Addressing the evolving challenge of β-lactamase mediated ... · β-lactamases are characterized...
Transcript of Addressing the evolving challenge of β-lactamase mediated ... · β-lactamases are characterized...
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Addressing the evolving challenge of β-lactamase mediated antimicrobial resistance:
ETX2514, a next-generation BLI with potent broad-spectrum activity against Class A, C and D enzymes
Alita Miller, PhD
Superbugs & Superdrugs USA, November 14-15, 2016, Iselin, NJ
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Overview of presentation
Co-evolution of β–lactams, β–lactamases and their inhibitors
Multidrug resistant Acinetobacter baumannii:
an unmet medical need
ETX2514 In vitro characterization
In vivo efficacy
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β-lactamases are characterized into four molecular classes
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Class A, C, and D have a serine at the active site and require water in the active site forβ-lactam hydrolysis
Class B are metalloenzymesthat require zinc at the active site
Drawz & Bonomo, (2010) Clin. Microbiol. Rev. 23: 160-201
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β-lactamases evolve after use of β-lactam antibiotics
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Cefazolin
1st Gen Cephalosporins
1980s 1990s 2000s 2010s 2020s1970s1960s
Class A
Class B
Class C
Class D
Cephalexin
Cefalothin
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β-lactamases evolve after use of β-lactam antibiotics:
1960 - 1970
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β-lactam drugs
1st Gen Cephalosporins
1980s 1990s 2000s 2010s 2020s1970s1960s
Class A
Class B
Class C
Class D
2nd Gen CephalosporinsCephamycins
TEM-1, SHV-1
Cefaclor
Cefotetan
Cefoxitin
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β-lactamases evolve after use of β-lactam antibiotics:
1970 - 1980
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β-lactam drugs
1st Gen Cephalosporins
1980s 1990s 2000s 2010s 2020s1970s1960s
Class A
Class B
Class C
Class D
2nd Gen CephalosporinsCephamycins
TEM-1, SHV-1AmpC overexpression
3rd Gen CephalosporinsMonobactam1st Gen BL/BLI combinations
Amoxicillin/Clavulanate, Ampicillin/Sulbactam
Ceftazidime
Cefotaxime
Aztreonam
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β-lactamases evolve after use of β-lactam antibiotics:
1980 - 1990
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β-lactam drugs
1st Gen Cephalosporins
1980s 1990s 2000s 2010s 2020s1970s1960s
Class A
Class B
Class C
Class D
2nd Gen CephalosporinsCephamycins
TEM-1, SHV-1AmpC overexpression
3rd Gen CephalosporinsMonobactam1st Gen BL/BLI combinations
ESBL TEM, SHV
ESBL CTX-M
Carbapenems2nd Gen BL/BLI
Imipenem
ESBL OXA
Plasmid AmpC
Meropenem
Piperacillin/tazobactam
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β-lactamases evolve after use of β-lactam antibiotics:
1990 - 2000
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β-lactam drugs
1st Gen Cephalosporins
1980s 1990s 2000s 2010s 2020s1970s1960s
Class A
Class B
Class C
Class D
2nd Gen CephalosporinsCephamycins
TEM-1, SHV-1AmpC overexpression
3rd Gen CephalosporinsMonobactam1st Gen BL/BLI combinations
ESBL TEM, SHV
ESBL CTX-M
Carbapenems2nd Gen BL/BLI
ESBL OXA
Plasmid AmpC
KPC carbapenemase
OXA carbapenemase
VIM
• No new β-lactams for Gram-negatives
• Therapy limited to colistin or tigecycline
NDM
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1980s 1990s 2000s 2010s 2020s1970s1960s
Class A
Class B
Class C
Class D
TEM-1, SHV-1AmpC overexpression
ESBL TEM, SHV
ESBL CTX-M
ESBL OXA
Plasmid AmpC
KPC carbapenemase
OXA carbapenemase
VIM NDM
Older β-lactamase inhibitors only work against a few classes of β-lactamases
Inhibited by Clavulanic Acid and Sulbactam
Amoxicillin-clavulanateTicarcillin-clavulanateAmpicillin-sulbactam
clavulanic acid
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1980s 1990s 2000s 2010s 2020s1970s1960s
Class A
Class B
Class C
Class D
TEM-1, SHV-1AmpC overexpression
ESBL TEM, SHV
ESBL CTX-M
ESBL OXA
Plasmid AmpC
KPC carbapenemase
OXA carbapenemase
VIM NDM
Older β-lactamase inhibitors only work against a few classes of β-lactamases
Inhibited by Tazobactam
Piperacillin-tazobactamCeftolozane-tazobactam (Zerbaxa)
tazobactam
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1980s 1990s 2000s 2010s 2020s1970s1960s
Class A
Class B
Class C
Class D
TEM-1, SHV-1AmpC overexpression
ESBL TEM, SHV
ESBL CTX-M
ESBL OXA
Plasmid AmpC
KPC carbapenemase
OXA carbapenemase
VIM NDM
Avibactam and other DABCO*s have broader spectra of inhibition than older β-lactamase inhibitors
Inhibited by Avibactam
*di-aza-bicyclo-octanone
• Ceftazidime-avibactam (AvyCaz)• Imipenem-relebactam (Ph III)• Zidebactam, RG6080 (Ph I)• Aztreonam-avibactam (Ph I)
MBL+ Enterobacteriaceae (Class B)(ATM is not degraded by MBLs; AVI inhibits serine BLs)
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1980s 1990s 2000s 2010s 2020s1970s1960s
Class A
Class B
Class C
Class D
TEM-1, SHV-1AmpC overexpression
ESBL TEM, SHV
ESBL CTX-M
ESBL OXA
Plasmid AmpC
KPC carbapenemase
OXA carbapenemase
VIM NDM
Very limited coverage of Class D -lactamases by avibactam
Inhibited by Avibactam
Addressed by ATM-AVI in Enterobacteriaceae
Gaps in Coverage
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• Gram-negative bacteria that causes infections in critically ill patients, with mortality rates as high as 43%1
• CDC Unmet Need Threat Level: Serious2
• 63% of A. baumannii isolates are considered multi-drug resistant, meaning at least three different classes of antibiotics no longer cure A. baumannii infections including carbapenems, often considered antibiotics of last resort
• Resistance to carbapenems in A. baumannii is associated with increasing prevalence of Class D -lactamases3,4
A. baumannii
1. Am. J. Respir. Crit. Care Med. 2011.1409; Int. J. Antimicrob. Agents 2009.5752. CDC. 2013. Antibiotic Resistant Threats in the US. pg. 58-603. M.M. Ehlers, et. al. 2012. InTech, DOI: 10.5772/303794. Potron, et al. 2015. Int. J. Antimicrob. Agents 45:568
Multi-drug resistant Acinetobacter baumannii
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Complexity of β-lactamase content in A. baumannii
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Whole-genome sequencing of 84 recent MDR A. baumannii strains provides insight into what is required for a successful next generation BL/BLI therapy
Inhibition of Classes A, C and D Required for Robust BLI Activity in A. baumannii
Class N %Most prevalent
variant(s)
A 45 53.6 TEM-1 (41/45)
B 1 1.3 IMP-1
Extended
spectrum C* 71 84.5ADC-30 (18/84)ADC-73 (18/84)
D 84 100
Multiple (70/84 encode two or more, (46/70
were OXA-23+OXA-51-like)
*all strains contain chromosomal adc gene
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The ultimate medicinal chemistry challenge
• How to selectively inhibit hundreds of bacterial enzymes?
• How to find the right balance between reactivity and hydrolytic stability?
• How to prepare synthetically challenging, diverse analogs to verify structural hypotheses?
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• A deep understanding of avibactam’s biology informed design of the next generation BLI
• Crystal structures provided insights to avibactam’s unique interactions with-lactamases
Avibactam-bound structures of CTX-M-15 at 1.1Å,From Lahiri et al (2013) AAC 57: 2496
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Discovery of ETX2514, a novel broad-spectrum serine BLI
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Using a combination of innovative chemistry and structure-based design
avibactam
Active site overlays of avibactam- (in grey, PDB: 4WM9) and an ETX2514 analog- (in green) bound OXA-24 structures. The water molecules are depicted as spheres. The hydrogen bonding network around the ETX2514 analog is shown in dashed lines.di-aza-bicyclo-octenone
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ETX2514 exhibits excellent β-lactamase inhibition across classes A, C and D
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IC50 after 5 min incubation (in µM)
Compound
Name9.2
Class A Class C Class D
E. cloacae
TEM-1
K. pneumoniae
CTX-M-15
E. cloacae
KPC-2
E. cloacae
P99
P. aeruginosa
AmpC
P. aeruginosa
OXA-10
A. baumannii
OXA-24/40
K. pneumoniae
OXA-48
Avibactam 0.011 0.0047 0.19 0.2 0.62 23 16 0.75
ETX2514 0.0012 0.00083 0.0043 0.0013 0.014 0.25 0.2 0.0063
Fold increase in potency 9X 6X 44X 154X 44X 92X 80X 119X
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Exceptional enzymatic spectrum translates into excellent activity across an isogenic panel of P. aeruginosa strains
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IC50 (in µM)
Compound
Name
Class A Class C Class D
E. cloacae
TEM-1
K. pneumoniae
CTX-M-15
E. cloacae
KPC-2
E. cloacae
P99
P. aeruginosa
AmpC
P. aeruginosa
OXA-10
A. baumannii
OXA-24/40
K. pneumoniae
OXA-48
Avibactam 0.011 0.0047 0.19 0.2 0.62 23 16 0.75
ETX2514 0.0012 0.00083 0.0043 0.0013 0.014 0.25 0.2 0.0063
Compound
Name
P. aeruginosa isogenic strains bearing corresponding -lactamases
Vector
aloneTEM-1 CTX-M-15 KPC-2 P99 AmpC OXA-10 OXA-24/40 OXA-48
Piperacillinalone
4 >1024 512 256 64 128 256 256 128
Piperacillin+Avibactam
4 8 4 8 4 16 128 128 8
Piperacillin+ETX2514
4 4 4 4 4 4 4 8 4
MIC (in mg/L)
BLI added at 4 mg/mL
No BLI
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Mecillinam(PBP2-selective
inhibitor)
Inhibition of PBP2 by ETX2514 results in intrinsic antibacterial activity vs. Enterbacteriaceae
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Control
ETX2514
Mecillinam
Aztreonam
ControlAztreonam
(PBP3-selective
inhibitor)
ETX2514
Pathogen
ETX2514 kinact/Ki in M-1s-1
PBP1a PBP2 PBP3
A. baumannii 180 1,800 3
P. aeruginosa 12 24 57
E. coli 120 17,000 2
Control
ETX2514
Mecillinam
Aztreonam
Morphology of antibiotic-treated E. coli
Linneas Bioscience
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ETX2514 restores β-lactam activity vs. multiple gram-negative pathogens
20
CompoundE. coli
n = 202
K. pneumoniae
n = 198
P. aeruginosa
n = 202
A. baumannii
n = 195
Imipenemalone 0.25 1 16 >64
+ ETX2514 ≤0.06 0.12 2 16
Meropenemalone ≤0.06 ≤0.06 16 >64
+ ETX2514 ≤0.06 ≤0.06 8 16
Aztreonamalone 32 32 64 >64
+ ETX2514 ≤0.06 ≤0.06 32 >64
Ceftazidimealone 16 >64 >64 >64
+ ETX2514 ≤0.06 ≤0.06 8 32
Sulbactamalone 64* >64¥ >64 64
+ ETX2514 ≤0.06* 0.12¥ >64 4
ETX2514 alone 1 8 >64 >64
*n = 21 strains ¥n = 20 strains
• Excellent activity vs E. coli & K. pneumoniae with all β-lactams tested
• Restores imipenem to MIC90 of 2 mg/L vs P. aeruginosa
• Restores sulbactam to MIC90 of 4 mg/L vs A. baumannii
MIC90 across recent clinical isolates (+/- ETX2514 at 4 mg/L)
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Intrinsic activity of sulbactam vs. A. baumannii
• Attributed to inhibition of PBP3
21Penwell et al.(2015) AAC 59:1680-89
+ sulbactamuntreated
• Frequency of resistance is low: 2-4x10-9 at 4X MIC• Resistance maps to residues near active site of PBP3• Resistant mutants are attenuated in fitness
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• Sulbactam:ETX2514* maintains excellent activity over time
MIC (mg/L) ≤0.06 0.12 0.25 0.5 1 2 4 8 16 32 >64
2011N=195
Cumul %susceptible
1 3.1 13.8 41.5 65.6 89.7 96.9 97.9 99.5 100 100
2012N=209
Cumul %susceptible
0 0.5 2.9 20.1 46.9 79 98.6 100 100 100 100
2013N=207
Cumul %susceptible
0 0 4.3 15.9 43.4 73.8 96.5 97.5 99 99 100
2014N=1131
Cumul %susceptible
1 1.6 7.8 27.9 63.7 88.9 99.6 99.6 99.7 100 100
Sulbactam:ETX2514: A novel combination against MDR A. baumannii
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MIC distributions for globally diverse A. baumannii clinical strains
*held at 4 mg/L
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Sulbactam:ETX2514 activity remains unchanged in carbapenem-resistant, colistin-resistant and MDR A. baumannii strains
23
0.2
5
0.5 1 2 4 8
16
32
64
12
8
0
1 0
2 0
3 0
4 0
5 0
M I C ( m g / L )
%
su
sc
ep
ti
bl
e
st
ra
in
s
s u l b a c t a m a l o n e v s . a l l ( N = 1 1 3 1 )
s u l b a c t a m : E T X 2 5 1 4 v s . a l l ( N = 1 1 3 1 )
s u l b a c t a m : E T X 2 5 1 4 v s . M E M - R ( N = 7 3 1 )
s u l b a c t a m : E T X 2 5 1 4 v s . C O L - R ( N = 5 6 )
s u l b a c t a m : E T X 2 5 1 4 v s . M D R ( N = 7 7 8 )
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Sulbactam:ETX2514 is active against A. baumannii encoding multiple β-lactamases
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drug N range MIC50 MIC90
imipenem 84 0.125 - >128 64 128
SUL-ETX2514
84 0.25 - 16 2 4
Summary of MICs (mg/L)
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Morphology of A. baumannii in the presence of sulbactam:ETX2514 suggests multi-target effects
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No Drug
SUL
ETX2514
SUL-ETX2514
A. baumannii ATCC 17978 was exposed to 1/2X MIC of drug for 3
hrs at 35° C and examined by light microscopy. Scale bar = 5 mm.
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Frequency of spontaneous resistance to sulbactam-ETX2514 is very low against clinical isolates of A. baumannii
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Strainβ-lactamase
contentFOR at 4X MIC Variant Protein affected
SUL-ETX2514
SUL MEM CAZ
ARC2058ADC-99-like;
OXA-95
Parent -- 1/4 4 0.5 4
2X-1 AspS [Q47P] 16/4 4 16 16
<9.0 x 10-10 2X-2 GltX [M240I] 16/4 4 8 4
2X-3 GltX [R117S] 64/4 4 32 8
2XL-1 PBP3 [V505L] 16/4 16 0.25 4
ARC2681ADC-42-like; TEM-1; OXA-40; OXA-
1327.6 x 10-10
Parent -- 2/4 8 32 256
4X-1 PBP3 [S390T] >64/4 >64 32 128
ARC2782ADC-79; TEM-1; PER-1; OXA-23;
OXA-66<9.0 x 10-10
Parent -- 0.5/4 32 16 >512
2X-1 PBP3 [T511A] 4/4 64 16 >512
MIC (mg/L)
• tRNA synthetase mutants are associated with the stringent response and are commonly seen with PBP2
inhibitors1
• Mutations in PBP3 affected the MIC of SUL-ETX2514 and sulbactam alone
Resistant mutants suggest sulbactam-ETX2514 works by inhibiting both PBP2 and PBP3
1Vinella et al. (1992) EMBO J. 11:1493-1501
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Sulbactam:ETX2514 exhibits excellent in vivo activity
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• Greater than 2-log kill achieved in both neutropenic mouse thigh and lung models of A. baumannii infections
7.40
9.40
8.408.03
6.636.19
4.854.61
4.19
2
3
4
5
6
7
8
9
10
Pre-treatment
Vehicle 2.5 /0.625
5 / 1.25 10 / 2.5 20 / 5 30 / 7.5 40 / 10 80 / 20
Log(
CFU
/g)
Lung
Stasis
sulbactam/ETX2514 (mg/kg) q3h
6.36
8.03 8.02
6.72
4.39 4.243.97 4.01 4.07
2
3
4
5
6
7
8
9
10
Pre-treatment
Vehicle 2.5 /0.625
5 / 1.25 10 / 2.5 20 / 5 30 / 7.5 40 / 10 80 / 20
Log(
CFU
/g)
Thigh
Stasis
sulbactam/ETX2514 (mg/kg) q3h
MDR A. baumannii ARC3486 (OXA-72, OXA-66, TEM-1, AmpC)
MIC(sulbactam) ≥ 32 mg/L, MIC(sulbactam/ETX2514) = 0.5 mg/L
Sulbactam/ETX2514 dose response (SC, 4/1 ratio)
Similar results obtained for 5 additional clinical isolates
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PK and safety of sulbactam:ETX2514
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• Rat and dog PK of ETX2514 showed low to moderate clearance and low volume of distribution translating to a projected half life of 1.1 hr in humans
• Excretion of unchanged drug was the predominant clearance mechanism with relatively low metabolism characterized in vitro and in vivo
• ETX2514 was well-tolerated in both rat and dog 14-day repeat dose toxicology studies up to 2000 mg/kg with no significant clinical findings after intravenous administration
no changes in ophthalmology, urinalyses, hematology parameters or organ weight
• In CV safety pharmacology studies, ETX2514 had no effects on qualitative electrocardiogram parameters, heart rates, or arterial pressures up to 2000 mg/kg
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Summary and Conclusions
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• ETX2514 is a potent inhibitor of a broad-spectrum of Class D β-lactamases while maintaining exquisite potency on Class A and C enzymes.
• ETX2514 potently restores the activity of multiple β-lactams in Gram-negative MDR pathogens.
• Sulbactam:ETX2514 is a novel BL:BLI combination to treat MDR A. baumannii infections, with an MIC90 = 4 mg/L (N = 1742 clinical isolates) and excellent in vivo activity.
• Currently in Phase I testing
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Acknowledgements
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• AstraZeneca Antibacterial Discovery• IHMA, Inc. • Linneas