Discovery of ETX2514, a novel, rationally designed inhibitor of Class A, C and D β-lactamases, for the

Transcription

1 Discovery of ETX2514, a novel, rationally designed inhibitor of Class A, C and D β-lactamases, for the treatment of Gramnegative infections Thomas Durand-Réville 253 rd ACS National Meeting MEDI 332 April 04, 2017

2 The relentless development of antimicrobial resistance Report Cover final for distribution.pdf (March 2017) 2

3 β-lactamases: major mechanism of resistance to β-lactam antibiotics β-lactamase expression Porin mutation/deletion Efflux pump overexpression Penicillin-binding protein (PBP) mutation β-lactamase inhibitors (BLI) restore β-lactam efficacy Approach clinically validated (e.g., Augmentin, Zosyn, Unasyn, Avycaz) MDR Gram-negative isolates consistently express multiple β-lactamases from Current Opinion in Microbiology (2010) 13, 55 3

4 Four families of β-lactamases β-lactamases use serine- or zinc-mediated hydrolysis to inactivate β-lactams 2,819 enzymes identified: Class A (1,155); Class B (361); Class C (693); Class D (610) Clavulanic acid Sulbactam Tazobactam Serine Enzymes b-lactamases Metallo Enzymes Class A Class C Class D Class B Emerging threat KPC Carbapenemase OXA Avibactam Relebactam Vaborbactam Major new opportunity Acinetobacter & Pseudomonas ETX (15 March 2017) 4

5 An unprecedented product profile Novel, best in class β-lactamase inhibitor (BLI), with coverage of Class A, C and D β-lactamases, to be combined with β-lactam antibiotics to treat MDR Gram-negative infections Product demonstrates improved in vitro coverage of contemporary clinical strains of MDR A. baumannii and P. aeruginosa I.V. product shows improved safety profile to current standard of care 5

6 The diazabicyclooctanone (DBO) avibactam: a great starting point Avibactam: stable to b-lactamase hydrolysis carbamoylation reversible X hydrolysis b-lactamase (active) b-lactamase (inactive) b-lactamase (active) Tazobactam: hydrolyzed by b-lactamases acylation irreversible hydrolysis Deep understanding of avibactam s biology Unique mechanism of inhibition of DBO Numerous β-lactamase co-crystal structures Informed design to discover the next generation BLI Ehmann D. et al., PNAS (2012) 109, Lahiri S. et al., AAC (2013) 57,

7 Three major challenges faced by the medicinal chemistry team MDR Gram-negative clinical isolates contain a combination of β-lactamases Design challenge: How to inhibit >2000 bacterial enzymes but no human ones? DBO β-lactamase inhibitors are covalent, reactive molecules Stability challenge: How to find the right balance between reactivity and hydrolytic stability? DBO complex structure is difficult to synthesize Synthetic challenge: How to prepare diverse analogs to verify our structural hypothesis? 7

8 Class D OXA β-lactamases possess strongly hydrophobic active sites a2-a3 loop loop Avibactam/OXA-48 (D) co-crystal structure Superimposed with: CTX-M-15 (A) TRU-1 (C) OXA-10 (D) OXA-24 (D) 8

9 DE (kcal/mol) A simplified quantum mechanics model to predict reactivity Rate of hydrolysis (M -1.s -1 ) r 2 = 0.81 DE -20 avibactam - Tetrahedral INT Density Functional Theory method M06-2X functional 6-31G** basis set Poisson-Boltzmann finite element solvation ETX2514 Model previously validated with set of lactam rings E ETX2514 avi = 2.68 kcal/mol would predict ~10 2 -fold increase enzyme k on Imming P. et al., J. Med. Chem. (2000) 43,

10 Exploring a unique chemical space around DBO scaffold Avibactam route cannot be used to make unsaturated analogs Original route: Long, linear synthesis Moderate yields Limited scope for analoguing Racemic Cis/trans separation Double bond migration New route is needed Stabilize double bond 10

11 Key chiral intermediate obtained using new route 4 Enone intermediate Stereoselective, highly efficient, reproducible, scalable, 6-step route to key intermediate Substituted vinyl Grignards introduce diversity at 4-position Enone functionality presents great potential for various downstream chemistries 11

12 High versatility of enone intermediate 3 4 Enone intermediate Facile installation of highly customizable side chains from enone intermediate Diastereoselective Luche reduction to install second stereocenter Xiong H. et al., ACS Med. Chem. Lett. (2014) 5,

13 New asymmetric route delivers novel diazabyclooctenone analogs Long, linear but asymmetric and modular route - overall yield < 1% Challenging protecting group strategy Highly polar and reactive products at end of synthesis 13

14 Careful rational design leads to discovery of ETX2514 BLI compound β-lactamase IC 50, (µm) Piperacillin MIC (mg/l) ± 4 mg/l BLI Hydrolysis rate constant k (h -1 ) KPC-2 (A) AmpC (C) OXA-24 (D) P. a. parent* KPC-2 (A) AmpC (C) OXA-24 (D) none 4 >64 >64 >64 avibactam > >64 >64 > NT NT NT NT NT 5 ETX NT NT NT NT NT NT NT NT NT NT >64 8 NT *P. aeruginosa parent strain PAO1 (ampc-, poxb-), MIC (BLI) > 64 mg/l ETX2514 selected based on biochemical and microbiological profile Broad spectrum β-lactamase activity likely due to combination of increased reactivity and tighter binding to Class D OXA enzymes McGuire H. et al., WO 2013/

15 ETX2514 binding mode similar the avibactam in AmpC ETX2514-AmpC (1.34Å) Avibactam-AmpC (PDB:4HEF, 1.86Å) Side view Top view ETX2514 covalently bound to the catalytic SER-90 of AmpC Conformation similar to avibactam 15

16 Covalent, reversible mechanism of inhibition by ETX2514 apo carbamoyl apo carbamoyl TEM TEM-1 + CTX-M TEM-1/ETX TEM-1/ETX CTX-M-15 (1h) 2.76e e e e e e4 CTX-M-15 Mass, Da TEM-1 Protein mass spectrometry confirmed the covalent reversible mechanism for ETX2514 between TEM-1 and CTX-M-15 Recyclization mechanism similar to avibactam 16

17 ETX2514 inhibits β-lactamases in whole cell assay β-lactamase class piperacillin alone +AVI +ETX2514 none CTX-M-15 A > GES-11 A PER-1 A SHV-2a A > TEM-1 A > VEB-1 A > KPC-2 A > KPC-3 A > KPC-3 D179Y A β-lactamase class piperacillin alone +AVI +ETX2514 NDM-1 B >64 >64 64 VIM-1 B >64 >64 >64 VIM-2 B >64 >64 >64 AmpC C > P99 C > OXA-10 D >64 >64 8 OXA-23 D > OXA-24 D >64 >64 8 OXA-48 D > OXA-58 D >64 >64 4 MIC in mg/l 4 mg/l AVI = avibactam, P. aeruginosa parent strain PAO1 (ampc-, poxb-), MIC (AVI) > 64 mg/l, MIC (ETX2514) > 64 mg/l ETX2514 restores piperacillin activity in a wide range of serine β-lactamase-containing P. aeruginosa isogenic strains Similar results obtained in combination with ceftazidime and aztreonam and also in the A. baumannii isogenic panel 17

18 ETX2514 inhibits Gram-negative bacterial PBPs Acylation rates against PBPs ETX2514 k inact /K i (M -1 s -1 ) Pathogen PBP1a PBP2 PBP3 A. baumannii 180 1,800 3 Control Morphology of antibiotic-treated E. coli (0.5x MIC) Control P. aeruginosa E. coli ,000 2 Mecillinam Mecillinam (PBP2-selective inhibitor) Significant PBP inhibition against Enterobacteriaceae results in intrinsic antibacterial activity Aztreonam Aztreonam (PBP3-selective inhibitor) PBP2-mediated MOA confirmed by cytological profiling ETX2514 ETX

19 A new route established to scale-up ETX2514 Synthetic route reduced from 18 to 11 steps Scalable, asymmetric synthesis 19

20 ETX2514 restores β-lactam activity in multiple Gram-negative pathogens Microbiological profile against recent Gram-negative clinical isolates (± ETX2514 at 4 mg/l) Compound (MIC 90, mg/l) Imipenem Meropenem Aztreonam Ceftazidime Sulbactam E. coli n = 202 K. pneumoniae n = 198 P. aeruginosa n = 202 A. baumannii n = 195 alone >64 + ETX alone >64 + ETX alone >64 + ETX >64 alone 16 >64 >64 >64 + ETX alone 64 >64 > ETX >64 4 ETX2514 alone 1 8 >64 >64 Excellent activity against E. coli and K. pneumoniae with all β-lactams tested ETX2514 restores imipenem and ceftazidime to their breakpoints vs. P. aeruginosa ETX2514 restores sulbactam to MIC 90 of 4 mg/l vs. A. baumannii Intrinsic activity for both K. pneumoniae and E. coli, including isolates containing Class B metallo-β-lactamases or the mcr-1 gene 20

21 MDR A. baumannii: a growing unmet medical need Between 60,000 and 100,000 infections per year in the U.S., ~130,000 per year in EU5 - Forecast to grow over the next decade A. baumannii causes infections among critically ill patients; mortality rates as high as 43%, 63% MDR 1 Class D β-lactamases are responsible for failure of many β-lactams 2-4 CDC Unmet Need Threat Level (2013): Serious WHO Priority Pathogens List (2017): Critical 1 Am. J. Respir. Crit. Care Med ; Int. J. Antimicrob. Agents Prevalence of Carbapenemases in Acinetobacter baumannii, Antibiotic Resistant Bacteria A Continuous Challenge in the New Millennium, InTech, DOI / Diversity, Epidemiology & Genetics of Class D β-lactamases. AAC Lancet ; J. Glob. Infect. Dis

22 Complexity of β-lactamase content in MDR A. baumannii Whole-genome sequencing of 84 recent MDR A. baumannii isolates β-lactamase Class N % Most prevalent variant(s) A TEM-1 (41/45) B IMP-1 Extended spectrum C* ADC-30 (18/84) ADC-73 (18/84) D Multiple (70/84 encode two or more, (46/70 = OXA-23+OXA-51-like) *all strains contain chromosomal adc gene Inhibition of Classes A, C and D required for robust activity against MDR A. baumannii 22

23 Sulbactam/ETX2514: a novel combination against MDR A. baumannii MIC distributions for globally diverse A. baumannii clinical strains MIC (mg/l) > * N= * N= * N= * N= # N=202 Cumul % Cumul % Cumul % Cumul % Cumul % *Study performed at IHMA Inc., # Study performed at JMI labs Sulbactam/ETX2514 maintains excellent activity over time Sulbactam/ETX2514 activity remains unchanged in carbapenem-resistant, colistin-resistant and extensive drug-resistant (XDR) A. baumannii strains 23

24 Sulbactam/ETX2514 exhibits best in class activity vs. A. baumannii Microbiological profile in recent A. baumannii clinical isolate collection (n=608), MIC in mg/l Compound or Combination * Max MIC 50 MIC 90 Sulbactam/ETX2514 > Meropenem (Merrem ) >32 >32 >32 Ceftolozane/tazobactam (Zerbaxa ) >32 32 >32 Ceftazidime/avibactam (Avycaz ) > >128 BL ±BLI combinations Tetracyclines Polymyxins Tigecycline (Tygacil ) >8 1 4 Minocycline (Minocin ) > Colistin (Coly-Mycin ) >8 1 2 * All BLIs tested at 4 mg/l Frequency of spontaneous resistance to sulbactam/etx2514 is very low against clinical isolates of A. baumannii (<9.0 x for 3 isolates) 24

25 ETX2514 PK profile is similar to avibactam Species/Dose Parameter avibactam ETX2514 Rat CL (ml/min/kg) (+/-5.0) 3mg/kg CLr (ml/min/kg) (+/-1.0) [15min-IV infusion in 5% dextrose in water] V ss (L/kg) (+/-0.07) t 1/2 (h) (+/-0.01) Dog AVI (0.43 mg/kg) & ETX2514 (0.4 mg/kg) cassette [15min-IV infusion in 5% dextrose in water] CL (ml/min/kg) CLr (ml/min/kg) V ss (L/kg) t 1/2 (h) 3.4 (+/-1.0) 2.9 (+/-1.0) 0.23 (+/-0.02) 0.78 (+/-0.18) 5.1 (+/-1.5) 3.6 (+/-1.0) 0.28 (+/-0.03) 0.80 (+/- 0.13) PK profile consistent with DBO class and compatible with β-lactam partners Renal excretion of unchanged drug was the predominant clearance mechanism Projected half-life of 1-2 hr in humans Avibactam rat PK data from CEFTAZIDIME-AVIBACTAM, NDA

26 Log(CFU/g) Log(CFU/g) Sulbactam/ETX2514 exhibits excellent in vivo activity 10 Thigh Sulbactam/ETX2514 dose response (IV, 4/1 ratio) MDR A. baumannii ARC3486 (OXA-72, OXA-66, TEM-1, ADC-30) MIC(sulbactam) 32 mg/l, MIC(sulbactam/ETX2514) = 0.5 mg/l 10 Lung Stasis Stasis Pretreatment Pretreatment Vehicle 2.5 / / / / 5 30 / / / 20 Sulbactam / ETX2514 (mg/kg) q3h 2 Vehicle 2.5 / / / / 5 30 / / / 20 Sulbactam / ETX2514 (mg/kg) q3h Greater than 2-log kill achieved in both neutropenic mouse thigh and lung models of MDR A. baumannii infection Similar results obtained for 5 additional clinical isolates 26

27 Excellent physicochemical properties and preclinical safety profile ETX2514 preclinical package: logd < 0, aqueous solubility 200 mg/ml Cardiac channel panel IC 50 > 1000 µm (herg, NaV1.5, CaV1.2, Iks, KV4.3) GLP Genetic toxicology Negative (Ames, mouse lymphoma assay, in vivo micronucleus) General toxicology (2-week regulatory tox studies) No target organ Rat NOAEL 2000 mg/kg Dog NOAEL 2000 mg/kg GLP CV study (dog telemetry) No CV effects (NOAEL 2000 mg/kg) 27

28 ETX2514: the next generation BLI 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 MIC 90 = 4 mg/l (N = 1742 clinical isolates) and excellent in vivo efficacy and safety ETX2514 is currently in Phase I clinical trials 28

29 Acknowledgements AstraZeneca Infection Discovery AstraZeneca Pharmaceutical Development Novexel Pharmaron, Syngene IHMA Inc., JMI Laboratories Linneas Bioscience 29