an NIH IP/CP for Topical Microbicides Stability of Mucosal Cytokine Profiles (Proteins) and Mucosal Mononuclear Lymphocyte Phenotypes Following Rectal Administration of UC-781 Microbicide Gel in a Phase 1 Safety Assessment (blinded data at 75% completion) D Cho, I McGowan, J Elliott, G Cortina, A Dominguez, K Tanner, EJ Johnson, T Saunders, A Adler, E Khanukhova, C Price, P Anton UCLA, NIH, CONRAD

NIH IP/CP Study Intent Safety assessment: using most detailed assays available WHAT are reasonable safety indices, outside of FDA requirement WHAT research assays may be important safety indices: “immunotoxicity indices” HPTN 056 (McGowan PI) attempted to investigate this in 4 groups of men (N=4/group) each 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: Histology (quantitative & qualitative), secreted Ig, cytokine mRNA from tissue, MMC phenotypes GOALS: to reduce #, identify preferred sensitive assays McGowan et al JAIDS 2007

NIH IP/CP Study Outline 75% Subjects studied thus far [19 men (70%), 8 women (30%)] with all samples collected 3 collection timepoints (baseline, single dose/30 minutes, 7 days) 3 dosing groups:(i) placebo (ii) high-dose UC-781 (0.25% gel) (iii) low-dose UC-781 (0.1% gel)

NIH IP/CP Trial Objectives and Indices Secondary Objectives: To determine whether use is associated with rectal mucosal damage (immunotox): Epithelial sloughing Histopathology Mucosal mononuclear cell phenotype (flow) Mucosal cytokine mRNA (tissue) Mucosal cytokines (secreted) Mucosal immunoglobulins Fecal calprotectin Explants- susceptibility to HIV infection

NIH IP/CP Trial Objectives and Indices Secondary Objectives: To determine whether use is associated with rectal mucosal damage (immunotox): Epithelial sloughing: No difference seen between subjects nor between visits Histopathology: No difference seen between subjects nor between visits Mucosal mononuclear cell phenotype (flow) Mucosal cytokine mRNA (tissue): Batched for end-of-study analysis Mucosal cytokines (secreted) Mucosal immunoglobulins: No difference seen between subjects nor visits Fecal calprotectin: No difference seen between subjects nor between visits Explants- susceptibility to HIV infection: reported previously

NIH IP/CP RM Phase 1 Trial Design Randomization: 0.1% UC-781, 0.25% UC-781, or placebo Visit 1 Visit 2Visit 3 Visit 4 Visit 5 Visit 6 ScreeningPhone interview Single-dose exam Safety; Given 7 daily doses 7-day exam Baseline <4 wk  1 wk ~ 8 days Week 0Week 2Week 5Week 6Week 8 flex

NIH IP/CP Sample Collection & Processing Mucosal mononuclear cells (MMC) for FACS: Mucosal biopsies collected pre routine, (large cup forceps) at 3 visits (Anton AIDS 2000) V2 (Baseline) preceded at intervention; V3 (single dose) and V5 (7-day) post-intervention MMC isolation and staining per routine (Shacklett J Immunol Meth 2003) MMC stained with combination of: CD4-FITC, HLA-DR-FITC, CD38-PE, CCR5-PE, CXCR4-APC) Rectal fluid collection for cytokines by Luminex™: Surgical sponges inserted for 5 minutes to collect samples at 3 visits V2 and V3 samples acquired before product introduced; data merged as baseline V3 and V5 sample acquired after exposure Rectal fluid eluted by centrifugation Luminex™ multiplex bead array quantified: IL-1 , IL-6, TNF , IFN- , MIP-1 , IL-12, RANTES (results: pg/ml)

NIH IP/CP Data Presentation Blinded data presented with laboratory numbers recoded to maintain blind…numerical sequence # are the same as seen in the Explant presentation Data presented as box plot showing 25-75% range (Interquartile range=IQR) with median identified; ‘whiskers’ reflect 1.5xIQR. “Outliers” are small circles Data presented as “grouped” at each visit Data next presented according to explant ‘responders’, ‘non-responders’ or ‘middle’ When available, HPTN 056 data presented to give context and evidence of reproducibility at baseline

NIH IP/CP MMC phenotypes by FACS: Does CCR5 on CD4 change? CCR5 expression on CD4+ MMC does not seem to change in group as a whole after single or 7-day exposure; Subset analysis based on explant responders: no changes. Mean similar to HPTN 056

NIH IP/CP MMC phenotypes by FACS: Do ‘double +s’ on CD4 change? CCR5/CXCR4 dual expression on CD4+ MMC does not seem to change in group as a whole after single or 7-day exposure; Subset analysis based on explant responders: no changes. Mean similar to HPTN 056

NIH IP/CP MMC phenotypes by FACS: Does activation on CD4 change? While trends in CD38 and HLA-DR on CD4 MMC may be suggested, there is no difference over time compared to baseline changes. HPN 056 data not immediately available.

Collection of rectal secretions for cytokines/chemokines with surgical sponges

V2V3V5V2V3V5V2V3V Boxplots of IL-6 Group IL-6 U19 (All)U19 (NON)U19 (Med)U19 (RESP) (n=27)(n=9) V2V3V5V2V3V5V2V3V5V2V3V5 Luminal cytokines by Luminex™: IL-1  or IL-6 No appreciable differences over time or within subgroups of IL-1  or IL-6. In general, IQR reasonably tight for whole group at baseline.

Luminal cytokines by Luminex™: IL-12 or IFN-  No appreciable differences over time or within subgroups of IL-12 or IFN- . IQR reasonably tight for IL-12; broad for IFN- .

Luminal cytokines by Luminex™: TNF-  or RANTES No appreciable differences over time/within subgroups of TNF-  or RANTES. IQR reasonably tight for TNF-  ; broad for RANTES.

Luminal cytokines by Luminex™: MIP-1  No appreciable differences over time or within subgroups of MIP-1- . In general, more variable IQR.

NIH IP/CP “Blinded” Interpretations MMC phenotypes: Based on FACS data, there appears to be no difference among the all subjects in the 3 groups (placebo, high- does, low-dose) over all visits on co-receptor expression or activation status. Rectal fluid cytokines: Using Luminex™ data from rectal sponges eluates, there does not appear to be any trend/significant differences among 7 measured cytokines/chemokines after single or 7 day exposure. Based on these still blinded data, if these assays (MMC phenotype and ‘secreted’ cytokines) are relevant and sensitive enough to assess early/mild immunoreactive changes to topical UC-781 exposure, we are not detecting such changes

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!