1. Future therapies Massimo Levrero Department of Internal Medicine - DMISM LEA INSERM U785 Life-Nanoscience Laboratory for Functional Genomics Sapienza University - Rome

2. Inhibitors of polyprotein processing Inhibitors of replication Potential Antiviral Targets and Approaches IFN IFN lambdaIFN alfa Immuno- modulators Direct-acting antivirals (DAAs) Therapeutic vaccines Host-targeting antivirals (HTAs) Replication, polyprotein processing and/or assembly Entry NS5B polymerase inhibitors NS3 protease inhibitors NS5A replication complex inhibitors Popescu C-L & Dubuisson J. Biol Cell 2009;102:63-74.

3. Preclinical Phase I Phase II Phase III Filed Boceprevir(MSD) Telaprevir(Vertex/JJ) TMC-435(Tibotec/JJ) MK7009(MSD) ITMN191/R7227(Roche/Intermune) BI201335(BI) BMS650032(BMS) GS9256(Gilead) MK5172(MSD) ABT450(ABT) ACH2684(Achillion) BMS 790052 (BMS) AZD-7295 (AZN) BMS 824393 (BMS) PPI-1301 EDP-239 (Enanta) GSK Vertex Idenix719 MSD Taribavirin (Valeant) IFN λ (Zymogen/Novartis) Debio025/ NIM811 (Novartis) Nitazoxamide (Romark) Silibinine Vitamine D BMS BI ROCHE Gilead R7128 (Roche /pharmasset) PSI 7977 pharmasset) BI Japon Tonbacco R0622 (Roche) Medivir (Tibotec) GLS9393 (GSK) PSI 938 (Pharmasset) Biocryst INX 189 (Inhibitrex) BMS791325 (BMS) Filibuvir (PFE) GS9190 (Gilead) ANA598 (Anadys) BI201127 (BI) Vx222 (Vertex) ABT333 ABT072 (ABT) IDX 375 (Idenix) IDX 184 (Idenix) SCY-835 PPI-461 VBY-376 VX-985(Vertex) VX-813(Vertex) GS9451(Gilead) RG7348 (Roche) TMC 647055 (Tibotec) A837093 (Abbott) VX-916 VX-759 Celgosivir Bavituximab AVL-181(Avila) AVL-192(Avila) PSI-661 (Pharmasset) ACH-2928 (Acillion) GS-5885 Vertex Abbott Pharmasset Nucleoside NS5B Polymerase Inhibitors (8) Nucleotide NS5B Polymerase Inhibitors (4) Non Nuc NS5B Polymerase inhibitors (12) NS3/4A Protease inhibitors (19) NS5A inhibitors (13) DAA combinations (17) Others (6) Cyclophilin. I (2) IDX 077 (Idenix) IDX 079 (Idenix) ABT267(ABT) HCV pipeline by mechanism of action

4. Preclinical Phase I Phase II Phase III Filed Boceprevir(MSD) Telaprevir(Vertex/JJ) TMC-435(Tibotec/JJ) MK7009(MSD) ITMN191/R7227(Roche/Intermune) BI201335(BI) BMS650032(BMS) GS9256(Gilead) MK5172(MSD) ABT450(ABT) ACH2684(Achillion) VBY-376 VX-985(Vertex) VX-813(Vertex) GS9451(Gilead) AVL-181(Avila) AVL-192(Avila) NS3/4A Protease inhibitors (19) IDX 077 (Idenix) IDX 079 (Idenix) A second wave and a second generation of NS3 / NS4A protease inhibitors

5. Viral genotype “still” determines the approach to HCV Therapy Genotypes 2 and 3 – Therapy today and in the near future will be pegIFN/RBV Genotype 1 – Additional option of an HCV protease inhibitor combined with pegIFN/RBV – Boceprevir and telaprevir approved in Europe in July- September 2011 Both indicated for untreated and previously treated HCV Different regimen formats Different criteria for shortening therapy

6. Phase III virological efficacy Boceprevir ( Victrelis®) or Telaprevir ( Incivo®) Boceprevir SVR increases from 38% to 63/66% Naive patients Increased SVR compared to Peg-IFN/RBV Telaprevir SVR increases from 44% to 72/75% Poordad F et al. N Engl J Med 2011: 364: 1195-1206 Sherman KE et al. Hepatology 2010; 52 (Suppl) : 401A. Jacobson IM et al. Hepatology 2010; 52 (Suppl) : 427A.

7. Boceprevir Relapsers SVR increases from 29% to 75% Partial-Responders SVR increases from 7% to 52% Treatment-experienced patients Increased SVR compared to Peg-IFN/RBV Telaprevir Relapsers SVR increases from 24% to 83/88% Partial-responders SVR increases from 15% to 54-59% Null-responders SVR increases from 5% to 29/33% Phase III virological efficacy Boceprevir ( Victrelis®) and Telaprevir (Incivo ®) Bacon BR., et al. N Engl J Med 2011; 364:1207-1217. Zeuzem S, et al. J Hepatol 2011; 54(Suppl) : S3

8. PROVE-2 [1] SVR (%) 0 20 40 60 80 100 BPR 48 Wk (No Lead-in) (n = 16) BP + Low-Dose R (48 Wk) (n = 59) SPRINT-1 [2] 36 50 36 60 Both PegIFN and RBV Required in Protease Inhibitor Combination Regimens 1. Hezode C, et al. N Engl J Med. 2009;360:1839-1850. 2. Kwo PY, et al. Lancet. 2010;376:705-716. Dosages not consistent between above studies. TPR 12 (n = 82) TP 12 (n = 78) SVR (%) 0 20 40 60 80 100

9. Patient groups with the greatest need for improved therapies: TVR and BOC SVR by Patient Type 1.Zeuzem S, et al. N Engl J Med. 2011;364:2417-2428. 2. Bacon BR, et al. N Engl J Med. 2011;364:1207-1217. 3. Jacobson IM, et al. N Engl J Med. 2011;364:2405-2416. 4. Poordad F, et al. N Engl J Med. 2011;364:1195-1206. 5. Zeuzem S, et al. EASL 2011. Abstract 5. 6. Vierling JM, et al. AASLD 2011. Abstract 931. 0 20 40 60 80 100 SVR (%) RelapserNaive White/ Nonblack Null Responder Naive BlackPartial Responder Cirrhotic Null Responder 68-75 [3,4] 53-62 [3-4] *Pooled TVR arms of REALIZE trial. 75-83 [1,2] 52-59 [1,2] 29-38 [1,6] 14 [5] *

10. Probability of Resistance during Triple Therapy Patients with virologic treatment failure during triple therapy (break-through, stopping rules) Jacobson et al., NEJM 2011; Zeuzem et al., NEJM 2011 On-treatment virologic failure 8% (13% with 8 weeksTVR) Telaprevir 1% 19% 52% Bacon et al., NEJM 2011; Poordad et al., NEJM 2011 9% n.a. Boceprevir 35%

11. Switch TVR ↔ BOC in case of break-through? Sarrazin et al., J Hepatol 2011 in press V36A/ M T54S/AV55AQ80R/ K R155K/T /Q A156SA156T/VD168A/E/G/ H/T/Y V170A/ T Telaprevir* (linear) Boceprevir* (linear) SCH900518* (linear) BILN-2061 ** (macrocyclic) Danoprevir* (macrocyclic) MK-7009* (macrocyclic) TMC435* (macrocyclic) BI-201335* (macrocyclic?) BMS-650032* (macrocyclic) GS-9451* (macrocyclic) ABT450* (macrocyclic) IDX320** (macrocyclic) ACH1625** (macrocyclic) MK-5172*** (macrocyclic) * mutations associated with resistance in patients ** mutations associated with resistance in vitro *** no viral break-through during 7 days monotherapy Sarrazin et al., J Hepatol 2011 in press * mutations associated with resistance in patients ** mutations associated with resistance in vitro *** no viral break-through during 7 days monotherapy

12. Baseline predictive factors of SVR in the era of the first HCV protease inhibitors Identical in triple-therapy than in SOC ( age, viral load, fibrosis, IL28B) but their weights are lower. Some baseline predictive factor of SVR in the SOC era are not anymore predictive factor of SVR in triple-therapy (insulino-resistance, weight) In case of poor baseline predictive factors of SVR ( non CC IL28B, severe fibrosis ) SVR is increased with triple therapy > 50% and the benefit is maximal. In Naive genotype 1 patients with poor baseline predictive factors of SVR, triple therapy is mandatory as first line treatment

13. Challenges of Using 1 st generation Protease Inhibitors in Clinical Practice Increase in adverse effects [TVR: anemia, rash] [BOC: anemia, dysgeusia] Regimen complexity, TID dosing, Pill burden Adherence Concerns over resistance Improved SVR rate but still room for improvement Use restricted to genotipe 1

14. 1st wave DAAs Efficacy Genotype dependency Barrier to resistance NS3/4A (protease inhibitors) +++++ – NS5A++++ – NS5B (nucleosides) + –+++ NS5B (non-nucleosides) + –++ Efficacy Genotype dependency Barrier to resistance NS3/4A (protease inhibitors) ++++++ 2 ++ 2 NS5A++++++ 3 ++ 3 NS5B (nucleosides) +++ 1 +++ NS5B (non-nucleosides) ++++ New DAAs 1 e.g. PSI-7977, PSI-938; 2 e.g. MK-5172, ACH-1625; 3 e.g. PPI-461

15. Protease Inhibitors in Clinical Development DrugCurrent Clinical Phase Doses per DayActive Against HCV Genotype Side Effects TMC-435III11,2,4,5,6Bilirubinemia BI 201335III11,2 ?Jaundice, Rash, Gastrointestinal DanoprevirII31,2 ?Gastrointestinal, neutropenia, ALT increase VaniprevirII21,2 ?Vomiting MK-5172I11,2,3,4,5,6Na Ciesek S et al, Clin Liver Dis 2011;15:597-609

16. New Agents Generally Maintain or Improve Upon Efficacy in GT1 Treatment-Naive Phase II Studies, Drug + PegIFN/RBV 0 20 40 60 80 100 SVR (%) 71-83 68-8565-85 75-86 61-84 BI 201335 [3] Danoprevir [4] Narlaprevir [5] TMC435 [6] Vaniprevir [7] BOC or TVR [1,2] 63-75 38-50 Filibuvir [8] 56 Tegobuvir [9] 42-83 Daclatasvir [10] 53-76 Alisporivir [11] 1.Poordad F, et al. N Engl J Med. 2011;364:1195-1206. 2. Jacobson IM, et al. N Engl J Med. 2011;364:2405-2416. 3. Sulkowski M, et al. EASL 2011. Abstract 60. 4. Terrault N, et al. AASLD 2011. Abstract 79. 5. Vierling JM, et al. AASLD 2011. Abstract LB-17. 6. Fried M, et al. AASLD 2011. Abstract LB-5. 7. Manns MP, et al. AASLD 2010. Abstract 82. 8. Jacobson I, et al. EASL 2010. Abstract 2088. 9. Lawitz E, et al. EASL 2011. Abstract 445. 10. Pol S. ICAAC 2011. Abstract HI-376. 11. Flisiak R, et al. EASL 2011. Abstract 4. Not head-to-head comparisons

17. New Pis Improve Responses in Difficult-to-Treat Patients Efficacy in Null Responders AgentTrial, PhasePts Meeting Efficacy Measure, % Telaprevir and Boceprevir [1,2]  BOC or TVR + PRREALIZE/PROVIDE, IIISVR: 29-38 Investigational Protease inhibitors  BI 201335 + PR [3] SILEN-C2, IIbSVR: 21-35  TMC435 + PR*ASPIRE, IIbSVR: 41-59  Vaniprevir + PR [4] IIbSVR: 40-80 1. Zeuzem S, et al. N Engl J Med. 2011;364:2417-2428. 2. Vierling JM, et al. AASLD 2011. Abstract 931. 3. Sulkowski M, et al. EASL 2011. Abstract 66. 4. Lawitz E, et al. AASLD 2011. Abstract LB-13.

18. Will newer PIs be backbone drugs for combination therapies At least additive antiviral activity No cross-resistance No drug-drug interaction No overlapping safety issues No combination of TID with BID drugs (= QID)

19. Quad Therapy: Do We Have an Ideal DAA Combination? Genotype DependencyAntiviral EfficacyBarrier to Resistance NS3A (PI)+++++/++ NS5A+/++++++/++ NS5B (NUC)++++/+++++ NS5B (non-NUC)++/+++ Combine potent antiviral efficacy with high genetic barrier Welzel T et al, Clin Liver Dis 2011;15:657-664

20. Two DAAs Alone or Combined With PegIFN Alfa/RBV in Null Responders Lok A, et al. AASLD 2010; abstract LB-8, Lok A, et al. EASL 2011; abstract LB1356. Null responders = patients who achieved <2 log 10 IU/mL decline in HCV RNA following 12 weeks of treatment with pegIFN alfa/RBV. BID = twice daily, PO = orally, SC = subcutaneously, QD = once daily. BMS-790052 (NS5A RCI) 60 mg PO QD BMS-650032 (NS3 PI) 600 mg PO BID PegIFN alfa-2a 180 µg SC once weekly RBV 1000-1200 mg daily according to body weight 19 of 21 patients had unfavourable IL28B genotypes (rs12979860 CT/TT) 20 Group A Group B BMS-790052 + BMS-650032 (n=11) BMS-790052 + BMS-650032 + pegIFN alfa/RBV (n=10) Follow-up x 48 weeks 24-week treatment Post-treatment Week 12: SVR12

21. n (%) Dual therapy (N=11) Quadruple therapy (N=10) HCV RNA <10 IU/mL at week 4 at EoT SVR12 7 (63.6%) 5 4 (36.4%) 6 (60%) 10 (100%) 10 (100%) Viral breakthrough60 Relapse10 BMS-790052 (NS5A) + BMS-650032 (PI) ± PegIFN/Rbv In Null Responders 19/21 had IL28B genotypes CT or TT Safety: – Diarrhoea was the most common AE (71.4%) (mainly mild-moderate) – 6 patients experienced ALT >3x ULN, all had total bilirubin <2x ULN – 6 patients (all receiving PR) experienced Grade 3/4 neutropenia – No SAEs/discontinuations due to AEs Lok A, et al. EASL 2011, oral

22.  Daclatasvir (BMS-790052) QD (NS5A inhibitor) + asunaprevir (BMS-650032) BID (NS3 protease inhibitor) ± pegIFN/RBV for 24 wks Combination Therapy for Null Responders 1. Lok A, et al. EASL 2011. Abstract 1356. 2. Chayama K, et al. AASLD 2011. Abstract LB-4. 100 80 60 40 20 0 36 Daclatasvir + Asunaprevir Daclatasvir + Asunaprevir + PR SVR24 (%) 90 90* N/A US Study [1] Japan Study [2] *all genotype 1b patients.

23. 0% 20% 40% 60% 80% 100% Undetectable HCV RNA* *HCV RNA not detected by Roche COBAS TaqMan HPS v2.0 (LLOD 10IU/mL) cEVR, undetectable HCV RNA at week 12, SVR12, undetectable HCV RNA at week 12 post-treatment. BMS-790052 + BMS-650032 64 7/11 60 6/10 RVR 46 5/11 100 10/10 EOT 36* 4/11 100 10/10 SVR12 No SAEs or discontinuation due to adverse events Six subjects experienced ALT >3x ULN (all had bilirubin < 2x ULN) Six subjects (all receiving pegIFN alfa/RBV) experienced Grade 3/4 neutropenia *2/9 genotype 1a, 2/2 genotype 1b Lok A, et al. AASLD 2010; abstract LB-8, Lok A, et al. EASL 2011; abstract LB1356. BMS-790052 + BMS-650032 + pegIFN alfa/RBV Two DAAs Alone or Combined With PegIFN Alfa/RBV in Null Responders

24. Dual therapy with the NS5A inhibitor BMS-790052 and the NS3 protease inhibitor BMS-650032 in HCV genotype 1b-infected null responders Chayama K, et al, Hepatology in press = 1 = 9 open-label, phase 2a study of 10 Japanese patients with HCV 1b chronic hepatitis and prior null response to PEG IFN and ribavirin (<2 log10 HCV RNA reduction after 12 weeks)

25. Dual therapy with the NS5A inhibitor BMS-790052 and the NS3 protease inhibitor BMS-650032 in HCV genotype 1b-infected null responders Chayama K, et al, Hepatology in press

26. Additional announced IFN-free study designs in treatment-experienced patients Combination Therapy for Null Responders Drug 1Drug 2Overall Regimen TMC435 QDPSI-7977 QD± RBV 12 or 24 wks ABT-450/RTV QD ABT-333 BID+ RBV Danoprevir/ RTV BID Mericitabine BID + RBV 24 wks Protease inhibitor Nucleos(t)ide analogue polymerase inhibitor Nonnucleoside polymerase inhibitor

27. Combination Regimens in GT1 Treatment-Naive Patients Strategy: protease inhibitor (telaprevir) + nonnucleoside polymerase inhibitor (VX- 222) [1] – ± pegIFN/RBV for 12 wks, then RGT Strategy: protease inhibitor (ABT-450/r) + nonnucleoside polymerase inhibitor (ABT-333 or ABT-072)* – + RBV for 12 wks – All 44 patients achieved cEVR – Of 10 patients tested thus far, 9 achieved SVR24 100 80 60 40 20 0 Patients (%) 93 82 87 83 VX-222 400 mg BID + TVR + PR VX-222 100 mg BID + TVR + PR SVR24; 12 total wks of therapy SVR12; 24 total wks of therapy 1. Nelson DR, et al. AASLD 2011. Abstract LB-14.

28. INFORM-1: An all Oral IFN Free Regimen of RG7128 + RG7227 RG7128 1000 mg BID + RG7227 900 mg BID Days 135791113 Limit of Detection Median Log 10 HCV RNA (IU/mL) 1 2 3 4 5 6 7 TF - Nulls Naïves LLOD: Lower limit of detection LLOQ: Lower limit of quantification Gane EJ, et al. Lancet 2010; 376(9751):1467-75. 50 25 0 10 20 30 40 50 60 70 80 90 100 <LLOQ<LLOD 88 63 Nulls Naïves EOT HCV RNA < LLOQ or LLOD (%)

29. SOUND-C1: BI 201335 (PI) plus BI 207127 (non- NUC) plus RBV in Naive HCV-1 Patients Proportion of patients with HCV RNA <25 IU/mL during treatment n/N (%)Day 8Day 15Day 22Day 29 400 mg tid BI 207127 + BI 201335 + RBV 4/15 (27)7/15 (47)10/15 (67)11/15 (73) G1a2/105/106/10 G1b2/5 4/55/5 600 mg tid BI 207127 + BI 201335 + RBV 3/17 (18)14/17 (82)17/17 (100) G1a2/88/8 G1b1/85/88/8 G6e0/11/1 Zeuzem S, et al. Gastroenterology in press The SOUND-C2 study: up to 59 % of SVR-12

30. Ongoing Research Evaluates Potential for All-Oral Therapy Several all-oral regimens under investigation Drug 1Drug 2Drug 3RBV BI 201335BI 207127N/A ± PSI-7977PSI-938N/A ± ABT-450/ RTV ABT-333 or ABT-072 N/A + PSI-7977DaclatasvirN/A ± GS-9256TegobuvirN/A ± GS-9451GS-5885± Tegobuvir± AsunaprevirDaclatasvirBMS-791325N/A Protease inhibitor Nucleos(t)ide analogue polymerase inhibitor Nonnucleoside polymerase inhibitor NS5A inhibitor All-oral regimens of single drug + RBV also under investigation. IFN-free regimens shown to be highly effective in GT2/3 [1]  Nucleotide analogue PSI-7977 + RBV for 12 wks – PegIFN included for 0, 4, 8, or 12 wks 100 80 60 40 20 0 SVR (%) 100 PSI-7977 + 0 wks PegIFN (IFN-free) PSI-7977 + 4 wks PegIFN PSI-7977 + 8 wks PegIFN PSI-7977 + 12 wks PegIFN 1. Gane EJ, et al. AASLD 2011. Abstract 34.

31. Planned or ongoing Phase III Trials TVR BID + PR PR Telaprevir in GT1 IL28B CC patients PSI-7977 + R PSI-7977 across genotypes TMC435 + PR PR TMC435 in GT1 patients BI 201335 + PR PR BI 201335 in GT1 patients BI 201335 + PR PR Daclatasvir + PR PR Daclatasvir in GT1/4 patients Daclatasvir + PR QUAD Telaprevir + VX-222 + PR in GT1

32. Can We Make Regimens Simpler? Can We Improve Adherence? Several Drugs in Development Are Dosed Once or Twice Daily *With RTV boosting. QD ABT-072 ABT-267 ABT 450* ACH-1625 BI 201335 Daclatasvir GS 5885 GS9451 IDX 184 INX-189 MK-5172 Narlaprevir* PSI-7977 PSI-938 TMC435 BID ABT-333 Asunaprevir BI 201335 BI 207127 BMS 791325 Danoprevir* Filibuvir GS9256 Mericitabine Setrobuvir Tegobuvir Vaniprevir VX-222 TID BI 207127 Danoprevir

33. Fewer AEs With Some Investigational Agents (Preliminary Data) Agent AEs More Frequent in Experimental Arm vs PegIFN/RBV Discontinuations due to AEs, % (Wk) Boceprevir/Telaprevir [1,2] Anemia, dysgeusia, neutropenia, rash, anorectal symptoms13-14 (48) ABT-072 [3] (N = 27)Headache0 (12) ABT-333 [3] (N = 18)None0 (12) ABT-450/r [4] (N = 30)None0 (12) Alisporivir [5] (N = 215)Transient hyperbilirubinemia5 (48) Asunaprevir [7] (N = 36)Fatigue11 (12) BI 201335 [6] (N = 355)GI events, jaundice, and rash8 (48) Daclatasvir [8] (N = 36)None8 (12) Danoprevir [9] (N = 194)ALT elevation, neutropenia, nausea, diarrhea4 (12) Mericitabine [10] (N = 81)None6 (36) PSI-7977 [11] (N = 95)None3 (12) Setrobuvir [12] (N = 63)Rash2 (12) TMC435 [13] (N = 309)Mild bilirubin increases7 (24) Vaniprevir [14] (N = 169)GI events6 (48) Studies displayed include those with data through at least 12 wks and with discontinuation rates lower than BOC/TVR. 1. Boceprevir [package insert]. May 2011. 2. Telaprevir [package insert]. May 2011. 3. Gaultier, et al. APASL 2011. 4. Lawitz E, et al. EASL 2011. Abstract 1220. 5. Flisiak R, et al. EASL 2011. Abstract 4. 6. Bronowicki JP, et al. EASL 2011. Abstract 1195. 7. Sulkowski M, et al. EASL 2011. Abstract 60. 8. Pol S, et al. EASL 2010. Abstract 1189. 9. Terrault N, et al. AASLD 2010. Abstract 32. 10. Pockros P, et al. EASL 2011. Abstract 1359. 11. Nelson D, et al. EASL 2011. Abstract 1372. 12. Lawitz E, et al. AASLD 2010. Abstract 31. 13. Fried M, et al. AASLD 2010. Abstract LB-5. 14. Lawitz E, et al. AASLD 2011. Abstract LB-13. Can We Make Regimens Simpler? Can We Improve Adherence?

34. Evolution of HCV Therapy 20012011Future PegIFN/RBV Protease inhibitor Nucleos(t)ide polymerase inhibitor Nonnucleoside polymerase inhibitor NS5A inhibitor Host targeting agent Next ? Majority of protease inhibitors have targeted genotypic coverage but MK-5172, TMC435 have broad coverage [1,2] Most nucleos(t)ide analogue polymerase inhibitors are pan-genotypic 1. Brainard DM, et al. AASLD 2010. Abstract 807. 2. Fried M, et al. AASLD 2010. Abstract LB-5. ?

35. Conclusions The next generation of protease inhibitors are in development (expected to be available in 2015 or even earlier) Different DAA combination therapies are being evaluated in early phase trials – Whether Peg-IFN and/or RBV will be needed in all patientsis unclear Final aim for a DAA combination regimen – All oral – QD – Safe and well tolerable – Pan-genotypic – IL28-independent – Limited resistance