S. aureus - Global Engage

Affinium Pharmaceuticals
Microbiome Considerations Of
A Novel, Specific Spectrum,
Anti-Staphylococcal Antibiotic
Ed Mascioli, MD
CEO, Affinium Pharmaceuticals
October , 2013
Microbiome/Microbiota
R&D and Business Collaboration Forum
Page 1
San Diego, October 7-8, 2013
Bacterial Fatty Acid Biosynthesis System (FASII) is a
Selective Bacterial Target
Mammalian Fatty Acid Synthase (FAS)
•
Large single protein has complete
set of 7 catalytic domains
Bacterial Fatty Acid Synthesis Cycle (FASII)
•
Discrete enzymes
Mammalian Enoyl-ACP Reductase (ER)
Bacterial Enoyl-ACP Reductase (e.g. FabI)
AFN-1252 Human FAS1 IC50 >100 µM
AFN-1252 S. aureus FabI IC50 = 14 nM
No homology between the human FAS and bacterial FASII systems
October , 2013
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Sequence homology of 6 pathogens
mapped to S. aureus FabI structure
•
FabI sequence from six
pathogens are overlaid
•
The amino acid sequence in
the inhibitor binding site is
conserved and clearly defined
•
However differences between
orthologs exist in the shape
and volumes of the binding
pocket
•
Potential for developing
additional specific-spectrum
compounds
absolutely conserved
average conservation
highly variable
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Diversity of Enoyl-ACP Reductase Defines the
Specific Spectrum of FabI Inhibitors
•
Phylogenetic mapping clearly delineates
the Enoyl-ACP reductase isoforms
•
Species in bold have multiple forms of
enoyl-ACP reductase
•
FabI target is essential only in those
bacteria that have a unique FabI
isoform
•
FabI is unique and essential in
staphylococci
FabI
Unique FabI’s from other species are
FabL divergent
•
FabV
FabK
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Essentiality of FabI in Gram-Negative Species
• Endogenous fatty acid synthesis is highly essential in Gramnegative bacteria
 No exogenous fatty acid supplementation bypasses inhibition
 Lipid moieties of the Gram-negative outer membrane such as LPS are
unique and are dependent on endogenous Gram-negative fatty acid
biosynthesis cycle
• Key Gram-negative pathogens that have a unique and essential
FabI include
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October , 2013
Neisseria, Acinetobacter, Moraxella
Helicobacter pylori
Many Enterobacteriaceae – e.g. Klebsiella, E. coli
Hemophilus influenzae
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FabI Opportunities: A Family of Specific Inhibitors of
Multiple Pathogens Allow for Selective Therapy
S. aureus FabI
AFN-1252
• Non-staphylococcal FabI ortholog programs
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P. acnes FabI
AFN-1630
•
Propionibacterium acnes: acnes vulgaris
E. coli: uncomplicated UTI
M. tuberculosis: tuberculosis
Helicobacter pylori: gastric ulcers
Neisseria gonorrhoeae (future)
Francisella tularensis: tularemia (biodefence)
Bacillus anthracis: anthrax (biodefence)
Initial leads, require medicinal chemistry
programs
E. coli FabI
AFN-1625
H. pylori FabI
AFN-1213
October , 2013
F. tularensis FabI
AFN-1059
B. anthracis FabI
AFN-1401
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Fatty Acid Synthesis (FASII) is a Novel and Valid
Anti-staphylococcal Target
S. pneumoniae
non-essential:
•
•
External fatty acids:
 Shut down
endogenous fatty
acid synthesis
Inhibition of FAS II in
the presence of
external fatty acids:
 Uptake of external
fatty acids
 No effect on growth
October , 2013
S. aureus
essential:
•
External fatty acids:
 Do not shut down
endogenous fatty
acid synthesis
 FASII cycle
continues to turn
•
Inhibition of FASII in
the presence of
external fatty acids:
 No uptake of
external fatty acids
 Accumulation of
toxic intermediates
 Growth inhibition
 Cell death
Parsons et al. 2011, Metabolic basis for the differential susceptibility of Gram-positive
pathogens to fatty acid synthesis inhibitors. PNAS, 108:15378-83
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AFN-1252 Mechanism of Action
AFN-1252
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Selective inhibitor of staphylococcal fatty acid
synthesis
•
•
Tightly bound in the S. aureus FabI pocket
•
•
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Little to no effects on DNA, RNA, protein and
cell wall synthesis
Forms multiple hydrogen bonds with the protein
and the co-factor (NADPH)
Forms aromatic ring stacking interactions with
the protein
Highly potent inhibitor of S. aureus FabI
•
October , 2013
Ki(app) = 12 nM
Kaplan et al. 2012, Mode of Action, In Vitro Activity, and In Vivo Efficacy of AFN-1252, a
Selective Antistaphylococcal FabI Inhibitor. Antimicrob. Agents Chemother. 56:5865-74 Page 8
AFN-1252 Has High Potency and Specific Spectrum
of Activity Against Staphylococcus
S. aureus
Non-staphylococci
Number
Range MIC50 MIC90
of Strains
Gram positive cocci
Streptococcus
648 4 - >4 >4
>4
741 4 - >4 >4
>4
Enterococcus
Bacterial Group/Genus
Gram negative non-fermentors
38 >4
Acinetobacter
70 2 - >4
Moraxella
510 >4
Pseudomonas
>4
>4
>4
>4
>4
>4
Gram negative fermentors
1,852 0.5 - >4
E. coli & P. mirabilis
1,036 1 - >4
Other Enterobacteriaceae
4
>4
>4
>4
>8
>8
>8
>8
Anaerobes
Gram positive species
20
Gram negative species
19
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S. aureus MIC90 of 0.016 µg/ml
Same potency against all drugresistance mechanisms tested including
MRSA, VISA, VRSA, LRSA, etc.
October , 2013
•
>8
>8
No activity against nonstaphylococcal species
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AFN-1252 Has a Low Propensity for
Resistance Selection
Multiple Passage Resistance Selection with
S. aureus 43300 MRSA
100
MIC (ug/ml)
10
1
0.1
0.01
0.001
0
6
12
Passage number (days)
Vancomycin
Ciprofloxacin
18
24
AFN-1252
• Multiple passage resistance selection for 24 days with AFN-1252
resulted in negligible 4X increase in MIC
 Same result with 4 strains including MRSA and MRSE
 Comparable to vancomycin (2X MIC increase)
 Superior to ciprofloxacin (64X MIC increase)
October , 2013
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Efficacy, Safety, and ADME of AFN-1252 in Animals
• Demonstrated efficacy in mouse MRSA models of:
 Pneumonia
– Accumulation in lung and ELF
 Thigh abscess
 Subcutaneous abscess
 Subcutaneous granuloma pouch
 Lethal acute peritonitis / septicemia
• 3-20 times more efficacious than linezolid on a mg/kg basis
• Safety–28 day rat & 28 day dog – at high exposures 300 mg/kg
 Rat adrenal hyperplasia, reversible
 Dog HR increase, BP decrease, normal histopathology
• Well distributed to all tissues (mouse autoradiography)
October , 2013
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Affinium Summary: AFN-1252/AFN-1720
Effective, Safe, Well-Tolerated, First-in-class, Staph-specific,
potent oral & iv anti-staphylococcal antibiotic
•
Effective, safe, and well-tolerated in >220 subjects
•
Both oral and iv formulations; prodrug rapidly and completely converted to
active drug
•
Little to no antibiotic-associated diarrhea observed
•
Novel mechanism of action
•
Nanomolar potency against MRSA and all other known resistant
Staphylococcus strains; 5400 strains and 11 antibiotic classes tested
•
Staphylococcus-specific antibacterial activity
•
3-20 times more efficacious than linezolid on a mg/kg basis
•
Low propensity for resistance development, less cross-resistance
•
Consistent with Inf Dis Soc Amer’s antibiotic stewardship policies promoting
use of most specific antibacterial agents
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Phase 2a Study Design in ABSSSI
•
Open-label, single arm study in N. America
•
Enrolled 103 patients ahead of schedule
•
New drug; no prior clinical efficacy; outpatient only; large lesions
•
Conforms to current FDA indication definition and efficacy endpoints
•
AFN-1252 dose 200 mg po BID for 5 to 14 days; average ~8 days
 Second antibiotic not covering Staph could be added
 Only 10% on ampicillin or amoxicillin
•
Inclusion criteria
 Adults with ABSSSI (lesion with surface area > 75 cm2 )
 Pain, Erythema, Induration, Warmth (+/- purulence, fluctuance)
 ↑WBC, ESR, or CRP
 Gram stain consistent with staphylococcal infection
•
ITT population: 54% with history of opiate abuse;
27% with HCV or HBV; 3% HIV; 4% TB
•
Mean age 40.6 years; 66% men
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Phase 2a ABSSSI Study Efficacy
• Cellulitis 27%; Wound infections 35%; Abscesses 39%
• Erythema: 30 – 1200 cm²; mean 300 cm²
• Cultures: Staph aureus 97%; 47% MRSA; 53% MSSA; Strept 3%
• 94.5% Day 3 improvement (no evidence of progression)
• Overall Cure = Day 3 Improvement + Cure at STFU
(lesion resolution)
• 93.2% Overall Cure
– MRSA 91.9% (37 patients)
– MSSA 92.3% (39 patients)
• Results are at upper end of range of historical efficacy data
October , 2013
– Day 3 improvement:
66 – 96%
– Overall Cure:
80 – 95%
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Phase 2a ABSSSI Study Safety
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69/103 patients reported TEAEs
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4 patients withdrew due to TEAEs
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4 patients with SAEs
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Most common TEAEs were HA (26%), N/V (21%)
•
HA at Tmax; should lessen with QD extended release formulation
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Laboratory results show no safety signals
•
QTc analysis reveals no signal
•
Diarrhea in only 3 of 103 subjects; one subject also on amoxicillin
•
Multiple ascending dose Phase 1 studies
 90% of AEs were mild to moderate in severity
 2 with headache (one with N&V)
 2 with cellulitis (streptococcal)
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October , 2013
No drug related treatment emergent SAEs
1 patient (heroin withdrawal syndrome)
1 patient with new neck abscess (ongoing IVDA) needed I&D
2 patients with cellulitis that required IV antibiotic therapy and hospitalization
 No diarrhea seen in 30 subjects on 200 – 400 mg po QD for 10 days
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S. aureus Prevalence at Various Human Body Sites
Conlan S, Kong HH, Segre JA. 2012. Species-level analysis of DNA sequence data
from the NIH Human Microbiome Project. PloS one 7:e47075.
October , 2013
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Human Microbiome Composition
Normal Populations
Mean non-zero abundance (size) and population prevalence (intensity) of microbial clades
• Shows Staphylococcal species to be low in prevalence and
abundance in normal human stool
Human Microbiome Project C. 2012. Structure, function and diversity of the healthy human
microbiome. Nature 486:207-214.
October , 2013
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Effects of Antibiotics on Gut Flora
Comparison of microbial community composition in the ceca of antibiotic-treated mice
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Antibiotic Treated: 10 days with amoxicillin, metronidazole, bismuth
Recovery: 14 days post antibiotics
Antonopoulos DA, Huse SM, Morrison HG, Schmidt TM, Sogin ML, Young VB. 2009. Reproducible community
dynamics of the gastrointestinal microbiota following antibiotic perturbation. Infection and immunity 77:2367-2375.
October , 2013
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Figure 8. Individualized Cp Responses
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3 human subjects
•
Treated with
ciprofloxacin
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500 mg bid x 5 d
• Stool sampled at
Days either before (-) or
after (+) ciprofloxacin
-60d, -6d, -1d, +5d, +33d,
+180d
Dethlefsen L, Huse S, Sogin ML, Relman DA (2008) The Pervasive Effects of an Antibiotic on the Human Gut Microbiota, as
Revealed by Deep 16S rRNA Sequencing. PLoS Biol 6(11): e280. doi:10.1371/journal.pbio.0060280
http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0060280
Antibiotic Administration Routes Significantly
Influence the Levels of Antibiotic Resistance in Gut
Microbiota
• Mice administered ampicillin or tetracycline
• Either p.o. or i.v.
• Measured antibiotic resistance in the stool after inoculation of:
 tetracycline resistance - tet(M)-carrying Enterococcus spp.
and
 ampicillin resistance – bla(CMY-2)-carrying E. coli
• Found much greater increase in antibiotic resistance genes in
stool after p.o. administration of antibiotics than from
intravenous administration
• Increase more pronounced with ampicillin since mostly renal
excretion
•
Zhang L, Huang Y, Buckley T et al. 2013. Antimicro Agents Chemo 57: 3659-3666.
October , 2013
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ADME Considerations of AFNM-1720 Prodrug
• Rapid bioconversion of the prodrug to active AFN-1252
after both IV and oral administration
• Oral administration to animals results in rapid appearance
of active drug in plasma
• No first pass metabolism
• Metabolized in liver
• Hepatic metabolites excreted through both liver and kidney
• No active drug found in urine
October , 2013
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Proposed Mouse Gut Microbiome Study
Comparing Multiple Antibiotics
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Clindamycin
Moxifloxacin
Linezolid
AFN-1720 Prodrug
Placebo
• Each is an active anti-Staph agent, including MRSA
• Will administer antibiotic p.o. for 7 - 10 days
• Fecal sampling @ Baseline, During dosing, After dosing
• 16S rRNA sequencing
• Comparative Antibiotic Microbiome Effects
October , 2013
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Clostridium difficile Independent Risk
Factors
• Barnes-Jewish Hospital, St. Louis, MO;
• 36,086 admissions; 382 cases of C. difficile during 2003
• Positive Risk Factors:
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Increasing age
Prior admission within 60 days
Hypoalbuminemia
Leukemia/lymphoma
Mechanical ventilation
Anti-motility drugs
Histamine-2 blockers
Proton pump inhibitors
Intravenous vancomycin
Fluoroquinolones
Cephalosporins – 1st, 3rd, 4th generation
• Metronidazole protective
October , 2013
Dubberke et al, CID 2007; 45:1543-9
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AFN-1252 Attributes of Greatest Importance - Overall
Use in Hospital and Community
Novel MoA
Respondents mentioned
2-4 attributes each
Bactericidal
Narrow Spectrum
Oral Bioavailability
Oral and IV
MRSA Activity/MIC
QD Oral Dosing
Lack of Cross Resistance
Safety/Sparing of Gut Flora
0
5
10
15
20
25
Number of Unaided Mentions
Source: Affinium Hospital & Community Qual survey, 2009
• Feedback was consistent across all four market studies showing the
following value drivers for use of AFN-1252 in hospital or community
October , 2013
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