Douche as Microbicide Vehicle: Safety, Pharmacokinetics, Acceptability of 3 Candidates

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 semipermeablemembrane (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 onlynon-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 analintercourse (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) • HYPERdistributes into rectosigmoid (further in this subject than in any other, most of which lost all signal or were restricted to rectum) • ISOdistributes 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 Sagittal MIP Coronal

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

11. 400 300 200 100 0 HYPER HYPO ISO 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). 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. 3 2.5 2 2 1.5 1 1 .5 0 0 Baseline Baseline HYPER HYPER HYPO HYPO ISO ISO Epithelial Changes Epithelial Denudation * Average score • HYPER enema significantly greater compared to baseline, HYPO, ISO. • Histologic score: % of fields with finding, 0% (Grade 0) to >67% (Grade 3) Lamina Propria Hemorrhage *HYPER v. each of the other 3, p<0.01 * Average score *HYPER v. each of the other 3, p<0.03

14. Permeability

15. Brief Acceptability

16. Product Acceptability

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