In this study, we examined the PK profile of two NRTI’s after microdose and standard therapeutic dosing regimens. For zidovudine, plasma PK showed linearity over a 3000-fold dosing range, but the normalized intracellular ZDV-TP was several folds higher with the microdose compared to the standard dosing regimen. For TFV, the normalized plasma AUCinf
was 1.5-times higher with the microdose regimen, possibly indicating higher oral bioavailability, but intracellular TFV-DP PK was linear over the 3000-fold dose range tested. As has been suggested previously, the ability to precisely predict human PK from a microdosing study will vary from molecule to molecule 18–20
The different results for intracellular ZDV-TP and TFV-DP may reflect the different kinases involved in their phosphorylation pathways. ZDV is phosphorylated sequentially by thymidine kinase, thymidylate kinase, and NDP kinase, with thymidylate kinase acting as a rate-limiting enzyme in this process21
. An earlier study showed that the phosphorylation of ZDV-MP to ZDV-DP is characterized by a high Km
and low Kcat
, and that intracellular ZDV-MP concentrations were much lower than the Km
of this enzyme after a 300 mg dose7
. Since the concentration of ZDV-MP is likely to be far below the Km
with either the standard 300 mg dose or the microdose (100 μg), the rate of conversion of ZDV to ZDV-TP should be similar in either case. However, ZDV-MP has been reported to have a substrate suppression effect on thymidylate kinase 21
. With microdose of ZDV, this suppression might be reversed and the enzyme may become more efficient. This could have contributed to the higher ZDV phosphorylation observed with the microdose regimen.
Tenofovir is phosphorylated by adenylate kinase to TFV-MP, and subsequently phosphorylated by nucleoside diphosphate kinase (NDPK) to TFV-DP. In vitro
data suggest that this second step can also be accomplished by pyruvate kinase and creatine kinase 22
. With TFV and TFV-MP as substrates, both phosphorylation steps are also characterized by low Kcat
and high Km22, 23
. In other words, phosphorylation is carried out at low velocity and high capacity, which may explain the slow accumulation of TFV-DP in cells and the linearity of PK across the 3000-fold range of doses we evaluated in this study.
Studying phosphorylation in the biological target cell subset in vivo may provide valuable information applicable to future drug use and development. It has been reported that ZDV-TP formation is lower in CD4+ cells than in total PBMCs, while lamivudine triphosphate concentrations are the same amongst different cell types9
. Here we compared the intracellular phosphorylated concentrations of ZDV and TFV in CD4+ cells versus total unfractionated PBMCs. ZDV-TP concentrations were lower in CD4+ cells in the standard dose regimen, while TFV-DP concentrations were not different in these subsets of cells.
The reason for the different phosphorylation of ZDV in specific cell types is not fully understood, and the result of this study supports the existence of a rate-limiting step, or nonlinear phosphorylation specific to ZDV in CD4+ cells. The difference of ZDV-TP concentrations in CD4+ cells compared to PBMCs was statistically significant with standard dosing (Wilcoxon signed rank test, p<0.05), but not with microdosing. The difference in ZDV-TP concentrations after microdose and standard dose regimens was larger in CD4+ cells compared to PBMC’s (11.9-fold versus 3.9-fold), unlike TFV-DP (similar concentration ratios in PBMC’s and CD4+ cells after the two dosing regimens). These observations further support the existence of a rate-limiting step in the formation of ZDV-TP in CD4+ cells.
AMS analysisof the intracellular concentrations of ZDV-TP and plasma TFV yielded excellent concordance with published data 11, 24–26
. The longer estimated half-life and higher AUCinf
of ZDV in plasma in this study was almost certainly because AMS measures the sum of ZDV and its metabolites, mainly ZDV-glucuronide. For TFV-DP, single dose PK for TFV-DP in PBMC has not been reported in the literature, and all PBMC TFV-DP data were collected from subjects who have reached steady state. It is possible to estimate the steady state Css
based on the PK profile after a single dose if the PK is linear and stationary. If we use Css
/tau, the derived mean Css
with 300 mg once daily dose is about 60 fmol/106
cells. This derived steady state TFV-DP concentration agrees with that reported in the literature 24,27–29
We observed a significant plateau phase for intracellular TFV-DP concentrations between 12–72 hours following a single dose, followed by a more conventional but prolonged elimination phase. A prolonged plateau phase suggests balanced formation and elimination of TFV-DP. Estimation of the T1/2
of TFV-DP is complicated by this multiphasic elimination, i.e., plateau phase followed by prolonged elimination phase, and this may explain the wide range of elimination half-lives reported in vitro
(50 h in resting T cells) 8,30
and in patients taking TDF (100–150 h) 28,31
. In our study, intracellular TFV-DP formation appeared to continue as plasma TFV concentrations fell to undetectable levels. A detailed calculation of the absolute ratio of concentrations of intracellular and extracellular TFV found that the positive gradient from outside the cell to inside the cell persisted during the TFV-DP formation and plateau phases and for up to 72 hours post dose (). This suggests that it is not necessary to posit a reservoir or accumulation of intracellular TFV-DP precursors such as TFV or TFV-MP. Recent data also proved little accumulation of TFV or TFV-MP in PBMC’s in vivo27
. Our single dose studies allowed demonstration of a hysteresis of TFV-DP formation relative to plasma TFV, and a biphasic elimination profile of TFV-DP elimination. This aspect of TFV-DP PK has not been reported previously in the literature. These findings support the observations of Patterson’s study, where TDF-DP concentrations were detectable in rectal and vaginal mucosal homogenates as long as 14 days following a single dose25
TDF is widely used for the treatment of HIV infection, and also shows promise in preexposure prophylaxis for HIV infection 32, 33
. Our findings may have potential implications for future prevention studies using TDF. The 12 hour time lag before the beginning of the TFV-DP plateau following a single dose suggests that the first dose of TDF for PrEP may need to be taken at least several hours prior to exposure in order to be effective depending on the effective TFV-DP concentration. On the other hand, the long plateau and slow elimination of TFV-DP indicates that a daily dosing regimen should be quite forgiving of missed doses if used continuously.
For the comparison of microdose and standard dosing regimens, we used a fixed sequence design with paired analysis to compensate for interindividual variability, our study was therefore informative despite the small number of subjects. Carryover of intracellular phosphates from the microdose phase was observed, but in only two subjects in each arm and the amount was too small to significantly influence the results of the standard dose phase. A fixed sequence design has the advantage over a crossover design of avoiding potential consequences of induction or inhibition of drug metabolizing enzymes, transporters, kidney function and other potential drug toxicities that might change PK after a standard dose of ZDV or TDF. AMS may facilitate early development of investigational nucleoside analogs through microdosing, but rate-limited metabolism complicates interpretation of intracellular metabolism with microdosing studies. When rate-limiting steps potentially present, caution should be exercised in extrapolating from microdose to standard dose.