Interspecies Differences in Tenofovir Alafenamide Fumarate Stability in Plasma

Tenofovir (TFV) alafenamide fumarate (TAF) is an antiretroviral that has been evaluated in alternative drug delivery systems in several species. The ex vivo stability of TAF was evaluated, and TAF was stable in dog-, sheep-, and macaque-spiked plasma. A negative bias was observed in TAF recovery in rabbit-spiked plasma; there was complete loss of TAF and corresponding overestimation of TFV in rodent-spiked plasma. These data highlight considerations when evaluating TAF and TFV concentrations in preclinical studies.

ABSTRACT

Tenofovir (TFV) alafenamide fumarate (TAF) is an antiretroviral that has been evaluated in alternative drug delivery systems in several species. The ex vivo stability of TAF was evaluated, and TAF was stable in dog-, sheep-, and macaque-spiked plasma. A negative bias was observed in TAF recovery in rabbit-spiked plasma; there was complete loss of TAF and corresponding overestimation of TFV in rodent-spiked plasma. These data highlight considerations when evaluating TAF and TFV concentrations in preclinical studies.

TEXT

Tenofovir disoproxil fumarate (TDF) and tenofovir alafenamide fumarate (TAF) are oral prodrugs of the nucleotide analog reverse transcriptase inhibitor tenofovir (TFV) (1). The addition of the alafenamide group to the antiviral compound prevents nonspecific esterase activity, resulting in a more stable, intact prodrug in systemic circulation (1, 2). Compared to TDF, TAF has shown improvements concerning bone density and renal function (3–5); however, TAF is also associated with increases in body mass index and elevations in cholesterol concentrations (6). Both TDF and TAF are approved for HIV treatment; based on its pharmacologic profile, TAF is currently approved for HIV treatment at a lower dose (25 mg) than TDF (300 mg). Further, studies have shown that TAF is not inferior to TDF for HIV prevention in cisgender men and transgender women and has been approved for HIV prevention as the once-daily fixed-dose formulation Descovy (25 mg TAF and 300 mg emtricitabine; Gilead Sciences) in these populations (7).

TAF has been the focus of a number of preclinical efforts, including those evaluating the safety and pharmacokinetics of subdermal and transdermal delivery devices (8–12). These studies have been conducted in several model systems, including mouse, dog, and macaque, among others. Consequently, the characterization of TAF across potential species is critical, particularly if pharmacologic findings will be scaled allometrically to humans. Although TAF is more stable in human blood and blood plasma than TDF, there are challenges with analyte stability ex vivo. Our group and others have demonstrated limited whole-blood and plasma TAF stability over 1 h and 24 h, respectively (13 and unpublished data). Further, species-based differences in TAF stability ex vivo have not been well characterized in the literature. This work focuses on assessing the stability of TAF spiked into plasma isolated from several model organisms and the ability to use bioanalytical methods validated for human samples in other species.

K₂EDTA plasma samples from dog, human, macaque, mouse, rabbit, rat, and sheep were acquired from BioIVT (Westbury, NY). TAF (Gilead Sciences, Foster City, CA) and TFV (Toronto Research Chemicals, Toronto, ON, Canada) were dissolved in dimethyl sulfoxide and water, respectively, and spiked into the aforementioned plasma samples at concentrations of 0.09 ng/ml, 3.00 ng/ml, and 130 ng/ml (TAF) and 3.00 ng/ml, 20.0 ng/ml, and 170 ng/ml (TFV). Drug-spiked plasma samples were incubated for 1 h at room temperature (22 to 26°C) before analysis. TAF and TFV were prepared and measured via a previously described liquid chromatographic-mass spectrometric (LC-MS) method and analyzed in triplicate (13). Assay analytical measuring ranges were 0.03 ng/ml to 150 ng/ml and 1 ng/ml to 200 ng/ml for TAF and TFV, respectively. The multiplexed LC-MS assay was validated in accordance with FDA guidance for industry and bioanalytical method validation recommendations (14). Calibration standards were prepared using human K₂EDTA plasma. Percent deviations from theoretical concentrations (%DEV) and percent differences from mean drug concentrations measured in human K₂EDTA plasma (%DIF) were used to evaluate the ability of the method to accurately quantify TAF and TFV in nonhuman species.

The structures of TAF and TFV are illustrated in Fig. 1. There were species-specific differences in TAF and TFV quantitation in K₂EDTA plasma when handled under equivalent conditions (Table 1). TAF- and TFV-spiked dog, sheep, and macaque plasma yielded average recoveries (%DEVs) of ≤±10.8%; further, no bias was observed. In comparisons of drugs spiked in dog, sheep, and macaque plasma to those in human plasma, average %DIFs were ≤±8.19%. At all concentrations evaluated, %DEVs and %DIFs were <±15%. We observed a negative bias in TAF measurements extracted from rabbit plasma. The average %DEV from target TAF concentrations was −18.1%; compared to human-spiked plasma, the average %DIF was −22.3%. At the three evaluated concentrations, TAF showed negative biases (%DEVs) of −22.5% (0.090 ng/ml), −24.8% (3.0 ng/ml), and −7.00% (130 ng/ml); conversely, TFV concentrations yielded positive biases of 7.54% (3.0 ng/ml), 9.62% (20 ng/ml), and 17.1% (170 ng/ml) compared to theoretical concentrations. While the negative bias of TAF was less pronounced at higher concentrations in rabbit plasma, the breakdown of TAF contributed to the positive bias observed in TFV measurements. TAF was nonquantifiable in mouse and rat plasma across the analytical measuring range of the assay. Conversely, TFV concentrations showed a significant positive bias, with %DEVs of >25% for both species. Further, there was a positive correlation between the amount of TAF spiked into mouse and rat plasma samples and observed TFV concentrations (Fig. 2). In TAF-spiked mouse and rat plasma at 130 ng/ml, TFV concentrations were 61.3% and 58.2% higher than the target concentration (170 ng/ml). Elevated concentrations (>100 ng/ml) of TFV were also observed in rodent plasma spiked with 130 ng/ml TAF alone, further illustrating the ex vivo conversion of TAF to TFV; much lower TFV concentrations were observed in plasma from rabbit (29 ng/ml) and other species (<13 ng/ml) (data not shown).

FIG 1.

Structures of tenofovir (TFV) (A) and tenofovir alafenamide fumarate (TAF) (B).

TABLE 1.

Recovery and differences in TAF and TFV concentrations in drug-spiked plasma

Plasma	%DEV and %DIF at each drug concna
	TAF					TFV
	0.090 ng/ml	3.0 ng/ml	130 ng/ml	Mean %DEV	Mean %DIF	3.0 ng/ml	20 ng/ml	170 ng/ml	Mean %DEV	Mean %DIF
Human	0.0924	3.17	127	2.11	NA	3.04	20.6	182	3.79	NA
Macaque	0.0937	3.34	127	4.51	2.22	2.84	20.4	168	−1.44	−5.19
Dog	0.0877	3.16	124	−0.527	−2.66	3.38	20.8	181	7.62	3.59
Sheep	0.0996	3.42	140	10.8	8.19	3.33	20.8	176	6.2	2.27
Rabbit	0.0697	2.26	121	−18.1	−22.3	3.23	21.9	199	11.4	7.04
Mouse	NQ	NQ	NQ	—	—	3.71	23.7	274	34.5	24.8
Rat	NQ	NQ	NQ	—	—	3.26	22.6	269	26.5	18.2

^a%DEV, [(mean − theoretical)/theoretical] × 100. %DIF = [(animal − human)/((animal + human)/2)] × 100. NA, not applicable, as human-spiked plasma is the comparator. NQ, nonquantifiable. —, TAF concentrations below the assay lower limit of quantitation (<0.030 ng/ml) at all evaluated concentrations.

FIG 2.

Correlation between theoretical and observed TFV concentrations (in nanograms per milliliter) in plasma from dog (A), macaque (B), rabbit (C), sheep (D), mouse (E), and rat (F).

These data suggest increased ex vivo hydrolysis of TAF to TFV postcollection in rabbit, mouse, and rat samples compared to that of other species, with a much more significant effect in rodent specimens. Increased hydrolysis can lead to the underestimation of TAF concentrations with a corresponding overestimation of TFV in biological samples postcollection. TAF conversion occurs much more rapidly in rodent and rabbit plasma than in plasma of canines, sheep, nonhuman primates, and humans. Therefore, preclinical data evaluating TAF and TFV pharmacokinetics in these species should be interpreted accordingly. The ex vivo instability of TAF may be mitigated by the addition of weak acids to plasma immediately postprocessing. Studies have shown that the addition of acetic acid or formic acid to plasma postcentrifugation can inhibit esterase enzymes and subsequent TAF hydrolysis (15, 16). Recently, Xiao and colleagues reported that the addition of formic acid after blood collection inhibited TAF hydrolysis to TFV, thus enhancing analyte stability postcollection to 6 h on wet ice and 24 h at 4°C (16). The acidification of plasma can inhibit endogenous esterases, thereby preventing enzyme activity and drug hydrolysis postcollection (17). However, the specific ratio of plasma to acid may depend on drug concentrations, anticoagulant additives, and species; the aforementioned acidification experiments were performed in human K₂EDTA plasma.

The differences observed across species may be attributed to increased gene expression or esterase enzyme activity in rodents and rabbits compared to that in larger animals. Studies have shown high expression of carboxylesterases in mice, rats, and rabbits, with lower relative expression in dogs, sheep, and primates; enzyme expression also correlated with drug hydrolysis (18). These data support the observed decreased recoveries of TAF in spiked plasma in these species. As bioanalytical LC-MS assays are typically validated using human specimen sources, important considerations must be analyzed when quantifying drugs (or prodrugs) from other species. The negative bias observed in TAF-spiked rabbit plasma had less of an effect on TFV concentrations than on those of rodent-spiked plasma, which may be due to the incomplete hydrolysis of TAF in rabbit plasma. This work shows that, under nonpretreated conditions, TAF cannot be quantified in mouse and rat plasma, and TFV concentrations are overestimated due to ex vivo TAF conversion.