Tenofovir, emtricitabine, and tenofovir diphosphate in dried blood spots for determining recent and cumulative drug exposure

Abstract

Tenofovir (TFV) disoproxil fumarate (TDF)±emtricitabine (FTC) are widely used for HIV treatment and chemoprophylaxis, but variable adherence may lead to suboptimal responses. Methods that quantify adherence would allow for interventions to improve treatment and prevention outcomes. Our objective was to characterize the pharmacokinetics of TFV-diphosphate (TFV-DP) and FTC-triphosphate (FTC-TP) in red blood cells (RBCs) and peripheral blood mononuclear cells (PBMCs); to extend the RBC analysis to dried blood spots (DBSs); and to model how RBC/DBS monitoring could inform recent and cumulative drug exposure/adherence. Blood samples were collected from 17 HIV-negative adults at 5 visits over a 30-day pharmacokinetics study of daily oral TDF/FTC. Dosing was discontinued on day 30 and blood was collected on days 35, 45, and 60 during the washout period. Plasma/RBCs/PBMCs/DBSs were all quantified by liquid chromatography/tandem mass spectrometry. DBSs were paired with RBCs and plasma for comparisons. The median (interquartile range) RBC TFV-DP half-life was 17.1 (15.7-20.2) versus 4.2 (3.7-5.2) days in PBMCs. At steady state, TFV-DP was 130 fmol/10(6) RBCs versus 98 fmol/10(6) PBMCs. FTC-TP was not quantifiable in most RBC samples. TFV-DP in RBCs versus DBSs yielded an r(2)=0.83. TFV-DP in DBSs was stable at -20°C. Simulations of TFV-DP in RBCs/DBSs, when dosed from one to seven times per week, demonstrated that each dose per week resulted in an average change of approximately 19 fmol/10(6) RBCs and 230 fmol/punch. TFV and FTC in plasma versus DBSs was defined by y=1.4x; r(2)=0.96 and y=0.8x; r(2)=0.99, respectively. We conclude that DBSs offer a convenient measure of recent (TFV/FTC) and cumulative (TFV-DP in RBCs) drug exposure with potential application to adherence monitoring.

FIG. 1.

Natural log tenofovir-diphosphate (TFV-DP) concentrations in red blood cells (RBCs) according to days after discontinuation of TDF-FTC from 17 HIV-seronegative participants. Two to five million RBCs were analyzed per sample.

FIG. 2.

(A) Tenofovir (TFV) in plasma versus DBSs and (B) emtricitabine (FTC) in plasma versus DBSs. DBSs and plasma were obtained from the same blood draw from three participants over two intensive pharmacokinetic studies of TDF-FTC. One pharmacokinetics visit was after the first dose and the other was after the last dose before drug discontinuation. The linear regression for TFV in plasma versus DBSs was 1.4x, r²=0.96 and that for FTC in plasma versus DBSs was 0.8x, r²=0.99. The regression did not account for repeated measures.

FIG. 3.

(A) Tenofovir-diphosphate (TFV-DP) in RBCs versus DBSs obtained from the same blood draw from five participants. The linear regression for TFV-DP in RBCs versus DBSs was 0.085x, r²=0.83. The regression did not account for repeated measures. This translates to approximately 12 million RBCs per 3-mm DBS punch. (B) Paired samples over the 30-day washout period were available from three participants. The TFV-DP half-lives were parallel in DBSs (fmol/punch) and RBCs (fmol/10⁶ RBCs).

FIG. 4.

The effect of storage conditions on tenofovir-diphosphate (TFV-DP) concentrations in DBSs over time. Samples stored at room temperature (circles), 4°C (squares), and −20°C (triangles) were compared with samples from the same DBS spot stored at −80°C. The dashed lines represent a 15% difference from −80°C. Samples stored at room temperature were >15% below the level at −80°C after approximately 2 weeks.

FIG. 5.

Simulated tenofovir diphosphate (TFV-DP) levels in DBSs and RBCs following different patterns of drug exposure. TFV-DP levels start from steady state associated with daily dosing to show the rate of change over time.