After tenofovir alafenamide dosing in vivo , tenofovir-dp was unquantifiable in most tissues (91%) although cervical and vaginal epithelial cells efficiently formed tenofovir-dp from tenofovir alafenamide in vitro . These findings warrant further investigation of tenofovir alafenamide's pharmacology.

Animal models and dose selectionThis analysis utilized three preclinical models from two species of humanized mice: stem cell hemopoietic/RAG 2-(hu-HSC-Rag, n=36) and bone marrow-liver-thymus (BLT, n=13) and one species of nonhuman primate (NHP): rhesus macaques (n=18).Humanization of the mice was by protocols that have been previously described (Melkus et al. 2006; Berges et al. 2006). The extent of humanization for the mouse models was assessed by quantifying the human T-cell populations using flow cytometry. All the humanized mice were female, while six (33.3%) NHPs were female. Half of the animals were left uninfected while the other half were infected with 200 μL of 2.1 x 10 6 IU/mL of HIVBal D7 intraperitoneally (hu-HSC-RAG) or 200 μL of 90,000 tissue culture infectious units (TCIU) of HIVJRcsf intravenously (BLT) or 10 4.5 median tissue culture infective dose (TCID50) RT-SHIV intravenously (NHPs) (North et al. 2005; North et al. 2010) for four weeks. The hu-HSC-RAG and BLT mice underwent humanization when aged three to six months and were then dosed with various ARV regimens for a period of ten days. Hu-HSC-RAG mice were dosed with EFV 10 mg/kg only (six uninfected and six infected animals) or atazanavir (ATZ) 140 mg/kg only (six uninfected and six infected animals) or a combination of tenofovir (TFV) 208 mg/kg, emtricitabine (FTC) 240 mg/kg, raltegravir (RAL) 56 mg/kg and maraviroc (MVC) 62 mg/kg (six uninfected and six infected animals). BLT mice were dosed with a combination of TFV, FTC, RAL, MVC and ATZ at equivalent doses but not EFV due to toxicity concerns. All drugs were administered by oral gavage. Dosing regimens for each of the animal models are summarized in Supplementary Table 1. Dosage selection for all drugs in the animal

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In a "kick and kill" strategy for human immunodeficiency virus (HIV) eradication, protective concentrations of antiretrovirals (ARVs) in the lymph node are important to prevent vulnerable cells from further HIV infection. However, the factors responsible for drug distribution and concentration into these tissues are largely unknown. Although humanized mice and nonhuman primates (NHPs) are crucial to HIV research, ARV tissue pharmacology has not been well characterized across species. This study investigated the influence of drug transporter expression, viral infection, and sex on ARV penetration within lymph nodes of animal models and humans. Six ARVs were dosed for 10 days in humanized mice and NHPs. Plasma and lymph nodes were collected at necropsy, 24 hours after the last dose. Human lymph node tissue and plasma from deceased patients were collected from tissue banks. ARV, active metabolite, and endogenous nucleotide concentrations were measured by liquid chromatography-tandem mass spectrometry, and drug transporter expression was measured using quantitative polymerase chain reaction and quantitative targeted absolute proteomics. In NHPs and humans, lymph node ARV concentrations were greater than or equal to plasma, and tenofovir diphosphate/ deoxyadenosine triphosphate concentration ratios achieved efficacy targets in lymph nodes from all three species. There was no effect of infection or sex on ARV concentrations. Low drug transporter expression existed in lymph nodes from all species, and no predictive relationships were found between transporter gene/protein expression and ARV penetration. Overall, common preclinical models of HIV infection were well suited to predict human ARV exposure in lymph nodes, and low transporter expression suggests primarily passive drug distribution in these tissues. SIGNIFICANCE STATEMENTDuring human immunodeficiency virus (HIV) eradication strategies, protective concentrations of antiretrovirals (ARVs) in the lymph node prevent vulnerable cells from further HIV infection. However, ARV tissue pharmacology has not been well characterized across preclinical species used for HIV eradication research, and the influence of drug transporters, HIV infection, and sex on ARV distribution and concentration into the lymph node is largely unknown. Here we show that two animal models of HIV infection (humanized mice and nonhuman primates) were well suited to predict human ARV exposure in lymph nodes. Additionally, we found that drug transporter expression was minimal and-along with viral infection and sex-did not affect ARV penetration into lymph nodes from any species.