concentration range
of the materials was used in the tests. Degradation products of PEA
were tested with MTT assay and, polyethylene imine (PEI) and polyL-lysine (PLL) were used as controls. ARPE-19 cells on a 96-well
plate were incubated for 5 hours with different concentrations of the
polymers (0.0001 - 5 mg/ml). Kinetic ocular modelling was carried
out using Stella software (ISEE systems 9.0). Kinetic simulation
model was built to predict the intravitreal concentrations of the PEA
degradation products and control polymers. In the model, the
intravitreal polymer dose was set at 10 mg, the dissolution took place
in sink conditions, and the degradation rate of free PEA was varied in
the simulations.
Results: The PEA degradation products did not show any signs of
toxicity in the MTT assay at test concentrations (0.0001 - 5 mg/ml),
but the control polymers showed toxicity (PEI: IC 50 = 6.3 ± 1.0
µg/ml; PLL: IC50 = 48.7 ± 11.0 µg/ml). For PEA borne materials the
pharmacokinetic model predicted a maximum total concentration
range of 0.2 - 4 mg/ml, for PEI 1.4 - 6 mg/ml and PLL 1.2 - 6 mg/ml.
Conclusions: Combination of kinetic modeling and cellular toxicity
testing indicates ocular toxicity of PLL and PEI. PEA and its
degradation are predicted to be safe after intravitreal administration.
Cytotoxicity screening and pharmacokinetic simulations are a
promising tool in the development of ocular drug delivery systems.
Commercial Relationships: Eva Tuominen, None; George Mihov,
DSM (E); Mengmeng Zong, DSM (E); Sanjay Sarkhel, None;
Aylvin A. Dias, DSM Biomedical (E); Arto Urtti, None
Support: PANOPTES (EU-FP7 246180)
Retinal Safety and Efficacy of a Dexamethasone Biodegradable
Implant to Treat Macular Edema Associated to Retinal Vein
Occlusion: A Phase I/II Clinical Trial
Rubens C. Siqueira1, Renato B. Cunha1, Andre Messias1, Armando S.
Cunha2, Silvia Fialho2, Rodrigo Jorge1. 1Retina, Sao Paulo
University, Sao Jose do Rio Preto, Brazil; 2Pharmacology, Minas
Gerais Federal University, Belo Horizonte, Brazil.
Purpose: To evaluate the safety and efficacy of a biodegradable
implant containing 350 µg of dexamethasone (DDS-25 gauge) for the
treatment of macular edema associated to retinal vein occlusion
(vME)
Methods: Prospective, nonrandomized, open-label, phase I/II clinical
trial, including 10 patients (n=10 eyes) with chronic vME, showing
ETDRS best-correct visual acuity (BCVA) of 20/50 or worse.
Evaluations included BCVA, spectral-domain optical coherence
tomography (OCT - Spectralis Heidelberg Engineering) for
determination of central macular thickness (CMT), full-field
electroretinography (ISCEV standard ERG), kinetic visual field
(Octopus 900), and fluorescein and indocyanine green angiography.
Evaluations were performed at baseline, and 1, 4, 12 and 24 weeks
after intravitreal DDS-25 insertion.
Results: The mean ± SE (range) CMT at baseline was 461.2 ± 41.3
μm (288 to 701 µm), and 439.6 ± 40.4 µm (259 to 631 µm), 442.5 ±
44.6 µm (255 to 632 µm), 354.6 ± 31.2 µm (228 to 537 µm), 316.5 ±
26.4 µm (226 to 441 µm) at 1, 4, 12, and 24 weeks respectively.
Showing a significant improvement of 144.7 ± 46.0 µm at 24 weeks
(P=0.0059; ANOVA). BCVA improved significantly in 0.14 ± 0.06
logMAR (7 ETDRS letters) at 24 weeks (P=0.0248), with 6 patients
improving between 1 to 4 ETRS lines. Mean rod b-wave amplitude
was 265.4 ± 30.4 µV at baseline and 255.6 ± 32.6 µV at 4 weeks
(P=0.375), and no significant changes were observed for any ERG
parameters, visual fields or angiography during follow-up. Related
adverse events included no significant IOP elevation of 3.2 ± 1.6
mmHg (P=0.071) at week 24.
Conclusions: Our data suggests that the DDS-25 is safe and efficient
for the treatment of vME in a short follow up time (6 months). A
larger prospective randomized study is warranted to confirm these
preliminary findings.
Commercial Relationships: Rubens C. Siqueira, None; Renato B.
Cunha, None; Andre Messias, None; Armando S. Cunha, None;
Silvia Fialho, None; Rodrigo Jorge, None
Therapeutic effect of stealth-type polymeric nanoparticles with
encapsulated cyclosporine A on experimental autoimmune
uveoretinitis
Tsutomu Sakai1, Kana Kuroyanagi1, Tsutomu Ishihara2, Kiichiro
Okano1, Hiroshi Tsuneoka1. 1Ophthalmology, Jikei Univ School of
Medicine, Setagaya-ku, Japan; 2Chemical biology and applied
chemistry, Nihon university college of engineering, Kooriyama,
Japan.
Purpose: The therapeutic effects of cyclosporine A encapsulated in
biocompatible and biodegradable blended nanoparticles of poly
(lactic acid) (PLA) homopolymers and PEG-block-PLA copolymers
(stealth nanocyclosporine) were examined in an experimental
autoimmune uveoretinitis (EAU) model in Lewis rats.
Methods: EAU was induced by S-antigen peptide in Lewis rats.
Accumulation of systemically administered Cy7-labeled stealth
nanoparticles in inflamed eyes of rats with EAU was assessed using
in vivo fluorescence imaging. And the therapeutic effect of stealth
nanocyclosporine or saline on EAU was examined. The eyes were
obtained 7 days after the treatment and the histological score was
determined. using pathological findings. The expression of
inflammatory cytokines including IL-6, IL-17, and VEGF was
determined immunohistochemically.
Results: Cy7-stealth nanoparticles accumulated in inflamed eyes of
rats with EAU and remained in situ for a 3-day period. Systemically
administered stealth nanocyclosporine reduced the clinical scores of
rats with EAU within 1 day and maintained the effect for 2 weeks.
This treatment also decreased the histological scores and the
expression of inflammatory cytokines in the retina of EAU.
Conclusions: The strong therapeutic benefit on EAU obtained with
the stealth nanocyclosporine may have been due to prolonged blood
circulation and targeting to the inflamed uvea and retina, in addition
to sustained release in situ.
Commercial Relationships: Tsutomu Sakai, None; Kana
Kuroyanagi, None; Tsutomu Ishihara, None; Kiichiro Okano,
None; Hiroshi Tsuneoka, None
Development of an in vitro pharmacokinetic model of the human
eye
Sahar Awwad1, 2, Alastair Lockwood1, 2, Abeer Mohamed Ahmed1, 2,
Garima Sharma1, 2, Ashkan Khalili2, Steve Brocchini1, 2, Peng T.
Khaw2. 1UCL School of Pharmacy, London, United Kingdom;
2
National Institute for Health Research (NIHR) Biomedical Research
Centre at Moorfields Eye Hospital NHS Foundation Trust and UCL
Institute of Ophthalmology, London, United Kingdom.
Purpose: Pharmacokinetics remains a major challenge in drug
development for the posterior segment. Accurate evaluation and
optimisation of vitreous drug distribution is hindered by differences
between humans and animal platforms. Furthermore, measurements
at serial time points necessitate use of many animals, which has both
cost and ethical implications. We aimed to design an in vitro flow
model that would mimic posterior segment fluid dynamics, and aid
the determination of vitreous drug distribution. Similar to the design
of simulated gastric solutions to mimic dissolution within the
intestinal tract, we are also focused on developing a simulated
vitreous solution that can be used in early preclinical studies.
Methods: Several prototype chambers (~4.2 mL internal volume )
were fabricated incorporating both a model anterior and posterior
segment. These were filled with simulated vitreous (a polymeric
combination of hyaluronic acid and agar) replicating the viscosity
and rheology of human vitreous. Aqueous flow input ports and an
output sampling port were used to provide fluid flow by introducing a
2.0 μl/min flow rate with phosphate buffered saline (pH 7.4) from the
anterior segment at room temperature (25°C). Dexamethasone
sodium phosphate (1.0 mg) was solubilised in distilled water (1.0 ml)
and this solution (50 μl) was injected into the model. Samples (100
μl) were taken over 5 days at 24 hourly intervals from different
anatomical regions. Analysis was performed using High Performance
Liquid Chromatography (HPLC) at 240 nm.
Results: The viscosity properties of the hyaluronic acid-agar
combination were found to be similar to that reported for human
eyes. This solution is thought to be a reliable simulation of in vivo
viscosity that can occur. A concentration of 1.03 mg/ml (±0.06
mg/ml) dexamethasone sodium phosphate was injected into the
model and sampled for 5 days. It was sampled at various points from
the model and release kinetics showed it was within the therapeutic
range (10-100 uM).
Conclusions: This simple dynamic posterior segment model may aid
the pharmacokinetic study of ocular drugs in early preclinical
development. Further studies to incorporate saccadic movements,
different forms of dexamethasone, temperature conditions and in
vivo-in vitro correlations are required including testing the release
kinetics of other anti-inflammatory and anti-fibrotic drugs.
Commercial Relationships: Sahar Awwad, None; Alastair
Lockwood, None; Abeer Mohamed Ahmed, Steve Brocchini
(WO09/063222) (P), Peng Khaw (WO09/063222) (P); Garima
Sharma, None; Ashkan Khalili, University College London (P);
Steve Brocchini, None; Peng T. Khaw, University College
Moorfields (P)
Support: NIHR Moorfields Biomedical Research Centre, UCL
School of Pharmacy, Grand Charity, Helen Hamlyn Trust, Fight for
Sight
Tunable sustained intravitreal drug delivery system for
daunorubicin using oxidized porous silicon
Huiyuan Hou1, Alejandra Nieto2, Feiyan Ma1, Su-Na Lee1, Kaihui
Nan1, William R. Freeman1, Michael J. Sailor2, 3, Lingyun Cheng1.
1
Department of Ophthalmology, Shiley Eye Center, UCSD, San
Diego, CA; 2Department of Chemistry and Biochemistry, UCSD, San
Diego, CA; 3Department of Bioengineering, UCSD, San Diego, CA.
Purpose: Daunorubicin (DNR) is a potent therapeutic agent for
unwanted proliferative ocular diseases. However, it has a narrow
therapeutic window and short vitreous half-life. We have previously
shown that covalent bonding DNR to functionalized porous silicon
(pSi) can extend DNR vitreous stay from days to months. Current
study is to investigate the feasibility and capacity of regulating DNR
release by quantitatively altering nano pore size of this unique
delivery system.
Methods: Porous silicon microparticles were prepared by
electrochemical etches followed by ultrasonication. Three different
etching parameters were used to acquire three nano-pore sizes (15nm,
26nm, and 43nm). pSi particles were oxidized at 800°C and in vitro
degradation was examined at 37°C in a closed system by quantitate
silicon using ICP-OES. For in vivo drug release study, the oxidized
pSi particles were further silanized for daunorubicin loading. DNR
was loaded into the pSi particles by covalent attaching. Three
milligrams of each type of DNR loaded particle was intravitreally
injected into 4 rabbits eyes. Only one eye of each animal was used.
After the injection, the eyes were examined at day 1, 3, 7 and 14 by
slit lamp biomicroscopy, indirect ophthalmoscopy, and tonometry.
All rabbits were sacrificed at day 14 and vitreous was dissected out.
The supernatant of the vitreous was subjected to HPLC/MS/MS for
daunorubicin quantitation.
Results: The 32-day in vitro degradation studies showed that
degradation rate of pSi was associated with pore sizes; with the larger
pore being faster degraded. The mean degradation rate of pSi with
46nm pores was significantly larger than that of the other two pore
sizes (44.2 vs 25.7 or 21.2 ug/mL, p<0.0001). In vivo drug release
study demonstrated that free daunorubicin in vitreous at postinjection day 14 was 12ng/mL for 43nm pore pSi, 3ng/mL for 26nm
pore pSi, and 1ng/mL for 15nm pore pSi. Pore expansion from 15nm
to 43nm led to a 12 folds increase of daunorubicin release
(p<0.0001). Clinical monitoring and fundus photographs did not
reveal any ocular toxicity or visually difference in amount of the
particles in rabbit vitreous.
Conclusions: The current study demonstrated the feasibility to
regulate daunorubicin release from porous silicon through pore size
manipulation. The capacity of this regulating seems promising and
further study is warrantied.
Commercial Relationships: Huiyuan Hou, None; Alejandra Nieto,
None; Feiyan Ma, None; Su-Na Lee, None; Kaihui Nan, None;
William R. Freeman, OD-OS, Inc. (C); Michael J. Sailor,
Spinnaker Biosciences (I); Lingyun Cheng, Spinnaker Biosciences
(C)
Support: NIH EY020617
3D Computational Fluid Dynamic Simulation of an Intravitreal
Brimonidine Implant in the Rabbit Eye
Julie E. Whitcomb1, Susan S. Lee2, Brandon D. Swift1, Josh Rowe3,
Mohammad R. Kazemi4, Jie Shen1. 1Pharmacokinetics & Drug
Disposition, Allergan, Irvine, CA; 2Ophthalmology Clinical
Research, Allergan, Irvine, CA; 3Bioanalytical Sciences, Allergan,
Irvine, CA; 4Independent Engineer Consultant, San Jose, CA.
Purpose: Drug delivery to the posterior segment of the eye is
challenging. Traditional methods of systemic and topical delivery are
ineffective due to numerous anatomical barriers (e.g. blood-retina
barrier, resistance of corneal epithelium, and rapid elimination from
aqueous humor). Intravitreal sustained-release implants provide
advantages by avoiding these barriers and reprieving the frequent
dosing burden. A mathematical model was developed to predict drug
distribution following an intravitreal dose of a sustained-release
implant.
Methods: An anatomically accurate 3D rabbit eye was developed to
simulate the advection-diffusion of brimonidine from an implant in
the vitreous. The diffusion coefficients of the ocular tissues were
defined using published ganciclovir data, which have similar
physicochemical properties to brimonidine. The similarity was
verified by experimental distribution profiles following an intravitreal
injection of a bolus mixture of the two compounds into rabbits. The
elution profile of brimonidine was used to calculate a flow rate and
mass flux across the implant domain. The simulation was compared
to vitreous and retina concentrations from a pharmacokinetic rabbit
ocular study with a sustained-release polymeric implant containing
brimonidine. A parameter sensitivity analysis was conducted to
examine the affect of different release rates and implant location on
drug distribution.
Results: The simulated concentration-time profile was in agreement
with the measured tissue concentrations using a scaling factor. The
concentration magnitude varied significantly between elution profiles
and the localized concentration distribution was dependent on the
implant location. This suggests that the release rate and implant
location are important factors when developing a sustained-release
implant.
Conclusions: Computational fluid dynamic modeling is a valuable
tool to predict ocular pharmacokinetics. Parameter sensitivity
analysis highlighted the importance of initial location and size of the
implant in determining drug distribution.
Observed and predicted brimonidine concentrations in the retina and
vitreous. A scaling factor of 200 was applied to the simulation data.
Symmetry plane contour plots of the simulated brimonidine
concentration (scalar) at t=6 days post dose. For implants with an
average release rate of 1.5 μg/day (left), 3 μg/day (middle) and 20
μg/day (right).
Commercial Relationships: Julie E. Whitcomb, Allergan (E);
Susan S. Lee, Allergan, Inc. (E); Brandon D. Swift, Allergan, Inc.
(E); Josh Rowe, Allergan (E); Mohammad R. Kazemi, None; Jie
Shen, Allergan (E)
Optimization of In-vitro DiffER model to study/compare the
effect of Formulations on Cross-Scleral transport of poorly
aqueous soluble drug
Thomas E. Rowe1, Kenneth W. Reed2, 1, Tuhin Bhowmik1.
1
Encompass Pharmaceutical Services, Norcross, GA;
2
Pharmaceutical Sciences, Belmont University, Nashville, TN.
Purpose: Ophthalmic drugs intended for the treatment of back of the
eye disorders often suffers the limitation of cross-scleral penetration
to achieve therapeutic efficacy. The purpose of this study was to
optimize the Diffusion-erosion in-vitro model (DiffER) to understand
the behavior of the drug diffusion/ transport from the formulation into
the sclera and out to the receptor media. This method will help us to
pre-screen various formulations at an early Formulation &
Development Phase to indicate the possibility of extending the
formulation to be tested on animal models.
Methods: Frozen mature rabbit scleras were thawed in diffusion
media and placed on specially adapted spherical diffusion Franz Cells
and dosed with 0.3 mg (about 2 drops) of active in three different
formulations (emulsion, aqueous suspension, and paraffin
suspension). For the dynamic experiments the pre corneal layer was
initially flushed with PBS at an increased flow rate immediately postdosage to simulate reflex tear flow then reduced to a basal flow rate
for the remaining duration of the experiment. To evaluate drug
retention and extent of absorption characteristics of each formulation,
eroded solution from the pre-corneal layer was collected at intervals
for analysis. Receiver chamber solution was also sampled at selected
intervals. Analysis of samples were performed via HPLC
Results: The results from the DiffER experiment were contrasting
and varied from formulation to formulation. The order of drug
transport in the formulation was quantified and permeability coefficients were calculated.
Conclusions: The DiffER may be efficiently utilized as an in-vitro
tool to discern the rate of drug transport through optic tissues
between different drug formulations.
Commercial Relationships: Thomas E. Rowe, Encompass (E);
Kenneth W. Reed, Encompass (C); Tuhin Bhowmik, None
Ocular bioavailability of brimonidine 0.1% BID in non-human
primates
Chinatsu Tosha, Sherri Decker, Guadalupe Ruiz, Octavio Avalos,
Werhner Orilla, Larry Gruber, Ton Lin, Alexandra S. Almazan,
Daniel W. Gil, James A. Burke. Biological Science, Allergan, Inc.,
Irvine, CA.
Purpose: To determine whether topically applied Alphagan 0.1%
BID in non-human primates would deliver sufficient brimonidine to
the central retina to activate α2-adrenergic receptors for a
neuroprotective effect.
Methods: Alphagan 0.1% (Allergan, Inc., Irvine, CA) was
administered to one or both eyes of six female cynomolgus monkeys
every 12 hours (BID; 9 eyes) for four weeks. Three contralateral eyes
served as controls and received placebo. Animals were euthanized 2
hours following the final dose. Then, ocular tissues from anterior
segments (aqueous humor (AH), iris and ciliary body (CB)) and from
posterior segments (conjunctiva, peripheral sclera, vitreous humor,
and tissues from a central 8 mm punch biopsy: neurosensory retina,
underlying RPE/choroid, and sclera) were collected and assayed for
brimonidine concentrations. The brimonidine plasma concentration
was also analyzed to examine the possible systemic transfer of the
drug.
Results: The brimonidine concentrations in anterior segments were
189 ± 132 nM (AH), 29147 ± 7526 nM (iris) and 322560 ± 73384
nM (CB). The concentrations in posterior segments were 11062 ±
6027 nM (conjunctiva), 9565.4 ± 3927 nM (peripheral sclera), 47 ±
11 nM (vitreous), 122 ± 26 nM (central retina), 3,352 ± 1,017 nM
(RPE/choroid) and 1,994 ± 465 nM (sclera). The plasma
concentration of brimonidine in the bilaterally treated monkeys was
0.49 ± 0.43 nM. The retinal brimonidine concentrations in control
untreated eyes were 16% of that of treated eyes. The brimonidine
concentrations in anterior segments of control eyes were 29 ± 44 nM
(AH), 1123 ± 523 nM (iris) and 15436 ± 10596 nM (CB). The
concentrations in posterior segments of control eyes were 1808 ±
1675 nM (conjunctiva), 807 ± 428 nM (peripheral sclera), 807 ± 428
nM (vitreous), 807 ± 428 nM (central retina), 985 ± 319 nM
(RPE/choroid) and 327 ± 295 nM (sclera).
Conclusions: This study indicates that Alphagan 0.1% BID delivers
pharmacologically sufficient brimonidine to the central retina of nonhuman primates. The distribution of plasma and ocular tissue
brimonidine concentrations suggests that delivery to the retina was
via the periocular pathway; not via systemic transfer. The strong
bonding of brimonidine to ocular melanin may allow a long retention
of brimonidine in the pigmented ocular tissues after the topical
administration. Melanin in choroid and RPE may play a major role in
facilitating the delivery of brimonidine to the central retina.
Commercial Relationships: Chinatsu Tosha, Allergan, Inc. (E);
Sherri Decker, Allergan (E); Guadalupe Ruiz, Allergan Inc. (E);
Octavio Avalos, Allergan (E); Werhner Orilla, Allergan, Inc. (F),
Allergan, Inc. (I), Allergan, Inc. (E), Allergan, Inc. (P); Larry
Gruber, None; Ton Lin, Allergan Inc (E); Alexandra S. Almazan,
Allergan (E); Daniel W. Gil, Allergan (E); James A. Burke,
Allergan, Inc (E)
Sustained Delivery of Prostaglandin from Drug-Containing
Depots Using Ocular Rings in Beagles
Kathryn S. Crawford1, Jeanne Y. Ellis2, Jack Rulander3, Stephen
Johnston3, Francis S. Lai3, Edward J. Ellis2, Charles D. Leahy2.
1
PharmOcu, Andover, MA; 2Vista Scientific, LLC, Andover, MA;
3
Massachusetts Medical Device Development Center, University of
Massachusetts, Lowell, MA.
Purpose: To evaluate the efficacy of sustained-release latanoprost
from depots in a topical ophthalmic drug delivery device
(TODDD™).
Methods: The right eyes of 8 ocular normotensive adult beagle dogs
were fitted with ocular ring devices, each containing 2 latanoprostdrug depots (cylindrical cores, 600 μg latanoprost). The depots were
matched in volume and surface release area to those of a humanconfigured device. A device with blank depots containing no
latanoprost was placed on the right eye of 1 additional animal. All
left eyes remained untreated. Clinical slit-lamp exams were
performed pre-placement on Day 1 and post-placement on Days 1, 8
and 17. Daily observations were performed to assess the presence of
the device (retention) and any ocular abnormalities. Intraocular
pressure (IOP) and pupil diameter were measured pre- and postplacement on Day 1, and on Days 4, 8, and 16. Plasma samples were
collected on Day 1 prior to, and approximately 4 hours after device
insertion, and on Day 8. Tear samples were collected on Day 16 by
Schirmer strip in the lower cul-de-sac. Tear and plasma samples were
analyzed by LC/MS/MS for latanoprost and latanoprost acid. The
lower limit of quantitation was 0.5 ng/mL.
Results: IOP reduction in the treated eye compared to the control eye
was approximately 3 mmHg on Day 4 (n=6) and Day 8 (n=4), and 7
mmHg on Day 16 (n=3) in the dogs that had retained the latanoprostloaded devices, representing a 37% reduction in normotensive IOP
from baseline. There was no effect on IOP in the animal wearing the
placebo device. The IOP returned to baseline levels in all eyes after
removal of the devices. A range of 25 - 215 ng/mL latanoprost was
recovered from the tears of animals wearing the drug devices. No
latanoprost was detected in plasma or in the tear samples from
untreated eyes.
Conclusions: Long-term retention of this ring device configuration
and dimensions in this species was not achieved, but this study
demonstrates the therapeutic feasibility of the sustained-release (nonring) TODDD™ configured for human ocular dimensions. In vitro
analysis of the worn devices and of the extended latanoprost release
profile from new devices relative to tear levels measured at Day 16
indicates that sustained IOP-lowering could be achieved over a 2-3
month time period.
Commercial Relationships: Kathryn S. Crawford, Vista
Scientific, Inc. (C); Jeanne Y. Ellis, Vista Scientific (I), Vista
Scientific (P); Jack Rulander, Vista Scientific LLC (F); Stephen
Johnston, Vista Scientific (F); Francis S. Lai, Vista Scientific (F);
Edward J. Ellis, Vista Scientific LLC (I), Vista Scientific LLC (P);
Charles D. Leahy, Vista Scientific LLC (I), Vista Scientific LLC (P)
Support: NIH Grant R44EY013479
Conclusions: Release of model drugs metformin and ciprofloxacin
shows that this material is suitable for prolonged release of both
hydrophobic and hydrophilic molecules. The data provides a basis for
predicting polymer & drug formulations that will produce a desired
release profile for an intra-vitreal device. The analysis method can be
extrapolated to examine and predict release from potentially any
biomaterial formulation. Cytotoxicity results support the use of the
materials intravitreally.
Evaluating & Predicting Drug Release from an Implantable
Biomaterial
Ivana Postic, Heather Sheardown. Chemical Engineering, McMaster
University, Hamilton, ON, Canada.
Purpose: Drug delivery from siloxane-based materials is attractive
due to its inherent properties and biocompatibility. The work
describes a siloxane-based matrix with polyethylene glycol (PEG)
additive, developed to act as an intra-vitreal implant for release of
therapeutics to the posterior segment of the eye. The effects of
various constituents on the release rate were examined in order to
develop predictable, long-term, controlled drug release.
Methods: PEG (1-10 wt%, 500-20,000MW) was blended with liquid
PDMS (Sylgard® 184 Kit) and 5-10wt% of drug (metformin
hydrochloride or ciprofloxacin). Platinum curing agent was added to
the entire mixture and cured at 70°C. Quarter-inch discs were
punched out and drug release (37°C, 100rpm) into 1ml of posphate
buffered saline was measured using UV spectroscopy. ProMV
software was used to apply multi-variate methods (ProSensusAncaster,Canada). Cytotoxicity testing was performed by incubating
PEG/PDMS discs for 48 hours with aRPE19, a retinal pigment
epithelium cell line. Cellular metabolism was measured via
fluorescence from PrestoBlue reagent.
Results: Standard analysis showed significant differences in release
between the two drug types (Figure 1). Release of hydrophobic
ciprofloxacin showed promise for very long-term release (less than
5% released at 17 days). Similar to the release of metformin, the
hydrophilicity of the material could be adjusted up to 10wt% and
500MW without significantly affecting the release rate from that of
the control. Increasing the concentration of ciprofloxacin from 5wt%
to 10wt% allowed for even further prolonged release. Analysis using
latent variable methods indicates good fit and predictability of release
within the experiments conducted. The drug delivery devices show
generally low cytotoxicity when cultured in the presence of aRPE19
cells.
A hollow metal microneedle reduce reflux in intravitreal
injections
SungHo Lee1, Dongkyu Lee2, Yong Song You2, Soon Hyun Kim2,
Chang Yeol Lee3, Hyungil Jung3, Sung Jin Lee4, Oh Woong Kwon2.
1
R&D Center, Lumieye Genetics Co., Ltd., Seoul, Republic of Korea;
2
Retina center, NUNE Eye Hospital, Seoul, Republic of Korea;
3
Biotechnology, Yonsei University, Seoul, Republic of Korea;
4
Ophthalmology, Soonchunhyang University Hospital, Seoul,
Republic of Korea.
Purpose: The study aimed to evaluate the reduction of reflux in
intravitreal injections using a minimally invasive hollow metal
microneedle.
Methods: Rabbit twenty eyes undergoing intravitreal injection were
allocated into two groups to compare the vitreal reflux after injection
of 0.02 ml of 0.1% sodium fluorescin (Alcon) using a 30G needle and
a hollow metal microneedle (five millimeter long, hundred outer
diameter, fifty inner diameter and fifteen degree beveled angle). The
amount of drug reflux was estimated by measuring amount of
fluorescin in an absorbent cotton swab and the width of
subconjunctival fluorescence staining area.
Results: The mean measured reflux of volume was statistically less
with hollow metal microneedle (0.27 SD ± 0.25 ng for swab absorbed
fluorescin; 3.4mm SD ± 0.5 for width of subconjunctival
fluorescence staining area) than undergoing the 30G needle injection
(1.51 SD ± 0.63 ng for swab absorbed fluorescin; 8.2 mm SD ± 0.7
for width of subconjunctival fluorescence staining area) .
Conclusions: Analyses of vitreal reflux showed that hollow metal
microneedle prevent vitreal reflux drugs after intravitreal injection
than 30G needle. Moreover, microneedle provide accuracies in drug
administration, lesser scleral damage and the risk of complication
after repeat injections.
Commercial Relationships: SungHo Lee, None; Dongkyu Lee,
None; Yong Song You, None; Soon Hyun Kim, None; Chang Yeol
Figure 1. Significant difference in release between hydrophobic
ciprofloxacin and hydrophilic metformin exists.
Commercial Relationships: Ivana Postic, None; Heather
Sheardown, Alcon (F), Alimera Sciences (F)
Lee, None; Hyungil Jung, None; Sung Jin Lee, None; Oh Woong
Kwon, None
504 Anterior Eye: Physiology and Pharmacology
Thursday, May 09, 2013 8:30 AM-10:15 AM
Contributing Section(s): Anatomy/Pathology
Induction of MAPK Phosphatase-1 by the Novel Selective
Glucocorticoid Receptor Agonist Mapracorat Suppresses
Inflammatory Signaling Pathways in Human Corneal Epithelial
Cells
Megan E. Cavet, Karen L. Harrington, Thomas R. Vollmer, Mary
Richardson, Jin-Zhong Zhang. Pharmaceutical R&D, Bausch &
Lomb, Rochester, NY.
Purpose: Prior studies demonstrated that mapracorat, a novel
selective glucocorticoid receptor agonist (SEGRA), potently inhibits
anti-inflammatory cytokine release in human corneal epithelial cells
(HCEpiC). The aim of this study was to determine the contribution of
an endogenous mitogen activated protein kinase (MAPK) regulator,
MAPK phosphatase-1 (MKP-1), to mapracorat’s anti-inflammatory
effects in HCEpiC.
Methods: HCEpiC were challenged with IL-β or hyperosmolarity
(480 mOsm) and the levels of MKP-1 protein and MAPK
phosphorylation in the presence of mapracorat were determined by
Western blotting. A range of mapracorat doses were tested and
dexamethasone (DEX) was used as the control. The effects of the
MKP-1 inhibitor triptolide on IL-β- and 480 mOsm-induced
inflammatory signaling in the presence of mapracorat was also
determined.
Results: Mapracorat alone did not stimulate the expression of MKP1 in HCEpiC. IL-1β or 480 mOsm media significantly increased
MKP-1 expression over basal levels. Following IL-β or hyperosmolar
challenge, mapracorat further significantly increased MKP-1 protein
levels at least 2 fold, with a peak expression at 1 h with IL-1β and 2-4
h with 480 mOsm media. The effects were not dose-dependent within
the tested mapracorat concentration range (30 - 300 nM). The
magnitude of increase in MKP-1 was comparable to that observed
with DEX. Concomitantly to the increase in MKP-1, mapracorat also
significantly reduced the levels of phosphorylated p38 and JNK
MAPKs induced by IL-1β or hyperosmolarity. Triptolide-induced
MKP-1 inhibition reversed the inhibitory effects of mapracorat on IL1β or 480 mOsm-induced MAPK signaling.
Conclusions: These data suggest that mapracorat exerts its antiinflammatory effects, at least in part, by increasing the expression of
the MAPK deactivator MKP-1 in human corneal epithelial cells.
Commercial Relationships: Megan E. Cavet, Bausch + Lomb (E);
Karen L. Harrington, Bausch + Lomb (E); Thomas R. Vollmer,
Bausch + Lomb (E); Mary Richardson, Bausch and Lomb (E); JinZhong Zhang, Bausch & Lomb, Inc (E)
Pirfenidone nanoparticles improve healing and prevent corneal
scarring following alkali burn
Sarbani Hazra1, Sushovan Chowdhury2, Rajdeep Guha2, Trivedi
Ruchit3, Uday B. Kompella3, Aditya Konar2. 1Veterinary Surgery &
Radiology, WBUAFS, Kolkata, India; 2Animal Facilities, Indian
Institute of Chemical Biology, Kolkata, India; 3Department of
Pharmaceutical Sciences, University of Colorado Anschutz Medical
Campus, Aurora, CO.
Purpose: This study was designed to evaluate the effects of
pirfenidone nanoparticles on corneal reepithelization and scarring,
major clinical challenges after alkali burn.
Methods: Effect of pirfenidone on collagen I and α-SMA synthesis
from TGFβ induced primary corneal fibroblast cells was assessed by
immunocytochemistry and immunoblotting. Pirfenidone loaded poly
(lactide-co-glycolide) (PLGA) nanoparticles were prepared,
characterized and their delivery in primary corneal fibroblast cells
was examined. Alkali burn was induced in one eye of Sprague
Dawley rats (n=18) followed by once daily topical treatment with
PBS (n=6), free pirfenidone (n=6), or pirfenidone nanoparticles (n=6)
containing equivalent amount of pirfenidone. Corneal reepithelization was assessed daily by flourescein dye test, absence of
stained area indicated complete re-epithelization and the time for
complete re-epithelization was determined. Corneal haze was
assessed daily for 7 days under slit lamp microscope and graded
using standard method (Fantes et al. 1990). After 7 days, collagen I
deposition in the superficial layer of cornea was examined by
immunohistochemistry.
Results: Pirfenidone prevented (P<0.05) increased collagen I and αSMA synthesis by TGFβ induced corneal fibroblasts in a dose
dependent manner. Pirfenidone could be loaded successfully within
PLGA nanoparticles, which entered the corneal fibroblast within 5
minutes. There was a significant (P<0.05) improvement in corneal reepithelization and reduction in corneal haze in corneas treated with
pirfenidone nanoparticle compared to PBS. Eyes treated once daily
with free pirfenidone did not show improvement
Conclusions: Pirfenidone decreases collagen synthesis and prevents
myofibroblast formation. Pirfenidone nanoparticles improve corneal
healing and prevent fibrosis. Pirfenidone nanoparticles are of
potential therapeutic value in treating corneal chemical burns and
other corneal fibrotic diseases.
Fantes, FE, Hanna, KD, Waring, GO 3rd, Pouliquen, Y, Thompson,
KP and Savoldelli, M (1990). Wound healing after excimer laser
keratomileusis (photorefractive keratectomy) in monkeys. Arch
Ophthalmol 108: 665-675
Commercial Relationships: Sarbani Hazra, None; Sushovan
Chowdhury, None; Rajdeep Guha, None; Trivedi Ruchit, None;
Uday B. Kompella, University of Colorado Denver (P), PCAsso
Diagnostics (C), NanoTrans Technologies, Inc. (F), Univesity of
Nebraska Medical Center (P); Aditya Konar, None
Support: Dept of Biotechnology,Govt of India
The effect of sulindac liposome on inflammation induced corneal
neovascularization
Jun-Sub Choi, Choun-Ki Joo. Ophthalmology & Visual Science,
Catholic Univ Korea Coll of Med, Seoul, Republic of Korea.
Purpose: To investigate the effect of topical sulindac liposome on
inflammatory induced corneal neovascularization.
Methods: Sprague-Dawley male (250g) rats were used for alkali
burn model. After alkali injuries using 1 N NaOH, the control group
(n=10) received topical PBS four times a day for 2 days. The other
study groups received topical sulindac-liposome. The corneal
neovascularization were evaluated using slit lamp biomicroscopy.
And tissue sections were analyzed for inflammatory cell infiltration.
Inflammatory cells were confirmed by immunohistochmistry for
F4/80.
Results: The sulindac liposome inhibited corneal neovasculariztion
and inflammatory response after corneal injury. In light microscopic
findings, the corneal thickness was increased and many
polymorphonuclear leukocytes infiltrated in the corneal stroma in the
control group. In immunohistochemical study, in sulindac-liposome
treated group, the F4/80 positive cells were detected lower than
control group (p<0.05, respectively).
Conclusions: The sulindac is anti-inflammatory drugs. The topical
sulindac liposome has beneficial effect on inflammatory induced
corneal neovascularization.
Supported: This study was supported by a grant of the Korean Health
Technology R&D Project, Ministry for Health & Welfare, Republic
of Korea. (A092258)
Commercial Relationships: Jun-Sub Choi, None; Choun-Ki Joo,
None
Support: Korean Health Technology R&D Project (A092258)
Application of phosphoproteomic analysis in myopic chick retina
Fengjuan Yu1, Kingkit Li1, Thomas Chuen Lam1, Chi-ho To1, 2.
1
Laboratory of Experimental Optometry, School of Optometry, the
HongKong Polytechnic University, Kowloon, Hong Kong; 2State
Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Centre,
SunYat-sen University, Guangzhou, China.
Purpose: Protein phosphorylation plays an important role in
regulating a wide range of cellular activities. However, its role in the
mechanism of eye growth has not been characterized. The purpose of
this study is to profile the phosphoproteins in lens induced ametropia
in chicks.
Methods: White leghorn chicks (4-day old) worn either -10 or +10D
lenses for 3 days to induce myopia and hyperopia respectively.
Refraction and ocular biometry were measured before and after lens
wear. After the lens treatment, the retina tissues were removed. It was
further homogenized, extracted and double digested with trypsin and
GluC. The retinal peptides were enriched with titanium dioxide and
then injected to a 1D nano-flow liquid chromatography tandem mass
spectrometry. Protein identifications are performed with ProteinPilot
software in IPI chick database.
Results: Expectedly, the chick eyes developed significant and
expected ametropia according to refraction and A-scan results after
three days of lens treatment. A total of 718 peptides in lens-induced
myopic/hyperopic retina were detected with phosphorylation. The
specific site of phosphorylation of each peptide was identified. These
phosphopeptides can be further divided into three groups: common
peptides in myopia and hyperopia (218 pairs); peptides that appear
only in myopia (129 peptides); peptides that appear only in hyperopia
(163 peptides).
Conclusions: The study demonstrated the feasibility of
phosphoproteomic analysis in ametropic chick retina in a highthroughput manner. Together with quantitative proteomic
approaches, it may provide the next level of understanding of the
protein changes that drive the differential eye growth process.
Commercial Relationships: Fengjuan Yu, None; Kingkit Li,
None; Thomas Chuen Lam, None; Chi-ho To, None
Support: by the Centre for Myopia Research (A360), the Myopia
Niche Area Fund (JBB7P), RGC General Research Fund (B-Q15Q
and B-Q29N) and PolyU PhD Studentship (RPEX)
Gender, Ethnicity, and Time After Awakening Affect Tear Film
Cytokine Concentrations
Meng C. Lin1, 2, Joycelyn O. Niimi1, Nancy A. McNamara2, 3, Yixiu
Zhou1, Jeremy Shumaker1, Heather M. French1, Tiana Leung1, Shiyin
L. Wang1, Karyn Siemasko4, Michael E. Stern4. 1School of
Optometry, Clinical Research Center, University of California,
Berkeley, Berkeley, CA; 2School of Optometry, University of
California, Berkeley, Berkeley, CA; 3Francis I. Proctor Foundation,
University of California, San Francisco, San Francisco, CA;
4
Allergan, Inc, Irvine, CA.
Purpose: To investigate how cytokine concentrations in the tear film
of normal eyes differ by gender, ethnicity, and time after awakening.
The potential impact of these factors on baseline cytokine levels in
healthy eyes will provide an important reference for deciphering
comparisons with ocular surface diseases.
Methods: 42 subjects (ages 18-30 years; 22 Asians, 20 non-Asians;
22 males, 20 females) with no prior history of contact lens wear and
no dry eye symptoms (OSDI score < 13) underwent an ocular surface
screening evaluation. Baseline tear samples were collected on all
subjects before sleep using capillary tubes held at the tear meniscus.
Of the 42 subjects studied, 14 subjects slept in the research lab and
their tear samples were collected immediately after awakening, and
again 3 hrs after. Tear samples from each eye were analyzed
separately with multiplex bead-based assays for the concentrations
(pg/ml) of 11 cytokines, including IFN-γ, IL-12p(70), TNF-α, IL-1α,
IL-1β, IL-6, IL-8, IL-17, IL-10, IL-13, IL-15. Correlations between
measurements on fellow eyes were accounted for using linear mixed
effects modeling.
Results: With the exception of IL-8 and IL-17, the average baseline
concentrations of all cytokines were higher in the tears of female
subjects compared with males and significant gender differences (pvalues <0.05) were found for IFN-γ, IL-1α, IL-1β, IL-6, IL-12p(70),
IL-13 and TNF-α. With the exception of IL-8 and IL-13, Asians had
lower concentrations compared with non-Asians and significant
ethnic differences (p-values <0.05) were found for IFN-γ, IL-1α, IL6, IL-10, IL-12p(70), and IL-17 in their tears compared with nonAsians. IL-8 was the only cytokine to exhibit an increase (p<0.05) in
concentration upon awakening, while all others decreased. The
concentrations of all cytokines recovered to baseline after 3 hrs of
awakening.
Conclusions: Baseline cytokine concentrations vary significantly by
gender and ethnicity. The impact of these differences must be
considered in studies that use tear cytokine levels as a quantitative
measure of inflammation during disease. It is also important to note
that eye closure significantly altered tear cytokine concentrations,
which subsequently recovered to baseline levels within 3 hrs of
awakening. The increased concentration of IL-8 in tears upon
awakening may be related to a substantial infiltration of PMN cells
into the tears during sleep.
Commercial Relationships: Meng C. Lin, TearLab Corporation (F),
Allergan, Inc. (F); Joycelyn O. Niimi, None; Nancy A. McNamara,
None; Yixiu Zhou, None; Jeremy Shumaker, None; Heather M.
French, None; Tiana Leung, None; Shiyin L. Wang, None; Karyn
Siemasko, Allergan, Inc. (E); Michael E. Stern, Allergan, Inc. (E)
Cross-Species and Cross-Age Comparison of Esterase Mediated
Metabolism in Vitreous Humor: Human versus Rabbit, Dog and
Monkey
Mayssa Attar, Jie Shen, Moon S. Kim, Que-Chau Radojicic. Drug
Safety Evaluation, Allergan, Irvine, CA.
Purpose: Little is known regarding the enzymatic capabilities in
vitreous humor across species and age groups. The objective of this
study was to compare esterase mediated metabolism and identify any
age effect in human vitreous humor relative to rabbit, dog and
monkey vitreous humor.
Methods: Latanoprost (13,14-dihydro-17-phenyl-18,19,20-trinorprostaglandin F2α-1-isopropyl ester) was used as a prototype
substrate to monitor esterase metabolism. Human vitreous humor was
obtained from individual donors ranging in age from 67 to 86 years.
Young and old vitreous humor was obtained from rabbit (<1 and >2
years old), dog (2-5 and >5 years old) and monkey (3-4 and > 5 years
old). Metabolite formation was measured following incubation at
37°C of 500 μL vitreous humor and 6 μM latanoprost with timepoints
taken at 0, 10, 20, 30 and 60 minutes. Liquid chromatography tandem
mass spectrometry (LC-MS/MS) was used to quantify latanoprost
disappearance and latanoprost acid formation.
Results: Latanoprost was metabolized to latanoprost acid in human
and animal vitreous humor indicating esterases are active in vitreous
humor. Rates of esterase metabolism were highest in human followed
by rabbit, followed by monkey, followed by dog. Esterase
metabolism appeared to decrease with increasing age except in rabbit.
Latanoprost was stable when incubated in buffer for 60 minutes at
37°C.
Conclusions: Esterase mediated latanoprost metabolism is active in
human and animal vitreous humor. Rate of esterase metabolism was
highest in human as compared to all animals tested. Among the
animal species tested rates of esterase metabolism in rabbit are most
similar to human. Esterase metabolism tended to decrease with age.
Commercial Relationships: Mayssa Attar, Allergan (E); Jie Shen,
Allergan (E); Moon S. Kim, Allergan (E); Que-Chau Radojicic,
Allergan (E)
Bicarbonate sensitive soluble adenylyl cyclase (sAC) generates
the cAMP in aqueous humor (AH)
Yong Lee1, Yong Lee1, Lavoisier Ramos-Espiritu3, Lihua Y.
Marmorstein1, Jochen Buck3, Lonny Levin3, Alan D. Marmorstein1, 2.
1
Ophthalmology and Vision Science, University of Arizona, Tucson,
AZ; 2Physiology, University of Arizona, Tucson, AZ;
3
Pharmacology, Weill Cornell College of Medicine, New York, NY.
Purpose: Recently we demonstrated that sAC a bicarbonate sensitive
enzyme that catalyzes the conversion of ATP to cAMP plays a role in
regulating outflow facility in mice. sAC is highly expressed in ciliary
body but cold not be detected in drainage tissues implying a
communicative pathway linking the cilliary body to regulation of
outflow facility. In this study we aimed to determine whether cAMP
is present in the AH of mice and whether it is generated by sAC.
Methods: AH was collected from the eyes of 3-6-month old sAC
knock-out (KO) mice or WT litermates by cannulation of the anterior
chamber with borosilicate glass microneedles. AH collection was
performed between 2:00 PM and 4:00 PM to avoid the influence of
circadian dynamics on our measurements. AH from both eyes of four
mice were pooled and cAMP concentration determined using a
commercial ELISA kit (Bio-Rad).
Results: Levels of cAMP in AH of sAC KO mice were 0.37 ± 0.59
pmol/ml (mean±sd, n=3) significantly different (p < 0.05) from the
2.67 ± 1.33 pmol/ml (mean±sd, n=3) measured in litermate controls.
Conclusions: cAMP levels in sAC KO mice were reduced ~7.25
fold. These findings suggest that cAMP in AH is generated primarily
by sAC. Further studies are necessary to understand the effects of
varying cAMP in AH on outflow facility.
Commercial Relationships: Yong Lee, None; Yong Lee, None;
Lavoisier Ramos-Espiritu, None; Lihua Y. Marmorstein, None;
Jochen Buck, None; Lonny Levin, CEP Biotech (I), CEP Biotech
(P); Alan D. Marmorstein, None
Support: NIH EY021153 and an unrestricted grant from Research
To Prevent Blindness
Beneficial Assessment of A Combined Tropicamide and Various
Oxime Treatments Against Miosis and Visual Dysfunction
Following Ocular Exposure to the Nerve Agent Sarin
Xxx Xxxx. Xxxxxx, Xxxxxx, MD.
Purpose: Eye exposure to the organophosphorus irreversible
acetylcholinesterase inhibitor sarin results in long-term miosis (a
reduction of at least 50% of pupil width) and reduction in visual
function. Anti-cholinergic drugs, such as atropine, are used topically
in order to counter these effects and obtain symptomatic relief.
Unfortunately, such compounds attenuate ocular discomfort at the
expense of producing mydriasis and partial cycloplegia symptoms,
which may worsen visual performance. This study was aimed to test
beneficial drugs in contradicting the sarin-induced miosis and visual
impairment, which will minimally affect vision.
Methods: Male Pigmented Long-Evans rats were topically exposed
to sarin (0.2-1 μg), and 20 min later were topically treated. Pupils
were illuminated with an infrared spotlight and images were digitally
recorded with a computerized infrared-capable video camera, thus
measuring pupil width. Pupil width was determined 15 min -8 h
following exposure and treatment. Visual function assessment was
performed using the Cued Morris Water Maze task, 15-35 min
following sarin exposure and treatment. In this version, cued
navigation involves finding a goal location by approaching a single
cue that marks the visible goal.
Results: Rats exposed topically to various sarin doses showed a
dose-dependent miosis, which partially recovered within 4-8 h.
Oxime treatments with or without the anti-cholinergic drug
tropicamide differentially improved the sarin induced miosis and the
resulting impairment in visual performance.
Conclusions: The miotic as well as the visual defects observed,
following topical sarin exposure are contradicted to various extent by
the treatments used.
Commercial Relationships: Xxx Xxxx, None
Dexamethasone Modifies Viability of Mitomycin C-Treated
Tenon’s Fibroblasts
Shu-Wen Chang1, 2, Wei-Ting Ho1, Tsan-Chi Chen1, San-Fong Chou3.
1
Department of Ophthalmology, Far Eastern Memorial Hospital, New
Taipei City, Taiwan; 2Department of Ophthalmology, National
Taiwan University Hospital, Taipei, Taiwan; 3Department of Medical
Research, Far Eastern Memorial Hospital, New Taipei City, Taiwan.
Purpose: To investigate whether dexamethasone modifies the effect
of mitomycin C (MMC) in human Tenon’s capsule fibroblasts
(HTFs) and to explore its molecular mechanism.
Methods: HTFs were treated with MMC for 5 minutes and incubated
in DMEM with 10 μM dexamethasone (DEX). Viability of the
treated HTFs was analyzed by WST-1 assay. The amount of IL-8
section in untreated, MMC-treated, or peroxisome proliferatoractivated receptor gamma (PPARγ)-silenced HTFs was determined
by enzyme-linked immunosorbent assay. Expression of PPARγ and
the apoptotic proteins was examined by immunoblotting.
Results: Recombinant IL-8 noticeably suppressed HTF cell
proliferation in a dose-dependent manner. MMC treatment
significantly upregulated IL-8 secretion and inhibited cell
proliferation in HTFs. Both effects were reversed by DEX
incubation. DEX upregulated PPARγ and Bcl-xL in untreated HTFs
and MMC-treated HTFs at 1 day and 2 days of incubation,
respectively. PPARγ silencing reduced Bcl-xL expression and
enhanced IL-8 secretion. However, MMC treatment and/or DEX
incubation did not alter the expression of two apoptotic indicators,
PARP-1 and pro-caspase-3.
Conclusions: DEX reversed the MMC-inhibited HTF cell
proliferation and diminished the MMC-upregulated IL-8 secretion via
upregulating the PPARγ expression (Figure 1).
Figure 1. Scheme depicting reverse regulation of dexamethasone
in MMC-induced cell death of HTFs.
Commercial Relationships: Shu-Wen Chang, None; Wei-Ting
Ho, None; Tsan-Chi Chen, None; San-Fong Chou, None
TRPV1 and corneal Pain
Eric Senning, Sharona E. Gordon. Physiology and Biophysics,
University of Washington, Seattle, WA.
Purpose: Current analgesic treatment for corneal damage or
following ophthalmic surgery falls short of desired goals. One
promising approach is the temporary deactivation of TRPV1
(transient receptor potential vanilloid 1) expressing nociceptors by
administration of the TRPV1 agonist resiniferatoxin. Here we
consider the molecular pathways of TRPV1 regulation.
Inflammatory signals increase the excitability of TRPV1-expressing
nociceptors, at least in part, by increasing the number TRPV1
channels in the plasma membrane (PM) of nociceptors. However, the
dynamics of TRPV1 in the PM have not been studied. We
hypothesize that TRPV1 properties are heterogeneous within a cell
and that channels are actively regulated as a function of local activity.
Methods: We used total internal reflection fluorescence (TIRF)
microscopy to explore the dynamics of single TRPV1 molecules in
living cells. Combining TIRF microscopy with whole-cell patch
clamp of isolated mouse sensory neurons and HEK293T/17 cells
allowed us to image both the localization of single TRPV1 molecules
within cells (TRPV1-eGFP) and their activity (capsaicin-activated
fluorescent “sparklets” at site of Ca2+ influx).
Results: A number of surprising findings came out of our singlemolecule experiments: (1) TRPV1 channels in isolated sensory
neurons and in cultured cells are mobile, with a range of Deff from
0.01 to 0.2 µm2/s; (2) a given Ca2+ sparklet arises from exactly one
channel, with two-state fluorescence reflecting capsaicin-activated
gating; (3) photobleaching and fluorescence intensity analysis
indicate that TRPV1 channels do not cluster in the PM; and (4) the
lateral mobility of TRPV1 in the plasma membrane decreased as a
function of its open duration.
Conclusions: Our data show that TRPV1 activates without forming a
higher order oligomer and that a subset of channels exhibit lateral
mobility as they gate. Although the mechanism by which TRPV1
activity and TRPV1 mobility are coupled and the role of mobility
changes in cell signaling remain to be determined, our data
demonstrate the power of single-molecule measurements to reveal
aspects of signaling not observable in macroscopic experiments.
Given previous work indicating that TRPV1 is part of a signaling
complex including the TrkA receptor for nerve growth factor and
PI3K, our data suggest that dynamic localization of these complexes
in response to TRPV1 agonists may constitute a new form of
regulation of local signaling.
Commercial Relationships: Eric Senning, None; Sharona E.
Gordon, None
Support: NH grant EY01730, EY017564
Different Roles of Carbonic Anhydrase in Human vs. Bovine
Corneal Endothelial Transport
Thomas M. Malikowski1, Michael E. Duffey2, Sangita P. Patel3, 4.
1
School of Medicine & Biomedical Sciences, University at Buffalo,
Buffalo, NY; 2Physiology & Biophysics, University at Buffalo,
Buffalo, NY; 3Ophthalmology, SUNY Eye Institute, University at
Buffalo, Buffalo, NY; 4Research Service, VAWNYHS, Buffalo, NY.
Purpose: Regulation of fluid homeostasis in the cornea is critical for
maintaining corneal clarity. Buffering by carbonic anhydrases plays a
central role in rabbit and bovine models of corneal endothelial fluid
transport. Topical carbonic anhydrase inhibitors (CAIs) in rabbits
result in decreased fluid efflux measured by increases in corneal
thickness. However, routine use of topical and systemic CAIs for
glaucoma in humans does not increase corneal thickness. The
hypothesis tested here is that there are species differences in the role
of carbonic anhydrases in corneal endothelial transport.
Methods: Protocols were approved by the R&D Committee (VAMC,
Buffalo, NY). Bovine eyes were obtained from local abattoirs. Deidentified human corneas not suitable for transplant were obtained
from the local eye bank. Corneal endothelial transport was measured
using the short-circuit current (Isc) technique. The recording solution
was (in mM): 111.6 NaCl, 29 NaHCO3, 4.8 KCl, 1.0 CaCl2, 0.8
MgCl2, 0.9 NaH2PO4, 10 HEPES, 5 glucose, bubbled with 5% CO2 /
95% air, pH 7.5. CAIs were added (500 µM acetazolamide, 100 µM
ethoxzolamide, 100 µM dorzolamide, or 100 µM brinzolamide) and
Isc measured. Water and DMSO were used as controls. Drug effect
was the percentage change of total Isc.
Results: All CAIs generated significant (p<0.0001) rapid declines in
Isc in bovine corneal endothelium compared to water or DMSO (mean
% change ± S.D.: ethoxzolamide −35.5 ± 12.9, n = 8; dorzolamide
−33.6 ± 7.2, n = 8; brinzolamide −35.5 ± 13.5, n = 9; acetazolamide
−21 ± 9.5, n = 8; water −1.6 ± 4.3, n = 7; DMSO +1.3 ± 4.8, n = 8).
In contrast, there was no rapid decline in human (mean % change ±
S.D.: ethoxzolamide −4.8 ± 10.3, n = 2; dorzolamide +8.0 ± 20.4, n =
3; brinzolamide +6.7 ± 13.9, n = 3; acetazolamide +2.7 ± 38.9, n = 6).
The Isc differences between bovine and human were significant (p <
0.05) for all CAIs except acetazolamide (p = 0.12).
Conclusions: Carbonic anhydrases support one-third of active ion
transport in bovine corneal endothelium but do not in human. This
finding suggests distinct mechanisms of ion transport are operational
in corneal endothelium in different species and supports the clinical
observation that therapeutic use of CAIs does not disrupt corneal
fluid homeostasis in humans. Such species differences will be
instrumental in guiding future research and development in corneal
endothelial disease.
Commercial Relationships: Thomas M. Malikowski, None;
Michael E. Duffey, None; Sangita P. Patel, Alcon Research
Institute (F)
Support: Alcon Research Institute Young Investigator Grant (SPP).
Funded in part by Ralph Hochstetter Medical Research Fund in honor
of Dr. Henry C. and Bertha H. Buswell (SPP). University at Buffalo,
School of Medicine, Summer Research Fellowship (TMM).
Unrestricted Grant, Research to Prevent Blindness, NY, NY.
ROCK Inhibitor Y-27632 Enhances the Mucin Secretion from
Goblet Cells Derived from Limbal Stem Cells
Won Seok Choi1, Eui Sang Chung2, Tae-Young Chung2, Joon Young
Hyon3, 4, Won Ryang Wee3, Young Joo Shin1. 1Ophthalmology,
Hallym University College of Medicine, SEOUL, Republic of Korea;
2
Ophthalmology, Sungkyunkwan University School of Medicine,
SEOUL, Republic of Korea; 3Ophthalmology, Seoul National
University College of Medicine, SEOUL, Republic of Korea;
4
Ophthalmology, Seoul National University Bundang Hospital,
SEOUL, Republic of Korea.
Purpose: To investigate the effect of Rho-associated coiled kinase
inhibitor (ROCK inhibitor) Y-27632 on Goblet cells derived from
corneal limbal stem cells.
Methods: Goblet cells were derived from human limbal stem cells
and cultured. The cells were treated with ROCK inhibitor. Periodic
acid-Schiff (PAS) staining and immunofluorescence staining of
ABCG2, CK-7 and MUC5AC was performed. Cell proliferation rate
was measured with BrdU labeling assay.
Results: The cultured cells were stained with ABCG2 and CK-7.
Goblet cell derived from limbal stem cells showed mucin secretion
by PAS staining and MUC5AC staining. Cell proliferation rate
increased with ROCK inhibitor.
Conclusions: Goblet cells were differentiated from limbal stem cells.
ROCK inhibitor enhanced mucin secretion and proliferation rate in
Goblet cells derived from limbal stem cells.
Commercial Relationships: Won Seok Choi, None; Eui Sang
Chung, None; Tae-Young Chung, None; Joon Young Hyon, None;
Won Ryang Wee, None; Young Joo Shin, None
Impact of Acute Exposure to High Altitude on Anterior Chamber
Geometry
M Dominik Fischer1, 2, Gabriel Willmann1, Andreas Schatz1, Kai
Schommer3, Ahmad Zhour1, Eberhart Zrenner1, Karl-Ulrich BartzSchmidt1, Florian Gekeler1. 1Centre for Ophthalmology, University
Hospital Tuebingen, Tuebingen, Germany; 2Nuffield Laboratory of
Ophthalmology, University of Oxford, Oxford, United Kingdom;
3
Department of Sports Medicine, Medical Clinic, University Hospital
Heidelberg, Heildelberg, Germany.
Purpose: This study aimed to quantify the impact of acute exposure
to high altitude on central corneal thickness and the geometry of the
anterior chamber angle. This work is related to the Tuebingen High
Altitude Ophthalmology (THAO) study.
Methods: Anterior segment spectral domain optical coherence
tomography was used to quantify changes of central corneal
thickness, anterior chamber angle and angle opening distance in 14
healthy subjects between baseline recordings (341 m) and during
acute exposure to high altitude (4559 m).
Results: Detailed longitudinal analysis revealed highly significant (p
< 0.0001) increased central corneal thickness (CCT) in healthy
subjects during acute altitude exposure (CCTbaseline = 517.53±28.28
μm vs. CCTaltitude = 539.87±31.28 μm; mean±sd). This change was
completely reversible upon descend and no subject demonstrated
persisting structural or functional sequels. Geometric measures of the
anterior chamber angle remained consistent with no significant
changes in angle opening distance (AOD) at 500 μm (AODbaseline =
695.96±190.00 μm vs. AODaltitude = 673.71±179.59 μm; p = 0.52)
and stable measurements of anterior chamber angle (ACA) in degree
(ACAbaseline = 37.85±6.53 vs. ACAaltitude = 36.29±5.81 μm; p =
0.34).
Conclusions: Significant changes of CCT occur in response to acute
exposure to high altitude in healthy control subjects. This might be
due to decreased atmospheric pressure and consequently decreased
blood oxygen saturation (SpO2) in non-acclimatized subjects and
constitute a mild corneal edema formation. Interestingly, AOD at 500
μm and ACA remained stable during the acute challenge to hypoxic
conditions at high altitude. This is the first time a quantitative
approach has been used to assess changes of the anterior segment
during acute, non-acclimatized high altitude exposure. As such, it
might provide a basis for the debate on changes of intraocular
pressure during exposure to high altitude.
Commercial Relationships: M Dominik Fischer, None; Gabriel
Willmann, None; Andreas Schatz, None; Kai Schommer, None;
Ahmad Zhour, None; Eberhart Zrenner, Retina Implant AG (F),
Retina Implant AG (I), Retina Implant AG (C), Retina Implant AG
(P), QLT Inc (C), Servier, Paris (C), Steinbeis GmbH&CoKG,
Stuttgart (I), Steinbeis GmbH&CoKG, Stuttgart (C), Neurotech, USA
(C), Pfizer, USA (C); Karl-Ulrich Bartz-Schmidt, Retina Implant
(P); Florian Gekeler, Retina Implant AG (F), Okuvision GmbH (F),
Retina Implant AG (C), Retina Implant AG (P)
Evaluation of Feature Detectors and Descriptors on the Iris
Sandro I. De Zanet1, 2, Michael Rueegsegger1, 2, Tobias Rudolph1, 2,
Sebastian Wolf2, Jens H. Kowal1, 2. 1Ophthalmic Technologies
ARTORG Center, University of Bern, Bern, Switzerland;
2
Department of Ophthalmology, University of Bern, Bern,
Switzerland.
Purpose: Characterizing and matching human iris patterns is
essential for video tracking in ophthalmic surgery as well as in iris
recognition. This study presents an evaluation of feature detection
and description algorithms. The evaluation is based on a database of
19 eyes captured in the infrared spectrum.
Methods: Repeatability and precision/recall were measured for
combinations of state-of-the-art feature detectors and descriptors:
SURF, SIFT, FAST, STAR and BRISK as detectors and SURF,
SIFT, FREAK, BRISK and BRIEF as descriptors. The algorithms
were tested with images of the iris in different geometric and
photometric transformations. A ground truth was provided by using
manually segmented and non-rigidly registered irides and masking
out reflections. Images were acquired from 40 human irides. The eyes
were illuminated by infrared LEDs and captured by a camera via a
beam splitter on a slit lamp microscope. To fixate the gaze of the
patients yellow LEDs were used to indicate different gaze angles.
Images where a good ground truth could not be found were discarded
which results in 19 testable irides.
Results: The most accurate detector/descriptor combination has been
found to be STAR/SU-BRISK which features fast detection in
combination with fast matching through the Hamming distance.
Especially the SU-BRISK descriptor has proven to be the most
distinctive independent from the used detector. Generally orientationand scale-variant descriptors performed far better than their invariant
counterparts. Additionally binary descriptors based on intensity
comparisons like BRIEF, BRISK or FREAK yield a more
discriminatory descriptor than real value based vectors. Through the
evaluation of detected point distributions it is evident that the region
from the collarette to the pupil border is the are with the most
features. Gaze change of 15° proved to decrease the matching
accuracy. However, decreasing the contrast with less intensive light
impaired accuracy more than gaze change. The area under the
precision-vs-recall-curve shows a range of 0.01 to 0.71 which
indicates the importance of isolating the best configuration for iris
images.
Conclusions: In this study a good candidate for real-time featurematching has been found which can be used in torsional eye tracking
used in beam therapy, namely the STAR detector with the scale- and
rotation-variant BRISK descriptor.
film (TF)).
Results: In-vivo: median lid margin thickness was 1.8mm (LL) and
1.9mm (UL), but not significant different (p=0.258). Lissamine green
drops stayed unaltered in all subjects. Median OB grade was 3. The
LL margin tightened in blinks by 1.2mm (median) but this was not
related to OB (r=-0.25; p=0.220). Median TMD ratio (TMD almost
closed eye / TMD opened eye) was 1.8 indicating an increasing
separation of the upper eyelid margin from the cornea. This small
effect was significantly correlated to OB (r=0.875; p<0.001). TM
appeared to retreat slightly behind the UL (in y-axis direction, toward
the cul-de-sac). In-vitro: When dynamically simulating OB the invitro TMD increased and the in-vitro TM retreated. TF was anchored
to the UL by surface tension and mixed with the LL TM even without
lid contact.
Conclusions: Central keratinized lid margins frequently do not touch
in spontaneous blinks and it appears that the central keratinized lid
margins are not aligned, as is traditionally presumed. This appears
not to impact tear film spreading.
Unfiltered matches of the STAR detector/SU-BRISK descriptor
Commercial Relationships: Sandro I. De Zanet, None; Michael
Rueegsegger, None; Tobias Rudolph, None; Sebastian Wolf,
Allergan (F), Allergan (C), Allergan (R), Bayer (F), Bayer (C), Bayer
(R), Novartis (F), Novartis (C), Novartis (R), Heidelberg Engineering
(C), Heidelberg Engineering (F), Hoya (F), Hoya (R), Optos (F),
Optos (C), Optos (R), Euretina (S); Jens H. Kowal, None
Figure 1: Grading scale of over-blink
Commercial Relationships: Heiko Pult, None; Britta H. RiedePult, None; Caroline A. Blackie, TearScience (E); Paul J. Murphy,
None; Donald R. Korb, TearScience (F)
Tear Film and Lid Margins in Over-Blink
Heiko Pult2, 1, Britta H. Riede-Pult2, 1, Caroline A. Blackie3, 4, Paul J.
Murphy5, Donald R. Korb3, 4. 1School of Optometry and Vision
Sciences, Cardiff University, Cardiff, United Kingdom; 2Dr. Heiko
Pult - Optometry and Vision Research, Weinheim, Germany;
3
TearScience Inc., MA, USA, Boston, MA; 4Korb Associates,
Boston, MA; 5School of Optometry and Vision Sciences, University
of Waterloo, Waterloo, ON, Canada.
Purpose: It is generally accepted that the eyelid margins touch each
other in spontaneous blinks while spreading the lipid over the tear
film in the upwards movement of the upper lid. In contrast, recent
research has demonstrated that the central upper lid (UL) and lower
lid (LL) do not touch in spontaneous blinks due to an over-blink
(OB). Furthermore a lissamine green drop, placed onto the anterior
portion of the central LL, was demonstrated to be unchanged in
spontaneous blinks (drop-test). The aim of this project was to
evaluate UL and LL margins movements and a possible tear film
spreading model based on the new knowledge.
Methods: In 15 subjects (9 female; median age= 45) lid-margin
thickness of the opened eye was measured by Pentacam. Drop-test
was performed over a period of 60sec. Blinks were filmed by highspeed video from a temporal-inferior view to analyse OB (Fig. 1) and
LL margin movement (z-axis). UL tear meniscus depth (UL-TMD;
horizontal width between UL margin and cornea; z-axis) was
observed in the opened eye and almost closed eye via a mirror and
slit lamp microscope. Image J Software was used for digital analysis
of TMD and LL movement. OB was simulated in vitro by a dynamic
blink model (glass-plates (=lids and cornea; saline solution = tear
The role of reactive oxygen species on cytotoxicity of
moxifloxacin and benzalkonium chloride on human corneal
epithelial cells
Ta-Ching Chen, Fung-Rong Hu. National Taiwan University
Hospital, Taipei, Taiwan.
Purpose: To investigate the role of reactive oxygen species (ROS)
and its mechanism on cytotoxicity of moxifloxacin and benzalkonium
chloride (BAK) on human corneal epithelial cells (HCECs).
Methods: Cultured HCECs were incubated with 0.5% moxifloxacin
as unpreserved solutions, and 0.001% benzalkonium chloride (BAC)
for 4 periods (5 min, 30 min, 1 h, and 3 h). Drug effects on cell
viability were evaluated by trypan blue exclusion assay and MTS
assay. Levels of reactive oxygen species (ROS) production were
evaluated using luminol- and lucigenin-enhanced chemiluminescence
assay. The protective effect of anti-oxidants (N-acetylcysteine and
3,5,4'-trihydroxy-trans-stilbene) on cytotoxicity of the tested drugs
was evaluated by MTS assay.
Results: A significant decrease in cell viability was observed
following incubation with 0.001% BAC and 0.5% moxifloxacin. Cell
recovery test revealed poor reversibility of cytotoxicity in both
moxifloxacin and BAC groups after more than 30 minutes of
exposure. ROS production was significantly increased after
incubation of HCECs in moxifloxacin and BAC solutions. Pretreatment of anti-oxidants may provide some protective effects
toward ROS-related cytotoxicity.
Conclusions: The cytotoxicity of moxifloxacin and BAK was related
to ROS production. Anti-oxidants offer protective effect toward
ROS-related cytotoxicity.
Commercial Relationships: Ta-Ching Chen, None; Fung-Rong
Hu, None
Growth factor secretion in human keratocyte cultures following
photodynamic inactivation (PDI)
Tanja Stachon1, Jiong Wang1, 2, Achim Langenbucher3, Berthold
Seitz1, Nora Szentmáry1. 1Department of Ophthalmology, Saarland
University Hospital, Homburg/Saar, Germany; 2Department of
Ophthalmology, Renmin Hospital of Wuhan University, Wuhan,
China; 3Experimental Ophthalmology, Saarland University,
Homburg/Saar, Germany.
Purpose: Photodynamic inactivation (PDI) may be an alternative
treatment option of infectious keratitis, with increasing resistance of
microorganisms to antibiotics. In previous studies we determined
viability, apoptosis, proliferation, CD34 and α-smooth actin
expression of keratocytes following PDI. The purpose of our present
study was to assess the secretion of KGF, VEGF, TGFβ1, HGF and
FGFb in human keratocytes following PDI, in vitro.
Methods: Primary human keratocytes were isolated by digestion in
collagenase A (1 mg/ml) from human corneal buttons, and cultured in
DMEM/Ham’s F12 medium supplemented with 10% fetal calf
serum. Five and twenty-four hours after PDI (100 nM chlorine e6,
illumination 13 minutes at 670 nm), the release of growth factors was
determined using enzyme-linked immunosorbent assay (ELISA). The
protein concentration of the cells was measured with Bradford Assay.
Results: Five hours following PDI, the secretion of HGF was 0.43
pg/µg protein, and FGFb expression was 3.47 pg/µg protein. The
secretion of HGF decreased (p < 0.01) and the release of FGFb
increased significantly (p < 0.0001) compared to controls. At this
time point TGFβ1 was not detectable and VEGF and KGF secretion
was not significantly different from control cultures. Twenty-four
hours after illumination expression of none of the growth factors was
significantly different compared to controls. Treatment only with
illumination or Ce6 did not show changes in the expression of growth
factors at any time point.
Conclusions: Five hours after PDI, HGF expression decreases and
FGFb expression increases in keratocyte cell cultures. However, 24
hours after treatment growth factor secretion seems to be normalized.
The altered secretion of HGF and FGFb may play a role in activation
of keratocytes and wound healing response after PDI of the cornea.
Commercial Relationships: Tanja Stachon, None; Jiong Wang,
None; Achim Langenbucher, None; Berthold Seitz, None; Nora
Szentmáry, None
Efficacy of a novel synthetic topical tetrapeptide on eliciting
analgesia subsequent to experimentally induced chemical corneal
injury
Bruce I. Gaynes, Michael Russo, David Goldmeier. Ophthalmology,
Loyola University Chicago, Maywood, IL.
Purpose: To ascertain analgesic action of a novel synthetic
tetrapeptide (SIS-ZEP04) analogue in reducing pain in a rat model of
experimentally induced chemical corneal injury.
Methods: Eight adult Sprague Dawley rats were utilized for study.
Following approval of the local animal use committee, animals
underwent treatment with 20 µL of 0.01 mg/mL of tetrapeptide in
0.01% ethanol vehicle twice daily in the right eye for 14 consecutive
days. A negative control was employed in the fellow eye. Efficacy of
the peptide as an analgesic was ascertained by the capsaicin eye
irritant test comprised of a dilute 0.02% solution of capsaicin.
Capsaicin administration to both eyes was performed at baseline, day
7 and day 14. The time required for resolution of blepharospasm, eye
wiping and squinting following administration of capsaicin was
recorded as a surrogate indicator of analgesic efficacy in relation to
the control eye.
Results: Mean time (seconds) for recovery from capsaicin induced
irritation in both right and left eyes at baseline was 38.0 +/- 7.8 and
52.2 +/- 9.8 seconds respectively. Following SIS-ZEP04 treatment
(day 14) mean time for capsaicin induced irritation recovery was
12.75 +/-5.6 and 24.4 +/- 14.69 seconds for right and left eyes
respectively. A statistically significant reduction in mean time to
capsaicin recovery was found for both right and left eyes (paired one
way t test, p=0.0125 and 0.0036 respectively, alpha=0.5). CochetBonnet aesthesiometry measurements did not deviate from baseline
levels at study conclusion.
Conclusions: The exogneous application of a synthetic tetrapeptide
appears to demonstrate significant efficacy in reducing ocular pain
and modifying pathways of nociception following experimental
chemical ocular injury. As analgesic action was apparent in both
treated and control eyes, it is unclear what effect, if any, the vehicle
exerted in minimizing ocular pain or rather if a central action induced
by systemic absorption of the peptide is in place. Moreover, the
analgesic action appears to occur without reduction in corneal
sensitivity. Further study is required to clarify the mechanism by
which this peptide exerts apparent analgesia in the mammalian eye.
Commercial Relationships: Bruce I. Gaynes, Shulov Institute of
Science Ltd (P), Shulov Institute of Science Ltd (F); Michael Russo,
None; David Goldmeier, None
Support: Shulov Institute of Science Ltd. Rehovot, Israel
Enantiomeric Separation, Ophthalmic Formulations and
Mydriatic Activity Oo Cyclopentolate Hydrochloride
Danilo Aleo1, Sergio Mangiafico1, Maria G. Saita1, Barbara Melilli1,
Melina G. Cro1, Sebastiano Mangiafico1, Nicola D'Antona2,
Giovanni Nicolosi2. 1R&D, Medivis, Catania, Italy; 2Istituto Chimico
Biomolecolare, CNR, Catania, Italy.
Purpose: Cyclopentolate Hydrochloride (CYP) is widely used as
mydriatic and cycloplegic agent. The product is marketed in the
racemic form, an equimolar mixture of the two enantiomers (+) and () Cyclopentolate. The aim of our study was the development of an
enantio-separation methodology, the preparation of two ophthalmic
formulation based on (+) CYP (MDV-D) and (-) CYP (MDV-L) as
well as the evaluation of their mydriatic activity in rabbits.
Methods: Optical resolution of the racemic mixture was performed
via diasteromic salt formation using (2R,3R) O-O’-di-p- toluoyltartaric acid (DPTTA) as resolving agent. A microemulsion was used
to formulate and to deliver 1% of (+) and 1% of (-) CYP. Nine
Albino rabbits were used to control mydriatic effect of MDV-D,
MDV-L, and of the commercial Cyclopentolate racemic solution
(Ciclolux, Allergan Inc.). The pupils were filmed during 35 minutes
with a video camera connected to an operation microscope, and the
mean pupil diameters were measured from the video recordings.
Results: A diastereoisomeric salt containing an enantiomeric excess
(E.E 90%) of (-) CyP (-) DPTTA has been achieved using (2R,3R) OO’-di-p- toluoyl-tartaric acid (DPTTA). This salt was then purified by
re-crystallization from ethanol (99% purity). (-) CYP (-) DPTTA was
dissolved in a 0,4N HCl and extracted with Methyl tert-Butyl Ether
(MTBE). Evaporation of aqueous phase gives (-) CyP (100% [α] 20D
= -32,8). The mother liquor containing (+)CyP (-)DPTTA was
extracted with MTBE and evaporation of aqueous phase gives (+)
CYP (100% [α] 20D = +32,8). 1% of (+) CYP (MDV-D) and 1% of
(-) CYP (MDV-L) microemulsion were instilled on the ocular surface
of rabbits and the mydriatic effect of both formulations was
performed. MDV-D and MDV-L showed the same effect on pupil
size when compared each other or with Ciclolux.
Conclusions: The comparison between the ophthalmic formulations
of the two enantiomers (MDV-D and MDV-L) with one another and
in comparison to the commercial formulation of the racemic product
(Ciclolux) showed no statistically significant differences in their
mydriatic activity. Studies on the known side effects in humans of
Cyclopentolate enantiomers (dizziness and mental confusion,
impaired coordination of movements, etc…) are in progress.
Commercial Relationships: Danilo Aleo, Medivis (E); Sergio
Mangiafico, Medivis (E); Maria G. Saita, Medivis (E); Barbara
Melilli, Medivis (E); Melina G. Cro, Medivis (E); Sebastiano
Mangiafico, medivis (E); Nicola D'Antona, None; Giovanni
Nicolosi, None
530 Gene Therapy and Delivery
Thursday, May 09, 2013 10:30 AM-12:15 PM
Oral delivery of bioencapsulated myelin basic protein ameliorate
amyloid burden and RGC loss in transgenic mouse model of
Alzheimer’s disease
Qiuhong Li1, Amrisha Verma1, Ping Zhu1, Pollob K. Shil1, Neha
Kohli2, Donevan Westerveld2, Henry Daniell2. 1Ophthalmology,
University of Florida, Gainesville, FL; 2University of Central Florida,
Orlando, FL.
Purpose: Increased deposition of amyloid beta 42 (Aβ42) is
associated with RGC apoptosis and retinal structural and functional
impairments. Myelin basic protein (MBP), a major structural protein
of CNS, also possesses intrinsic protease activity capable of
degrading Aβ amyloid, binds Aβ amyloid and inhibit Aβ fibril
formation. We aimed to investigate the protective effect of oral
delivery of bioencapsulated MBP fused with the transmucosal carrier
cholera toxin B subunit (CTB) in triple transgenic (3xTg) mouse
model for Alzheimer's disease (AD).
Methods: CTB-MBP fusion protein was expressed in chloroplasts of
tobacco. CTB-GFP expressed from chloroplasts of tobacco was also
used as control. The 3xTg AD mice (12-14 months old) were fed
with CTB-MBP bioencapsulated in plant cells for 3 months. Aβ42
aggregates were evaluated by ELISA and western blotting of
fractionated brain and retinal proteins, as well as
immunofluorescence of frozen sections using antibodies specific to
Aβ42. Retinal morphology, apoptosis and RGC density were
evaluated from fixed sections.
Results: Orally delivered CTB- GFP bioencapsulated in plant cells
was detected in brains and retinae of healthy mice. Brain Aβ levels
were reduced in 3xTgAD mice fed with bioencapsulated CTB-MBP,
especially the Aβ-42 insoluble fraction. The amyloid plaque intensity
was reduced in a concentration dependent manner by CTB-MBP
incubation with human AD and 3xTgAD mice brain sections. CTBMBP oral delivery reduced Aβ-42 accumulation in retinae and
prevented loss of retinal ganglion cells. Lyophilization of leaves
increased CTB-MBP concentration by 17-fold and facilitated long
term storage at room temperature in capsules.
Conclusions: Orally delivered CTB-MBP was able to reduce Aβ42
aggregates in the brain and retina and prevent RGC loss in the aged
3xTg AD mouse. Bioencapsulation protected fusion protein from
acids and enzymes in the digestive system and CTB fusion facilitated
uptake by target cells including brain and retina, thus this technology
provides a novel, more efficient, environmentally friendly and costeffective delivery of therapeutic proteins free of human or animal
pathogens to treat neurodegenerative diseases.
Commercial Relationships: Qiuhong Li, None; Amrisha Verma,
None; Ping Zhu, None; Pollob K. Shil, None; Neha Kohli, None;
Donevan Westerveld, None; Henry Daniell, None
Support: American Diabetes Association, American Heart
Association, Research to Prevent Blindness, NIH grants EY021752
and EY021721 to Li; NIH R01 HL 109442 and NIH R01 HL 107904,
Bill and Melinda Gates Foundation Global Health grant OPP
1031406 and the Juvenile Diabetes Research Foundation grant 172011-286 to Dr. Henry Daniell
Treatment of Patients with Leber Congenital Amaurosis Type 2
with an AAV Vector Expressing RPE65
Tim Stout1, Richard G. Weleber1, Maureen McBride1, David J.
Wilson1, Dawn Peters1, Margaret R. Humphries3, Terence R. Flotte3,
Lauren J. Jensen1, Andreas Lauer1, Jeffrey D. Chulay2.
1
Ophthalmology, Casey Eye Institute-OHSU, Portland, OR; 2AGTC
Inc, Alachua, FL; 3School of Medicine, University of Massachusetts,
Worcester, MA.
Purpose: To evaluate the safety and efficacy of rAAV2-CB-hRPE65
in patients with Leber congenital amaurosis (LCA) caused by
mutations in the RPE65 gene.
Methods: Twelve patients (aged 6-39y) with RPE65 mutations were
treated with a single, unilateral subretinal dose of 1.8 x 1011 or 5.4 x
1011 viral genomes in 450 μL. Serial postoperative examinations
included measurements of acuity, static and kinetic perimetry, optical
coherence tomography, fundus photography, luminance sensitivity
and visual quality-of-life function.
Results: All subjects tolerated the surgery and study agent
administration well without significant or unexpected complications.
No instances of persistent subretinal fluid or ocular inflammatory
disease were observed. Visual acuities were transiently depressed in
the treated eye of all patients during the first 1 to 2 weeks after
surgery, but returned to baseline or better in all of but two patients to
date. Improvements in visual acuity after treatment were seen in the
four youngest patients (age 6 to 11 years - with better baseline visual
acuities of 40 to 62 ETDRS letters) with increases ranging from 6 to
12.5 ETDRS letters in the treated eye. For the three subjects with
visual acuity of 20 to 31 ETDRS letters, one had a 2.5 letter increase
and two had a 6.5 or 12 letter decrease in the treated eye. GATE total
and central 30O hill of vision analysis trended towards improvement
in treated eyes when compared to baseline values. The five subjects
with the poorest baseline visual acuity (0 or 1 ETDRS letters) had
little or no change in their visual acuity over time, but for the four
subjects followed for at least 6 months three had a small but
statistically significant increase in kinetic perimetry visual field area
with the V4e target in the treated eye compared to baseline. An
improvement in visual functioning and quality of life was noted by
most patients.
Conclusions: Treatment of LCA2 patients with rAAV2-CB-hRPE65
is safe and appears effective. The greatest improvements in visual
acuity were observed in younger patients who presented with better
baseline visual acuity.
Commercial Relationships: Tim Stout, Clayton Foundation (P),
Oxford Biomedica (C), AGTC (F), Peregrine Pharmaceuticals Inc
(C), Stem Cells Inc (C); Richard G. Weleber, AGTC (C), VFMA
patent application (P), Pfizer (C), Oxford Biomedica (F); Maureen
McBride, None; David J. Wilson, None; Dawn Peters, None;
Margaret R. Humphries, None; Terence R. Flotte, None; Lauren
J. Jensen, None; Andreas Lauer, Oxford Biomedica (F), Acucela
(F), NIH (F); Jeffrey D. Chulay, AGTC (E)
Support: AGTC Clinical Trial Support, Foundation Fighting
Blindness ORDC Center Grant (Weleber, C-CL-0711-0534OHSU01)
Sustained Therapeutic Reversal of Canine Bestrophinopathy
with Gene Therapy using Recombinant AAV2
Karina E. Guziewicz1, Andras M. Komaromy2, Simone Iwabe1, Artur
V. Cideciyan3, Emily V. Dutrow1, Barbara Zangerl4, William A.
Beltran1, Samuel G. Jacobson3, William W. Hauswirth5, Gustavo D.
Aguirre1. 1Clinical Studies, University of Pennsylvania, Philadelphia,
PA; 2Department of Small Animal Clinical Sciences, Michigan State
University, East Lansing, MI; 3Scheie Eye Institute, University of
Pennsylvania, Philadelphia, PA; 4Centre for Eye Health, University
of New South Wales, Kensington, NSW, Australia; 5Department of
Purpose: Canine multifocal retinopathy (cmr), a spontaneous animal
model of BEST1-associated retinopathies in man, recapitulates the
spectrum of clinical and molecular features observed in human
bestrophinopathies, and is an important translational model for the
development and testing of therapeutic strategies. We have
previously shown that rAAV2-mediated BEST1 gene delivery
controlled by human VMD2 promoter (hVMD2) specifically targets
RPE cells, and is well tolerated in the wild-type canine retina. The
aim of these studies was to assess the safety, efficiency and
therapeutic potential of rAAV2-mediated BEST1 transgene
expression in cmr-affected dogs.
Methods: Thirteen cmr-affected dogs carrying R25X, R25X/P463fs
or P463fs mutations in BEST1 and exhibiting bilateral focal or
multifocal lesions were subretinally injected with rAAV2 expressing
either canine (0.136-1.59x1011vg/ml) or human (7.648.82x1011vg/ml) BEST1 regulated by the hVMD2 promoter. Each
treated and control (non-injected or BSS-injected) eye was monitored
clinically and imaged serially in vivo using cSLO/SD-OCT. For
morphological studies, treated and control tissue samples were
collected at 1 - 15 months post injection (p.i.).
Results: In all cases, the transient retinal detachment associated with
vector delivery completely resolved within 24h p.i. Based on the SDOCT analyses, some lesions within the treated regions disappeared as
early as 8 weeks and all resolved within 3 months after gene therapy,
and the treated areas remained asymptomatic thereafter. In cases with
advanced disease, hyperfluorescent FAF signals were still detectable
and likely localized to RPE. The untreated regions of the treated eye
or the contralateral control eye remained unchanged or developed
lesions. Specific Best1 immunolabeling was detected only within the
treated area, and comparable efficacy was noted with canine and
human cDNAs. Both the in vivo imaging and immunohistochemical
evaluation revealed no apparent adverse effects in RPE or retina
secondary to the BEST1 gene augmentation therapy.
Conclusions: rAAV2-mediated BEST1 gene augmentation therapy
shows great potential to reverse characteristic BEST1 lesions and
reverse pathology in cmr models up to 15 months p.i., and carries a
large translational promise as a first specific-treatment for human
bestrophinopathies.
Commercial Relationships: Karina E. Guziewicz, None; Andras
M. Komaromy, None; Simone Iwabe, None; Artur V. Cideciyan,
None; Emily V. Dutrow, None; Barbara Zangerl, None; William
A. Beltran, None; Samuel G. Jacobson, None; William W.
RetroSense (C); Gustavo D. Aguirre, None
Support: FFB, MVRF, NEI/NIH EY06855, EY17549, Van Sloun
Fund, Hope for Vision
Vertical Gene Transfer of Mutant Human G11778A ND4 in Next
Generation Mito-Mice
Hong Yu, Sacide S. Ozdemir, Tsung-Han Chou, Vittorio Porciatti,
John Guy. Ophthalmology, Bascom Palmer Eye Inst, Univ of Miami,
Miami, FL.
Purpose: To describe vertical gene transfer of the mutant human
ND4 (hmutND4) bred from founder mice generated by mitochondria
targeting sequence AAV infection of mouse embryonic stem cells.
Methods: hmutND4 with a FLAG epitope and mitochondrial
encoded mCherry under control of a mitochondrial promoter was
packaged into mito-targeted AAV2 containing the COX8 leader
sequence inserted into the VP2 capsid, then microinjected into the
mouse blastocyst. Retina, brain, optic nerve, heart, liver and skeletal
muscle were assessed by PCR, sequencing, two-dimensional blue
native polyacrylamide gel electrophoresis (2D BN-PAGE), complex I
activity and histopathology. Visual function was monitored by serial
pattern electroretinography (PERG), retinal structure by spectral
domain optical coherence tomography(SD-OCT) and mitochondrial
gene expression by confocal laser scanning ophthalmoscopy (CLSO)
of mCherry fluorescence.
Results: 60 transgenic mice were generated that contained varying
expression of in vivo mCherry fluorescence. Three females with the
highest levels of mCherry expression in the retina were mated with
males of the same strain and produced a total of 137 pups over 4
generations. Red fluorescent particles were seen in the RGC layer
and optic nerve head in 77% of mice, and cells with fluorescence
increased as mice aged suggesting replication of the transgene.
hmutND4 was detected by PCR in all tissues from the F0 founder
mice to their F1 to F4 progeny. Characteristic of mitochondrial
heteroplasmy, transgene levels differed significantly between tissues
(p<0.001) with the highest levels found in the optic nerves.
Expression of hmutND4 was confirmed by immunostaining and it
assembled into mouse complex I. Activity of complex I decreased
10% to 60% and was inversely correlated to the amount of hmutND4.
PERGs showed a progressive decline in amplitudes from 3 months
after birth to noise levels by 11 months of age. SD-OCT confirmed
progressive thinning of the RGC+IPL(p=0.00089). Ultrastructural
analysis showed severe loss of axons in the optic nerve,
demyelination and mitochondrial abnormalities in the brain and
degeneration of few fibers in skeletal muscle, but none in the heart.
Conclusions: Vertical transmission of mutant human ND4 into
subsequent generations of transgenic mito-mice results in the
characteristic hallmarks of human LHON affecting predominantly the
optic nerve despite its presence in all mouse tissues.
Commercial Relationships: Hong Yu, None; Sacide S. Ozdemir,
None; Tsung-Han Chou, None; Vittorio Porciatti, None; John
Guy, None
Support: R01 EY007141
Gene Therapy with the Mitochondrial Heat Shock Protein 70
(mtHSP70) Chaperone Supresses Neurodegeneration and
Irreversible Visual Loss in Experimental Optic Neurits
Venu Talla, Sacide S. Ozdemir, Tsung-Han Chou, Vittorio Porciatti,
John Guy. Ophthalmology, Bascom Palmer Eye Institute, University
of Miami, Miller School of Medicine, Miami, FL.
Purpose: To rescue visual loss and optic neuropathy in the
experimental autoimmune encephalomyelitis (EAE) mouse model
using gene therapy with the mtHSP70 chaperone responsible for
unfolding and import of most proteins into the mitochondria.
Methods: EAE was induced in female DBA/1J (n=20) mice by
subdermal injection of 0.1 ml homologous spinal cord emulsion in
CFA. Ten mice were rescued by intravitreal injection of ssAAVmtHSP70-Flag, 10 EAE and 10 unsensitized mice injected with
scAAV-Cox8-mCherry served as controls. Visual function was
assessed by pattern electroretinograms (PERG). High resolution
spectral domain OCT evaluated the thickness of the inner plexiform
layer + nerve fiber layers at 1, 3 and 6 months post injection (MPI).
All mice were euthanized 6MPI. Retinas and ONs were dissected for
histological and ultrastructural evaluation. Expression of
mtHSP70Flag in the retina and ONs was evaluated 15d PI by RTPCR, immunofluorescence (IF) and western blotting (WB).
Results: IF revealed a typical punctate and perinuclear expression of
Flag-HSP70 which colocalized with mitochondrial porin and thy1.2
labeled RGCs. RT-PCR and WB confirmed HSP70 expression in the
retina and ON. PERG amplitude at 3M and 6MPI showed 42% and
45% reduction in EAE-mCherry compared to control mCherry
(p<0.005,) mtHSP70 rescued mice also showed reduced amplitudes
(26% and 27% respectively, p<0.05) though it rescued the by 37%
and 41% respectively (p>0.05). PERG latency was delayed by 15%
and 21% in EAE-mCherry compared to unsensitized mCherry
controls (p<0.05), whereas the mtHSP70 injected mice rescued the
delay by 100% and 83% respectively at 3M and 6MPI. OCT showed
a thinning in EAE-mCherry compared to mCherry control at 3M
(16%) and 6MPI (15%) p<0.05, whereas mtHSP70 rescued this
thinning by 90% and 100% respectively (p<0.05). Ultrastructural
analysis of the EAE ONs demonstrated different levels of
degeneration in axons and myelin. Degenerated axons showed varied
mitochondrial number, shape and morphology. mtHSP70 ONs
showed relatively normal axons and myelin with organized
microtubules and mitochondria.
Conclusions: mtHSP70 gene therapy preserves vision by limiting
neurodegeneration of the ONs that causes permanent and untreatable
disability that is unaltered by commonly used disease modifying
drugs that target inflammation in multiple sclerosis.
Commercial Relationships: Venu Talla, None; Sacide S. Ozdemir,
None; Tsung-Han Chou, None; Vittorio Porciatti, None; John
Guy, None
Support: NIH-NEI R01 EY07982, NIH-NEI RO1 EY019077 and
NIH- P30-EY014801
AAV-mediated gene therapy restores retinal function and vision
in the RPGRIP1-deficient dog
Lolita Petit1, Elsa Lhériteau1, Michel Weber2, Guylène Le Meur2,
Jack-Yves Deschamps3, Philippe Moullier1, 4, Fabienne Rolling1. 1Inst
de Recherche Therapeutique 1, Lab de Therapie Genique INSERM
UMR 1089, Nantes, France; 2Service d'Opthalmologie, CHU-Hôtel
Dieu, Nantes, France; 3Emergency and Critical Care Unit, ONIRIS,
Nantes-Atlantic College of Veterinary Medicine-Food Science and
Engineering, Nantes, France; 4Department of Molecular Genetics and
Microbiology, College of Medicine, University of Florida,
Gainesville, FL.
Purpose: Recently, gene therapy targeting photoreceptors was
successfully used in canine models of rod-cone dystrophies,
supporting the translation of this therapy to the clinic. In contrast,
evidence of the efficacy of photoreceptors gene therapy in a large
animal model of cone-rod dystrophies is still lacking. The aim of this
study was to evaluate gene addition therapy in the retinitis
pigmentosa GTPase regulator interacting protein 1 (RPGRIP1)deficient miniature longhaired dachshund, a well characterized dog
model of cone-rod dystrophy.
Methods: We generated AAV2/5 and AAV2/8 vectors carrying the
canine Rpgrip1 cDNA under the control of the photoreceptor-specific
rhodopsin kinase promoter. These vectors were subretinally injected
to 1 eye of 4 RPGRIP1-deficient dogs at 1 month of age (AAV2/5,
n=2 - AAV2/8, n=2). One AAV2/5-treated dog was sacrificed at 1
month postinjection and Rpgrip1 expression was assessed by RTPCR on both treated and untreated retinas. In the 3 remaining treated
dogs, funduscopy, optical coherence tomography, full-field
electroretinography and behavioral tests were used to evaluate
outcomes of AAV-mediated gene transfer.
Results: RT-PCR analysis demonstrated an efficient expression of
Rpgrip1 in the AAV2/5-treated RPGRIP-/- retina. Morphological
analysis revealed a significant preservation of the retinal thickness
and vasculature in both AAV2/5- and AAV2/8 -treated retinas. In all
treated eyes, substantial cone-derived ERG responses were recorded
as soon as 1 month postinjection (30 to 70% of those recorded in
normal eyes) and remained stable for at least 12 months, whereas
cone function was undetectable in untreated contralateral eyes. This
rescue of cone function was accompanied by a significant
preservation of rod function in 2/3 dogs at 12 months post-treatment
(17 and 47% of those recorded at 1 month postinjection). At this age,
rod function was totally lost in untreated contralateral eyes. Most
importantly, both bright and dim-light vision was restored in all
treated dogs.
Conclusions: These results provide for the first time compelling
evidence that gene therapy can effectively restore retinal function and
vision in a large animal model of cone-rod dystrophy. It represents a
key step toward the clinic.
Commercial Relationships: Lolita Petit, None; Elsa Lhériteau,
None; Michel Weber, None; Guylène Le Meur, None; Jack-Yves
Deschamps, None; Philippe Moullier, None; Fabienne Rolling,
None
Support: Association Française contre les Myopathies, Agence
Nationale pour la Recherche, INSERM, Université de Nantes,
Fondation pour la Thérapie Génique en Pays de la Loire, Ministère
Français de l'Enseignement Supérieur et de la Recherche
Gene therapy in Leber congenital amaurosis due to rpe
mutations: results of the first six patients included in a clinical
trial
Guylene Le Meur1, Pierre Lebranchu2, Yann Péréon3, Sebastien
Schmitt4, Stéphane Bézieau5, Philippe Moullier6, Fabienne Rolling7,
Michel Weber8. 1CHU hotel dieu, Nantes, France; 2CHU hotel Dieu,
Nantes, France; 3CHU hotel Dieu, Nantes, France; 4CHU hotel Dieu,
Nantes, France; 5CHU hotel Dieu, Nantes, France; 6INSERM UMR
1089, Nantes, France; 7INSERM UMR 1089, Nantes, France; 8CHU
hotel Dieu, Nantes, France.
Purpose: To present the preliminary data of an AAV4 gene therapy
clinical trial in patients with rpe65 retinal degeneration
Methods: A phase I/II clinical trial assessed the safety and the
efficiency of a subretinal injection with AAV2/4.rpe65.hrep65 vector
in the worse eye of patient with rpe65-/- retinal dystrophy. We report
the result of the first six treated patients. The first three patients
received up to 400 µL and the three following patients up to 800 µL
of a vector solution at 6.1010 vector genome/mL. The primary safety
endpoints are evaluated by biomicroscopy, laser flare meter, fundus
photography, fluorescein angiography, OCT and a tolerance
questionnaire. Secondary efficacy endpoints are evaluated by global
ERG, multifocal ERG, visual field, near and far visual acuity, color
vision test, pupillometry, microperimetry, visual mobility test,
functional MRI and fundus autofluorescence.
Results: Patients, 20 to 42 years old, were treated between october
2011 and september 2012. The injected volume was between 200 μL
and 770 μL of the vector solution. No adverse effects or ocular
inflammation are noticed one year to 2 months after subretinal
injection. EDTRS score change on average of 5,8 letters after
subretinal injection (0,15 Log MAR). A decrease of the nystagmus is
noticed in all patients. A decrease of the moving time in the mobility
test in scotopic brightness is found with the treated eye. Visual field
modifications in the subretinal area are noticed in patients.
Conclusions: The preliminary data of this AAV4 gene therapy
clinical trial in patients with rpe65 retinal degeneration suggested a
good tolerance and a functional efficency. This results may be
confirmed by the pursuit of the follow up of the first six patients and
by the
data analysis of next included three patients.
Commercial Relationships: Guylene Le Meur, None; Pierre
Lebranchu, None; Yann Péréon, None; Sebastien Schmitt, None;
Stéphane Bézieau, None; Philippe Moullier, None; Fabienne
Rolling, None; Michel Weber, None
Support: PHRC07-09K
545 Retina: Physiology and Pharmacology
Thursday, May 09, 2013 10:30 AM-12:15 PM
Program #/Board # Range: 6314-6365/D0185-D0236
Contributing Section(s): Biochemistry/Molecular Biology, Visual
Neuroscience
Program Number: 6314 Poster Board Number: D0185
Functional Expression Of TRPV Channels In The Retinal
Endothelium
Jennifer E. McNaughten, Mary McGahon, Graham McGeown,
Timothy M. Curtis. Queens University Belfast, Belfast, United
Kingdom.
Purpose: Transient receptor potential vanilloid (TRPV) channels are
part of a superfamily of non-selective cation channels with a vast
range of physiological functions. Our aim was to identify the
functional TRPV channel subtypes expressed in the retinal
endothelium.
Methods: Retinal microvascular endothelial cells (RMECs) were
cultured from bovine arterioles. Fura-2-based Ca2+ microfluorimetry
was used to test for the functional expression of TRPV1, V2, V3 and
V4 in RMECs using a selection of agonists and antagonists.
Results: The ultrapotent TRPV1 agonist resiniferatoxin(10nM)
caused a transient rise in calcium in 50% of cells tested. These
responses were abolished in the presence of two different TRPV1
antagonists, capsazepine(5uM) and AMG9810(100nM). Functional
expression of TRPV2 was evident from calcium peaks in response to
Δ9-THC(10uM). The responses were reduced after incubation with
tranilast(75uM), a TRPV2 inhibitor. Carvacrol(100uM) provided
evidence for the presence of TRPV3 in RMECs causing consistant
calcium responses across a number of cells. Both tranilast and
carvacrol were used in the presence of the TRPA1 antagonist HC030031(10uM). The well known TRPV4 channel was tested for using
GSKA(100nM) and 4αPDD(1uM). Both elicited transient rises in
calcium. The specific TRPV4 antagonist HC067047(1uM) blocks
responses caused by GSKA. The aforementioned TRP channels were
tested for in EGTA(1mM) buffered calcium free solution. Under
these conditions none of the agonists initiated a rise in calcium
suggesting the responses were mediated by calcium influx from the
extracellular medium.
Conclusions: This study provides evidence for the functional
expression of TRPV1-V4. This warrants further research to elucidate
the physiological significance of TRP channels in endothelial cell
function.
Commercial Relationships: Jennifer E. McNaughten, None; Mary
McGahon, None; Graham McGeown, None; Timothy M. Curtis,
None
Support: Department for Employment and Learning
Further Studies On The Role Of Arachidonic Acid Metabolites
In The Regulation Of Potassium-Induced [3H]D-Aspartate
Release From Isolated Bovine Retinae By 5-epi-5-F3t-Isoprostane
Jamal Jamil1, Pratik Bankhele1, Ankita Salvi1, Thierry Durand3, Jean
Galano3, Alexandre Guy3, Ya Fatou Njie-Mbye2, Sunny E. Ohia2,
Catherine A. Opere1. 1Creighton.edu, Omaha, NE; 2Texas Southern
University, Houston, TX; 3Universities of Montpellier I and II,
Montpellier cedex, France.
Purpose: We have evidence that eicosapentanoic acid (EPA)-derived
F3-isoprostane (F3-IsoP), 5-epi-5-F3t-IsoP inhibits excitatory amino
acid neurotransmitter release in bovine retina. In the present study,
we investigated the role of arachidonic acid metabolites in the
inhibitory action of 5-epi-5-F3t-IsoP on K+-induced [3H]D-aspartate
release from bovine retina, in vitro.
Methods: Isolated neural retina were incubated in oxygenated Krebs
solution containing 200 nM of [3H]D-aspartate and then prepared for
studies of neurotransmitter release. Release of [ 3H]D-aspartate was
evoked by K+ (50 mM) stimuli applied at 90 mins (S1) and at 108
mins (S2) after the onset of superfusion. F3-IsoP was added 8 min.
before S1 while antagonists were present before and during S1 and S2.
Results: 5-epi-5-F3t-IsoP (0.1 nM - 0.1 µM) elicited an inhibitory
action on K+-evoked [3H]D-aspartate release in a concentrationdependent manner, achieving a maximum inhibition of 46.9% at 0.1
µM (IC30 of 1 nM). Pretreatment of retinal tissues with the
cyclooxygenase (COX) enzyme inhibitor, flurbiprofen (3 µM)
unmasked a biphasic action, being inhibitory at lower (0.1 pM-10pM)
and stimulatory at higher (0.1nM-0.1µM) concentrations of the IsoP.
All the antagonists used exhibited no effect on K+-induced [3H]Daspartate release. Similarly, SC 19220 (1 µM; EP 1) and AH 6809 (10
µM; EP1-3/DP1) had no effect on 5-epi-5-F3t-IsoP (0.1 pM)-induced
inhibition of the neurotransmitter release. On the contrary, other
receptor antagonists, BAY-u3405 (10 µM; TP/DP), SQ 29548 (10
µM; TP) and ozagrel (10 µM; Tx-synthase) reversed the stimulatory
action of the F3-IsoP (0.1 µM) on neurotransmitter release.
Conclusions: The EPA-metabolite, 5-epi-5-F3t-IsoP attenuates K+induced [3H]D-aspartate release via COX enzyme-dependent
mechanisms. Furthermore, the presence of COX unmasks a TPdependent stimulatory action of this F3-IsoP on K+-induced [3H]Daspartate release.
Commercial Relationships: Jamal Jamil, None; Pratik Bankhele,
None; Ankita Salvi, None; Thierry Durand, None; Jean Galano,
None; Alexandre Guy, None; Ya Fatou Njie-Mbye, None; Sunny
E. Ohia, None; Catherine A. Opere, None
Protective effects of an PPAR-γ agonist on retinal
ischemia/reperfusion injury in rats
Xiaoyan Zhang, Yi-qin Xiao, Yu Zhang, Jiaying zhang, Wen Ye.
Ophthalmology, Huashan Hospital affiliated to Shanghai Fudan
University, Shanghai, China.
Purpose: To investigate the protective effects of an PPAR-γ agonist
in the rat retina after ischemia/reperfusion (I/R) injury.
Methods: Retinal ischemia was induced in rats by increasing the
intraocular pressure to 110mmHg for 60 minutes. The PPAR-γ
agonist was delivered by periocular injection and intraperitoneal
injection before I/R. Seven days after I/R injury, retinal damage was
quantified by measuring the thickness of the retina, the functional
changes of VEP and ERG, and the RGC number. The expression of
GFAP, NF-κB p65 in the retina was determined by western blot, realtime polymerase chain reaction (PCR), and immunohistochemistry.
Results: I/R caused severe disruption of the retinal construction and
integrity. In I/R group without treatment the number of RGCs was
reduced by 53%. The PPAR-γ agonist either delivered by periocular
injection or by intraperitoneal injection preserved the thickness of the
retina after I/R, and the survival of RGC was 77% and 74%
respectively. Pretreated with the PPAR-γ agonist also attenuate the
destruction of VEP and ERG caused by I/R. The amplitudes of the
ERG b-waves and the VEP P1-N2 component were significantly
lower in the I/R group than in the groups pretreated with the PPAR-γ
agonist (p<0.01). After ischemia, expression of GFAP, NF-κB p65
subunit was upregulated and high phosphoration level of p65 subunit
was detected in retina. The PPAR-γ agonist pretreatment suppressed
NF-κB activation and reduced the GFAP overexpression.
Conclusions: The PPAR-γ agonist promotes the survival of RGCs
and protect retina histological integrity as well as retinal function
after retinal I/R. The protect effect appears to act through the antiinflammatory effects towards the inhibition of retinal glia activation,
suggesting that the PPAR-γ agonist may have a therapeutic potential
for the prevention of retinal diseases associated with I/R.
Hematoxylin and eosin staining of retina . (A) shows the retina of I/R
group.The PPAR-γ agonist pretreatment maintained the thickness of
the whole retina, IPL and INL after I/R (B,C). (D) shows the retina
tissue of the normal control group. ip., intraperitoneal injection; po.,
periocular injection.
Retrograde labeled RGCs in wholemount retinas 7 days after ocular
ischemia. ip., intraperitoneal injection; po., periocular injection.
Commercial Relationships: Xiaoyan Zhang, None; Yi-qin Xiao,
None; Yu Zhang, None; Jiaying zhang, None; Wen Ye, None
Support: Supported by the National Natural Science Foundation of
China (Grant No. 30901651)
SPECTROSCOPIC INVESTIGATION OF AGONISTINDUCED REARRANGEMENTS OF CYCLIC
NUCLEOTIDE-REGULATED ION CHANNELS
William N. Zagotta1, Michael C. Puljung1, Stefan Stoll2. 1Physiology
and Biophysics, University of Washington, Seattle, WA; 2Chemistry,
University of Washington, Seattle, WA.
Purpose: Cyclic nucleotide-gated (CNG) channels are expressed in
the outer segment of rod and cone photoreceptors and are responsible
for the primary electrical response to light. Along with
hyperpolarization-activated cyclic nucleotide-modulated (HCN) ion
channels, CNG channels are part of a sub-family of ion channels that
are activated by the direct binding of cyclic nucleotides, e.g.
adenosine 3’,5’-cyclic monophosphate (cAMP) and guanosine 3’5’cyclic monophosphate (cGMP), to a conserved, cytoplasmic domain.
The structure of the cyclic nucleotide-binding domain (CNBD) is
similar to those found in other cyclic nucleotide-activated proteins,
including the kinases PKA and PKG, the transcription factor CAP,
and the guanine nucleotide exchange factor Epac. The core of this
structure contains an eight-stranded β-roll followed by two helices
(the B- and C-helices). Cyclic nucleotides initially bind to residues in
the β-roll. Subsequent to binding, the C-helix of HCN and CNG
channels undergoes a translation toward the binding pocket as well as
a stabilization of its helical structure. This conformational
rearrangement is coupled to opening of the ion channel pore.
Knowledge of the structural mechanisms of channel activation is
critical for understanding the molecular underpinnings of vision.
Methods: In this study we use transition metal ion fluorescence
resonance energy transfer (tmFRET) and electron paramagnetic
resonance (EPR) spectroscopy to examine cyclic nucleotidedependent structural transitions in the purified C-terminal domain of
HCN2.
Results: We have extended our previous tmFRET studies to
demonstrate that the C-helix undergoes a coiled-to-helix transition
associated with agonist binding. Furthermore, we used EPR
spectroscopy on the spin-labeled HCN2 C-terminus to investigate the
reorientation and stabilization of the CNBD induced by cAMP
binding.
Conclusions: These studies further extend our knowledge of the
conformational changes in the CNBD of HCN and CNG channels
and may provide a general picture of the activation of other families
of cyclic nucleotide-regulated proteins.
Commercial Relationships: William N. Zagotta, None; Michael C.
Puljung, None; Stefan Stoll, None
Support: NIH R01 EY010329
Evaluation of homocysteine and its metabolic cofactors in
patients with non proliferative and proliferative diabetic
retinopathy
Giulia Malaguarnera1, Caterina Gagliano1, Mario D. Toro1, Filippo
Drago2, Teresio Avitabile1. 1Department of Ophthalmology,
university of Catania, Catania, Italy; 2Department of Clinical
Biomedicine, university of Catania, Catania, Italy.
Purpose: Homocysteine, a well-known inducer of vascular
endothelial cell damage has been associated with extracellular matrix
changes. Many studies demonstrated that high levels of this
aminoacid in diabetic patients increases significantly the risk of the
development of this pathology. This study has been undertaken to
investigate the role of homocysteine and its cofactors during the
progression of the diabetic retinopathy.
Methods: We measured the plasma levels of homocysteine, folic
acid, vitamin B6 and vitamin B12 in 113 diabetic type 2 patients with
non proliferative retinopathy (NPDR), 52 with proliferative diabetic
retinopathy (PDR) and 50 healthy subjects used as control group.
Results: We found higher plasma levels of homocysteine in NPDR
group compared to the control group (p<0.001). Also in the PDR
group we detected an increase of homocysteine compared to control
group (p<0.001) and NPDR group (p<0.01). The severity of DR was
associated with lower folic acid and vitamin B6 levels in all groups
but the lowest levels were observed in PDR (p<0.05). On the
contrary, vitamin B12 plasma levels were lower in both NPDR and
PDR compared to control (p<0.001) without significant difference
between PDR and NPDR groups.
Conclusions: These findings demonstrated that homocysteine, folic
acid, vitamin B6 and vitamin B12 may play a role in the development
and progression of diabetic retinopathy.
Commercial Relationships: Giulia Malaguarnera, None; Caterina
Gagliano, None; Mario D. Toro, None; Filippo Drago, None;
Teresio Avitabile, None
Support: PON 01-00110 UNIVERSITY OF CATANIA
Increased Expression of Endothelin B Receptors Precedes
Retinal Ganglion Cell Death in a Rodent Model of Glaucoma
Alena Z. Minton1, 2, Nitasha Phatak1, 2, Dorota L. Stankowska1, 2,
Shaoqing He1, 2, Hai-Ying Ma3, Raghu R. Krishnamoorthy1, 2. 1Cell
Biology & Anatomy, University of North Texas Health Science
Center, Fort Worth, TX; 2North Texas Eye Research Institute, Fort
Worth, TX; 3Pharmacology & Neuroscience, University of North
Texas Health Science Center, Fort Worth, TX.
Purpose: ETB receptors have been shown to be involved in the
pathogenesis of glaucoma. However, the precise sequence of
molecular events involving ETB receptor expression and retinal
ganglion cell death is not completely understood. This study was
aimed at investigating whether the expression of ETB receptors
precedes retinal ganglion cell loss in vivo in the Morrison’s elevated
intraocular pressure (IOP) model of glaucoma in rats.
Methods: Intraocular pressure (IOP) was elevated in one eye of
retired breeders Brown Norway rats using the Morrison’s method
(injection of hypertonic saline through episcleral veins), while the
contralateral eye served as control. Retinas were collected after 1 and
2 weeks of IOP elevation, sectioned and stained for ETB receptor
expression by immunohistochemistry. In a separate set of
experiments, retired breeders Brown Norway rats were used for
retrograde labeling of retinal ganglion cells (RGCs) with Fluoro-gold.
Following retrograde labeling, IOP was elevated in one eye using the
Morrison’s method, while the contralateral eye served as control.
After IOP was elevated, rats were maintained for 2 weeks and
sacrificed. Fluoro-gold labeled retinas were isolated, flat-mounted,
and images taken using a Zeiss LSM-510 confocal microscope.
Fluoro-gold-labeled RGCs were counted in three eccentricities from
the optic nerve head. In addition, optic nerves were isolated from
Brown Norway rats following 2 weeks of IOP elevation, sectioned
and stained using p-phenylenediamine. Confocal images were taken
and morphology of optic nerve axons was compared.
Results: Immunohistochemical analysis of retinal sections showed
no appreciable change in ETB receptor immunostaining following 1
week of IOP elevation. However, IOP elevation for 2 weeks
produced increased expression of ETB receptor in the RGCs, inner
plexiform layer (IPL) and inner nuclear layer (INL). In addition, IOP
elevation for 2 weeks resulted in 25% loss of RGCs in the first
eccentricity, while no significant loss was seen in the second and
third eccentricities. Moreover, there was no appreciable damage
observed in the optic nerve axons after 2 weeks of IOP elevation.
Conclusions: Early increase in expression of ETB receptors (at 2
weeks of IOP elevation) could contribute to RGC loss and damage to
the optic nerve axons.
Commercial Relationships: Alena Z. Minton, None; Nitasha
Phatak, None; Dorota L. Stankowska, None; Shaoqing He, None;
Hai-Ying Ma, None; Raghu R. Krishnamoorthy, None
Support: NEI: 1RO1 EY0199952-01
Vitreous penetration of orally administered acetazolamide in
humans
Armin Afshar, Rama Jager. Ophthalmology & Visual Science,
University of Chicago, Chicago, IL.
Purpose: The goal of this study was to determine if orally
administered acetazolamide, a carbonic anhydrase inhibitor,
penetrates the vitreous humor in humans. Carbonic anhydrase
inhibitors are thought to modulate the polarized distribution of
carbonic anhydrase at the level of the retinal pigment epithelium via
extracellular pH gradients, and enhance fluid absorption from the
retina. If orally administered acetazolamide penetrates the vitreous, a
quantification study of vitreous concentrations of the drug could help
establish safe dosages for intravitreous injection of the drug to treat
diabetic macular edema.
Methods: This was a prospective, non-randomized interventional
case series of eyes that underwent pars plana vitrectomy for tractional
retinal detachment repair. All patients except one control were given
500 mg of oral acetazolamide to take the night prior to surgery and
upon awakening the morning of surgery (approximately 3 hours prior
to surgery). During surgery, prior to beginning vitrectomy, 0.3 mL of
vitreous fluid was obtained by clamping the infusion line and
attaching the vitrector to a syringe via a short length of tubing.
Vitreous samples were packed on ice and brought to the laboratory
for analysis. Vitreous study specimens were extracted with
acetonitrile, centrifuged to remove proteins and analyzed using a
tandem mass spectrometer equipped with a high performance liquid
chromatographer.
Results: All 11 patients who took oral acetazolamide had detectable
concentrations of acetazolamide in the vitreous. The control sample
had no drug in the vitreous. Six specimens had acetazolamide in the
100-400 microgram/mL range, 2 specimens had acetazolamide in the
50-100 microgram/mL range and 3 specimens were measured under
1 microgram/mL.
Conclusions: Orally administered acetazolamide penetrates the
vitreous in humans. This study paves the way for additional research
investigating intravitreous injection of carbonic anhydrase inhibitors
and pharmacotherapeautics for diabetic macular edema.
Commercial Relationships: Armin Afshar, None; Rama Jager,
None
Support: Illinois Society for the Prevention of Blindness
Quality of fixation in major depressive disorder and the influence
of antipsychotic medication
Serena Fragiotta1, Pier Luigi Grenga2, Daniela Domanico2,
Alessandro Cutini1, Stefano Valente1, Vittoria De Rosa1, Enzo M.
Vingolo1. 1University of Rome La Sapienza, Latina, Italy;
2
S.M.Goretti Hospital, latina, Italy.
Purpose: To evaluate retinal function and fixation stability in major
depressive disorder (MDD) and to investigate the influence of
antipsychotic therapy on quality of fixation, using the MP-1
microperimeter (Nidek Technologies).
Methods: 28 patients with MDD (62.5±12.51 years) with logMAR
acuity ≤ 0.04 and 30 matched healthy subjects (HS) (63.62± 14.14
years) with logMAR acuity ≤ 0.0 were enrolled. According to the
Diagnostic and Statistical Manual of Mental Disorders 4th edition
(DSM-IV), single (296.2) or recurrent episode (296.3) of MDD were
included. Patients with any other diagnosed mental illness or ocular
disease were excluded. Retinal sensitivity, fixation stability, fixation
points within 2 and 4 degree, and bivariate contour ellipse area
(BCEA) were obtained from the MP-1 in MDD and HS. Moreover
patients were divided according to therapy: 12 patients with (group
A) and 16 without (group B) atypical antipsychotic drugs. Mean
BCEA (deg2) was normalized by logarithmic transformation
(Shapiro-Wilk test, p<0.05), and first standard deviation (68.2%) was
considered. Statistical analysis was performed using ANOVA with
Scheffé’s comparison and Pearson’s correlation.
Results: Mean retinal sensitivity was 15.21±4.04 dB in MDD
patients and 17.29±0.86 dB in HS (p=0.01).
Fixation points within 2 degree was 78.25±21.62% and 93.32±8.21%
within 4 degree, 17 MDD patients had a stable fixation, 10 relatively
unstable and 1 unstable fixation. Mean BCEA values in MDD
patients were significantly larger than HS (0.83±0.97 deg2 vs
0.29±0.25 deg2,p=0.02). There was a positive relationship between
logBCEA and age in HS (r=0.46, P=0.01), but not in MDD patients
(r=0.28,p=0.14). There was significant differences in BCEA between
groups (F=9.34,p=0.001). Comparison between group A and group B
showed a more restricted BCEA (p=0.01) in group A, whereas no
differences were observed between groups A/HS (p=0.91). Moreover
in group B mean BCEA was significantly larger than HS (p=0.001).
Conclusions: Patients with MDD had a reduction in retinal function
and fixation stability compared to HS. MDD patients treated with
antipsychotic drugs showed a significantly better fixation stability
respect to patients without antipsychotic therapy. In fact no
significant differences were found when group A was compared with
HS. This suggests the possible influence of antipsychotic drugs in
improving quality of fixation in MDD patients.
Commercial Relationships: Serena Fragiotta, None; Pier Luigi
Grenga, None; Daniela Domanico, None; Alessandro Cutini,
None; Stefano Valente, None; Vittoria De Rosa, None; Enzo M.
Vingolo, None
Translational Regulation of RPE65 Expression by microRNA
William Samuel1, R K. Kutty1, Todd Duncan1, Cynthia Jaworski1,
Toshifumi Hara2, Ashish Lal2, T. Michael Redmond1. 1Laboratory of
Retinal Cell and Molecular Biology, National Eye Institute / National
Institutes of Health, Bethesda, MD; 2Genetics Branch, National
Cancer Institute / National Institutes of Health, Bethesda, MD.
Purpose: The retinal pigment epithelium (RPE) performs functions
critical to the process of vision. In particular, RPE65, retinol
isomerohydrolase, plays an important role in the regeneration of
chromophore 11-cis retinal. Expression of both RPE65 mRNA and
protein is high in fresh RPE cells. However, RPE65 protein
expression is lost in RPE primary culture while RPE65 mRNA
remains. MicroRNAs (miRNAs) can regulate gene expression in
eukaryotes by targeting mRNA for translational repression by
binding to the 3’ UTR region. We wished to determine whether
miRNAs play a role in the translational repression of RPE65 in RPE
primary cultures by binding to the 3’UTR of RPE65.
Methods: The full-length 3’-UTR of human RPE65 was cloned into
psiCHECK2 reporter vector 3’ to the Renilla Luciferase coding
region. The possible binding of miRNAs to target sites in the 3’-UTR
of human RPE65 was identified by TargetScan. RPE65 3’-UTR
reporter construct was transiently transfected into human RPE cells
(ARPE-19) grown in 24-well cell culture plates. Human miRIDIAN
miRNA mimics (Dharmacon) based on TargetScan were also
transfected along with human RPE65 3’-UTR construct. psiCHECK2
was used as a control vector. The luciferase assay was performed
after 48 hr to identify miRNAs that target RPE65 3’-UTR.
Results: Several miRNAs were predicted to bind to the 3’-UTR of
human RPE65 by TargetScan bioinformatics analysis. These included
miR-210, miR-181 and miR-374. We have found these miRNAs to
be expressed in bovine RPE primary cultures. The human RPE65 3’UTR reporter construct showed luciferase activity when the reporter
vector was transfected into ARPE-19 cells. Of tested miRNA mimics,
miR-210 decreased the luciferase activity of the reporter when cotransfected with the human RPE65 3’-UTR reporter construct. In
addition, a moderate decrease in the luciferase activity of RPE65 3’UTR reporter construct was also observed with miR-181a among the
miR-181 family.
Conclusions: We show that miR-210 and 181a decrease the
luciferase activity of the RPE65 3’-UTR reporter construct in ARPE19 cells, suggesting that these miRNAs could potentially regulate the
expression of RPE65 by translational repression by binding to its 3’UTR. These results may explain why RPE65 protein disappears in
RPE primary cultures, though its mRNA persists.
Commercial Relationships: William Samuel, None; R K. Kutty,
None; Todd Duncan, None; Cynthia Jaworski, None; Toshifumi
Hara, None; Ashish Lal, None; T. Michael Redmond, None
Support: Intramural Research Program of the National Eye Institute,
National Institutes of Health
Decreased Protein Expression of P2X2 in the Retina of Type 1
Diabetic Rats Treated with Suramin and PPADS
Juan E. Gallo2, 1, Jorge Mancini2, 1, Gustavo A. Ortiz2, Juan O.
Croxatto3. 1Ophthalmology, Hospital Universitario Austral, Pilar,
Argentina; 2Nanomedicine & Vision Group, Universidad Austral,
Pilar, Argentina; 3Ocular Pathology, Fundacion Oftalmologica
Argentina "Jorge Malbran", Buenos Aires, Argentina.
Purpose: We have previously shown that inflammatory markers and
P2X2 were up-regulated in the retina of diabetic rats. The aim of this
study was to evaluate the protein expression of P2X2 in the retina of
diabetic animals treated by purinergic antagonists
Methods: Sixteen male Wistar rats of 250gr were treated with an
intraperitoneal injection of 45mg/kg of streptozotocin. Only animals
with glycemia levels above 200mg/dl were included in the study. The
treated animals were divided into three groups of 4 rats. These were
intraperitoneally injected with: 1) Suramin, 2) PPADS, 3) Suramin +
PPADS at 9 and 26 weeks of diabetes in each group. Four remaining
diabetic animals received no treatment. Twelve non-diabetic rats with
the same age of diabetics were used as a control group. Animals were
sacrificed after 38 weeks of diabetes. Retinas were analyzed by
Western Blot using a primary antibody against P2X2.
Results: Protein expression of P2X2 was much higher in diabetics
without treatment than in controls. Diabetic rats treated with
Suramin, PPADS and the combined compound had a significantly
lower expression of P2X2 than that found in diabetic animals without
treatment.
Conclusions: P2X2 receptor seems to play a role in diabetic
retinopathy development. This purinergic receptor could be a target
for the treatment of this disease.
Commercial Relationships: Juan E. Gallo, EP2186529 B1 (P);
Jorge Mancini, None; Gustavo A. Ortiz, None; Juan O. Croxatto,
None
Intracellular pH of Retinal Ganglion Cells During Systemic
Hypoxia
Verleen K. McSween, Suresh Viswanathan, Joseph A. Bonanno,
Stephen A. Burns, Shimin Li. School of Optometry, Indiana
University, Bloomington, IN.
Purpose: Previously we showed that intracellular pH of retinal
ganglion cells (RGCs) decreases as a result of acute elevation of
intraocular pressure (IOP) (McSween et al. ARVO 2012). The
purpose of the current study was to provide confirmatory data by
examining other perturbations, such as systemic hypoxia, that should
also affect RGC pH.
Methods: RGCs were retrograde labeled in anesthetized adult male
Brown Norway rats by superior colliculus injection of 10kD Alexa
Fluor 790 dextran and the pH sensitive dye 2'-7'-bis (carboxyethyl)5(6)-carboxyfluorescein (BCECF) dextran (30 mg/ml, 20 µl). Retinal
fluorescence was measured with a confocal scanning laser
ophthalmoscope (HRA2, Heidelberg Engineering®) before, during
and after breathing 10% oxygen (90% nitrogen) for 4 minutes via a
nose cone at 2 weeks following dye injections. The retinal images
were processed using custom MATLAB software and the
fluorescence of individual cells was analyzed using the MetaMorph®
image analysis software.
Results: The fluorescence intensity of 517 double labeled cells was
tracked. The intensity of Alexa790 labeling was not significantly
different before, during and after 10% oxygen breathing. The
intensity of BCECF labeling reduced by 19+11% during 10% oxygen
breathing relative to breathing room air (p<1x10-10) and returned to
97+14% of control intensity on restoration to breathing room air
(p<4x10-7).
Conclusions: Retinal hypoxic changes arising from systemic hypoxia
can be reliably measured in vivo. The changes seen are reversible and
support our previous report that acute elevation of IOP decreases
RGC intracellular pH.
Commercial Relationships: Verleen K. M

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group

Transcription

Similar documents

Practice Brochure - Arizona Retina Institute

Bionic Eyes - Hansma Lab

Retinal Toxicity - Healthcare Professionals

Trauma for the OD: A Case Management Approach

arvo ojala - Inga Ojala

Retinal Arterial Macroaneurysm

Retinal Manifestations in Familial Lipoprotein Lipase Deficiency

LECTURE 9 Posterior Pole retina pathology_HTN_DM retinopathy

the monaciano symposium - Fondo Studenti Italiani

Installation of Scrolling Ceiling