Drugs commonly administered to patients infected with the human immunodeficiency virus (HIV) have been studied for their propensity to alter the intracellular phosphorylation of the anti-HIV nucleoside analog stavudine (2',3'-dideoxy-2',3'-didehydrothymidine; d4T) in peripheral blood mononuclear cells (PBMCs) and U937 cells in vitro. PBMCs isolated from the blood of healthy volunteers were stimulated by the mitogen phytohemagglutinin (10 microg/ml) for 72 h. Stimulated PBMCs (3 x 10(6) cells/plate) were then incubated with [3H]d4T (0.65 microCi; 3 microM) and either acyclovir, dapsone, ddC, ddI, fluconazole, foscarnet, ganciclovir, itraconazole, lobucavir, ranitidine, ribavirin, rifampin, sorivudine, sulfamethoxazole, trimethoprim, lamivudine (3TC), zidovudine, or thymidine (30 and 300 microM) for 24 h. Doxorubicin and drugs showing some evidence of inhibition were also studied at 0.3 and 3 microM. Cells were extracted overnight with 60% methanol prior to analysis by radiometric high-performance liquid chromatography. Additional data for nine of the drugs were obtained by incubation with [3H]d4T in U937 cells for 24 h. The effect of d4T (0.2 to 20 microM) on zidovudine (0.65 microCi; 0.018 microCi) phosphorylation was also studied. Zidovudine significantly reduced d4T total phosphates in PBMCs and U937 cells (in PBMCs to 33% [P < 0.001] and 17% [P < 0.001] of that in control cells at 3 and 30 microM, respectively). A small reduction in zidovudine phosphorylation was seen with d4T but only at d4T:zidovudine ratios of 100 and 1,000. Of the other compounds screened, only thymidine, ribavirin, and doxorubicin produced inhibition of d4T phosphorylation in both PBMCs and U937 cells. However, doxorubicin was cytotoxic at 3 microM. The decrease in d4T phosphorylation in the presence of ribavirin is consistent with previous findings with zidovudine. Although ddC significantly inhibited the phosphorylation of d4T in PBMCs, this was not seen in U937 cells, and it is probable that the findings in PBMCs are related to mitochondrial toxicity [based on 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide cytotoxicity assay]. The only drugs screened which may interfere with d4T phosphorylation at clinically relevant concentrations were zidovudine, ribavirin, and doxorubicin.

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Mycophenolic acid (MPA) enhances the in vitro activity of abacavir (ABC) and other nucleoside analog reverse transcriptase inhibitors (NRTIs) against sensitive and NRTI-resistant HIV-1. This may occur via depletion of intracellular deoxyguanosine triphosphate (dGTP). Mycophenolate mofetil (MMF) 500 mg twice daily was added as a single agent to the antiretroviral regimens of five patients failing maximal available therapy. Therapy included ABC, and in most cases didanosine (DDI) and tenofovir (TDF). At entry, mean plasma HIV-1 RNA (VL) was 5.02 log copies/mL (median 4.78, range 4.71-5.63) and mean CD4 count was 106/microL (median 117, range 11-174). MMF was well tolerated. CD4 cell counts did not change significantly from baseline for up to 60 weeks of follow-up. Three of five subjects had VL declines of >0.5 log copies/mL immediately after adding MMF; a fourth subject had a sustained decline of >0.5 log copies/mL after week 8. Declines of >0.5 log copies/mL were lost in two patients at 6 and 8 weeks, and persisted in two patients at 36 and 60 weeks of follow-up, respectively. An increase in the ratio of carbovir triphosphate (CBV-TP), the active antiviral metabolite of ABC, to dGTP was documented in 3 of 4 subjects in temporal association with decreased VL. Trough plasma MPA levels ranged from 0.26-1.67 microg/mL; peak levels 90 minutes after dosing from 1.20-7.77 microg/mL. AUC of MPA appeared little changed when measured over 28 weeks of therapy. Declines in VL were observed in association with measurable changes in the CBV-TP/dGTP ratio in some patients, whereas MPA AUC was below the 30-60 microg*hr/mL range targeted in organ transplantation. The possibility that MMF may enhance the effect of selected NRTIs and be tolerated in late stage HIV disease deserves careful randomized study.

In this paper, we describe the development and use of enzymatic assays to determine intracellular lamivudine triphosphate (3TCTP) and carbovir triphosphate (CBVTP) concentrations in peripheral blood mononuclear cells (PBMCs) from human immunodeficiency virus (HIV)-infected patients. The assays involve inhibition of HIV reverse transcriptase (RT), which normally incorporates radiolabeled deoxynucleoside triphosphates into a synthetic template primer. For the 3TCTP assay, a preincubation procedure was added whereby 3TCTP becomes incorporated before [ 3 H]dCTP. At a 1:400 template primer dilution, control product formation was reduced by 88.0% with 0.8 pmol of 3TCTP. Standard 3TCTP inhibition curves were performed using this procedure. For the CBVTP assay, 0.1 pmol of CBVTP inhibited control product formation with and without the use of a preincubation step, so inhibition curves were constructed using both procedures. However, reduced template primer stability with assays using preincubation steps led to a single-incubation procedure being adopted for future studies. The presence of PBMC extracts interfered with the 3TCTP assay. However, this was overcome by the addition of CuSO 4 . PBMC extracts did not interfere with the CBVTP assay. Intracellular 3TCTP and CBVTP concentrations were determined in PBMCs from HIV-infected patients over 24 h or greater. Peak concentrations were obtained 6 to 8 h after dosing, and the half-lives of the anabolites suggested the possibility of once-daily dosing. These assays are currently being used for determination of 3TCTP and CBVTP concentrations in clinical studies.Previous analytical techniques have employed a combined high-pressure liquid chromatography-radioimmunoassay (HPLC-RIA) procedure for the measurement of intracellular dideoxynucleoside (ddN) phosphates in vivo (1,2,8,11,12,15,16,19,20). However, this methodology has the problem of requiring a large volume of blood from the patient (Ͼ20 ml per time point). Furthermore, the combined HPLC-RIA method is very time-consuming, with only a small number of samples processed daily. More recently, ddN phosphates have been determined by liquid chromatography tandem mass spectrometry (LC-MS-MS) (17). While this method has many advantages, there is a requirement for specialized and expensive equipment and timeconsuming sample preparation.An alternative approach is to measure the concentrations of the active ddN triphosphate (ddNTP) using an enzymatic assay. This procedure involves the inhibition of human immunodeficiency virus (HIV) reverse transcriptase (RT) enzyme that normally incorporates radiolabeled dNTPs into a specific synthetic template primer. By using known standard amounts of the ddNTP inhibitor, an inhibition curve can be derived. Therefore, the concentrations of the respective ddNTP present in cell extracts can be determined from the standard curve.Initial studies performed by Robbins et al. (14) using this type of procedure involved the determination of zidovudine TP (ZDVTP). Although the method was primarily used to ...

Hydroxyurea does not affect measurable pools of endogenous nucleosides in vivo. Decreased lamivudine phosphorylation over time may provide a novel pharmacological explanation for the mechanism of resistance to this drug.