1. DAIDS IPCP-HTM NIH/NIAID: 1 st IPCP on Rectal Microbicides Developments (2004-2011) Two Phase 1 Clinical Trials: RMP-01: Topical UC781 (single & 7-day topical) RMP-02 / MTN-006: Tenofovir (single oral, single & 7-day topical) Peter Anton, UCLA: PI Ian McGowan, MWRI/University of Pittsburgh: co-PI Ian McGowan, MWRI/University of Pittsburgh: PI NIH/NIAID: 2 nd IPCP on Rectal Microbicides Developments (2009-2014)

2. NIH IP/CP Outline  Intent  Assays for use in RM trials  1 st Phase 1 RM Clinical Trial utilizing assays: RMP-01 (UC781)  2 nd Phase 1 RM Clinical trial: RMP-02/MTN-006 (Tenofovir) context

3. Assay Optimization: selected for Phase 1 Assay factors addressed prior to trial (some still to address) : Apply to both vaginal and rectal samples Anticipate effect of standard clinical trial / home use such as enemas/douches/gels (osm; pH; dilution effect) Sequence of sample collection: inherent confounder Multi-site trials: determine where samples (Flow, explants etc) collected versus where/when processed (fresh, frozen, O/N, batched: 1 lab?) Semen/seminal fluid alter results/drug delivery? Same with sexual activity / trauma Relevance for interpreting “biopsy infection” experiments Compartment dilution effect on delivered drug concentrations? Quantify [microbicide] likely exposed to tissue. Challenge much greater in rectal than cervicovaginal explants NORMATIVE VALUES: to assess implications/actions of “out-of- range” values (and then: clinical relevance) NIH IP/CP

4. HPTN 056: Characterization of Baseline Mucosal Indices of Injury and Inflammation in Men for Use in Rectal Microbicide Trials Pre-Phase 1 rectal safety study:  normative ranges; inherent variability Primary Objective  Determine variability of mucosal immunological, virological and histopathological parameters in biopsies from 10cm & 30cm in the recto-sigmoid colon in 4 defined study groups (n=16): I.HIV-/RAI- II.HIV-/RAI+ III.HIV+/RAI+: high PVL IV.HIV+/RAI+: high PVL Secondary Objective  Determine within group stability of defined measures over time (3 flexible sigs over 6 weeks) & biological variability between groups Assays  Histopathology (qual/quant); flow cytometry, tissue cytokine mRNA, rectal secreted Ig, tissue VL) Total: 48 procedures; 1,440 biopsies McGowan et al, HPTN 056 JAIDS 2007

5. HPTN-056: Data Analysis  Data analyzed by group means  Subject variability around means explored  Definitions: Sig: within subject standard deviation Tau: between subject standard deviation Intra-class correlation (ICC) [ICC = Tau^2/ (Tau^2 + Sig^2)] ICC thresholds –>0.75 shows strong stability –>0.5 shows moderate stability McGowan et al, HPTN 056 JAIDS 2007

6. Stability of Flow Cytometry Data MeanG1G2G3G4SigTauICC CD3%686674705.59.80.76 CD4%424112294.08.00.81 CD8%292862455.211.90.84 CD31%686673705.69.90.76 CD19%323026377.48.30.56 ICC: Intra-class correlation >.75 shows strong stability McGowan et al, HPTN 056 JAIDS 2007

7. Stability of Cytokine Data MeanG1G2G3G4SigTauICC RANTES (log 10 ) 4.14.54.94.10.280.490.75 IFN-  (log 10 ) 2.73.23.33.00.290.500.74 IL-10 (log 10 ) 2.73.22.62.80.280.430.70 McGowan et al, HPTN 056 JAIDS 2007 ICC: Intra-class correlation >.75 shows strong stability

8.  Colorectal (10cm)  Colonic (30cm)  Colorectal (higher than 10cm)  Upper intestinal tract (systemic delivery to targets) Where to sample…”explants” When to sample…explants  Real-life factors: bowel movements, prep, pre/post sexual prep, menses  Drug trial factors: how long post exposure, frequency, # samples Safe to sample…? NIH IP/CP

9. X X Colorectal: Biopsy location NIH IP/CP

10. Colorectal Biopsy safety and location NIH IP/CP

11. Success Concentration Ano-Rectal Distance Anus Failure HIV RectumColon Microbicide RM Clinical Relevance: Selection Site for Explants Diagram courtesy: C Hendrix) ~10cm + effect? ~30cm no effect?

12. Rectal Explant Model used in RM Clinical Trials: “ex vivo biopsy infection” experiments Explant media soaked collagen rafts b)1 cm 2 raft placed atop the explant and inverted. HIV-1 infected and washed explants a) Explants transferred by transfer pipet to a dry petri-dish, cut-side down Mounted explants transferred to well of 24- well plate containing 500ul of explant medium Media 100% exchanged q 3 days for ~2 weeks (D1/D4/D7/D11/D14) Supernatant frozen for batched p- 24 ELISA (= qPCR of HIV RNA ) Fletcher et al AIDS 2006

13. A PHASE 1 SAFETY AND ACCEPTABILITY STUDY OF THE UC-781 MICROBICIDE GEL APPLIED RECTALLY IN HIV SERONEGATIVE ADULTS: RMP-01 P Anton, T Saunders, A Adler, C Siboliban, E Khanukhova, C Price, J Elliott, K Tanner, Ana Ventuneac, Alex Carballo-Dieguez, J Boscardin, Y Zhao, W Cumberland, AM Corner, C Mauck, I McGowan UCLA, NIH, CONRAD submitted

14. NIH IP/CP Rectal Biopsy Infections ex vivo: RMP-01  Samples acquired endoscopically (large-cup forceps): 14 biopsies at each site (10cm and 30cm)  NO DRUG ADDED TO EXPLANTS (except at V2): all drug applied in vivo  To laboratory and set up within 2 hours  HIV (pre-determined strain, titers) applied to explants, incubated 2 hrs  Biopsies washed (>3-5 times), then mounted on gelfoam, placed in well of 24-well plate; incubated for 12-14 days.  Controls: media (uninfected control); UC781 at baseline visit only to demonstrate in vitro efficacy  Supernatants for p24 taken every 3 days (100%); each time point is mean of 2 biopsies pooled; cumulative p24 graphed

15. HEC PlaceboUC781 0.10%UC781 0.25% 10cm 30cm NIH IP/CP (V2 = baseline) (V3 = single topical application UC781) Confidence from RMP-01 UC781 RM trial (n=36; 3 arms) in vivo exposed ex vivo infected Bx infection data (V2 vs V3) 10 4 virus titer

16. NIH IP/CP HEC PlaceboUC781 0.10%UC781 0.25% 10cm 30cm Baseline variability looks same…. but only 60% infected with this titer (V2 = baseline) (V3 = single topical application UC781) RMP-01 UC781 RM trial: now 10 2 virus titer

17. Lessons learned thus far for these types of trials: Do infectibility results from 30cm and 10cm differ: NO Need infectibility of ‘all’ baseline sample explants: YES Compare 10cm vs. 30cm for all subjects at baseline (V2) Paired t-test 10 2 viral dose0.8600 10 4 viral dose0.5228 No difference seen when using 36 subjects’ baseline data: “OK” to use 1 site in next trial Baseline infectibility NIH IP/CP Use higher titer virus for explant infection studies

18. DAIDS IPCP-HTM RMP-02/MTN-006: A Phase 1, Placebo-Controlled Trial of Rectally Applied 1%Vaginal Tenofovir Gel with Comparison to Oral Tenofovir Disoproxil Fumarate P Anton 1, R Cranston 3, A Carballo-Dieguez 4, A Kashuba 5, E Khanukhova 1, J Elliott 1, L Janocko 6,8, N Richardson-Harman 7, W Cumberland 9, C Mauck 10, C Hendrix 2,8, I McGowan 6,8 UCLA: Dept. of Medicine 1 & School of Public Health 9, Johns Hopkins University 2, University of Pittsburgh: Dept. of Medicine 3 & Magee-Womens Research Institute 6, Columbia University 4, UNC CFAR & School of Pharmacy 5, Alpha StatConsult LLC 7, MTN 8, CONRAD 10 Presented: CROI 2011, Boston, MA

19.  Rectal intercourse is commonly practiced by men and women  HIV transmission during receptive anal intercourse (RAI) is significantly greater per sexual act  CAPRISA 004 demonstrated 39% reduction in HIV infection using 1% tenofovir gel formulated for vaginal use  Given the prevalence of RAI and the success of this agent, this Phase 1 trial aimed to evaluate safety of the hyper- osmolar, vaginally formulated 1% tenofovir gel when used rectally; in addition, aims were to investigate acceptability, mucosal injury and multi-compartment PK Study Rationale

20. Baseline Evaluation Open label Oral tenofovir (N = 18) Single rectal tenofovir (N = 18) 2:1 7 Day Rectal tenofovir (N = 18) 2:1 Safety, PK / PD, acceptability RMP-02/MTN-006 Study Design  3 stage trial at 2 sites (UCLA/MWRI): open label TDF (oral) followed by 2:1 randomization of tenofovir: HEC placebo (for single rectal topical dose; 7-day rectal topical dosing)  Each dosing stage with 2 weeks of sampling (then: 2 weeks wash-out/healing)  Product: Tenofovir Disoproxil Fumarate (TDF) 300mg tablet, Tenofovir 1% gel (vaginal formulation/applicator) or HEC placebo gel  8.5 months; 3.5 months/participant  all 18 enrolled → completed. 100% retention (78% male; 22% female) Each participant completed 12 visits with 8 flexible sigmoidoscopies in 3.5 months → ~2300 bx >10,000 study samples

21. Clinical Unit UCLA/MG Clin Lab (Safety labs, HIV, RPR HSV 1 & 2) Rectal Swabs Blood Rectal Sponges Stool Sample Biopsies UCLA/MG Plasma PBMC MTN PK Lab Magee NAAT GC, CT UCLA/MG Cytokines UCLA/MG Genova (calprotectin) Rectal Lavages UCLA/MG (Sloughing Assay) UCLA/MG UCAL/MG (Explant Culture, Immunophenotyping PK processing UCLA Research Pathology (Qualitative ) MTN Micro (rectal microflora) MTN PK Lab Vaginal Sponge UCLA/MG MTN PK Lab Magee-WRI (gram stain,pH) Urine Magee NAAT GC/CT MTN PK Lab UCLA/MG Beta HcG & U/A Subject’s Samples Workflow: ‘chain of custody’ DAIDS IPCP-HTM

22. Primary Objective: Safety of 1% vaginally-formulated tenofovir gel, applied rectally Endpoint: ≥ Grade 2 AE Secondary Objectives: Acceptability, Mucosal Immunotoxicity, PK Endpoints - Acceptability: - % of those at last visit liking product - likely to use the candidate in the future if helps Endpoints - Mucosal Injury: - fecal calprotectin - rectal microflora - secreted rectal cytokines - rectal epithelial sloughing - rectal histology - rectal mucosal CD4+ T cell phenotype/activation Endpoints - PK: Tenofovir/diphosphate concentrations (9-10 compartments): Blood: plasma, PBMC, PBMC subsets (CD4+/CD4-) Fluids: rectal fluid, vaginal fluid (sponges) Tissue: Whole biopsy homogenates, isolated mucosal mononuclear cells (MMC) & CD4+/CD- subsets Exploratory Objective: ex vivo infectibility of in vivo exposed rectal tissue biopsies Endpoint: cumulative HIV-1 p24 levels in colorectal explant supernatants at 14d Study Endpoints

23. All Adverse Events n GI System Events SubjectsEventsSubjectsEvents ORAL SD 181537715 RECTAL SD Placebo 651339 RECTAL SD Tenofovir 12817610 RECTAL 7-D Placebo 65926 RECTAL 7-D Tenofovir 12 471237 Safety p=0.10 p=0.002 p=0.001 p=0.002 RECTAL 7-D Tenofovir 122525 Clinical Grade 3 Events

24. Acceptability 75% likely to use TFV in the future if they felt it might be helpful, despite relatively more dislike and discomfort.

25. DAIDS IPCP-HTM Mucosal Injury Indices

26.  With RECTAL dosing: plasma TFV Cmax & AUC: 2% of ORAL  ‘7-day’ RECTAL dosing: no TFV accumulation in plasma PK: TFV Exposure in Plasma

27. 7/1810/1212/12 PK: TFV-DP in Rectal Tissue (30 minutes post dose) Single ORAL: (i) TFV DP Tmax ≥ 24h post-dose. (ii) At 10 days, TFV DP detectable in 55% subjects Single RECTAL dose: (i) Tissue TFV DP Cmax was 112x > single ORAL and AUC all was 1.5x > single ORAL. (ii) At 10 days, TFV DP detectable in 80% ‘7-day’ RECTAL dosing: Tissue TFV DP accumulated: Cmax was 5x > single RECTAL dose

28. Biopsy Infection ex vivo of 7-day RECTAL dosing in vivo: Significant suppression seen ANCOVA p=0.005 (HIV-1 BaL TCID 50 =10 4 at ~10 cm) effect size: 0.80 Single ORAL dosing: no significant changes (p=0.65) Single RECTAL dosing: no significant changes (p=0.12)

29. Dose-Response Relationship: rectal tissue TFV DP with rectal biopsy infectibility Virus inhibition correlated with increasing tissue TFV-DP, even with small study n Feasible to assess both dose and response following in vivo exposure to drug

30. CONCLUSIONS  The vaginal formulation of 1% TFV gel was sub-optimal for clinical safety and acceptability when rectally applied.  Despite extensive mucosal indices of injury, none were seen with product use.  Consistent with other studies, the systemic absorption of rectally-delivered TFV was ~2% of oral dose.  Rectal dosing was associated with 100x more active TFV-DP in the target mucosa than oral dose.  Rectal tissue biopsy infection ex vivo was significantly reduced compared to control; may be a potential biomarker of efficacy.  PK/PD: Dose/response correlations were evaluable and significant in this intensive small trial.

31. Acknowledgments  Support from MTN: funded by NIAID (5U01AI068633), NICHD, NIMH, all of NIH.  U19 Integrated Preclinical-Clinical Program for HIV Topical Microbicides (IPCP-HTM) grant funded by DAIDS, NIAID, NIH grant (AI060614) DAIDS IPCP-HTM  Participants  Study Teams at each site: UCLA MWRI, University of Pittsburgh  NIH/DAIDS  MTN Network Support  MTN Microbiology Laboratory  MTN Clinical Pharmacology Analytical Lab at JHU  CONRAD  Gilead  Alpha StatConsult LLC

32. DAIDS IPCP-HTM QUESTIONS ?