A Phase 1 Randomized, Double-Blind, Placebo-Controlled Rectal Safety and Acceptability Study of Tenofovir 1% Gel Ian McGowan, Craig Hoesley, Ross Cranston, Philip Andrew, Laura Janocko, James Dai, Alex Carballo-Dieguez, Ratiya Kunjara Na Ayudhya, Jeanna Piper, Florian Hladik, Ken Mayer, and the MTN-007 Protocol Team. 19 th Conference on Retroviruses and Opportunistic Infections Seattle 6 th March, 2012 MTN-007

Background  Receptive anal intercourse is a high risk but common sexual behavior among men and women in the developed and developing world.  Non human primate studies have demonstrated that an antiretroviral rectal microbicide (RM) can reduce the acquisition of SIV #.  RMP-02/MTN-006 demonstrated a favorable PK/PD profile for rectal tenofovir (TFV) 1% gel but suboptimal safety profile* Gastrointestinal symptoms #Cranage M et al. PLoS Med, 2008; *Anton PA et al. CROI 2011

Rationale for MTN-007  To characterize the safety and acceptability of a reduced glycerin (RG) formulation of TFV 1% gel Original TFV 1% gel: 3111 mOsmol/kg RG TFV 1% gel: 836 mOsmol/kg Iso-osmolar:290 mOsmol/kg  To evaluate the utility of a broad range of biomarkers of mucosal safety

Study Population  Healthy, receptive anal intercourse abstinent, HIV-1 uninfected men and women  Key exclusion criteria Rectal or reproductive tract STI Hepatitis B surface antigen positive Bleeding tendency Pregnancy or breastfeeding

Study Design N=65 HEC (N=16) 1% TFV (N=16) 2% N-9 (N=17) Single dose 7 day daily doses 7-14 day interval Endoscopy Safety/behavioral assessment Screening No Treatment (N=16) Baseline Evaluation 7-14 day interval

Study Endpoints  Primary Grade 2 or higher adverse events  Secondary The proportion of participants who at their final clinic visit report via the acceptability questionnaire that they would be very likely to use the candidate microbicide during receptive anal intercourse Changes in mucosal parameters

Mucosal Safety Endpoints  Histopathology  Epithelial sloughing  Fecal calprotectin  Rectal microflora  Mucosal mononuclear cell phenotype by flow cytometry  Cytokines and chemokines qRT-PCR Luminex  Gene expression by microarray

Baseline Demographics by Arm All ArmsTFV GelN9 GelHECNo Rx N Mean Age Gender Male (%)45 (69%)10 (63%)13 (76%)12 (75%)10 (63%) Female (%)20 (31%)6 (38%)4 (24%)4 (25%)6 (38%) Hispanic6 (9%)1 (6%)2 (12%)2 (13%)1 (6%) Race Black (%)11 (17%)3 (19%)2 (12%)5 (31%)1 (6%) White (%)44 (68%)10 (63%)13 (76%)9 (56%)12 (75%) Other (%)10 (15%)3 (19%)2 (12%)2 (13%)3 (19%)

Incident Adverse Events by Arm All ArmsTFV GelN9 GelHECNo Rx N Grade 1 30 (46.2%)7 (43.8%)10 (58.8%)7 (43.8%)6 (37.5%) Grade 2 18 (27.7%)3 (18.8%)7 (41.2%)4 (25.0%) Grade 3 2 (3.1%)0002 (12.5%) Grade 4 1 (1.5%)001 (6.3%)0 Grade Total 51 (78.5%)10 (62.5%)17 (100.0%)12 (75.0%)

Incident GI Adverse Events GI Adverse Events in the Tenofovir Arm MTN-007 (N = 16) RG Formulation N% Abdominal pain319% Rectal urgency00% Bloating00% Nausea00% Diarrhea16% Flatulence638% Proctalgia16% Other319% Total956%

Gastrointestinal Adverse Events GI Adverse Events in the Tenofovir Arm MTN-007 (N = 16) RG Formulation RMP-02/MTN-006 (N = 12) Original Formulation N%N% Abdominal pain316%650% Rectal urgency00%542% Bloating00%542% Nausea00%433% Diarrhea16%758% Flatulence638%325% Proctalgia16%00% Other425%542% Total956%12100%

Acceptability Product (N)Intention to Use (%) RG Tenofovir (15)87% HEC Placebo (15)93% N-9 (16)63%

Mucosal Safety Assays  No significant changes were seen in histology, fecal calprotectin, and epithelial sloughing after single dose (SD) or 7 daily doses (7D)  Suggestive evidence of change (▲ and ▼) were seen with flow cytometry, qRT- PCR, and Luminex assays at SD and 7D  The majority of changes were seen when comparing N-9 to the other arms of the study

Mucosal qRT-PCR 9 cm15 cm BL SD 7D

Mucosal Safety Parameters (1) qRT-PCRSD7DSD + 7DComparisonP Value (s) IL-1  (9 cm) ▲ N9 vs. No Rx1.3e-3 ▲ N9 vs. HEC6.8e-4 ▼ TNF vs. N-96.1e-4 IL-6 (9 cm) ▲▲ N9 vs. HEC3.0e-4; 8.4e-5 ▼▼ TNF vs. N-91.3e-5; 1.8e-4 IL-6 (15 cm) ▼ N9 vs. HEC1.8e-3 IL-8 (9 cm) ▲ N9 vs. HEC2.8e-4 ▼▼ TNF vs. N-94.2e-5; 1.4e-3 MIP-1  (9 cm) ▼ TNF vs. N-92.7e-4

Mucosal Safety Parameters (2) Flow CytometrySD7DSD + 7DComparisonP Value (s) CD3+/CD8+ ▼▼ N9 vs. No Rx1.6e-3; 4.0e-4 Luminex RANTES ▲▲ N9 vs. HEC1.3e-3; 2.7e-4

Microarray Data  Gene array studies were performed on a subset of 8 participants per study arm (9 cm and 15 cm samples)  Labeled cRNA were hybridized to Illumina Human-HT12 v4 gene expression BeadChips.  The Benjamini Hochberg method was used to control for multiple testing (false discovery rate).  Fold change was calculated as the fold difference between treatment over baseline.

N-9 gel HEC gel No Treatment Tenofovir gel 9 cm Gene Expression at Day 7

Summary of Microarray Data N9TNFHECNo Rx Gene expression 9 cm rectum following 7 days of product application

Summary  The reduced glycerin formulation of TFV 1% gel was safe and acceptable and should move forward to Phase 2 development  The majority of mucosal safety signals were associated with the use of N-9  TFV 1% gel associated changes in gene expression require further evaluation

Key Issues Moving Forward  Determine whether gene array signals are important  Validation initial observations MTN-007 & Project Gel  Linked to hyperosmolar formulations: CHARM-01  Linked to tenofovir: MTN-017  Implications for vaginal microbicides

Acknowledgements  The MTN-007 study participants  CONRAD  MTN is funded by NIAID (5UM1AI068633), NICHD and NIMH, all of the United States National Institutes of Health