PROJECT SUMMARY Problem. Transgender women (TGW) have a 49-fold increased risk of becoming infected with HIV, yet they have been underrepresented in HIV prevention and treatment research. Feminizing hormone therapy (FHT) can alter the pharmacology of the nucleos(t)ide reverse transcriptase inhibitors (NRTIs) used for HIV pre-exposure prophylaxis (PrEP) through complex physiologic mechanisms, but the clinical implication of this drug interaction is unknown. Our Overarching Goal: is to determine whether FHT diminishes PrEP's potency in TGW on FHT by decreasing concentrations of the active metabolites (TFVdp/FTCtp) relative to their competing deoxynucleotides (dATP/dCTP). We will use these data to construct a population PK model describing this ratio in HIV transmission sites and predict PrEP efficacy in a transfeminine population taking and not taking FHT given different scenarios of PrEP adherence and intermittent dosing. Study Design: In Aim 1, we will conduct a study in 10 premenopausal women taking PrEP to determine how high and low estradiol exposure impacts nucleotide concentrations in different cellular populations of the lower gastrointestinal (GI) tract. We will measure TFVdp, FTCtp, dATP, and dCTP in total rectal cells collected by cytobrush and CD4 cells isolated from tissue biopsies by LC-MS/MS. These data will be used to validate that total rectal cells can be used as a pharmacology surrogate of isolated CD4+ T cells regardless of estradiol exposure and to inform our sampling procedures for subsequent study. In Aim 2, we will leverage the San Diego sites for a currently enrolling, multi-site PrEP demonstration project (PI Morris; NCT03086200) to co-enroll TGW on PrEP taking and not taking FHT into our pharmacology study (N=10 each). We will collect blood (plasma, serum, and peripheral blood mononuclear cells; PBMCs) as well as total rectal cells via anoscopy-assisted cytobrush. We will measure sex hormones (estradiol, progesterone, and testosterone) in serum and TFVdp, FTCtp, dATP, and dCTP in PBMC and total rectal cells by LC-MS/MS. In Aim 3 we will use these PK data to build a population PK model of PrEP exposure in the lower GI tract and simulate PrEP exposure under different dosing scenarios in 1000 TGW taking or not taking FHT. We will predict effectiveness of these simulations using previously published PrEP efficacy targets. Expected Outcomes: We believe these data will confirm our preliminary observations of ~7-fold decreased concentrations in TFVdp relative to dATP in TGW taking FHT. Our population PK model to describe the impact of FHT on PrEP pharmacology in HIV transmission sites will allow us to determine the minimal adherence requirements to protect 99% of the transfeminine population taking FHT. These pharmacology data will support an R01 application to explore dose taking and risk behavior and investigate PrEP outcomes in a cohort of TGW.