The recent observation that coitally-related TFV gel dosing provides partial protection against HIV acquisition is the first indication that vaginal microbicides have the potential to stem the HIV epidemic. These encouraging findings also underscore the need for more predictive biomarkers of microbicide PD and safety for future clinical trials, which will likely become more complex as we strive to achieve higher levels of protection with alternative dosing, varying formulations, and combinations of drugs. The current study indicates that TFV gel does not interfere with soluble mucosal immunity, a biomarker of safety, and that measurement of anti-HIV activity and drug levels in CVL may provide simple and inexpensive assays of PD and PK near or at the site of drug action. Measurements of PK and PD in CVL are directly applicable to drugs that act luminally or for products in which the Compound Library citations intracellular and extracellular drug pools are in equilibrium. Importantly, results obtained in this study suggest that this approach may also provide insight into drugs such as TFV, which is only active after intracellular modifications and for which the low permeability of the intracellularly charged nucleotide leads to accumulation of phosphorylated drug with a long half-life in the intracellular pool. The anti-HIV activity in CVL samples collected after gel use provides a direct measurement of the bioactivity of extracellular drug, which may serve as a reservoir to protect newly recruited immune cells. The importance of this is highlighted by non-human primate studies, which illustrate the central role of these cells in the establishment of infection. Virus crosses the mucosal epithelial barrier within hours to establish a small founder population of infected cells. This founder population then undergoes local expansion during the first week of infection to generate sufficient virus and infected cells to MK-0683 disseminate and establish a systemic infection. Because the number of immune targets within the vagina and cervix is relatively small and spatially dispersed, recruitment and activation of additional target cells is critical to the establishment of infection. Thus, it is important that a sufficient reservoir of extracellular drug be available to block infection of recruited target cells. The current study did not include biopsy sampling, but PK data obtained in recently completed clinical studies should provide data to determine whether concentrations of drug in intracellular tissue and extracellular compartments correlate. If this proves true, then CVL sampling could be added to clinical trials and may provide a more realistic and reproducible approach than biopsies for measuring PK and PD in clinical trials. These highly feasible assays could also provide an objective measure of adherence to gel product.