1. Douche as Microbicide Vehicle: Safety, Pharmacokinetics, Acceptability of 3 Candidates Craig W. Hendrix, MD Johns Hopkins University NIH Grant IP/CP U19 AI060614

2. Objectives Determine a relatively safe enema for use in future rectal microbicide development Explore the feasibility of an enema as rectal microbicide delivery vehicle

3. Definitions Osmosis: movement of solvent molecules (molecules in which solute is dissolved) through selectively-permeable membrane toward higher solute concentration, equalizing the solute concentrations Osmotic pressure: pressure needed to prevent the inward flow of water across a semipermeable membrane (e.g., cell, colon lining) Osmole: number of moles of a chemical that contribute to a solution's osmotic pressure (e.g., NaCl, 1 mole, but 2 osmoles) Osmolarity: osmoles per liter Osmolality: osmoles per kilogram Tonicity: takes account of only non-penetrating solutes (osmolarity considers both penetrating & non-penetrating solutes) Hyperosmolar: causes cells to shrink, draws water into fluid Iso-osmolar: no change in cell water, no water flow into fluid Hypo-osmolar: causes cells to swell, water flow out of fluid

4. Background Rectal douching (enema) prior to receptive anal intercourse (RAI) common among men who practice unprotected RAI (Carballo-Dieguez 2007, Hylton 2007) Rectal douching associated with increased risk for HIV transmission (Coates 1988, Moss 1988) Tap water (hypo-osmolar) and Fleet (hyper-osmolar) enema most commonly used (Hylton 2007) Tap water & hyper-osmolar enemas show colonic epithelium damage (Meisel 1977, Schmelzer 2004) Rectal hyper-osmolar gels induce greater epithelial loss than iso-osmolar gels (Fuchs 2007)

5. Study Design Randomized, blinded, comparative study Population: 9 HIV negative MSM Study Products: 125 mL rectal douche (enema) varying osmolality: Fleet (HYPER), Tap water (HYPO), Normosol-R™ balanced salt solution (ISO) Doses: 1 inpatient radiolabeled dose and 3 outpatient doses (used in the context of RAI) for each of the 3 products

6. Outcome Measures Distribution/retention: SPECT/CT imaging of radiolabeled enema 2 and 24 hours after dose Toxicity Colon biopsy 2 hours after dose Permeability for 24 hours after dose Acceptability: questionnaires after each dose, each product phase, and end of study structured interviews at study end

7. SPECT/CT Distribution Single Photon Emitted Computed Tomography/CT Radiolabeled products appear in color (SPECT) Anatomy appears in grayscale (CT) Views: coronal (front), sagittal (from left), axial (below) HYPER distributes into rectosigmoid (further in this subject than in any other, most of which lost all signal or were restricted to rectum) ISO distributes throughout the distal colon up to the splenic flexure (seen with ISO product in most subjects).

8. HYPER ISO

9. SPECT Distribution MIP: Maximal intensity projection, full thickness of SPECT scan unlike single slice in prior views “Coronal” view from subject’s front “Sagittal” view from subject’s left Distribution: –begins in rectum –up, forward through sigmoid colon –left and up through the descending colon –up to the splenic flexure. MIP Coronal MIP Sagittal

10. HIV Distribution Cell-free HIV Surrogate 99m Tc-Sulfur Colloid Cell-Associated HIV Surrogate 111 In-Lymphocytes MIP SPECT/CT Same distribution of cell-free and cell-associated surrogates Consistently within rectum and lower sigmoid colon

11. Enema Volume Volume of enema on SPECT @ 2 hrs Boxplots: median (bar), middle 50% (box) Significantly greater retained volume of ISO compared to HYPER. Quantitative assessments for proximal distribution, volume retained, and residual concentration were all significantly greater for ISO compared to HYPER with HYPO intermediate (data not shown). 0 100 200 300 400 HYPERHYPO ISO p<0.05

12. Colon Biopsy Iso-osmolar (10x, H&E) Healthy epithelial lining with single columnar cells rich with mucin across the surface (Grade 0). Hyper-osmolar (10x, H&E) Right section indicates loss of epithelial columnar cells, except in crypts, leaving exposed lamina propria cells (Grade 3).

13. Epithelial Changes 0.5 1 1.5 2 2.5 BaselineHYPER HYPOISO Average score * * HYPER v. each of the other 3, p<0.03 Lamina Propria Hemorrhage 0 1 2 3 BaselineHYPER HYPOISO Average score * * HYPER v. each of the other 3, p<0.01 Epithelial Denudation HYPER enema significantly greater compared to baseline, HYPO, ISO. Histologic score: % of fields with finding, 0% (Grade 0) to >67% (Grade 3)

14. Permeability Hyper- osmolar Hypo- osmolar Iso- osmolar C max (x10 7 ) 2.2 (1.3, 3.0) 3.6 (2.8, 10.0) 1.7 (0.6, 2.9) AUC (x10 7 ) 9.5 (8.6, 14.5) 21.4 (17.3, 53.7) 19.7 (7.1, 28.6) T max (hr) 2.0 (1.7, 3.4) 2.2 (1.4, 2.7) 3.7 (2.6, 6.0)

15. Brief Acceptability Sexual satisfactionHyper-osmolarHypo-osmolarIso-osmolar (% of occasions)M (SD)RangeM (SD)RangeM (SD)Range decreased11.1 0 no effect37.029.637.5 increased51.959.362.5 Acceptability rating 3.33 (.68)2 – 43.26(.94)1 – 43.33 (1.01)1 – 4

16. Product Acceptability Hyper-osmolarHypo-osmolarIso-osmolar M (SD)rangeM (SD)rangeM (SD)range Liked douche overall7.75(2.44)2 – 107.67(2.83)2 – 107.56(2.60)2 – 10 Sexual enjoyment after product use7.67(2.35)3 – 108.22(1.79)5 – 108.78(1.30)*6 – 10 Reports of RAI being worse after douching (n)120 Liked application process6.89(2.47)3 – 107.78(2.95)1 – 108.33(1.58)*5 – 10 Likelihood of future use8.56(1.24)7 – 108.78(1.48)6 – 109.33(1.00)7 – 10 Likelihood of use without condoms9.86 (.38)9 – 109.75 (.46)9 – 109.57 (.53)9 – 10 Likelihood of use if a 30- minute wait is required8.11(1.76)4 – 108.00(2.96)1 – 108.44(2.83)1 – 10

17. Summary & Impact HYPER –greatest epithelial loss and hemorrhage –concern for transmission risk? HYPO –greatest permeability –enhance active drug ingredient tissue penetration? ISO –greatest distribution/retention/concentration –least toxicity –trend to more favorable acceptability –candidate for microbicide delivery vehicle?

18. Acknowledgments Johns Hopkins –Edward Fuchs –Elizabeth Purdy –Rahul Bakshi –Francisco Leyva –Kathleen Truelove –Linda Lee –Michael Torbenson –Brian Caffo Microbicide Development Program –Peter Anton –Elena Khanukhova NIH (IPCP U19 AI060614) –Jim Turpin –Jeanna Piper –Cherlynn Mathias –Roberta Black