Abstract HIV/AIDS continues to be a major global health issue, and chronic liver disease has become a major cause of HIV-mortality. Several contributing factors are recognized including hepatotoxicity of anti-HIV drugs, coinfection of hepatitis viruses and widespread alcohol use. The likelihood of concurrent use of other addictive substances such as fentanyl is high. Excessive alcohol use has been linked directly to increased HIV viral load, engagement in HIV-risk behaviors, and poor adherence in HIV prevention interventions. In addition, alcohol and fentanyl are known to alter the functionality of drug metabolizing enzymes and transporters, critical determinants for the efficacy and safety of anti-HIV drugs. Indeed, we have shown that ethanol (alcohol) exposure decreased the hydrolysis of tenofovir alafenamide fumarate (TAF) and produced a new metabolite: ethyl TAF. This metabolite is a hybrid molecule with part from TAF and part from ethanol. TAF is a new version of TDF (tenofovir disoproxil fumarate) with decreased organ toxicity, and both tenofovir prodrugs are hydrolyzed and followed by phosphorylation to produce therapeutically active metabolite. TAF is hydrolyzed by carboxylesterase-1 (CES1), while TDF is hydrolyzed predominantly by CES2. TAF and TDF are listed in ~75% anti-HIV regimens used for both pre-exposure prophylaxis (PrEP) and treatment as prevention (TasP). Our Preliminary Study has also shown that fentanyl increased the expression of chemokine co- receptors and led to increased HIV infection. The central hypothesis of this project is that metabolism-based interactions of tenofovir drugs with alcohol +/- fentanyl produce signature changes that reliably indicate efficacy and safety of tenofovir regimens and serve as a foundation for developing adherence-monitoring and PrEP/TasP intervention strategies. The Specific Aims are: (1) to characterize metabolite signatures and mechanistic biomarkers, and (2) to ascertain the significance of alcohol +/- fentanyl-altered metabolism. To determine the metabolite signatures specific to alcohol +/- fentanyl, human primary hepatocytes and peripheral blood mononuclear cells (PBMCs) will be treated with a tenofovir-regimen in the presence or absence of alcohol (+/- fentanyl); the metabolite signatures will be determined by LC-MS/MS. The altered metabolite signatures will be ascertained by in vivo studies. Transcriptome analysis will be performed at both tissue (e.g., PBMC) and single cell level (selected samples) to identify mechanistic biomarkers. To establish the significance, hepatocytes and PBMCs will be incubated with a tenofovir-regimen with or without alcohol +/- fentanyl, and viral load and cytotoxicity will be monitored. The connection of metabolite signatures and biomarkers with efficacy and safety will be ascertained in vivo. Finally, PBMCs from HIV-negative patients exposed to PrEP, PrEP+alcohol or PrEP+alcohol/fentanyl will be tested against HIV infection ex vivo. The combination of ...