Studies of daily emtricitabine-tenofovir disoproxil fumarate (FTC-TDF) for HIV preexposure prophylaxis (PrEP) in men who have sex with men (MSM) modeled intracellular tenofovir-diphosphate (TFV-DP) in dried blood spots (DBS) to assess adherence and corresponding PrEP outcomes. We conducted a prospective, randomized, crossover pharmacokinetic study of TFV-DP in DBS during 33%, 67%, or 100% of daily dosing under directly observed therapy (DOT). Participants were assigned to two 12-week dosing regimens, separated by a 12-week washout. Forty-eight adults (25 women) from Denver and San Francisco were included. TFV-DP exhibited a median half-life of 17 days, reaching steady state in 8 weeks. TFV-DP was dose proportional with mean (SD) steady-state concentrations of 530 (159), 997 (267), and 1,605 (405) fmol/punch for the 33%, 67%, and 100% arms, respectively. Prior work in MSM demonstrated clinically meaningful TFV-DP thresholds of 350, 700, and 1,250 fmol/punch, which were estimated 25th percentiles for 2, 4, and 7 doses/week. In the present study, corresponding TFV-DP was within 3% of the prior estimates, and subgroups by site, race, and sex were within 14% of prior estimates, although males had 17.6% (95% confidence intervals [CIs], 6.5, 27.4%) lower TFV-DP than females. The thresholds of 350, 700, and 1,250 fmol/punch were achieved by 75% of men taking ≥1.2, 3.2, and 6 doses/week and 75% of women taking ≥0.6, 2.0, and 5.3 doses/week, indicating that lower dosing reached these thresholds for both sexes. In conclusion, TFV-DP arising from DOT was similar to previous estimates and is useful for interpreting PrEP adherence and study outcomes. (This study has been registered at ClinicalTrials.gov under identifier NCT02022657.).

O ral preexposure prophylaxis (PrEP) using coformulated tenofovir disoproxil fumarate and emtricitabine (TDF-FTC) has proven effective in preventing HIV infection in high-risk individuals (1-7). Unfortunately, PrEP efficacy has not been consistent across all studies, mostly due to variations in drug adherence (8)(9)(10). Multiple studies have demonstrated that sustained drug adherence and exposure are the main factors that determine success in PrEP (1, 5). However, despite its importance, no gold standard measure of antiretroviral adherence is currently available in routine clinical practice, and adequately quantifying adherence continues to be a challenge.Plasma and intracellular tenofovir (TFV) and TFV-diphosphate (TFV-DP) levels have been shown to be powerful markers of adherence to PrEP (1, 5, 11). In particular, TFV-DP in red blood cells (RBCs), measured using dried blood spots (DBS), was found to be a strong marker of cumulative adherence to TDF-FTC and highly predictive of PrEP efficacy in men who have sex with men (MSM) (5,7,12). This is due to the uniquely long intracellular half-life (17 days) of TFV-DP in RBCs (and DBS), which leads to high accumulation with optimal adherence, so that adherence gradients can be estimated. This is informative about cumulative TDF dosing (adherence) over an extended period (6). However, because of its long half-life, TFV-DP in DBS is unable to discriminate between patterns of recent versus remote dosing and cannot adequately detect variations in very recent dosing. Similar to TFV, FTC (the other component of the currently approved PrEP regimen) is also phosphorylated and trapped inside RBCs as FTC-triphosphate (FTC-TP) (13), with the advantage that it can be simultaneously quantified in DBS, along with TFV-DP. Although the pharmacokinetics of TFV-DP in DBS have been defined (6), our current knowledge about the disposition of FTC-TP in this matrix is limited. In addition, it remains unknown whether FTC-TP in DBS can provide adherence information complementary to that provided by TFV-DP.In this study, we aimed to characterize the pharmacokinetics of FTC-TP in DBS and to evaluate its utility as a marker of recent dosing with TDF-FTC.

The pharmacokinetics (PK) of tenofovir-diphosphate (TFV-DP) and emtricitabine-triphosphate (FTC-TP), the active anabolites of tenofovir disoproxil fumarate (TDF), and emtricitabine (FTC) in blood, genital, and rectal compartments was determined in HIV-positive and seronegative adults who undertook a 60-day intensive PK study of daily TDF/FTC (plus efavirenz in HIV positives). Lymphocyte cell sorting, genital, and rectal sampling occurred once per subject, at staggered visits. Among 19 HIV-positive (3 female) and 21 seronegative (10 female) adults, TFV-DP in peripheral blood mononuclear cells (PBMC) accumulated 8.6-fold [95% confidence interval (CI): 7.2-10] from first-dose to steady-state concentration (Css) versus 1.7-fold (95% CI: 1.5-1.9) for FTC-TP. Css was reached in ∼11 and 3 days, respectively. Css values were similar between HIV-negative and HIV-positive individuals. Css TFV-DP in rectal mononuclear cells (1,450 fmol/10 cells, 898-2,340) was achieved in 5 days and was >10 times higher than PBMC (95 fmol/10 cells, 85-106), seminal cells (22 fmol/10 cells, 6-79), and cervical cells (111 fmol/10 cells, 64-194). FTC-TP Css was highest in PBMC (5.7 pmol/10 cells, 5.2-6.1) and cervical cells (7 pmol/10 cells, 2-19) versus rectal (0.8 pmol/10 cells, 0.6-1.1) and seminal cells (0.3 pmol/10 cells, 0.2-0.5). Genital drug concentrations on days 1-7 overlapped with estimated Css, but accumulation characteristics were based on limited data. TFV-DP and FTC-TP in cell sorted samples were highest and achieved most rapidly in CD14 compared with CD4, CD8, and CD19 cells. Together, these findings demonstrate cell-type and tissue-dependent cellular pharmacology, preferential accumulation of TFV-DP in rectal mononuclear cells, and rapid distribution into rectal and genital compartments.