Protease and Polymerase Inhibitors for the Treatment of Hepatitis C Tarik Asselah MD, PhD Service d’Hépatologie & INSERM U773, CRB3 Hôpital Beaujon, Clichy tarik.asselah@bjn.aphp.fr

Protease and Polymerase Inhibitors for the Treatment of Hepatitis C Unmet Needs Mechanisms of Non Response Protease Inhibitors Polymerase Inhibitors Conclusion

Progress in the Treatment of Hepatitis C 47% 63% 35 43% 18 23% 6 16% Recently, advances have been made, with the combination ofpegylated interferon (PEGIFN) and ribavirin leading to a sustained virological response (SVR) in approximately 55% of cases. In genotypes 2 or 3, SVR rates reach 80%; in genotype 1 SVR rates is 50%. Despite this progress, treatment failure still occurs in about halfof the patients. Furthermore, therapy results in several side effects and high costs. These limitations have led to important development of novel compounds under the name of specifically targeted antiviral therapy for HCV (STAT-C). IFN PEG-IFN IFN+Riba PEG-IFN+Riba 1989 2009

Incidence of Therapeutic Failure PEG-IFN-a + Ribavirin Incidence of Therapeutic Failure 58% PEG-IFN-a 2a + ribavirin (Fried et al., 2002) 54% 48% PEG-IFN-a 2a + ribavirin (Hadziyannis et al., 2004) 24% PEG-IFN-a 2b + ribavirin (Manns et al., 2001) 18% 16% Genotype 1 Genotypes 2/3 Manns et al. Lancet 2001; Fried et al. NEJM 2002; Hadziyannis et al. Ann Intern Med 2004.

How These Drugs will be Evaluated ? Patterns of Virological Response Baseline Treatment Nonresponder Breakthrough HCV RNA Partial responder Relapser Detection limit Sustained responder (cure) HCV RNA Undetectable 6 months Time

Response-guided Therapy Requires Precise Definitions of on-Treatment Response RVR* HCV RNA negative at week 4 EVR** Complete EVR HCV RNA positive at week 4 but negative at week 12 Partial EVR HCV RNA positive at week 4 and 12 but  2 log10 drop from baseline at week 12 Non-EVR < 2 log10 drop from baseline at week 12 * RVR = rapid virological response ** EVR = early virological response Marcellin et al. AASLD 2007

Asselah T et al. Liver International 2009 The management of chronic hepatitis C must include better knowledge of viral cycle and mechanisms of non response. Hepatitis C virus (HCV) viral cycle. Potentially, each step of the viral cycle is a target for drug development. The HCV lifecycle starts with virion attachment to its specific receptor (not clearly identified). The HCV RNA genome serves as a template for viral replication and as a viral messenger RNA for viral production. It is translated into a polyprotein that is cleaved by proteases. Then, viral assembly occurs. Asselah T et al. Liver International 2009

For the discovery of new markers of treatment response Knowledge of antiviral actions of Interferon is crucial. Together, these findings suggest that HCV modulation of IFN induction and signaling attenuates the expression of IFN-stimulated genes, allowing HCV to evade the antiviral actions of the host response. Liver gene expression profiling has recently been studied in chronic hepatitis C according to the response of therapy. Figure 1: Hepatitis C Virus Infection and Immune Response. Hepatitis C virus can induce several signaling pathways. (A) Toll-like receptor (TLR) signaling. Activation of TLR3 leads to the recruitment of IκB kinase (IKK)-related kinases, TANK-binding kinase 1 (TBK1, also known as T2K and NAK), and IKKi (also known as IKKε). These kinases, together with adaptors TANK and NAP1, catalyze the phosphorylation of IFN regulatory factor-3 (IRF-3). TLR3 engagement also results in the activation of transcription factors AP-1 and NF-κB. Phosphorylated IRF3 forms a dimer, translocates into the nuclei, binds to DNA, and regulates the expression of IFN β (IFNβ) in collaboration with AP-1 and NF-κB. The HCV NS3-4A serine protease may block the phosphorylation and effector action of IRF-3. (B) RIG-I like RNA helicase signaling. After recognition of viral RNA, retinoic acid-inducible gene-I (RIG-I) and also Mda5 (not shown) recruit IFNβ promoter stimulator-1 (IPS-1, also known as MAVS, Cardif, and VISA) via CARD-CARD (caspase recruitment domain) interaction. IPS-1 is localized to mitochondria and acts as an adaptor that plays a critical role in the activation of IRF-3 and IRF-7 in a TBK1- and IKKi-dependent manner. IPS-1 also interacts with the Fas-associated death domain protein (FADD), which is required for the activation of IRF-3 and NF-κB. IRF-7 forms a dimmer, translocates into the nucleus to induce IFN-alpha/beta; homodimers of IRF-3 collaborate with NF-κB to induce IFN-beta. IPS-1 is targeted and inactivated by NS3-4A, a serine protease from HCV known to block IFN-beta production. (C) IFN signaling. Endogenous IFN-α/β bind to a common receptor expressed at the surface of target cells. Receptor engagement leads to the activation of STAT1 and STAT2, which, together with ISGF3G/IRF-9, bind to cis-acting IFN-stimulated response elements (ISREs), thereby activating the transcription of IFN-α/β-inducible genes such as those encoding RNaseL and protein kinase R (PKR) which degrade viral RNAs and block their translation. Also, the HCV core protein has been shown to induce the expression of SOCS-3, which can suppress Jak–STAT signalling events and block the IFN-induced formation of ISGF3 (ref. 58). Asselah T et al. GUT 2009

Prediction of Non response PEG-IFN-a + Ribavirin Interferon Stimulated Genes NR IFI6 IFI27 ISG15 IL8 OAS.. SVR Time Asselah T et al. GUT 2008 Feld et al. Hepatology 2007 Chen et al. Gastroenterology 2005

Protease and Polymerase Inhibitors for the Treatment of HepatitisC Unmet Needs Mechanisms of Non Response Protease Inhibitors Polymerase Inhibitors Conclusion

Asselah T et al. Liver International 2009 Hepatitis C virus (HCV) genome and potential drug discovery targets. The HCV, identified in 1989, is an enveloped Flavivirus with a 9.6 kb single-strand RNA genome. The HCV RNA genome serves as a template for viral replication and as a viral messenger RNA for viral production. It is translated into a polyprotein that is cleaved by proteases. All the HCV enzymes – NS2-3 and NS3-4A proteases, NS3 helicase and NS5B RdRp – are essential for HCV replication, and are therefore potential drug discovery targets. The knowledge of the structures of HCV protease and HCV polymerase has allowed structure-based drug design to develop inhibitors to these enzymes. Asselah T et al. Liver International 2009

Polymerase Inhibitors Enzyme Inhibitors Protease Inhibitors Telaprevir (Vertex-Tibotec) Boceprevir (Schering Plough) BI 201335 (Bohringer) ITMN-191 (Intermune) Polymerase Inhibitors R7128 (Pharmasset-Roche)

Telaprevir (Vertex-Tibotec) 1 -1 Peg-IFN + placebo -2 Reduction of viral load (Log 10 IU/mL) -3 -4 VX-950 Rapid decline of HCV RNA in patients treated with Telaprevir. In a phase I–II trial in 34 patients with genotype 1, at a dose of 750 mg, 3 times/day, a marked reduction in viral load of approximately 4–5 log has been observed [(33)]. -5 VX-950 + PEG-IFN -6 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Duration of treatment (days) Reesink et al. Gastroenterology 2006

Placebo + PEG-IFNα-2a + RBV Telaprevir : Prove 2 PR48 (n = 82) Placebo + PEG-IFNα-2a + RBV T12/PR24 (n = 81) TVR PEG-IFNα-2a RBV PEG-IFNα-2a RBV TVR PEG-IFNα-2a RBV T12/PR12 (n = 82) TVR PEG-IFNα-2a T12/P12 (n = 78) 12 24 48 72 Zeuzem et al. AASLD 2008 – A243

Telaprevir : Prove 2 SVR Relapse (%) % p = 0.004* p = 0.12* % 48 50 80 69 40 60 NS* 60 46 30 30 36 22 40 20 14 20 10 38/82 56/81 49/82 28/78 10/45 8/57 19/63 22/46 PR48 T12/PR24 T12/PR12 T12/P12 (no RBV) PR48 T12/PR24 T12/PR12 T12/P12 (no RBV) * vs PR48

Telaprevir : Side Effects Rash ( 10 % discontinuation for severe rash) Pruritus Anaemia Nausea, Diarrhoea Headaches Fatigue

Boceprevir : Sprint 1 P + R P + R + B FU 44 w. P + R P + R + B Lead-in P + R P + R + B FU 24 w. n = 103 P + R + B FU 44 w. n = 107 No Lead-in P + R + B FU 24 w. n = 103 When this antiprotease is used in combination with PEG-IFN, a reduction in viral load of more than 4 log has been observed at day 14. (B, C) Results of telaprevir in Prove 1 and Prove 2 studies. In the randomized, double-blind, placebo-controlled phase II Prove-1 (USA) and Prove-2 (Europe) trials, telaprevir is being administered for 12 weeks with PEG-IFN a-2a (180 mg/week) plus RBV (1000 or 1200 mg/day) (36, 37). Preliminary data from these trials shows that the triple-therapy regimen increases the rate of rapid virological response at week 4 and sustained virological response. PEG-IFN, pegylated interferon; RBV, ribavirin; SOC, standard of care. P + R (LD) + B FU 24 w. Low dose RBV SOC P + R (SOC) FU 24 w. n = 104 4 weeks 28 48 72 Kwo et al. AASLD 2008 –A LB16

Boceprevir : Sprint 1 Virological Response 12 to 24 weeks after end of treatment (ITT) 100 74 80 66 55 56 60 % patients with HCV RNA undetectable 38 40 20 n = 104 n = 107 n = 103 n = 103 n = 103 P/R 48 w (n = 104) P/R/B 28 w (n = 107) P/R/ 4 w → P/R/B 24 w (n = 103) P/R/B 48 w (n = 103) P/R 4 w → P/R/B 44 w (n = 103) Kwo et al. AASLD 2008 –A LB16

Boceprevir : Side Effetcs Fatigue, Nausea, Headache Dysgeusia Anaemia (45 % receiving erythropoietin)

BI 201335 (Bohringer) Manns et al. AASLD 2008 –A 1849 BI 201335 or placebo BI 201335 + PEG-IFNα-2a + RBV BI 201335 1 20 mg /j 48 mg /j 120 mg /j 240 mg /j -1 Placebo -2 HCV RNA Log 10 UI/ml -3 -4 -5 1 2 3 4 6 10 14 21 28 Days Manns et al. AASLD 2008 –A 1849

PEG-IFNα + RBV treatment-experienced patients with Genotype 1 BI 201335 108 PEG-IFNα + RBV treatment-experienced patients with Genotype 1 107 106 105 104 103 102 25 Limit of detection 1 1 2 3 4 6 10 14 21 28 Manns et al. AASLD 2008 –A 1849

(NR) : non-responders to PEG-IFNα + RBV ITMN-191 (Intermune) 7 6 Placebo 100 mg/12h 5 100 mg/8h HCV RNA log10 (UI/ml) 4 300 mg/12h (NR) 200 mg/8h 3 200 mg/12h 2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Jours (NR) : non-responders to PEG-IFNα + RBV Forestier et al. AASLD 2008 –A1847

R7128 (Pharmasset-Roche) HCV GT 1 Naifs , RVR Weeks of Treatment 88% 100% 88% 85% 80% 75% HCV RNA <15 UI/mL 60% 45% 40% 30% 20% 20% 10% 10% 10% 0% 1 2 3 4 Weeks of Treatment PR PR+R 500mg/12h PR+R 1000mg/12h PR+R 1500mg/12h Lalezari et al. EASL 2008. Pharmasset. Press Release Sept 2008

R7128 G 2 & 3 NR or Relapsers -1 -2  Mean HCV RNA -3 -4 -5 -6 1 2 3 4 -1 G 2 & 3 NR or Relapsers -2  Mean HCV RNA -3 -4 -5 -6 1 2 3 4 Weeks SOC 1 500 mg 2x/j + SOC Gane EJ et al. AASLD 2008 –A LB10

Serine protease domain RNA-dependent RNA polymerase HCV resistance NS2–NS3 proteinase NS3 protease C E1 E2 p7 NS2 NS3 4A NS4B NS5A NS5B Core Envelope Serine protease domain RNA-dependent RNA polymerase VX-950; SCH 503034 T54 R155 A156 D168 V36 R1479 (R1626) S96 N142 S282 C316 M414 M419 P495 T423 VX-950; BILN 2061 Valopicitabine VX-950; BILN 2061; SCH 503034 HCV-796 BILN 2061 Nonnucleosides VX-950 Sarrazin et al. Gastroenterology. 2007. Tong et al. Antiviral Res. 2006. De Francesco and Migliaccio. Nature. 2005. Le Pogam et al. Virology. 2006. Villano et al. Hepatology. 2006.

Combination of Enzyme Inhibitors in the Replicon System Protease Inhibitors Polymerase Inhibitors Replicon System SCH 503034 HCV -796 Increase Anti-viral Decrease Resistance NM 107 VX-950 R1479 + + + EASL 2007- Howe AY - Kenilworth, USA, Abstract 432 EASL 2007- Ralston R - Kenilworth, USA, Abstract 793 EASL 2007- McCown M – Palo Alto, USA, Abstract 790

Potential Antiviral Targets and Approaches Immune System Enzymes Protease Inhibitors Polymerase Inhibitors Ribavirine PEG-IFN Potential use in Combination

Enzyme Inhibitor + SOC (PEG-IFN + RBV) in Genotype 1 Naïve Patients Conclusion Enzyme Inhibitor + SOC (PEG-IFN + RBV) in Genotype 1 Naïve Patients Increase SVR from 50 to  70 % Shorten the Duration of Treatment New Definitions of Response Resistance occurs Rapidly Toxicity Concern The development of new molecules such as viral enzyme inhibitors (protease and polymerase) is ongoing. Promising results have been reported with two protease inhibitors (telaprevir and boceprevir) that are currently in phase III. Several other protease and polymerase inhibitors are under development. In the near future, it is likely that IFN-based therapy plus RBV will remain the backbone of the treatment of chronic hepatitis C. PEG-IFN and RBV are needed inorder to prevent HCVresistance to STAT-C drugs, and subsequently increase SVR. Genotypic and phenotypic resistance tests will also enter the therapeutic arena. Once several STAT-C agents become available, treatment strategies will include a combination of several drugs with different mechanisms of action (protease inhibitors plus polymerase inhibitors) that could hopefully result in IFN- and/or RBV-sparing regimens. In the future, there might be combinations of antivirals having additive potency, lacking cross resistance and with a good safety profile.

Combination of Enzyme Inhibitors Perspectives Combination of Enzyme Inhibitors Increase SVR Decrease Treatment Duration Side Effects Minimize Resistance Avoid Ribavirin, avoid PEG-IFN, When ? Studies in other Populations : NR, other Genotypes, HIV-HCV Coinfected patients, Liver Transplant patients…