Acceleration of hematopoietic reconstitution with a synthetic

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1996 87: 581-591
Acceleration of hematopoietic reconstitution with a synthetic
cytokine (SC-55494) after radiation-induced bone marrow aplasia
AM Farese, F Herodin, JP McKearn, C Baum, E Burton and TJ MacVittie
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Acceleration of Hematopoietic Reconstitution With a Synthetic Cytokine
(SC-55494) After Radiation-Induced Bone Marrow Aplasia
By Ann M. Farese, Francis Herodin, John P. McKearn, Charles Baum, Earl Burton, and Thomas J. MacVittie
The synthetic cytokine (Synthokine) SC-55494 is a high-affinity interleukin-3 (11-3) receptor ligandthat stimulates
greater in vitro multilineage hematopoietic activity thannative IL-3, while inducing no significant increase in inflammatory activityrelative t o native IL-3. Theaim of this
study was
t o investigate the in vivo hematopoietic response of rhesus
monkeys receiving Synthokine after radiation-induced marrow aplasia. Administration schedule and dose of Synthokine were evaluated. All animals were total-body irradiated
gamma radiation on day 0. Beginning
(TBI) with 700 cGy 8oCo
on day 1, cohorts of animals (n = 5) received Synthokine
subcutaneously (SC) twice daily with 25 p g l k g l d or 100 p g l
kg/d for23 days or 100 p g l k g l d for 14 days. Control animals
(n = 9) received human serum albumin SC once daily at 15
p g l k g l d for 23 days. Complete blood counts were monitored for60 days postirradiation andthe durations of neutropenia (NEUT; absolute neutrophil count IANCI<500/pL) and
thrombocytopenia (THROM; plateletcount
<2O,OOO/pL)
were assessed. Synthokine significantly ( P < .05) reduced
the duration ofTHROM versus the HSA-treated animals regardless of dose or protocol length. The most strikingreduction was obtained in theanimals receiving 100 p g l k g l d for
23 days (THROM = 3.5 W 12.5 days in HSA control animals).
Although theduration of NEUT was not significantly altered,
the depthof the nadir was significantly lessened in all animal
cohorts treated with Synthokine regardless of dose versus
schedule length. Bone marrow progenitor cell cultures indicated a beneficial effect of Synthokine on the recovery of
granulocyte-macrophage colony-forming units thatwas significantly higher a t day 24 post-TB1 in both cohorts treated
at 25 and 100 p g l k g l d for 23 days relative t o the control
animals. Plasma pharmacokinetic parameters were evaluated in both normaland irradiated animals. Pharmacokinetic
analysis performed in irradiated animals after 1 week of
treatment suggests an effect of repetitive Synthokine schedule andlor TB1 on distribution andlorelimination of Synthokine. These data show that the
Synthokine, SC55494, administered
therapeutically
post-TBI, significantly enhanced
platelet recovery and modulated neutrophil nadir and may
be clinically useful in the treatment of the rnyeloablated
host.
This is a US government work. There are no restrictions on
its use.
I
gand that has shown 10- to 20-fold greater biologic activity
than native IL-3 in human hematopoietic cell proliferation
and marrow colony-forming unit assays. Synthokine priming
of normal human peripheral blood leukocytes only induced a
twofold increase in histamine release and sulfidoleukotriene
(LTC,) synthesis versus human IL-3. The functional selectivity of Synthokine may be attributed to the greater binding
affinity for the (Y subunit of the (YIPIL-3 receptor complex
that Synthokine exhibited relative to native 1L-3.30The aim
of this study was to investigate the therapeutic efficacy of
Synthokine on hematopoietic reconstitution in a nonhuman
primatemodel
of high-dose, radiation-induced marrow
aplasia.
NTERLEUKIN-3 (IL-3) or multi-colony-stimulating factor (CSF) is a pivotal T-cell-derived cytokine in the
stimulation of hematopoiesis. In vitro studies have shown
that IL-3 stimulated the proliferation of early multipotential
progenitors as well as the growth and differentiation of lineage-restricted colony-forming cells.”8 Thus, IL-3 has been
reported to stimulate both megakaryocyte progenitor cells
and megakaryocytes. Variable responses to native IL-3 in
normal monkeys predicted an equivocal response in preclinical and clinical evaluations in situations of marrow failure
syndromes and radiation or chemotherapy-induced marrow
a~lasia.’”~
Gillio et all5 showed that, although IL-3 enhanced
myeloid recovery in cyclophosphamide- or 5-fluorouracil
(W)-treated nonhuman primates, platelet recovery could
not be shown. In contrast, it has been shown that IL-3 enhanced regeneration of platelets and reduced the duration of
thrombocytopenia while showing no effect on recovery of
neutrophils in a nonhuman primate model of radiation-induced marrow aplasia.I6.l7Recently, Winton et all8 showed
that once daily administration of IL-3 in a nonhuman primate
model of hepsulfam-induced pancytopenia had no demonstrable effect on regeneration of platelets or neutrophils.
Clinical studies using IL-3 in marrow failure syndromes or
after chemotherapy with or without bone marrow transplantation have shown modest but variable activity in accelerating hematopoietic
Therefore, despite its
therapeutic potential, native IL-3 is characterized by a relatively narrow therapeutic index due to the intrinsic inflammatory activity of this cytokine. The structure activity relationships of IL-3 have been explored to perform rational
design of clinically useful derivatives that would enhance
the therapeutic index by reducing hematopoietic cytopenia
as well as associated t o x i ~ i t y . * The
~ - ~ ~synthetic cytokine,
Synthokine (SC-55494),
is a high-affinity IL-3 receptor liBlood, Vol 87, No 2 (January 15). 1996: pp 581-591
MATERIALS AND METHODS
.4nimals
Domestic-bornmalerhesusmonkeys,
Macaca mulatta, with a
mean weight 3.9 2 0.2 kg, were housed in individual stainless steel
From Experimental Hematology. Armed Forces Radiobiology Re.search Institute, Bethesda, MD; the University of Maryland Cancer
Center, Baltimore, MD; Centre de Recherches Du Service de Santd
Des Armdes, Grenoble, France; and Searle R & D, Monsanto CO,
St Louis, MO.
Submitted May 26, 1995; accepted September 1, 1995.
Address reprint requests to AnnM. Farese, MS, University of
Maryland Cancer Center, I O S Pine St, MSTFRoom G-05, Baltimore, MD 21201-1192.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
This is a US government work. There are no restrictions on its me.
0006-4971/96/8702-0030$0.00/0
58 1
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582
cages in conventional holding rooms at the ArmedForces Radiobiology Research Institute (AFRRI) in an animal facility accredited by
the American Association for Accreditation of Laboratory Animal
Care. Monkeys were provided with 10 air changes per hour of 100%
fresh air, conditioned to 72°F ? 2°F with a relative humidity of SO%
? 20% and were maintained on a 12-hour lighudark full spectrum
light cycle, with no twilight. Monkeys were provided with commercial primate chow, supplemented with fresh fruit and tap water ad
libitum. Research was conducted according to the principles enunciated in the Guide for the Care and Use of Laboratory Animals,
prepared by the Institute of Laboratory Animal Resources, National
Research Council.
FARESE ET AL
Hemutologic Evaluations
The animals were randomly assigned to one of four treatment
groups composed of a minimum of five animals. Each animal was
irradiated on day 0. On day 1, groups of animals received Synthokine
(100 pg/kg/d, SC, twice daily [BID] for 14 consecutive days or 100
pgkgld or 25 pg/kg/d, SC, BID for 23 consecutive days) or HSA
( I S pg/kg/d, SC, every day [QD] for 23 consecutive days).
Peripheral blood. Peripheral blood was obtained from the saphenous vein to assay complete blood (Model S Plus 11; Coulter Electronics, Hialeah, FL) and differential counts (Wright-Giemsa Stain;
Ames Automated Slide Stainer, Elkhart, IN). Baseline levels (BL)
were obtained before irradiation. These parameters were monitored
for 60 days after irradiation and the durations of neutropenia (absolute neutrophil count [ANC] <500/pL) and thrombocytopenia
(platelet [PLT] count <20,00O/pL) were assessed. Whole blood
transfusions could have possibly altered the ANC and PLT count;
therefore, when determining the durations of neutropenia and thrombocytopenia, ANC and PLT counts had to be maintained for 3 consecutive days above threshold levels after the first increase for a true
recovery to be noted.
Bone marrow. Approximately 2 mL ofbonemarrowwas aspirated from the humerus and/or iliac crest of anesthetized primates
(ketamine, 10 mgikg administered intramuscularly with a 2 I -guage
needle in 0.3 mL volume) into preservative-free heparinized syringes. Low-density (<1.077 g/mL) mononuclear cells (MNCs) were
separated using Histopaque (Sigma, St Louis, MO) and resuspended
in Iscove's modified Dulbecco's medium (IMDM; GIBCO, Grand
Island, NY). The clonogenicity of bone marrow progenitor cells was
assayed in a short-term culture assay. Culture medium contained
0.9% methylcellulose (Methocult H4230; Stem Cell Tech, Vancouver, British Columbia, Canada) in IMDM with 30% fetal calf serum
(Hyclone Laboratories, Logan, UT). In addition, a combination of
recombinant human cytokines (granulocyte colony-stimulating fdctor [G-CSF] at S ng/mL; Amgen, Thousand Oaks, CA), mastcell
growth factor (MGF; SO ng/mL; Immunex Corp. Seattle, WA),
eythropoietin (EPO; 2 UlmL; Behringwerke, Marburg, Germany),
1L-3 (20 ng/mL), granulocyte-macrophage colony-stimulating factor
(GM-CSF; S ng/mL), and IL-6 (40 nglmL; Sandoz Pharmaceuticals,
East Hanover, NJ), and transferrin ( I O pg/mL; Sigma) were added.
MNCs were cultured at a plating density of 5 X IO4 cells/mL (days
0,24, and 46 postirradiation) or I X 10' cells/mL (day 16 postirradiation). Cells were incubated for I O days at 37°C with 5 % COz in air
in a fullyhumidified incubator. Granulocyte-macrophage colonyforming unit (GM-CFU)- and burst-forming unit-erythroid (BFUe)derived colonies (>50 cells) were expressed as the number of CFU/
10' MNCs.
Pharmacokinetic Study Design
Pharmacokinetic Analysis
Normal rhesus monkeys (n = 2/group) received 25 p g k g intravenous (IV) bolus doses of either Synthokine or native human IL-3
via a spahenous or cephalic vein, and plasma samples were collected
at intervals over a S-hour time frame. A second pharmacokinetic
study was performed on animals in the therapeutic study before (day
-6) and day 7 postirradiation. These animals (n = 2) received 25
pglkg, SC bolus doses of Synthokine, and plasma samples were
collected at intervals over an 8-hour time frame.
Affinity-purified goat anti-Synthokine antibody and affinity-purified goat antinative human IL-3 antibody wereused in sandwich
enzyme-linked immunosorbent assays (ELISAs) to determine Synthokine and native human IL-3 plasma levels. Synthokine or native
human IL-3 in plasma was bound in microtiter plate wells precoated
with the appropriate goat antibody. The plates were washed, incubatedwith additional goat antibody conjugated with peroxidase,
washed again, and allowed to react with peroxidase substrate and
the optical denisty (OD) of the resulting peroxidase productwas
determined. The plasma determinations were performed using nonvalidated assays with standard curve range lower limits of 1 ng/mL.
Irradiation
Monkeys placed in an Lucite restraining chair, after a prehabituation period, were bilaterally, total-body irradiated (TBI) with cobalt60 gamma radiation atthe AFRRI Cobalt-60 Facility to a total
midline dose of 700 cGy at a dose rate of 40 cGy/min. Dosimetry
was performed using a paired 0.5-mL tissue equivalent ionization
chamber, whose calibration was traceable to the National Institute
of Standards and Technology.
Recombinant Cytokines
Synthokine, SC-55494, is an IL-3 receptor agonist consisting of
1 12 amino acids. This Escherichia coli-derived protein has 21 amino
acids deleted from the N- and C-terminal regions, in addition to 27
amino acid position changes relative to native IL-3.3"The Synthokine
orthe control protein (human serum albumin [HSA]; Miles Inc,
Cutter Biological. Elkhart, IN) was administered as a l-mL bolus
subcutaneous (SC) injection.
Radiation Study Design
Clinical Support
An antibiotic regimen was initiated prophylactically when the
white blood cell count (WBC) was less than I,M)O/pL andcontinued
daily until the WBC was greater than 1,000lpL for 3 consecutive
days. Gentamicin (Lyphomed, Deerfield, IL; 10 mg/d, SC, QD) and
rocephin (Roche, Nutley, NJ; 250 mg/d, SC, QD) were administered.
Fresh, irradiated (1,500 cGy '"CO gamma radiation) whole blood
(approximately 30 mL/transfusion) from a random donor pool (monkeys of > 10 kg body weight) was administered when the PLT count
was less than20,00O/pLandthe
hematocrit was less than18%.
Transfusions and antibiotics were required to ensure 100% survival
in HSA-treated animals (unpublished results).
Detection of Rhesus Monkey-nti-Synthokine I g
Antibody titers in plasma samples from Rhesus monkeys dosed
with 100 p g k g BID Synthokine were determined by capture ELISA.
Samples were tested from days 0; 6, 7, 8, or 9; 13 or 14; 20; and
29,30, or 3 1. Ninety-six-well microtiter platers (Dynatech-Immuion
11, Chantilly, VA) were coated with goat-anti-Synthokine affinitypurified polyclonal antibody. Eachwell received 150 pL of 100
mmoVL NaHCO, (Fisher Scientific, Pittsburgh, PA), pH 8.2, containing 5 pg/mL goat-anti-Synthokine polyclonal antibody. Plates
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583
SC55494, SYNTHOKINE THERAPY IN IRRADIATED PATIENTS
Table 1. Plasma Pharmacokinetic Parametersfor Synthokine and
Native IL-3 After Intravenous Administration of 25 pglkg
to Rhesus Monkeys
Pharmacokinetic Parameters*
Synthokine
Elimination half-life b i n )
5.51
Clearance (mUmin/kg)
V, (Ukg)
46.1
4.38
0.058
0.282
95.8
V~ma
AUC (ng/mUhr)(0-4
77.6
.V
Abbreviations: V,, volume of central compartment; ,
AUC.
*Average values for three monkeys.
using the Dunnett’s Test. A log transformation was used to correct
for variance differences. These tests were performed using the software package SYSTAT (SYSTAT, Inc. Evanston, IL).
Native IL-3
52.5
0.078
0.408
dose/
(Ke~NminatNon)
were incubated overnight at room temperature in a chamber maintaining 100% humidity. The following morning, wells were emptied
and remaining reactive binding sites on the plastic surface of each
were well blocked for 1 to 1.5 hours at 37°C (Precision Gravity
Convection Incubator, Chicago, IL) in 100% humidity with 200 pL
of 10 mmol/L phosphate-buffered saline (PBS; GIBCO), pH 7.4,
containing 3% bovine serum albumin (BSA; Sigma) and 0.5% polyoxyethylene-sorbitan monolaurate (Tween 20; Sigma). Wells were
emptied and washed by filling each well four times with 150 mmoV
L NaCl containing 0.05% Tween 20 (wash buffer). For each sample
evaluated, one setof duplicate wells received 150 pL of dilution
buffer (10 mmol/L PBS, pH 7.4, 0.1% Tween 20) containing 1 pgl
mL of pharmaceutical grade recombinant human G-CSF (rhG-CSF
Amgen) as a control. A second set of duplicate wells received 150
pL of dilution buffer containing 1 pg/mL Synthokine. Plates were
incubated for 2.5 hours in a humidified chamber at 37°C. Wells were
emptied and each well was washed four times withwash buffer.
Dilutions of plasma samples were then added to their respective
plates. Rows A through G of each plate received 150 pL of dilution
buffer. Row H received 200 pL of plasma diluted 1: 100. Fifty microliters was removed from row H and titered 1:4 through row A in a
serial fashion to cover a dilution range from 1:100 to 1:1,638,400.
Plates were incubated for 1.5 hours in a humidified chamber at 37°C.
Plates were emptied and each well was washed four times with wash
buffer. Each well received 150 pL of dilution buffer containing
0.1 pg/mL of peroxidase-conjugated goat-anti-rhesus monkeyIg
(Nordic Immunologicals, Capistrano Beach, CA). Plates were incubated for 1.5 to 2 hours at 37°Cin a humidified chamber. Plates
were emptied and each well was washed four times with washbuffer.
Each wellreceived 150 pL of TMB peroxidase substrate (Kirkegaard
and Perry, Gaithersburg, MD). Plates were developed at room temperature for 10 minutes and read on an ELISA reader (Titertek; ICN
Medical, Costa Mesa, CA) at a test wavelength of 650 nmol/L.
Plasma antibody titers were calculated for each sample after subtracting the average OD of duplicate wells receiving rhG-CSF plus
plasma from the corresponding average OD of duplicate wells receiving Synthokine plus plasma.
Statistical Analysis
The Normal Scores Test was used to make painvise comparisons
of the durations of neutropenia and thrombocytopenia. The test was
performed using the software package StatXact (Cytel Software
C o p , Cambridge, MA). The Mann-Whitney test was used to evaluate the statistical significance between the nadirs. Bone marrowderived clonogenic activities were analyzed by two methods. Comparisonof treatment days post-TB1 toa historical baseline were
made as aone-sided randomization t-test and a Bonferroni correction
factor of 3 was applied to the P values. The comparison of treated
groups versus the time-matched, HSA-treated controls were made
RESULTS
Pharmacokinetics of Synthokine in Normal and Irradiated
Monkeys
The results indicated similar pharmacokinetic behavior of
Synthokine and native IL-3 after IV injection in the rhesus
monkey. The half-life was somewhat shorter (TlI2= 46.1
minutes v 52.5 minutes), but clearance was slower (4.38 mLl
midkg v 5.51 mL/min/kg) for Synthokine compared with
native IL-3 (Table 1).
The bioavailability of Synthokine administered SC in the
rhesus monkey in this study cannot be calculated directly
because the IV and SC doses were administered to separate
groups of monkeys. However, comparison of the IV and
SC area under the curve (AUC) values indicates a bioavailability of approximately 100%. Synthokine was detected
in the plasma collected from 0.25 to 8.0 hours after SC
administration of 25 pglkgldose. The average CMAX in normal monkeys was 25.7 ng/mL, whereas the average TMAX
and AUC were 1.3 hour and 106 ng/mL/h (0- to 8-hour
period; Table 1).
Plasma concentrations after SC administration to irradiated animals (measured at day 7 post-TBI) were between
two and three times higher than values determined in the
same animals before irradiation (Table 2). The average CMAX
on day 7 after TB1 was 55.9 ng/mL, whereas the average
TMAX and AUC were 1.S hour and 285 ng/mL/h, respectively,
relative to values for CMAX of 25.7 ng/mL, TMAX of 1.3 hour,
and AUC of 106 ng/mL/h for Synthokine administered to
animals before radiation exposure.
ELISA Determination of Rhesus Monkey-Anti-Synthokine
Plasma Ig
There were no antibody titers to Synthokine detected at
any evaluated time points post-TB1 in the animals receiving
100 pg/kg/d for either the 14-day or 23-day schedule.
Modulation of Thrombocytopenia
Thrombocytopenia (PLT <2O,OOO/pL) was evident in
HSA-treated control animals for an average of 12.5 days
(days 10 through 22) consequent to 700 cGyTB1 (Fig 1A
Table 2. Plasma Pharmacokinetic Parametersfor Synthokine
(SC-5545)After SC Administration of 25 pglkg toRhesus Monkeys
Pharmacokinetic Parameters”
Dose W k g ) 25
55.9
Cmax (ng/mL)
Tmax (h)
285 h)
AUC (ng/mUh)(0-8
Day -6
25
25.7
1.3
106
Day + l
1.8
* Average values for 2 monkeys. Monkey received TB1 with @‘CO
gamma radiation to a total midline dose of 700 cGy on day 0. Each
animal received a 25 pglkg SC dose of Synthokine on day -6 (before
TBI) and day +7 (after TBI). Each animal also received 25 pg/kg SC
doses of Synthokine twice daily on days 1 through 6.
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-
584
A
0-0
1000 E
FARESE ET AL
Synthokine(100pg/kg/d) (dl-23)
Synthokine (100pg/kg/d) (dl -14)
HSA (1 5 pg/kg/d)
100
20
10
1 II
01
I
I l l
4 6 8
I
l l l l l l l l l l l l l l l l l l l l / , l l l / ~
12 14 16 18
20
22
24
26
28
30
40
60
"""""""
Cytokine Administration
8
0-0
1000 F
-
Synthokine (l00 pg/kg/d) (dl-23)
Synthokine (25 pg/kg/d) (dl -23)
HSA (15 pg/kg/d)
100
20
10
1
L
-6b
b\
I
' 112' i4'
S
I 'I's' do' d2' ii l 6
Cytokine Administration
Time (days) Post Irradiation
and Table 3). Administration of Synthokine at 25 or 100 pgl
kg/d for 23 consecutive days post-TB1 significantly reduced
the duration of thrombocytopenia relative to HSA-treated
controls to 7.4 days ( P = .019 ) and 3.5 days (P = .OOl),
respectively (Fig 1A and Table 3), whereas the 100 p&g/
d dose administered for only 14 consecutive days post-TB1
reduced the thrombocytopenic period to 6.2 days ( P = .003;
Fig 1B and Table 3). The administration of Synthokine for
23 days at the 100 pg/kg/d dosage produced a reduction in
the duration of thrombocytopenia thatwassignificantly
greater than both the 14-day protocol at the same dose (3.5
v 6.2 days, P = .048)and the same 23-day protocol at the
lower dosage of 25 pg/kg/d (3.5 v 7.4 days, P = ,028). There
i 0O<*'
Fig 1. (A and B) Regeneration
of the circulating platelets after
sublethal irradiation ofrhesus
monkeys treated with HSAor
Synthokine. Synthokine was administeredat dosagesof 100 p g l
kgld for either 14 days (A) or 23
days (A and B) or 25 pglkgld for
23 days (B). HSA was administered for 23 days (A and B). The
profilesfor platelet responses to
Synthokine at 100 pg/kg/d for
23 days and HSA are repeated in
IB) for comparison.Counts are
the mean f SEM.
was no significant ( P = .187) difference in the duration of
thrombocytopenia when Synthokine was administered for 14
days at 100 pg/kg/d (6.2 days) versus 25 pgkgld dose for
23 days (7.4 days).
The administration of Synthokine, regardless of treatment
protocol, accelerated platelet recovery, with preirradiation
baseline levels being attained within approximately 28 days
after exposure. In contrast, HSA-treated controls required
approximately 40 days to attain baseline levels (Fig 1A).
The platelet nadir was significantly decreased relative to that
of the HSA-treated controls only in the cohort of animals
treated with Synthokine at 100 pg/kg/d for 23 consecutive
days.
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SC55494.SYNTHOKINETHERAPY
585
IN IRRADIATEDPATIENTS
Table 4. The Effect of Synthokine Administration onthe
Transfusion Requirements of Rhesus Monkeys Irradiated
at 700 cGy "CO
Table 3. The Effect of Synthokine Administration on the Duration
of Thrombocytopenia and Neutropenia in Rhesus Monkeys
Irradiated at 700 cGy "C0
HSA
Synthokine
d 1-23 BID
Thrombocytopenia
Neutropenia
d 1-14 BID
d 1-23 BID
d 1-23 QD
6.2d'
15.7 d
7.4 d*
14.2 d
12.5 d
14.9 d
3.5dit
14.1 d
25pg.
d 1-23
25 pglkgld
15
pglkgld
~-
100 pglkgld
Monkeys whole body irradiated with %
' o gamma radiation were
treated with control protein (HSA) or Synthokine according to protocol. Neutropenia is defined as an ANC less than 5001bL. Thrombocytopenia is defined as a platelet count of less than 20,OOOlpL.
x Statistically significant difference from the HSA-treated controls.
t Statistically significant difference from the 100 pglkgld, 14-day
protocol and the 25 pglkgld, 23-day protocol.
Modulation of Red Blood Cells and Transfusion
Requirements
Synthokine administration at 100 pgkgld for 23 days induced a significant increase (P s .05)in the nucleated red
blood cell count (NRBC) between days 20 and 23 post-TB1
versus the HSA-treatedcontrols (Fig 2). The 14-day administration of Synthokine at 100 pgkg/d significantly increased
the NRBC between days 18 and 23 post-TBI. There was no
evidence of clinical bleeding in any particular group of animals. All animals did not require transfusions. The HSA and
25 pgkgld Synthokine groups received an average of 1.5
transfusions per animal, whereas the two100 pgkgld cohorts
Synthokine Synthokine
Synthokine
Total no. of transfusions
215
per group
No. of animals receiving
transfusions per 215
group
No. of transfusions per
0.4
animal
100pg.
d 1-23
715
719
415
1.4
1.6
1OOpg.
d 1-14
HSA
415
14B
415
0.8
Monkeys whole body irradiated with mCo gamma radiation were
treated with control protein (HSA) or Synthokine according to protocol. Animals received transfusion of fresh, irradiated (1,500 cGy %o)
whole blood (approximately
30 mUtransfusion) froma random donor
pool when the platelet count was less than 20,OOO/hL and the hematocrit was less than 18%.
treated for 14 or 23 consecutive days received an average of
less than one transfusion per animal (Table 4).
Modulation of Neutropenia
Although the duration of neutropenia (ANC <500/pL)
was not significantly altered with Synthokine treatment ( P
> SO), the depth of neutrophil nadir was lessened in all
cohorts of animals treated with Synthokine relative to the
HSA-treated control animals (Fig 3A and B and Table 3).
The mean duration of neutropenia was 14.9 days in HSAtreated animals versus 14.2 and 14.1 days in the 25 pg/kg/
-
HSA
70 -
*
60 0 50
0 40
0
.
r
30 73
m
[r
z 20
10 -
Synthokine
(100 @kS/d, d l -23)
O-"O
Synthokine
(100 pS/kg/d, d l -14)
5
Fig 2. Number of NRBCper
100 WBCs in the peripheral
blood of irradiated rhesus monkeys treated with HSAorSynthokine. Synthokine was administered at dosages of either 100
pglkgld for either 14 days or 23
days IAl or 25 pglkgld for 23
days 16). HSA was administered
for 23 days. Counts are mean values.
-
0
01
I
4 6 8
12
14 16 18 20
22
24
26
Cytokine Administration
" " " _"
30
34
Time (days) Post Irradiation
38
42
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586
FARESE ET AL
c" -
100
W
0-0
10
h
Synthokine (100 pg/kg/d) (dl -23)
Synthokine (100 yg/kg/d) (dl -14)
HSA (15 pg/kg/d)
3.
2
0
v
X
1
0.5
W
a
0.01
I I l l
0 tI I
01 64
8
I
I
I
I
l l l l I l I I 1
12 14 16 1826
24
22
20
I l l
'
28 30
/
,
I
40
60
"""""""
Cytokine Administration
Synthokine (100 pg/kg/d) (dl-23)
Synthokine (25 pg/kg/d) (dl -23)
H HSA (15 pg/kg/d)
H
O0
F
0-0
B
10
c
'
=L
?c
0
X
1
0.5
W
0
z
a
0.1
0.01
-
J
50
Cytokine Administration-
Time (days) Post Irradiation
d and 100 yglkgld animals treated for 23 consecutive days
and 15.7 days for the cohort treated with 100 yglkgld for
only 14 consecutive days. Treatment with Synthokine did
not accelerate recovery of neutrophils to preirradiation ievels
relative to the HSA-treated animals (Fig 3A, B) and did not
modify the antibiotic requirements during the neutropenic
period.
Effect of Synthokine Administration on Bone Marrow
Progenitor Cell Recovery
The bone marrow-derived clonogenic activities for BFUe
and GM-CFU were signifcantly ( P < .05) depressed through
day 46 post-TB1 in the HSA-treated controls (Fig 4A and
B). In contrast, the BFUe and GM-CFU values for all Synthokine treatment protocols recovered to BL levels no later
Fig 3. (A and B) Regeneration
of the circulating neutrophils
after sublethal irradiation of rhesus monkeys treated with HSA
or Synthokine. Synthokine was
administered at dosagesofeither 100 pglkgld for either 14
days (A) or 23 days (A and B) or
25 pglkgld for 23 days (B). HSA
was administered for 23 days (A
and B). The profiles for neutrophil responses to Synthokine at
100 pglkgld for 23 days and
HSA are repeated in (B) for comparison. Counts are the mean f
SEM.
than day 46 post-TB1 (Fig 4A and B). The 23-day administration of synthokine at either 25 or 100 yg/kg/d significantly
increased ( P 5 .OS) the GM-CFU activity versus the timematched, HSA-treated controls by day 24 post-TB1 (Fig 4B).
The animals receiving the shortened Synthokine protocol (14
days) also displayed increased GM-CFU activity at day 24
post-TB1 versus the time-matched controls ( P = .06); however, a significant increase ( P = .03) wasnotnoteduntil
day 46 post-TB1 (Fig 4B). The BFUe activity in all three
Synthokine treatment groups was significantly ( P < .OS)
increased above the time-matched, HSA-treated controls at
day 46 post-TB1 (Fig 4A).
DISCUSSION
Hematopoietic growth factors such as GM-CSF, IL-3, IL6, IL-11, leukemia inhibitory factor (LIF), and megakaryo-
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SYNTHOKINE SC55494,
PATIENTS
IN IRRADIATED
587
t
BFUe
T
HSA
Fig 4. (A and B) Bone marrow-derived concentration of BFUe and GM-CFU after sublethal irradiation
of rhesus monkeystreated with HSA or Synthokine.
The (A) BFUeand (B) GM-CFU concentrations observed in normal rhesus primates baseline (BL) and
irradiated animals receiving Synthokine or HSA as
described in the Materials and Methods on days 16,
24, and 46 post-TBI.
Clonogenic
concentrations
are
reported as the mean 2 SEM of the CFUper lo5
MNCs.
25
t
T
100
(dli23)
100
(dl-23)
(dl-23)
(dl-23)
(dl-1 4)
Dose Synthokine (pg/kg)
to structure function relationships and improving its clinical
therapeutic index.
The synthetic cytokine, Synthokine, is a high-affinity IL3 receptor ligand that has shown greater in vitro multilineage
growth factor activity than native L - 3 while inducing no
CSF and GM-CSF have shown therapeutic efficacy in accelsignificant increase of inflammatory activity. The enhanced
erating production of n e ~ t r o p h i l s . ~Thrombocytopoietic
binding to the CY subunit has been implicated as a key deter.~~
efficacy in clinical situations of marrow failure has not been
minant of Synthokine's increased in vitro biologic activity.
realized for GM-CSF, whereas IL-3 and L 6 efficacy has
The purpose of this study was to evaluate the efficacy of
been questionable due to their low therapeutic indices and
Synthokine in decreasing the time to recovery from neutroIL-I 1 is currently in clinical
penia and thrombocytopenia in a nonhuman primate model
of radiation-induced marrow aplasia. The protocols evaluThe demonstrated in vitro efficacy of IL-3 as a growth
ated two doses of Synthokine (25 v 100 pg/kg/d) adminisfactor for stimulation of multipotent and committed myeloid
progenitors suggested that itmayplay
a major role as a
tered over 23 consecutive days postirradiation, as well as
primer by increasing the action of more lineage-restricted
one dose (100 pg/kg/d) of Synthokine administered over
cytokines such as G-CSF, GM-CSF, L - 6 , and IL-I 1. Such
both 14-day and 23-day schedules relative to HSA-treated
control animals.
efficacy has recently been shown in preclinical models of
We showed that Synthokine was able to ameliorate the
radiation or drug-induced marrow apasia for the combination
clinically significant, radiation-induced nadirs in ANC and
of IL-3 with GM-CSF or L - 6 and forecast the efficacy of
PLT, as well as tosignificantly decrease the duration of
the IL-3/GM-CSF fusion protein, PIXY321.'6"8,77,78
In addition, L - 3 has been shown to promote survival of primitive
thrombocytopenia without significant toxicity or morbidity
murine and human hematopoietic progenitor ~ e l l s . ~ ~ - relative
~*
to HSA-treated controls. Neither the dose of SynBrandt et a1" recently showed that addition of IL-3 to culthokine nor the protocol schedule affected the duration of
tures of highly enriched human committed progenitor cells
neutropenia or recovery of ANC to baseline, preirradiation
delayed the appearance of morphologic changes and DNA
values. This occurs despite the fact that Synthokine adminisfragmentation patterns associated with apoptosis. The demtration accelerated the postirradiation recovery of marrowonstrated efficacy of L 3 in promoting increased proliferaderived GM-CFU progenitors relative to the control retion as well as survival of hematopoietic progenitor cells
sponse. This apparent discrepancy between clonogenic data
warranted further analysis of the growth factor with regard
and peripheral blood leukocyte counts could be explained
cyte growth and development factor (MGDF) or thrombopoietin (Tpo) have been shown to stimulate megakaryocyte
differentiation in vitro and enhance platelet production in
preclinical models of normal and radiation or drug-induced
marrow ap~asia.i.14.16-1X.31-63 The lineage-specific cytokines G-
From www.bloodjournal.org by guest on October 15, 2014. For personal use only.
FARESE ET AL
588
by the fact that the enhanced bone marrow GM-CFU recovery was observed from day 24 post-TBI. It would take approximately 10 days for these progenitors to differentiate
into mature leukocytes. At that time (day 30 to 3 1 postirradiation), the ANC appeared to be greater in the Synthokinetreated animals (100 pg/kg/d for 23 days) relative to the
HSA group. IL-3-stimulated proliferation of primate and
human marrow progenitors has been shown with administration schedules as short as 7 consecutive days, whereas longer
treatment schedules were required to stimulate variable levels of circulating neutrophils and platelet^.^.'^.^^.'^ These results suggested that the increase in mature cell lineages is
dependent on the interaction of IL-3-primed progenitors
with lineage-specific cytokine~.'~In
two previous studies, we
were unable to show the efficacy of native IL-3 in reducing
neutropenic duration or recovery of ANC to preirradiation
level^.^^,^' In contrast, Gillio et a l l 5 reported that IL-3 reduced
both neutrophil nadir and duration of neutropenia in cyclophosphamide-and 5-FU-treated cynomolgus primates,
whereas no increase in platelet production could be shown.
Winton et a1,I8 using hepsulfam-induced pancytopenia in
rhesus primates, could not show therapeutic efficacy of native IL-3 for restoring platelets or neutrophils. However, the
administration protocol (a single daily, SC injection) may
account for the ineffectiveness of the native IL-3 in this
model." It is of interest that our pharmacokinetic analysis
of Synthokine versus native IL-3 showed similar values and
suggests a BID protocol for effective SC administration. It
is also of interest that plasma concentrations of Synthokine
were between two to three times higher at day 7 after TB1
compared with their preirradiation values. These results indicate an effect of repetitive Synthokine administration (7 consecutive days) and/or of TB1 on distribution and/or elimination of Synthokine.
Administration of Synthokine significantly enhanced the
recovery of PLT to preirradiation levels and reduced the
duration of thrombocytopenia at both doses and schedules
used, as well as reduced the nadir of PLT at the highest dose
administered over the 23-day schedule compared with the
HSA-treated controls. We previously showed the efficacy of
native IL-3 in accelerating PLT recovery and reducing the
thrombocytopenic p e r i ~ d . ~Two
~ , ~ doses
'
(25 and 100 pg/kg/
d) of Synthokine were evaluated in this model because of
noted modest hematopoietic activity of native IL-3 and Synthokine in rhesus primate bone marrow-derived cell cultures
(attributable to 81% and 64% respective homology to rhesus
IL-3; unpublished results). van Gils et alX5have also shown
that native human IL-3 does not bind efficiently to rhesus
marrow-derived target cells. The 100 pg/kg/d dose reduced
the duration of thrombocytopenia to 3.5 days versus 7.4 days
for the 25 pg/kg/d dose relative to 12.5 days for the HSAtreated controls treated on the 23-day injection schedule.
These data showed that Synthokine significantly enhanced
platelet recovery and modulated neutrophil nadir after radiation-induced marrow aplasia and suggests thatit may be
clinically useful in the treatment of the myeloablated host.
The 14-day treatment schedule at 100 pg/kg/d was not significantly more efficient than a 23-day therapeutic schedule
at 25 pg/kg/d in terms of both the duration of thrombocyto-
penia (7.4 days in the 25 pg/kg/d for 23-day group v 6.2
days in the 100 pg/kg/d for 14-day group) and bone marrow
clonogenic activity. According to our data, the duration of
treatment (especially beyond day 14 post-TBI) could be of
critical importance to sustain recovery. We can hypothesize
that, 2 weeks postirradiation, the compartment of radiationdepleted hematopoietic progenitors such as granulocyte, erythroid, macrophage, monocyte CFU, GM-CFU, andBFUe
entered an active period of self-renewal and differentiation
to replenish the peripheral blood mature cell compartment.
The committed precursors should therefore be in a strong
cytokine-dependent state. Any interruption of the treatment
might induce a phenomenon of deprivation, similar to that
observed in vitro withbonemarrow-derived
cells, lL-3dependent cell line cultures, or hematopoietic progenitor
cells when removing IL-3 or kit-ligand, which could interfere
with hematopoietic r e c o ~ e r y . ~ " ~ This
' ~ ' ~ consideration
~~"
has
to be taken into account for the design of future clinical
trials. These data also suggest that combined protocols of
Synthokine with lineage-restricted cytokines such as G-CSF
or GM-CSF would provide the desired effect of enhancing
hematopoietic production of both platelets and neutrophils.
The demonstrated therapeutic efficacy of combined, concurrent native humanIL-3 and GM-CSF aswell as theIL3/GM-CSF fusion protein PIXY321 in a similar model of
radiation-induced marrow aplasia would forecast efficacy for
Synthokine and G-CSF.'h,7x
ACKNOWLEDGMENT
The authors thank Lisa Grab, Namita Varma, Richard Brandenburg, David Matlick, Michael Flynn, and Nelson Fleming for their
superb technical assistance; Dr Marie Rock for Synthokine and native IL-3 ELISA and antibody detection assays; and William Jackson
for assistance with the statistical analysis.
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