1. 4 th Annual Clinical Care Options for Hepatitis Symposium: HCV Highlights

2. clinicaloptions.com/hep 4 th Annual Clinical Care Options for Hepatitis Symposium: HCV Highlights Raymond T. Chung, MD Director of Hepatology Massachusetts General Hospital Associate Professor of Medicine Harvard Medical School Boston, Massachusetts Interferon and Ribavirin: Mechanisms of Action, Resistance, and Why It Matters

3. clinicaloptions.com/hep 4 th Annual Clinical Care Options for Hepatitis Symposium: HCV Highlights IRF-3 activation Innate Immunity: An IFN Amplification Loop Adapted from Gale M Jr, et al. Nature. 2005;436:939-945. RIG-I STAT 2 STAT 1 IKK-  TBK1 IRF-7 P P P P IRF-3 P P P P IRF-9 Tyk2 Jak1 IFN-  IFN-  IFN-  STAT 1 P TLR3 Hepatitis Virus Nucleus Cytoplasm Viral PAMP: dsRNA STAT 2 P ISGF3 JAK-STAT pathway VRE IFN-  PRD ISRE IFN-stimulated genes: OAS, IRF-7, PKR, ISG56 etc IFN-  production IFN-  /  IFNAR-2 IFNAR-1 IFN signaling Jak-STAT ISG expression; IFN amplification loop IFN- 

4. clinicaloptions.com/hep 4 th Annual Clinical Care Options for Hepatitis Symposium: HCV Highlights The Adaptive (Cellular) Immune Response Finishes the Job Adapted from Liang TJ, et al. Ann Intern Med. 2000;132;296-305. HCV Viral entry MHC II TCR MHC I B cell CD4+ Th cell CD8+ CTL CD8+ CTL CD4+ Th cell Clonal expansion (Th1 or Th2) Th2 cytokines (IL-4, IL-5, IL-6, IL-9, IL-10, IL-13) Activation, differentiation Th1 cytokines Lysis Neutralizing HCV antibodies Clonal expansion Hepatocyte IFN-  (IFN-γTNF-α)

5. clinicaloptions.com/hep 4 th Annual Clinical Care Options for Hepatitis Symposium: HCV Highlights Viral Kinetics After IFN Therapy Adapted from Feld JJ, et al. Nature. 2005;436:967-972. Viral kinetics IFN (efficacy =  HCV RNA (log IU mL -1 ) Days After Start of Therapy Two phases of viral decline 1st phase: antiviral efficacy (  ) (innate) 2nd phase: clearance of infected hepatocytes (  ) (adaptive) 0 -2 -3 -4 -707142128

6. clinicaloptions.com/hep 4 th Annual Clinical Care Options for Hepatitis Symposium: HCV Highlights Ribavirin  Initially developed as an antiviral–guanosine analogue  No antiviral activity but improved ALT when given as monotherapy  Combination with IFN improved ETR but greatly enhanced SVR rates by decreasing relapse –Does not alter 1st phase kinetics appreciably –Modest  of PEG-IFN antiviral effect (0.5-1.0 log)  Mechanistic models must explain clinical observations

7. clinicaloptions.com/hep 4 th Annual Clinical Care Options for Hepatitis Symposium: HCV Highlights Ribavirin: Proposed Mechanisms of Action Adapted from Feld JJ, et al. Nature. 2005;436:967-972. Inhibition of HCV RdRp (1st phase) Immunomodulation (2nd phase) Defective HCV particles (decreased fitness) Hepatocyte RDPRTPRMP Ribavirin (-) IMP IMPDH GMP GTP HCV RNA TH1TH1 CTL RNA Mutagen RdRp Replication IFN- , TNF-  Inhibition of IMPDH (1st phase) RNA mutagenesis (2nd phase) Ribavirin TH2TH2

8. clinicaloptions.com/hep 4 th Annual Clinical Care Options for Hepatitis Symposium: HCV Highlights HCV NS3-4A Blocks IFN Induction at Multiple Levels Adapted from Gale M Jr, et al. Nature. 2005;436:939-945. IRF-3 P P P P P CBP/p300 IRF-3 IKK-  TBK1 NS3/4A NF-  l  TRIF TLR3 IRF-3 NF-  RIG-I MDA5 RIP-1 HCV NS3/4A IKK l  P FADD TRAF6 IRF-3/NF-  Target genes (IFN-  ) IPS-1 mitochondria

9. clinicaloptions.com/hep 4 th Annual Clinical Care Options for Hepatitis Symposium: HCV Highlights HCV Blocks IFN Signal Transduction Adapted from Gale M Jr, et al. Nature. 2005;436:939-945. STAT 2 STAT 1 SOCS-3 IRF-9 Tyk2 Jak1 STAT 1 P Nucleus Cytoplasm STAT 2 P ISGF3 IFN-  /  IFNAR-2 IFNAR-1 ISRE PP2A PIAS SOCS-1 ISG expression attenuated Core IFN-  IFN-  IFN-  Core HCV proteins SOCS Inhibition of Jak-STAT signaling Block STAT function IL-8 NS5A Inhibit P-STAT1 Degrade STAT1

10. clinicaloptions.com/hep 4 th Annual Clinical Care Options for Hepatitis Symposium: HCV Highlights IFN-Stimulated Genes as the Antiviral Workhorses Adapted from Samuel CE. Clin Microbiol Rev. 2001;14:778-809. Microarray studies have identified > 100 IFN-stimulated genes Antiviral Actions of Interferon IFN Protein kinase PKR Inactive Oligoadenylate synthetase OAS dsRNA ssRNA Protein kinase PKR Active (Ribosome associated) Oligoadenylate synthetase OAS Active (multiple forms: nuclear and cytoplasmic) Initiation factor elF-2  Phosphorylated initiation factor elF-2  P Phosphatase (Soluble) Pi mRNA translation inhibition ATP AMP 2’, 5’-oligo adenylic acid (2, 5 A) Phosphodiesterase RNase L Inactive RNA degradation RNase L Active Inactive NS5A E2 HCV Paucity of recognition sites

11. clinicaloptions.com/hep 4 th Annual Clinical Care Options for Hepatitis Symposium: HCV Highlights Treatment of Hepatitis C in Blacks: SVR 1. Muir AJ, et al. N Engl J Med. 2004;350:2265-227. 2. Jeffers LJ, et al. Hepatology. 2004;39:1702-1708. 3. Conjeevaram H, et al. AASLD 2005. Abstract 199. 26% 19% 39% 52% 0 20 40 60 80 100 Muir et al [1] Jeffers et al [2] Black White Virologic Response Rates (%) n = 100 n = 78n = 28 P <.001 PEG-IFN  -2b 1.5  g/ kg/wk x 48 weeks + RBV 1000 800 mg/d 100% genotype 1 PEG-IFN  -2a 180  g/ wk + RBV 1000- 1200 mg/d x 48 wks 98% genotype 1 52% 28% Virahep-C [3] n = 196 n = 205 P <.001 PEG-IFN  -2a 180  g/ wk + RBV 1000- 1200 mg/d x 48 wks 100% genotype 1

12. clinicaloptions.com/hep 4 th Annual Clinical Care Options for Hepatitis Symposium: HCV Highlights Impaired Host Antiviral Responses as the Basis for Inferior SVR Rates?  Differences between A-A and C-A appear to reside primarily in 1st phase viral decay  These findings suggest intrinsic defects in innate immunity (signal transduction or ISGs)  Search for genetic polymorphisms in innate immunity underway (Virahep-C)

13. clinicaloptions.com/hep 4 th Annual Clinical Care Options for Hepatitis Symposium: HCV Highlights Obesity and Impaired Antiviral Response Rates  Obesity also associated with impaired antiviral response rates (likely related to steatosis)  SOCS-3 as key mediator –Upregulated in obesity –Increased by HCV –Promotes degradation of IRS1 and IRS2  insulin resistance Kawaguchi T, et al. Am J Path. 2004;165:1499-1508. Walsh MJ, et al. Gut. 2006;55:529-535.

14. clinicaloptions.com/hep 4 th Annual Clinical Care Options for Hepatitis Symposium: HCV Highlights Higher-Dose IFN or Further Refinements in IFN PK  Theory: overcome intrinsic blocks to IFN action with higher doses of exogenous IFN  High dose PEG-IFN + RBV: recent studies showing modest SVR rates in prior PEG-IFN/RBV nonresponders  Albumin-IFN: extend half-life of IFN to permit extended dosing interval  Limitations: tolerability, toxicities, downstream block

15. clinicaloptions.com/hep 4 th Annual Clinical Care Options for Hepatitis Symposium: HCV Highlights Strategies to Improve or Replace RBV  IMPDH inhibitors –Likely not major mechanism of RBV against HCV –Antiviral effect may be offset by immunosuppressive effects  Higher RBV doses –Further decreases in relapse among genotype 1 patients?  Prodrugs –Permit targeted dosing of RBV (viramidine)

16. clinicaloptions.com/hep 4 th Annual Clinical Care Options for Hepatitis Symposium: HCV Highlights New Therapies for Chronic Hepatitis C: Rational Drug Design Gary Davis, MD Director, Division of Hepatology Baylor University Medical Center Medical Director, Liver Transplantation Baylor Regional Transplant Institute Dallas, Texas

17. clinicaloptions.com/hep 4 th Annual Clinical Care Options for Hepatitis Symposium: HCV Highlights Role of New Agents in Treating HCV  Primary aim should remain eradication  Chronic suppression may be achievable  Interferon likely to remain foundation of therapy  Combination therapy will be key  Other agents may allow lower doses or shorter duration of poorly tolerated drugs  New agents will be able to target different processes of the HCV replication cycle

18. clinicaloptions.com/hep 4 th Annual Clinical Care Options for Hepatitis Symposium: HCV Highlights Target: Infection of the Hepatocyte

19. clinicaloptions.com/hep 4 th Annual Clinical Care Options for Hepatitis Symposium: HCV Highlights Therapeutics: Infection of the Hepatocyte  Polyclonal preparations [1] –Neutralize infectious inoculae ex vivo –Inhibit or prevent infection in chimps –Studies in man disappointing to date  Monoclonal antibodies [2] –Anti-E2 human monoclonal XTL –Mild HCV RNA suppression with daily dosing  Vaccine-derived anti-E1E2 [3] –Neutralizing antibody –In vitro inhibition of CD81 and VSV pseudovirions 1. Davis, et al. Liver Transpl 2005. Willems, et al. J Hepatol. 2002. 2. Schiano, et al. Hepatol. 2005. 3. DiBisceglie, et al. Hepatol. 2005.

20. clinicaloptions.com/hep 4 th Annual Clinical Care Options for Hepatitis Symposium: HCV Highlights Therapeutics: Infection of the Hepatocyte (cont’d)  N-glycans in envelope glycoprotein (E1E2) are essential for protein folding, secretion/assembly, antigenicity, receptor binding, and cell entry  Mutations in E2 eliminate infectivity [1] –E2N2, E2N4 by blocking cell entry  MX-3256 (celgosivir, Migenix) [2] –In vitro synergy with interferon-ribavirin –No reduction in HCV RNA 1. Goffard, et al. J Virol. 2005. 2. Yoshida, et al. Gastroenterology. 2006.

21. clinicaloptions.com/hep 4 th Annual Clinical Care Options for Hepatitis Symposium: HCV Highlights Target: RNA Transport to the ER 3’ 5’

22. clinicaloptions.com/hep 4 th Annual Clinical Care Options for Hepatitis Symposium: HCV Highlights Therapeutics: RNA Transport to the ER  Oligonucleotides –Ribozymes –Antisense oligos –siRNA

23. clinicaloptions.com/hep 4 th Annual Clinical Care Options for Hepatitis Symposium: HCV Highlights Therapeutics: RNA Transport to the ER (cont’d) mRNA Antisense oligo Ribozyme Translation arrest Endogenous ribonucleases destroy ineffective mRNA

24. clinicaloptions.com/hep 4 th Annual Clinical Care Options for Hepatitis Symposium: HCV Highlights Therapeutics: siRNA  Ancient host process of gene silencing, probably evolved for antiviral defense/genome protection  siRNA –Exogenous short synthetic ds nucleic acid molecules –Highly modified to increase stability (nuclease resistance), prolong half-life, and reduce nonspecific (off-target) effects –Incorporated into RNA-induced splicing complex (RISC) which pairs it with target mRNA –Destruction of mRNA by RNase

25. clinicaloptions.com/hep 4 th Annual Clinical Care Options for Hepatitis Symposium: HCV Highlights Target: Translation and Protein Processing

26. clinicaloptions.com/hep 4 th Annual Clinical Care Options for Hepatitis Symposium: HCV Highlights Therapeutics: Translation and Protein Processing HCV polyprotein C E1E2p7 NS2NS3NS4ANS4BNS5ANS5B Serine protease (trans) Serine protease (cis)

27. clinicaloptions.com/hep 4 th Annual Clinical Care Options for Hepatitis Symposium: HCV Highlights Therapeutics: Translation and Protein Processing (cont’d)  Conclusions –Potent antivirals –Orally bioavailable –Well tolerated –Synergy with IFN; increased IFN sensitivity –Require maintenance of trough concentration –May be able to shorten course of therapy –Other PI in development: ITMN B (InterMune)

28. clinicaloptions.com/hep 4 th Annual Clinical Care Options for Hepatitis Symposium: HCV Highlights Target: Viral RNA Transcription -RNA Subcellular Membrane 3’ 5’ +RNA 5’ 3’ Subcellular Membrane -RNA 3’ 5’ +RNA 5’ 3’ +RNA 5’ 3’ +RNA 5’ 3’

29. clinicaloptions.com/hep 4 th Annual Clinical Care Options for Hepatitis Symposium: HCV Highlights Therapeutics: Viral RNA Transcription  HCV polymerase inhibitors in development –NM-283 (valopicitabine, Idenix) –R1626 (Roche) –HCV-796 (Viropharma)

30. clinicaloptions.com/hep 4 th Annual Clinical Care Options for Hepatitis Symposium: HCV Highlights Target: Virus Assembly

31. clinicaloptions.com/hep 4 th Annual Clinical Care Options for Hepatitis Symposium: HCV Highlights Therapeutics: Virus Assembly  N-glycans in envelope glycoprotein (E1E2) are essential for protein folding, secretion/assembly, antigenicity, receptor binding, and cell entry  Imino sugars inhibit α-glucosidases and prevent proper glycosylation of viral envelope proteins; may inhibit secretion and infectivity of viruses –Zitzmann, et al. PNAS 1999; Mehta, et al. FEBS Ltr 1998