Mechanism of Action of the Cyclophilin-inhibitor Class of Compounds. Katyna Borroto-Esoda

Transcription

1 Mechanism of Action of the Cyclophilin-inhibitor Class of Compounds Katyna Borroto-Esoda

2 Prototype of Cyclophilins (Cyps): Cyclophilin A (CypA) Originally discovered as a specific ligand for the immunosuppressive drug, cyclosporin A Abundant cytosolic protein (.1% of total cellular proteins) Ubiquitously expressed in eukaryotic cells Possesses a peptidyl-prolyl cis-trans isomerase activity Has been proposed to act as a chaperone in protein trafficking or as a catalyst of protein folding Slide 2

3 History of Cyclophilin Inhibitors in HCV Therapy Treatment with cyclosporin A improves fibrosis scores of chimpanzees chronically infected with non-a non-b hepatitis Teraoka et al, 1988, Transplant Proc 2: 868 Cyclosporin-based immunosuppressive regimens increase the likelihood of sustained virological response to interferon therapy in recurrent hepatitis C infection following liver transplantation Van Thiel et al, 1991, Transplant Proc 23: 352 Firpi et al, 26, Liver Transpl 12: 51 ReVis-TC Group, 211, Transplantation 92: week treatment with interferon + cyclosporin A produces significantly higher rates of sustained virological response than treatment with interferon alone 55% versus 32%; 24 weeks after completion of treatment Inoue et al, 23, J Gastroenterol 38: 567 Slide 3

4 CypA is the Main Cyclophilin Member Involved in HCV Replication Yang et al. J Virol 28; Chatterji et al. J Biol Chem 29; Kaul et al. PLoS Pathog 29 Slide 4

5 Development of HCV Resistance to Cyclophilin Inhibitors In Vitro Selection for resistance to and its analogues by passage in subgenomic replicon cells results in mutations primarily in NS5A and NS5B: however, mutations in NS3 have also been reported The most frequently observed mutation which results in the greatest resistance has been the D32E substitution in the NS5A In Vivo Resistance is typically slow to develop compared to DAAs The D32E mutation was observed following up to 48 weeks of therapy with Alisporivir in combination with Peg/RBV Fernandes et.al., Hepatology 21 Coelmont et.al., PlosOne 21 Hopkins et.al., J Hep 21 Puyant et.al., AAC 21 Li et.al., Hepatology 211 Slide 5

6 Cyclosporin Inhibits Cellular Cyclophilins Peptidyl-Prolyl Cis-Trans Isomerases Cyclosporin A Cyclophilin Binding (required for antiviral activity) Calcineurin Binding (required for immunosuppression) O N N O O N HO H N O O N O CyP-A N O O O N N N N O Immunosuppressive activity dependent on calcineurin inhibition N O CN A subunit CN B subunit Slide 6

7 Proposed Mechanism of Action of Against HCV Replication CypA is a component of the HCV replication complex The PPIase activity of CyPA is required for HCV replication inhibits the PPIase activity of CyPA and interferes with recruitment of CyPA by the replication complex Slide 7

8 Candidate HCV Compound 3,4 di-substituted cyclosporin analog which retains cyclophilin binding but lacks the calcineurin binding and associated immunosuppressive activity Viral resistance is rare and weak in in vitro selections 2-Step synthesis from cyclosporin A Good phys-chem properties: Water soluble, protein binding ~75% Good pharmacokinetic properties Orally bioavailable Low potential for drug-drug interactions: low inhibition of transporters, P45s Slide 8

9 Displays Similar Antiviral Activity Against Various Subtypes of HCV In Vitro was tested in vitro against the HCV genotype 1b (Con1), 1a (H77) and 2a (JFH-1) replicons Assays were performed in the HuH7 cell line at SRI demonstrated similar activity against all three HCV genotypes Replicon Con1(1b) H77 (1a) JFH-1 (2a) EC 5 ( M).9 ±.3.9 ±.8.5 ±.1 (n) (>4) (2) (3) Preliminary studies indicate that has nanomolar activity against HCV genotype 3 Slide 9

10 Proof of Concept - Study SCY Phase 1 randomized, double-blind, placebo-controlled, dose-escalation HCV genotype 1 subjects randomly assigned to 15 consecutive days of orally administered or placebo in a 6:1 (active:placebo) ratio 3 mg/day (1 mg t.i.d.) 6 mg/day (2 mg t.i.d.) 9 mg/day (3 mg t.i.d.) Intensive PK sampling on Days 1-3 Hopkins et.al., J Hep 212 Slide 1

11 Comparison of Median Plasma Concentration-Time Profiles for Cohorts 4, 5 and 6 1 Plasma Concentration (ng/ml) mg tid 2 mg tid 3 mg tid EC 9 (476 ng/ml) EC 5 (132 ng/ml) Time (hour) Slide 11

12 -14: Mean and Median HCV RNA Profiles for Cohort 6 vs. Pooled Placebo 1. Log1 Change in HCV RNA from Baseline Mean Cohort 6 Response Mean Placebo Response* Median Cohort 6 Response Median Placebo Response* * Mean and median placebo responses determined from placebo subjects in Cohorts 4, 5, and 6 (n=3) Study Day Slide 12

13 Anti-Viral Effects of Mimic an IL-28B-Dependent Interferon Response CT Slide 13

14 Viral Infection Triggers Interferon Production and the Innate Immune Response dsrna IL28/IL29 = IFNλ M. Gale, E.M. Foy; Nature, 25 Slide 14

15 Comparison of 2 5 OAS and Neopterin Responses in Subjects with the TT and CC IL28B Genotypes Day 1, Subject 72 (TT Genotype) Day 2, Day 3, Fold Change in OAS Concentration from Baseline Day 1, Subject 72 (TT Genotype) Day 2, Day 3, Fold Change in Neopterin Concentration from Baseline OAS Concentration Neopterin Concentration Day 1, Subject 71 (CC Genotype) Day 2, Day 3, Fold Change in OAS Concentration from Baseline Day 1, Subject 71 (CC Genotype) Day 2, Day 3, Fold Change in Neopterin Concentration from Baseline OAS Concentration Neopterin Concentration Slide 15

16 Comparison of Interferon α and Interferon β Responses in Subjects with the TT and CC IL28B Genotypes Day 1, Subject 72 (TT Genotype) Day 2, Day 3, Interferon Alpha Concentration (pg/ml) Day 1, Subject 72 (TT Genotype) Day 2, Day 3, Interferon Beta Concentration (pg/ml) Interferon Alpha Concentration Interferon Beta Concentration Day 1, Subject 71 (CC Genotype) Day 2, Day 3, Interferon Alpha Concentration (pg/ml) Day 1, Subject 71 (CC Genotype) Day 2, Day 3, Interferon Beta Concentration (pg/ml) Interferon Alpha Concentration Interferon Beta Concentration Slide 16

17 Comparison of Interferon ʎ1 (IL29) and ʎ3 (IL28) Responses in Subjects with TT and CC IL28B Genotypes Day 1, Subject 72 (TT Genotype) Day 2, Day 3, Interferon Lambda 1 (IL29) Conc. (pg/ml) Day 1, Subject 72 (TT Genotype) Day 2, Day 3, Interferon Lambda 3 (IL28B) Conc. (pg/ml) Interferon Lambda 1 (IL29) Concentration Interferon Lambda 3 (IL28B) Concentration Day 1, Subject 71 (CC Genotype) Day 2, Day 3, Interferon Lambda 1 (IL29) Conc. (pg/ml) Day 1, Subject 71 (CC Genotype) Day 2, Day 3, Interferon Lambda 3 (IL28B) Conc. (pg/ml) Interferon Lambda 1 (IL29) Concentration Interferon Lambda 3 (IL28B) Concentration Slide 17

18 Healthy Volunteers Do Not Produce Interferon in Response to Treatment with (-15 Subject 21) Subject 21 Day 7, Interferon Alpha Concentration (pg/ml) Subject 21 Day 7, Interferon Lambda 1 Concentration (pg/ml) Interferon Alpha Concentration Interferon Lambda 1 (IL29) Concentration SCYNEXIS Proprietary and Confidential Information Slide 18

19 Concordance between Concentrations and Changes in the Plasma Protein Concentrations of Endogenous Interferons and 2 5 -OAS Subject IFN IFN IFN 1 IFN OAS Pearson correlation coefficients between the plasma concentrations of and the various analytes. Slide 19

20 Cyclophilin Inhibitors Relieve the Innate Immunity Blockade in HCV Infected Cells HuH7-Con1b IFN- IFN- 2'5'-OAS pg/mg protein 2 1 pg/mg protein 1 5 pg/mg protein pg/mg protein day IFN- pg/mg protein day HuH7- parental IFN- pg/mg protein day '5'-OAS SCY635 Alisporivir day day day P. Gallay, Scripps HuH7 stock IL28B genotype = CC (IL28B SNP rs ) Slide 2

21 Interferons Activate Innate Immune Response in Cultured HCV(+) PBMCs controls-il29-normalized controls-oas1-normalized Slide 21 IFNa2a IFNa2b IFNb IFNa1 IL28B IFNa2a IFNa2b IFNb IFNa1 IL28B IFNa2a IFNa2b IFNb IFNa1 IL28B IFNa2a IFNa2b IFNb IFNa1 IL28B TT1 CT1 CT2 CT IL-29 (pg/mg protein) IFNa2a IFNa2b IFNb IFNa1 IL28B IFNa2a IFNa2b IFNb IFNa1 IL28B IFNa2a IFNa2b IFNb IFNa1 IL28B IFNa2a IFNa2b IFNb IFNa1 IL28B TT1 CT1 CT2 CT3 OAS (pmol/mg protein) No induction of IFNα or IFNβ by IL28B. IL28B SNP rs

22 Cyclophilin Inhibitors Can Modulate Release of Innate Immunity Markers by HCV(+) PBMCs IFNa-normalized IFNb-normalized DeBio25 DeBio25 DeBio25 DeBio25 TT1 CT1 CT2 CT IFN (pg/mg protein) DeBio25 DeBio25 DeBio25 DeBio25 TT1 CT1 CT2 CT3 IFN (pg/mg protein) IL29-normalized DeBio25 DeBio25 DeBio25 DeBio25 TT1 CT1 CT2 CT OAS1-normalized IL-29 (pg/mg protein) DeBio25 DeBio25 DeBio25 DeBio25 TT1 CT1 CT2 CT3 OAS (pmol/mg protein) IL28B SNP rs Slide 22

23 PBMCs from HCV+ Donors Release Interferons Following Ex Vivo Exposure to IFN (pg/ml sup) TT (n=2) CT (n=6) CC (n=1) IL29 (pg/ml sup) TT (n=2) CT (n=6) CC (n=1) TT CT IL28 B CC TT CT IL28 B CC IL28B SNP rs Slide 23

24 inhibits HCV through at least two distinct mechanisms: Inhibition of viral recruitment of cyclophilin A Increased production of endogenous interferon alpha which in turn activates the Jak-STAT pathway The apparent association between IL28B and antiviral response to may be explained by: Restoration of the Innate Immune Response Contributes to Antiviral Activity -concentration-dependent production of interferon alpha and lambda in HCV-infected patients Individual antiviral response as determined by host factors regulating the innate responsiveness of the Jak-STAT pathway. These observations link the response to and the IL28B pre-treatment predictor of response to exogenous interferon alpha therapy Slide 24

25 Contributors Chemistry Mike Peel Bob Fogelsong Keqiang Li Andrew Scribner SCYNEXIS Biochemistry Ann Sluder Richard Harris Bernard Scorneaux Sarah Moser Clinical Sam Hopkins Pam Rusnak Betty DeMassimo Yves Ribeill (CEO) Mike Garrett (IP) Collaborators Gunter Fischer (Max-Planck) Philippe Gallay (Scripps) Jim Baugh Jose Garcia Koichi Watashi (NIID, Tokyo) Takuji Daito DMPK Steve Wring Ryan Randolph Craig Smitley Slide 25