The mechanism of phosphorylation of anti-HIV D4T by nucleoside diphosphate kinase.
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The last step in the intracellular activation of antiviral nucleoside analogs is the addition of the third phosphate by nucleoside diphosphate (NDP) kinase resulting in the synthesis of the viral reverse transcriptase substrates. We have previously shown that dideoxynucleotide analogs and 3'-deoxy-3'-azidothymidine (AZT) as di- or triphosphate are poor substrates for NDP kinase. By use of protein fluorescence, we monitor the phosphotransfer between the enzyme and the nucleotide analog. Here, we have studied the reactivity of D4T (2',3'-dideoxy-2',3'-didehydrothymidine; stavudine) as di- (DP) or triphosphate (TP) at the pre-steady state. The catalytic efficiency of D4T-DP or -TP is increased by a factor of 10 compared with AZT-DP or -TP, respectively. We use an inactive mutant of NDP kinase to monitor the binding of a TP derivative, and show that the affinity for D4T-TP is in the same range as for the natural substrate deoxythymidine triphosphate, but is 30 times higher than for AZT-TP. Our results indicate that D4T should be efficiently phosphorylated after intracellular maturation of a prodrug into D4T-monophosphate. (+info)
Excision of beta-D- and beta-L-nucleotide analogs from DNA by the human cytosolic 3'-to-5' exonuclease.
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The cytosolic 3'-to-5' exonuclease from chronic lymphocytic leukemia cells was highly purified, and its ability to remove beta-D- and beta-L-nucleotide analogs from the 3'-end of DNA was determined. The relative rate of excision of beta-D-ddCMP, beta-L-ddCMP, beta-L-FddCMP, beta-L-SddCMP, beta-L-Fd4CMP, and beta-L-OddCMP from the 3'-end of a single-stranded oligonucleotide primer or a primer annealed with complementary DNA and/or RNA templates was assessed. The rate of excision of beta-D-nucleotides from the 3'-end of DNA was higher than that of beta-L-nucleotides, which could be partly attributable to the affinity of the enzyme to beta-D-nucleotide-terminated DNA being 5-fold higher compared with that of beta-L-nucleotide-terminated DNA. The rate of removal of beta-L-Fd4CMP and beta-L-OddCMP from the 3'-end of DNA was at least 8 to 10 times lower compared with that of beta-L-SddCMP. HIV reverse transcriptase could elongate DNA primers after the removal of chain terminators by the cytosolic exonuclease. Concentrations of nucleoside 5'-monophosphate analogs that inhibit the cytosolic exonuclease by 50% were estimated. Among the nucleoside 5'-monophosphate analogs examined, beta-L-Fd4CMP appeared to be the most effective inhibitor of the cytosolic exonuclease, with an ID(50) value of 38 microM. (+info)
A randomized, controlled, phase II trial comparing escalating doses of subcutaneous interleukin-2 plus antiretrovirals versus antiretrovirals alone in human immunodeficiency virus-infected patients with CD4+ cell counts >/=350/mm3.
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A total of 73 patients with baseline CD4+ cell counts >/=350 cells/mm3 who were receiving combination antiretroviral therapy (ART) were randomized to receive subcutaneous interleukin-2 (IL-2; n=36) in addition to ART or to continue ART alone (n=37). Subcutaneous IL-2 was delivered at 1 of 3 doses (1.5 million international units inverted question markMIU, 4.5 MIU, and 7.5 MIU per dose) by twice-daily injection for 5 consecutive days every 8 weeks. After 24 weeks, the time-weighted mean change from baseline CD4+ cell count was 210 cells/mm3 for recipients of subcutaneous IL-2, compared with 29 cells/mm3 for recipients of ART alone (P<.001). There were no significant differences between treatment groups for measures of plasma human immunodeficiency virus RNA (P=.851). Subcutaneous IL-2 delivered at doses of 4.5 MIU and 7.5 MIU resulted in significant increases in CD4+ cell count (P=.006 and P<.001, respectively), compared with that seen in control patients. These changes were not significant in the 1.5 MIU dose group compared with that in the control patients (P=.105). Side effects that occurred from subcutaneous IL-2 administration were generally low grade, of short duration, and readily managed in an outpatient environment. (+info)
Early virological failure in naive human immunodeficiency virus patients receiving saquinavir (soft gel capsule)-stavudine-zalcitabine (MIKADO trial) is not associated with mutations conferring viral resistance.
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The MIKADO trial was designed to evaluate the efficacy of stavudine-zalcitabine-saquinavir (soft gel capsule) [d4T-ddC-SQV(SGC)] in 36 naive patients (-3.3 log(10) units at week 24 [W24]). Among the 29 patients remaining on d4T-ddC-SQV(SGC) until W24, 10 harbored a virological failure (viral load of >200 copies/ml at W24) (group 1). To determine the reasons for therapeutic failure, genotypic and phenotypic resistance test results and SQV concentrations in plasma were analyzed and compared to those in successfully treated patients (viral load of <200 copies/ml at W24) (group 2). Reverse transcriptase and protease genotypic analyses in group 1 revealed the acquisition of only one SQV-associated mutation (L90M) in only two patients. There was no significant increase in the 50 or 90% inhibitory concentration of SQV in patients with or without the L90M mutation. However, the fact that two patients developed an L90M mutation only 4 weeks after relapse points to the need for genotypic resistance testing in the context of an initial failure of the antiretroviral regimen. At W24, the median SQV concentration in group 1 (71 ng/ml) was significantly lower than in group 2 (475 ng/ml), and the plasma SQV concentration was correlated with the viral load at W24 (r = -0.5; P<0.05) and with the drop in viral load between day 0 and W24 (r = -0.5; P<0.01). These results and the fact that the plasma SQV concentrations in the two groups prior to relapse (W12) were not significantly different strongly suggest that the early failure of this combination is not due to viral resistance but to a lack of compliance, pharmacological variability, and drug interactions or a combination of these factors. (+info)
In vivo antagonism with zidovudine plus stavudine combination therapy.
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Human immunodeficiency virus (HIV)-infected subjects receiving zidovudine were randomized either to add stavudine (d4T) or didanosine (ddI) to their current regimen or to switch to ddI or d4T monotherapy. After 16 weeks of therapy, the mean reduction in HIV RNA from baseline was 0.14 log(10) copies/mL in patients receiving d4T or zidovudine plus d4T. In subjects receiving ddI or ddI plus zidovudine, reductions were 0.39 and 0.56 log(10), respectively. CD4 cell counts remained stable or showed modest increases in all arms except the zidovudine plus d4T arm. Patients receiving zidovudine plus d4T showed progressive declines in CD4 cell counts with a median of 22 cells/mm(3) below baseline by 16 weeks. Examination of intracellular levels of d4T-triphosphate in 6 subjects was consistent with previous in vitro studies demonstrating pharmacologic antagonism between zidovudine and d4T. Analysis of these data suggests that zidovudine and d4T should not be prescribed in combination and that ddI provides greater antiviral activity than d4T in zidovudine-treated patients. (+info)
Pharmacokinetics of single-dose oral stavudine in subjects with renal impairment and in subjects requiring hemodialysis.
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Two open-label studies assessed the pharmacokinetics of single orally administered doses of 40 mg of stavudine in subjects with renal impairment. In one study (study I), 15 subjects with selected degrees of renal impairment, but not requiring hemodialysis, were stratified into three groups of five subjects each according to creatinine clearance (CL(CR)) normalized by body surface area (ml/min/1.73 m(2)): mild (CL(CR), 60 to 80), moderate (30 to 50), and severe (/= 90) were also enrolled. The stavudine area under the curve from 0 h to infinity (AUC(0-infinity)) increased nonlinearly with declining renal function: 1,864, 2,215, 3,609, and 5,928 ng. h/ml for normal renal function and for mild, moderate, and severe renal impairment, respectively (P = 0.0001 between renal impairment groups). The following stavudine dosage recommendations for renal impairment were proposed for subjects weighing >/=60 kg: CL(CR) of >50 ml/min/1.73 m(2), 40 mg every 12 h; CL(CR) of 21 to 50 ml/min/1. 73 m(2), 20 mg every 12 h; and CL(CR) of 10 to 20 ml/min/1.73 m(2), 20 mg every 24 h. For subjects weighing <60 kg, the proposed doses were 30, 15, and 15 mg, respectively, with the same dosing intervals specified above. In a second study (study II), 12 subjects with end-stage renal disease requiring hemodialysis three times a week were enrolled in a randomized, open-label crossover study (dialysis 2 h after dosing and lasting 4 h or dosing without dialysis). There were no statistically significant differences for AUC(0-infinity), AUC(2-6), time to maximum concentration of drug in serum, half-life, or apparent oral clearance when the two treatment dosage regimens were compared. As a result of study II, the recommended dosing rate for subjects requiring hemodialysis was the same as that proposed for those with severe renal impairment not requiring hemodialysis; however, dosing was recommended to follow hemodialysis and to occur at the same time each day. (+info)
Structural basis for activation of alpha-boranophosphate nucleotide analogues targeting drug-resistant reverse transcriptase.
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AIDS chemotherapy is limited by inadequate intracellular concentrations of the active triphosphate form of nucleoside analogues, leading to incomplete inhibition of viral replication and the appearance of drug-resistant virus. Drug activation by nucleoside diphosphate kinase and inhibition of HIV-1 reverse transcriptase were studied comparatively. We synthesized analogues with a borano (BH(3)(-)) group on the alpha-phosphate, and found that they are substrates for both enzymes. X-ray structures of complexes with nucleotide diphosphate kinase provided a structural basis for their activation. The complex with d4T triphosphate displayed an intramolecular CH.O bond contributing to catalysis, and the R(p) diastereoisomer of thymidine alpha-boranotriphosphate bound like a normal substrate. Using alpha-(R(p))-boranophosphate derivatives of the clinically relevant compounds AZT and d4T, the presence of the alpha-borano group improved both phosphorylation by nucleotide diphosphate kinase and inhibition of reverse transcription. Moreover, repair of blocked DNA chains by pyrophosphorolysis was reduced significantly in variant reverse transcriptases bearing substitutions found in drug-resistant viruses. Thus, the alpha-borano modification of analogues targeting reverse transcriptase may be of generic value in fighting viral drug resistance. (+info)
Rat multispecific organic anion transporter 1 (rOAT1) transports zidovudine, acyclovir, and other antiviral nucleoside analogs.
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Organic anion transporter 1 (OAT1) is a p-aminohippurate/dicarboxylate exchanger that plays a primary role in the tubular secretion of endogenous and exogenous organic anions. OAT1 is located in the basolateral membrane of the proximal tubular cells and mediates the uptake of various organic anions from the peritubular fluid. In this study, we investigated the transport of antiviral nucleoside analogs via rat OAT1 (rOAT1) using a heterologous expression system in Xenopus laevis oocytes. Oocytes injected with rOAT1 cRNA showed significantly higher uptake of zidovudine (AZT) and acyclovir (ACV) than control oocytes. rOAT1-mediated uptake of AZT and ACV was probenecid-sensitive and increased by the outwardly directed gradient of glutarate. The affinity of rOAT1 for AZT and ACV was determined to be 68 and 242 microM, respectively. Five other antiviral agents that we studied (zalcitabine, didanosine, lamivudine, stavudine, and trifluridine) were also shown to be transported by rOAT1, whereas foscarnet, a phosphate analog, was not. The aforementioned nucleoside analogs lack a typical anionic group and are not very hydrophobic. This study demonstrates extension of the substrate spectrum of rOAT1 and provides a molecular basis for the pharmacokinetics of antiviral nucleoside analogs. (+info)