Levels of Intracellular Phosphorylated Tenofovir and Emtricitabine Correlate With Natural Substrate Concentrations in Peripheral Blood Mononuclear Cells of Persons Prescribed Daily Oral Truvada for HIV Pre-exposure Prophylaxis
Successful clinical trials of antiretroviral pre-exposure prophylaxis (PrEP) to prevent HIV infection have been reported in heterosexual men and women, men who have sex with men (MSM), and injection drug users.1 A daily oral drug regimen containing the nucleoside reverse transcriptase inhibitor (NRTI) tenofovir disoproxil fumarate (TDF) alone or in combination with emtricitabine (FTC) has been effective when participant adherence is high.1 Analysis of PrEP dosing patterns estimates taking at least 4 TDF doses per week provides 96% protection from HIV infection and at least 2 doses per week provides 76% protection in MSM and transgender women,2 although some estimate more frequent dosing in heterosexual women may be needed.3 TDF and FTC require phosphorylation in HIV target cells to tenofovir diphosphate (TFV-DP) and emtricitabine triphosphate (FTC-TP) to provide PrEP protection as competitive analogs of naturally occurring deoxyadenosine triphosphate (dATP) and deoxycytidine triphosphate (dCTP), respectively. However, it is unclear if physiological conditions that increase deoxynucleoside triphosphate (dNTP) concentrations can affect pharmacokinetics (PK) and pharmacodynamics (PD) of NRTIs. This may be particularly relevant when cellular dNTP pools in HIV target cells increase in response to immune activation. Decreased ratios of phosphorylated NRTIs to their respective dNTPs have been associated with cell activation in vitro and in nonhuman primate studies,4,5 yet this observation remains unexplored in PK and PD studies that measured intracellular drug.6–8 The study presented here compared dATP and dCTP concentrations to TFV-DP and FTC-TP in peripheral blood mononuclear cells (PBMCs) of persons using daily oral Truvada during a successful HIV PrEP study. We further examined if variations among intracellular NRTI:dNTP ratios were related to lymphocyte activation.
PBMCs were isolated from 12 persons before (T0) and 2, 4, and 8 hours after an observed single daily dose of Truvada during their 3-month study visit for the TDF2 PrEP Trial, so samples should be at pseudo-steady state.9 Prescribed dosing at bedtime resulted in assumed T0 sampling approximately 18–24 hours after the previous dose. Intracellular active drug metabolites TFV-DP, FTC-TP, and their natural substrates, dATP and dCTP, respectively, were measured in methanol extracts of PBMCs by high-performance liquid chromatography–tandem mass spectrometry as previously described.5,10 A wide range of presumed steady-state intracellular concentrations was observed for TFV-DP (median = 64 fmol/106 PBMC; range: 20–387 fmol/106 PBMC) and FTC-TP (median = 6451 fmol/106 PBMC; range: 1361–30,310 fmol/106 PBMC), yet concentrations of TFV-DP and FTC-TP were highly correlated (Spearman Rank Order, ρ = 0.860, P < 0.001).
To estimate adherence to PrEP dosing in our substudy, we compared our intracellular TFV-DP and FTC-TP concentrations to those reported in the HIV Prevention Trials Network (HPTN) 066 study of observed Truvada dosing.7 This comparison estimated that all of our substudy participants were taking 4 or more Truvada doses per week (TFV-DP > 19 fmol/106 PBMC, FTC-TP > 600 fmol/106 PBMC), whereas 10 had TFV-DP concentrations (≥36 fmol/106 PBMC) and 11 had FTC-TP concentrations (≥2200 fmol/106 PBMC) consistent with daily dosing. Within the 8-hour sampling window after the observed Truvada dose, all 12 participants' TFV-DP and FTC-TP concentrations were at or above those reported for observed daily dosing in HPTN 066.
There were also wide ranges of concentrations for both dATP (median = 430 fmol/106 PBMC; range: 89–2375 fmol/106 PBMC) and dCTP (median = 776 fmol/106 PBMC; range: 226–1635 fmol/106 PBMC) in the PBMC samples. However, these nucleotide concentrations were highly correlated with their respective intracellular drug analog concentrations in the predose sample (T0) (TFV-DP and dATP, ρ = 0.881, P < 0.001; FTC-TP and dCTP, ρ = 0.909, P < 0.001). The ratio of phosphorylated drug to dNTP in PBMCs was lower for TFV-DP:dATP (median, 0.