Copyright © 2007 Merck & Co., Inc., Whitehouse Station, New Jersey, USA All rights Reserved Resistance to the HIV-Integrase Inhibitor Raltegravir: Analysis of Protocol 005, a Phase 2 Study in Patients with Triple-Class Resistant HIV-1 Infection D.J. Hazuda, M. D. Miller, B. Y. Nguyen and J. Zhao for the P005 Study Team Merck Research Labs, North Wales PA 19454

Copyright © 2007 Merck & Co., Inc., Whitehouse Station, New Jersey, USA All rights Reserved Integrase strand transfer inhibitor (IC 50 ~ 10 nM) –hydroxypyrimidinone carboxamide Antiviral IC 95 ~ 31  20 nM (50% NHS) Active across diverse clinical isolates and HIV-2 Potent antiviral effect in treatment naïve patients –Phase II 24 week data (Markowitz et. al. IAS 2006) Robust activity in HIV-1 patients with triple class resistance – Phase II 24 week data (Grinsztejn et al ICAAC 2006) – Phase III data (Cooper et al & Steigbigel et al CROI 2007) MK-0518: Raltegravir

+ OBT § GSS = Genotypic sensitivity score by Phenosense GT Grinsztejn et al. Lancet 2007; 369: PN005 Phase 2 Study in Treatment Experienced Patients: Baseline Characteristics MK-0518*Placebo* 200 mg N= mg N= mg N=45N=45 Median Age (yrs) Male84%89%91%89% Mean log 10 HIV RNA Mean CD4 Count (/mm 3 ) Median Years of Prior ARTs OBT: Median # of ARTs 4444 GSS § : 0 to all ARTs 27 (63%) 38 (84%) 35 (78%) 28 (62%) GSS § : 0 to PI # pts with enfuvirtide as new OBT 38 (88%) 13 (30%) 45 (100%) 8 (18%) 41 (91%) 13 (29%) 39 (87%) 10 (22%) Copyright © 2007 Merck & Co., Inc., Whitehouse Station, New Jersey, USA All rights Reserved

Raltegravir Phase 2 PN005 Efficacy: Percent (95% CI) of Patients with Viral Load < 400 copies/mL and <50 copies/mL * BID + OBT Noncompleters = Failure approach Grinsztejn et al. Lancet 2007; 369: Number of Patients

Copyright © 2007 Merck & Co., Inc., Whitehouse Station, New Jersey, USA All rights Reserved Methods: PN005 Phase 2 Integrase Genotyping/Phenotyping Analysis Genotyping –population sequencing –first point identified as failure (non-responder and rebound) –compare sequence taken at baseline –cross sectional analysis –limited longitudinal studies (ongoing) Phenotyping –site directed mutants –limited data on patient derived sequences –single cycle infection assays with LTR- responsive reporter cell lines

Copyright © 2007 Merck & Co., Inc., Whitehouse Station, New Jersey, USA All rights Reserved Analysis of raltegravir resistance in PN005: Phase 2 Study in patients w/triple class resistance: Genotype of first time point, 24 week results Virologic failure was observed in 38/133 (28.6%) patients on RAL Integrase mutations were observed in 35 of 38 patients failing RAL RAL failure was generally associated with either of two genetic pathways (N155 or Q148) Additional mutations were frequently observed with both pathways Q148 containing mutants were more common than N155 Most common pattern Q148H/G140S N155H pathway Total N = 14 Q148 pathway Total N = 20 N155H (n = 2)Q148H, G140S (N=13) N155H, L74L/M (n = 1)Q148R (n = 1) N155H, E92Q (n = 1)Q148R, G140S (n = 2) N155H, T97A (n = 3)Q148K, E138K (n = 1) N155H, Y143H (n = 1)Q148R, E138E/K (n = 1) N155H, G163K (n = 1)Q148R, L74L/M, E138A (n = 1) N155H, E92Q, T97A (n = 1)Q148H/R, G140S (n = 1) N155H, V151I (n = 1) N155H, G163G/R (n = 2*) N155H, D232N (n = 1)Y143R (n = 1)

Copyright © 2007 Merck & Co., Inc., Whitehouse Station, New Jersey, USA All rights Reserved Profile of Patients in PN005 with virologic failure on raltegravir Similar number of patients with mutations at amino acids 148 or 155 across 200, 400, and 600 mg treatment groups Factors decreasing likelihood of developing mutations at a.a. 148 alone, 155 alone, either 148 or 155 (hazard ratio < 1): –Lower viral load (≤ 100,000 copies/mL vs. > 100,000 copies/mL) –New use of enfuvirtide in OBT –PSS > 0 Baseline Characteristics Patients with virologic failure on RAL vRNA > 100,000 53% vRNA < 100,000 47% CD4 <200 58% CD4 >200 42% GSS = 0 68% GSS ≥ 1 32%

Copyright © 2007 Merck & Co., Inc., Whitehouse Station, New Jersey, USA All rights Reserved Mutations observed in PN005 are similar to those selected with different integrase inhibitors in cell culture

Copyright © 2007 Merck & Co., Inc., Whitehouse Station, New Jersey, USA All rights Reserved Effect of signature and secondary mutations on raltegravir resistance Q148 PathwayN155 Pathway

Copyright © 2007 Merck & Co., Inc., Whitehouse Station, New Jersey, USA All rights Reserved Effect of signature and secondary mutations on infectivity Q148H/K/R and N155H single mutants display reduced infectivity. Among all mutants examined, Q148H/G140S has highest replication capacity in both infectivity (shown) and multiple cycle replication assays (data not shown). Q148 PathwayN155 Pathway

Copyright © 2007 Merck & Co., Inc., Whitehouse Station, New Jersey, USA All rights Reserved Summary of integrase genotyping and phenotyping results in PN005 Signature mutations Q148H/R/K and N155H engender: –>10-fold reduced susceptibility to raltegravir –decreased viral replication capacity Secondary mutations were usually observed: –consistently enhanced the level of raltegravir resistance –sometimes improved viral replication capacity (subset of combinations) Q148-containing combinations displayed higher level resistance than N155H-containing combinations –Q148 mutations were more prevalent in PN005 The G140S/Q148H variant was the most resistant and most fit variant –Resistance >100x; replication capacity near WT –Most common combination observed in PN005 Resistance is the primary driver for the acquisition of additional mutations, is there a pharmacologic threshold?

Copyright © 2007 Merck & Co., Inc., Whitehouse Station, New Jersey, USA All rights Reserved Longitudinal analysis of subjects with early N155H genotype: evidence of ongoing evolution to acquire higher level resistance

Copyright © 2007 Merck & Co., Inc., Whitehouse Station, New Jersey, USA All rights Reserved The Q148 pathway may be preferred Combinations containing Q148 mutations were more frequent in PN005 Resistance to raltegravir was generally higher with combinations containing Q148 mutations Patients who fail with Q148 combinations return to baseline vRNA –Some patients with N155H viruses appear to retain lower vRNA set point, partial activity or fitness? More longitudinal analysis is required

Copyright © 2007 Merck & Co., Inc., Whitehouse Station, New Jersey, USA All rights Reserved Mutations can confer cross resistance to structurally diverse integrase inhibitors IC 50 ratio of mutants vs. WT HIV-1 in infectivity assay 399 / 838 / 2194X 502X

Copyright © 2007 Merck & Co., Inc., Whitehouse Station, New Jersey, USA All rights Reserved Conclusions Raltegravir failure <30% in triple class resistant patients in PN005 –GSS=0 in >68% of virologic failures Raltegravir failure was associated with integrase mutations in two distinct genetic pathways defined by two or more mutations including –A signature mutation at either Q148(H/R or K) or N155(H) and –one or more secondary mutations unique to each pathway Signature mutations at Q148 and N155 conferred >10X reduced susceptibility to raltegravir and resulted in decreased viral replication capacity Secondary mutations appear to serve primarily to enhance the level of resistance to raltegravir –Although mitigation of effects on replication capacity were observed for a subset of combinations (eg. G140S/Q148H the most common variant), all combinations evaluated to date displayed increased resistance –Despite >10X resistance, viruses with a single mutation at N155 evolved to acquire increased resistance suggesting a pharmacologic threshold Mutations at Q148 and N155 can confer cross resistance to structurally diverse integrase inhibitors

Copyright © 2007 Merck & Co., Inc., Whitehouse Station, New Jersey, USA All rights Reserved AcknowledgementsInvestigators B. GrinsztejnBrazil C. KatlamaFrance J. GatellSpain A. LazzarinItaly D. VittecoqFrance C. GonzalezUSA J. Sierra Mexico G. CarosiItaly S. LittleUSA M. MoroniItaly J. Rockstroh Germany M. Kozal USA R. LiporaceUSA E. Jones-LopezUSA B. Clotet Spain S. StaszewskiGermany Merck Research Labs B-Y. Nguyen J. Chen R. Isaacs C. Harvey H. Teppler L. Wenning M. Miller D. Hazuda R. Danovich M. Rowley V. Summa J. Vacca J. Wai D. McMahonUSA P. KumarUSA C. LeeMalaysia K. SquiresUSA M. Opravil Switzerland N. ClumeckBelgium J. LennoxUSA J. EronUSA J. GallantUSA M. NelsonUK S. BrownUSA K. TashimaUSA V. SorianoSpain C. CrumpackerUSA D. KuritzkesUSA All patients in Protocol 005