Increased susceptibility to genital herpes in medroxyprogesterone-treated mice may provide a surrogate of increased HIV risk and a preclinical biomarker of topical preexposure prophylaxis safety. well tolerated and did not result in any increase in HSV-2 susceptibility but guarded HCL Salt mice from herpes simplex virus 2 (HSV-2) disease compared to mice treated with the HEC placebo gel. No significant increase in inflammatory cytokines or chemokines in vaginal washes or switch in cytokine chemokine or mitochondrial gene expression in RNA extracted from genital tract tissue was detected. To further evaluate efficacy mice were treated with gel once daily beginning 12 h prior to high-dose HSV-2 challenge or 2 h before and after viral challenge (BAT24 dosing). The 0.3% TDF gel provided significant protection compared to the HEC gel following either daily (in 9/10 versus 1/10 mice < 0.01) or BAT24 (in 14/20 versus 4/20 mice < 0.01) dosing. In contrast 1 tenofovir (TFV) gel guarded only 4/10 mice treated with either regimen. Significant protection was also observed with daily 0.03% TDF compared to HEC. Protection was associated with greater murine cellular permeability of radiolabeled TDF than of TFV. Together these findings suggest that TDF is usually safe may provide substantially greater protection against HSV than TFV and support the further clinical development of a TDF ring. INTRODUCTION The majority of new human immunodeficiency computer virus (HIV) infections are transmitted through heterosexual contact with young women bearing a disproportionate burden. However efforts to prevent transmission in this populace have met with limited success. Oral preexposure prophylaxis (PrEP) was effective in serodiscordant partners (where one partner is usually seropositive for HIV-1 contamination and one is seronegative) (1) but not in other female populations presumably reflecting difficulties with adherence (2 3 Adherence has also been a problem in vaginal gel PrEP trials. The CAPRISA 004 trial a randomized placebo-controlled trial evaluating 1% tenofovir (TFV) vaginal gel for the prevention of HIV when it was applied before and after sexual intercourse (but no more than two doses in a 24-h period; referred to as BAT24 dosing) was the first topical PrEP study to demonstrate significant efficacy (4). TFV gel reduced HIV acquisition by an estimated 39% overall and by 54% in women with high gel adherence. Even though CAPRISA 004 trial was not designed to HCL Salt evaluate whether TFV gel guarded against herpes simplex virus 2 (HSV-2) a 51% reduction in HSV-2 incidence was observed in the tenofovir arm (5). However a subsequent study with daily TFV gel (Microbicide Trials Network study MTN 003) failed to show protection a result that was most likely linked to low adherence as evidenced by analysis of plasma drug levels (3). The development of delivery systems that overcome some of the barriers to adherence such as intravaginal rings (IVRs) is usually a priority for HIV prevention (6). To address this need we recently developed an IVR formulation of tenofovir disoproxil fumarate (TDF) (7). We selected TDF a prodrug of tenofovir (TFV) because it is usually more potent than TFV toxicity in cell and tissue culture and unlike other microbicides had little effect on the epithelial barrier in a polarized dual-chamber cell HCL Salt culture model (9 10 Topical delivery of drugs has resulted in unanticipated toxicities which have been linked to an increased risk of HIV acquisition in large clinical trials. For example nonoxynol-9 which has been safely used for decades as a contraceptive was found to increase the risk of HIV MUC1 possibly reflecting disruption of the epithelial barrier and induction of an inflammatory response (11 -15). Similarly 6 cellulose sulfate gel was also associated with a pattern toward increased HIV risk which resulted in early termination of the clinical trial (16). We subsequently found that mice were significantly more susceptible to low doses of HSV-2 administered 12 h after 7-daily intravaginal gel applications of 6% cellulose sulfate or numerous formulations of nonoxynol-9 than mice that received a hydroxyethylcellulose (HEC) placebo gel (17 18 The increased susceptibility was associated with modest increases in inflammatory mediators in the genital tract and disruption of the epithelial barrier. In contrast 1 TFV gel 0.5% PRO 2000 and 0.1% griffithsin gel did not increase the risk of HSV infection in this model (15 17 19 These findings suggest that this relatively inexpensive small-animal model may provide a biomarker of microbicide safety. Thus the current studies were designed to test HCL Salt topically delivered TDF in the murine security model. To.