1. an NIH IP/CP for Topical Microbicides The Evolving Design of Rectal Microbicide Safety Studies: a Perspective from Two Trials Peter Anton Center for HIV Prevention, UCLA AIDS Institute UCLA/McGee-U Pitt/CONRAD/NIH

2. NIH IP/CP New? definition of ‘safety’? what to compare to? What’s “normal” first populations to study? where to look: anus, rectum, rectosigmoid, colon, SI?? what assays? how to interpret changes that have clinical relevance? Always more questions than answers

3. NIH IP/CP Distinct ‘rectal-compartment’ issues fragile epithelia – single cell increased absorptive potential/resistance profiles minimal benefit of endoscopic appearance in healthy subjects tube with constant cleansing flow untested, still developing safety indices suspected but not proven gender differences sequence of rectal fluid, tissue sampling critical to avoid confounders ‘preparatory enema’ injurious itself? rectal-specific applicator-avoid trauma/AE

4. NIH IP/CP assumption: N9-induced epithelial sloughing dangerous assumption: biopsy sites increase risk, alter absorption assumption: preparatory enemas safe assumption: not many heterosexuals have RAI, it’s an MSM issue fact: gut is continuously sloughing fact: lining is both exquisitely fragile and extremely resilient, reparative fact: physiologically inflamed (cells, cytokines, trafficking…) fact: rectal tissue is a rapid portal to systemic areas fact: in health, much injury and repair is ongoing… So, how do we determine “not safe” without knowing what to measure and what are normative “ranges”? Not clear what is “not safe” in RM

5. Rectosigmoid mucosa following mild endoscope trauma..raises the question of how long infection remains ‘a mucosal disease’...raises the question of how quickly are injury and repair reflected as inflammation.

6. NIH IP/CP first effort to try to define these normative ranges…“immnuotoxicity” for primarily financial and some scientific reasons, limited to men HPTN 056 (McGowan PI) attempted to quantify these ranges in 4 groups (N = 4/group); each subject seen every 2 weeks for 6 weeks to evaluate reproducibility and stability of readouts. Groups:HIV- men, no hx of RAI HIV- men, + hx RAI HIV+ men, undetectable PVL HIV+ men, PVL >5000 copies/ml Indices…at 2 sites (10cm and 30 cm):  Histology (quantitative & qualitative)  secreted IgG and IgA  cytokine mRNA from tissue  MMC phenotypes  not endoscopic appearance Pivotal: HPTN 056 McGowan et al JAIDS 2007

7. NIH IP/CP at least at this stage, not a huge difference in readouts between 10cm and 30cm (to be tested) quantitative histology far too variable in health HIV+ have some (predictable) baseline differences from HIV- HIV- with RAI no different from HIV- without RAI (? frequency) rectal Ig readouts have large intra-subject, intra-group variability cytokines and cell phenotypes quite stable caveats to 056: not very sexually-active population, older, men What did we learn from HPTN 056?

8. NIH IP/CP first effort: as first RM trial, many potholes to avoid first effort: first interventional test of HPTN 056 indices preferential assays for ‘safety’? Others (calprotection, fluid cytokines)? how interpret findings without “positive control”? IND study; great collaboration with Biosyn, now CONRAD Pre-PSRC very helpful in preparation for PSRC Critical first steps by NIAID to adapt toxicity tables for AE reporting to enable efforts to distinguish anticipated procedure-related findings 1 st trial: UC-781 RM

9. A Phase I Randomized, Blinded, Placebo-Controlled Safety and Acceptability Study of the UC-781 Vaginal Microbicide Gel Formulation Applied Rectally in HIV-1 Seronegative Adults  Sponsored by CONRAD (previously Biosyn) with NIAID’s U19 IP/CP  Single site: UCLA  NNRTI evaluated in 36 seronegatives (men and women)  2 concentrations/placebo with single and 7d exposures  UC-781 gel:  Universal Placebo: (not excipient; same as vaginal trials)  Acceptability  pilot PK

10. NIH IP/CP “routine” clinical sn/sx and laboratories rectal swabs for STI, microflora rectal sponges for secreted IgG, IgA and cytokines * stool calprotectin * rectal lavage for epithelial sloughing * tissue at 10cm and 30cm for: Histology (qualitative)(quantitative dropped) Cytokine mRNA MMC for phenotype by FACS Explant infection studies * plasma pK (to 24 hours) Indices/Assays used in UC-781 RM safety trial * Not in HPTN 056

11. RM UC-781: Phase 1 Trial Design Randomization: 0.1% UC-781, 0.25% UC-781, or placebo Visit 1Visit 2Visit 3 Outpatient Visit 4Visit 5 Screening Phone interview Single-dose Clinical Eval 7 daily doses Clinical EvalBaseline <4 wk  1 wk ~ 8 days Week 0Week 2Week 5Week 6Week 8 flex

12. ‘post-biopsy’ appearance UCLA IRB # 02-05-001; approved for 30 bx per visit Multiple publications, Near 100% adherence No AE

13. Participants will apply small amount of lubricant to applicator for insertion. Not to use OTC lubricants as they may cause toxicity and interact with the study product. Not optimal for rectal application, but tolerated. Applicator (vaginal form) issues

14. NIH IP/CP Sequence of sampling important type and timing of enema important collect rectal fluid (sponges/swabs) prior sampling of epithelia should follow RM introduction (post enema) and prior to scope trauma rapid coordination for explant experiments (9 bx gives 2 viral doses) while safe to bx within a few days, if assessing for inflammation, may want longer interval…

15. NIH IP/CP Findings thus far in UC-781..still blinded nearly completed; end study date mid 3/08 intensive recruiting/scheduling (~80 phone calls/emails per subject): HIGH adherence no significant AE or procedure problems lessons from blinded results that have guided next trial: no apparent difference between 10cm and 30cm; therefore, only 10cm in next trial. Can anal bx with stool and sponges suffice? Ig variabilty even greater in this trial that in HPTN 056..therefore, dropped as future immunotox/safety assay in next trial

16. 0 50000 150000 250000 350000 Group IgA (ng/ml) UC-781 (All) U19 (Low)U19 (Med)U19 (High) (n=27)(n=9) HPTN056 (n=8,v=24) V2V3V5V2V3V5V2V3V5V2V3V5 NIH IP/CP Boxplots comparing range of IgA at UC-781 baseline (V2) with HPTN 056 Boxplots of IgA

17. NIH IP/CP Boxplots comparing range of IgG at UC-781 baseline (V2) with HPTN 056

18. A two-site, Phase 1, double-blind, placebo-controlled safety and pharmacokinetic trial of topical, vaginally-formulated 1% tenofovir gel applied rectally with an exploratory pharmacokinetics study comparing topical with oral tenofovir levels in rectal tissue, rectal fluid, and blood.  Sponsored by CONRAD, Gilead with NIAID’s U19 IP/CP  Sites: UCLA & McGee/U Pitt  Tenofovir (oral/topical) evaluated in 18 seronegatives (men and women)  Oral: all get 300 mg tablet (single exposure only)  Topical: Randomized 2:1 (1% drug: Universal placebo)(single; 7-day)  pilot PK: plasma  exploratory pK: 5 comparments NIH IP/CP

19. “routine” clinical sn/sx and laboratories rectal swabs for STI, microflora rectal sponges for secreted IgG, IgA and cytokines * stool calprotectin * rectal lavage for epithelial sloughing * tissue at 10cm and 30cm for: Histology (qualitative)(quantitative dropped) Cytokine mRNA MMC for phenotype by FACS Explant infection studies * plasma pK (to 24 hours) Indices/Assays used in UC-781 Tenofovir RM safety trial * Not in HPTN 056

20. Week: 012345678 9 1011 Visit:1234 A/B5 A/B67 A/B8 A/B910 Phone call Baseline Oral Single Topical 7-day Topical ScreenFlex Flex 50% Flex Flex 50% Flex Randomization: 2:1 (drug:placebo) Group A or Group B (50:50) ALL get 6 flex sigs Oral/Topical Tenofovir RM trial

21. NIH IP/CP Lessons and questions RM trials are fast evolving. Asset for VM trials. can we rationally continue to reduce (or add) assays? how to determine if CHANGE in assays means anything clinically, even in those studied for stability in HPTN 056? using the best we have at present, are we now erring on the side of being too sensitive? May be tough to actually INCREASE risk…Sx best guide? back to original question: given reparative capacity of gut, WHAT is “not safe” and how to capture that? biopsies are safe explants may be biomarkers of efficacy other assays may be bio-indicators of injury although N9 may not be the optimal + control, still worth it

22. an NIH IP/CP for Topical Microbicides NIH NIAID U19 IP/CP #AI060614: “Microbicide Development Program” Biosyn, Inc Anne Marie Corner Linda Knapp Linda Kristekas UCLA Ian McGowan (U Pitt) Chomchay Siboliban Amy Adler Terry Saunders Elena Khanukhova Charlie Price Julie Elliott John Boscardin Ying Zhao Daniel Cho Karen Andrews Elizabeth Johnson Alexis Dominguez Julia Klein NIH Jim Turpin Jeanna Piper Cherylnn Mathias Grace Chow Consultants Alex Carballo-Dieguez Ana Vetuneac CONRAD Henry Gabelnick Christine Mauck Tim McCormick Marianne Callahan VOLUNTEERS!