Effect of mycophenolate mofetil on the pharmacokinetics of antiretroviral drugs and on intracellular nucleoside triphosphate pools.

OBJECTIVE: To study the effect of mycophenolate mofetil therapy on the pharmacokinetic parameters of a number of antiretroviral drugs, on intracellular pools of deoxycytidine triphosphate (dCTP) and deoxyguanosine triphosphate (dGTP), and on intracellular concentrations of the triphosphate of lamivudine (3TCTP). DESIGN: Randomised pharmacokinetic study. PARTICIPANTS: Nineteen HIV-1-infected patients. METHODS: Antiretroviral-naive men starting treatment with didanosine 400 mg once daily, lamivudine 150 mg twice daily, abacavir 300 mg twice daily, indinavir 800 mg twice daily, ritonavir 100 mg twice daily and nevirapine 200 mg twice daily were randomised to a group with or without mycophenolate mofetil 500 mg twice daily. After 8 weeks of therapy, the plasma pharmacokinetic profiles of mycophenolic acid (the active metabolite of mycophenolate mofetil), abacavir, indinavir and nevirapine, and triphosphate concentrations (dCTP, dGTP and 3TCTP) in peripheral blood mononuclear cells, were determined. RESULTS: Nine of the 19 patients received mycophenolate mofetil. There was no difference in plasma clearance of indinavir or abacavir between the two groups. The clearance of nevirapine was higher in patients using mycophenolate mofetil (p = 0.04). In 12 patients, of whom five also received mycophenolate mofetil, intracellular triphosphates were measured. There was no significant difference in intracellular dCTP, dGTP or 3TCTP concentrations between the two groups. CONCLUSION: In this small cohort of patients, mycophenolate mofetil therapy reduced the plasma concentration of nevirapine but had no effect on plasma concentrations of indinavir and abacavir. There were no consistent effects of mycophenolic acid on the intracellular concentrations of dCTP, dGTP or 3TCTP.

Enzymatic assay for measurement of intracellular DXG triphosphate concentrations in peripheral blood mononuclear cells from human immunodeficiency virus type 1-infected patients.

DXG ([2R-cis]-2-amino-1,9-dihydro-9-[2-[hydroxymethyl]-1,3-dioxolan-4-yl]-6H-purin-6-one) and its prodrug DAPD ([2R-cis]-4-[2,6-diamino-9H-purin-9-yl]-1,3-dioxolane-2-methanol; amdoxovir) are novel 2',3'-dideoxynucleosides (ddNs) displaying activity against human immunodeficiency virus type 1 (HIV-1). In this paper, we describe the development of an enzymatic assay for determining the intracellular active metabolite of DXG and DAPD, DXG triphosphate (DXGTP), in peripheral blood mononuclear cells (PBMCs) from HIV-infected patients. The assay involves inhibition of HIV reverse transcriptase (RT), which normally incorporates radiolabeled deoxynucleoside triphosphates (dNTPs) into a synthetic template primer. DXGTP (0.6 pmol) inhibited control product formation with or without a preincubation step. Inhibition was greatest when the template primer was most diluted. DAPDTP inhibited control product formation only at very high levels (50 pmol) and when a preincubation procedure was used. However, reduced template primer stability in assays using preincubation steps, coupled with potential interference by DAPDTP, led to the current assay method for DXGTP being performed without preincubation. Standard DXGTP inhibition curves were constructed. The presence of PBMC extracts or endogenous dGTP did not interfere with the DXGTP assay. Intracellular DXGTP and dGTP concentrations were determined in PBMCs from HIV-infected patients receiving oral DAPD (500 mg b.i.d.). Peak concentrations of DXGTP were obtained 8 h after dosing and were measurable through 48 h postdose. Levels of endogenous dGTP were also determined over 48 h. No direct relationship was observed between concentrations of DXGTP and dGTP. Quantification of DXGTP concentrations in PBMCs from patients receiving a clinically relevant dose of DAPD is possible with this enzymatic assay.

Time-dependent changes in HIV nucleoside analogue phosphorylation and the effect of hydroxyurea.

BACKGROUND: Nucleoside analogues are activated to their triphosphates, which compete with endogenous deoxynucleoside triphosphate (dNTP) pools to inhibit HIV reverse transcriptase. Hydroxyurea has been administered with nucleoside analogues to modulate intracellular dNTP pools and thus the ratio of drug triphosphate:endogenous triphosphate. OBJECTIVES: To examine changes in drug activation over time and investigate the effects of hydroxyurea on intracellular phosphorylation of antiretroviral nucleoside analogues. PATIENTS: A total of 229 HIV-infected individuals receiving abacavir, lamivudine and zidovudine were randomly assigned to receive or not nevirapine and hydroxyurea. Twenty-four patients were recruited to an observational substudy measuring intracellular drug triphosphate and dNTP concentrations at 0, 2, 6, 12, 24 and 48 weeks. METHODS: Drugs were extracted from isolated peripheral blood mononuclear cells before analysis of endogenous dNTP and drug triphosphates by primer extension assays. RESULTS: Twenty-two out of 24 patients were followed to completion of the substudy. Hydroxyurea had no demonstrable effect on endogenous dNTP or drug triphosphate levels at any timepoint. However, the ratio of zidovudine triphosphate to endogenous deoxythymidine triphosphate was significantly increased with hydroxyurea. A significant decrease in lamivudine triphosphate (3TCTP) and the 3TCTP:endogenous deoxycytidine triphosphate ratio was seen over 48 weeks. In five patients who failed therapy in the first 24 weeks, significantly reduced 3TCTP was seen. CONCLUSION: 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.

The addition of mycophenolate mofetil to antiretroviral therapy including abacavir is associated with depletion of intracellular deoxyguanosine triphosphate and a decrease in plasma HIV-1 RNA.

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.

Development of enzymatic assays for quantification of intracellular lamivudine and carbovir triphosphate levels in peripheral blood mononuclear cells from human immunodeficiency virus-infected patients.

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.