1. Extracellular and Intracellular Tenofovir DF and Emtricitabine Exposure in Mucosal Tissue after a Single Dose of Fixed-Dose TDF/FTC: Implications for Pre-exposure Prophylaxis Kristine B Patterson, Heather A Prince, Eric Kraft, Amanda Jones, Sunita Paul, Nicholas J Shaheen, Melissa Spacek, Paris E Heidt, Sunila Reddy, Jim Rooney, Julie B Dumond, Myron S Cohen, and Angela DM Kashuba University of North Carolina, Chapel Hill, NC USA Gilead Sciences, Foster City, CA USA

2. Truvada ® for prevention Tenofovir disoproxil fumarate (TDF) + Emtricitabine (FTC) Daily oral dosing of TDF +/- FTC being evaluated. There are no PK data in mucosal tissue after a single oral dose of TDF and FTC. Clinical trials evaluating EPISODIC ORAL dosing are being planned.

3. Objectives Primary –To characterize tenofovir and emtricitabine (TFV/FTC and TFV-DP/FTC-TP) exposure in multiple biological compartments in both men and women after a single oral dose of Truvada ®. Secondary –To analyze the decay characteristics (t 1/2 ) of TFV/FTC and TFV-DP/FTC-TP concentrations in multiple biological compartments in both men and women after a single oral dose of Truvada ®.

4. Methods Single site, open-label trial Healthy HIV-negative men and women (ages 18-40) –Comprehensive STD screening –Sexually abstinent –Using contraception Single observed dose of Truvada ®

5. Pharmacokinetic Sampling Cervical (CT) and Vaginal Tissue (VT) Rectal Tissue (RT) Sampling Time (Days) 1 2 5 7 10 14 Blood Plasma (BP), PBMC, Cerv-Vag Fluid (CVF)

6. Analyses Sample Analyses: –LC/MS/MS –TFV and FTC: LLOQ 0.1 ng/mL –TFV-DP and FTC-TP: LLOQ 2-10 fmol Data Analyses: –Non-compartmental PK (WinNonlin 6.0) (composite) –Summary statistics (SAS 9.1.3) AUC TFV and FTC: ng*days/mL or gm AUC TFV-DP and FTC-TP: fmol*days/10 6 cells or 0.2uL Penetration ratios (AUC Ratios); –CVF or tissue AUC 0-14d ÷ BP AUC 0-14d

7. Subject Demographics Median (range)Females (n=7)Males (n=8) Age (yrs) 22 (21-25) 26 (19-37) BMI (kg/m 2 ) 24.8 (21.2-28.6) 23.5 (18.8-28.1) Race 6 White 1 Black 5 White 2 Asian

8. Median Blood Plasma Concentrations Can Be Quantified Up to 14 Days Post-Dose TenofovirEmtricitabine AUC (ng*days/mL) BP 89 t ½ (hr) 56BP 60 AUC (ng*days/mL) t ½ (hr) 67

9. TenofovirEmtricitabine rectal tissue Median Rectal Tissue Concentrations Are Higher Than Blood Plasma Up to 14 Days Post-Dose AUC BP 89 AUC RT:BP 33 t½t½ 42 56 RT 266 BP 60 AUCAUC RT:BP 4.3 t½t½ 87 67 RT 2981

10. Tenofovir DiphosphateEmtricitabine Triphosphate Median Intracellular Rectal Tissue Concentrations Can Be Quantified 2-14 Days Post-Dose AUC RT 6,495 PBMC 10,813 AUC Ratiot½t½ 45 56 RT 199 PBMC 10,832 AUCAUC Ratio NA t½t½ 67 87 NA

11. Tenofovir Emtricitabine cervicovaginal fluid Median Cervicovaginal Fluid Concentrations Are Higher Than Blood Plasma Up to 14 Days Post-Dose AUC CVF 233 BP 89 AUC CVF:BP 2.6 t½t½ 53 56 CVF 2520 BP 60 AUCAUC CVF:BP 42 t½t½ 43 67

12. TenofovirEmtricitabine Vaginal and Cervical Tissue Concentrations Are Similar To, or Higher Than, Blood Plasma Up to 14 Days Post-Dose AUC CT 510 AUC Ratio 5.8 t½t½ 24 AUCAUC Ratio CT 2496 42 t½t½ 57 VT 50 BP 89 110 56 VT 419 BP 60 32 67 0.6 7 cervical tissue vaginal tissue cervical tissue vaginal tissue

13. Tenofovir DiphosphateEmtricitabine Triphosphate Median Intracellular Vaginal and Cervical Tissue Concentrations Can Be Detected 1-14 Days Post-Dose AUC CT 132 AUC Ratio NA t½t½ 148 AUCAUC Ratio CT 167 NA t½t½ VT 1171 PBMC 10,813 43 VT 1492 PBMC 10,832 86

14. Summary and Implications Preferential penetration of TFV/FTC seen in this study supports quantification of other ARVs in all mucosal tissues as part of early development strategies for oral PrEP. Differential drug terminal elimination (“Tail”) emphasizes consideration for combination therapy esp. episodic dosing. TFVTFV-DPFTCFTC-TP Blood, PBMCs 14 d 10 d Rectal Tissue AUC RT:BP 33 14 d - 14 d 4.3 14 d - 2 d Cervicovaginal Fluid AUC CVF:BP 2.6 14 d 42 14 d Vaginal Tissue AUC VT:BP 0.6 14 d - 14 d 7 10 d - 2 d Cervical Tissue AUC CT:BP 5.8 7 d - 14 d 42 10 d - 1 d

15. Acknowledgements Study Volunteers Gilead Sciences UNC CFAR Clinical Pharmacology and Analytical Chemistry Laboratory (P30 AI50410) National Institute of Health (K23 AI077355) UNC Clinical Translational Research Center (RR025747)