Anti-human immunodeficiency virus activity, cross-resistance, cytotoxicity, and intracellular pharmacology of the 3'-azido-2',3'-dideoxypurine nucleosides.

Although the approved nucleoside reverse transcriptase (RT) inhibitors (NRTI) are integral components of therapy for human immunodeficiency virus type 1 (HIV-1) infection, they can have significant limitations, including the selection of NRTI-resistant HIV-1 and cellular toxicity. Accordingly, there is a critical need to develop new NRTI that have excellent activity and safety profiles and exhibit little or no cross-resistance with existing drugs. In this study, we report that the 3'-azido-2',3'-dideoxypurine nucleosides (ADPNs) 3'-azido-2',3'-dideoxyadenosine (3'-azido-ddA) and 3'-azido-2',3'-dideoxyguanosine (3'-azido-ddG) exert potent antiviral activity in primary human lymphocytes and HeLa and T-cell lines (50% inhibitory concentrations [IC50s] range from 0.19 to 2.1 microM for 3'-azido-ddG and from 0.36 to 10 microM for 3'-azido-ddA) and that their triphosphate forms are incorporated as efficiently as the natural dGTP or dATP substrates by HIV-1 RT. Importantly, both 3'-azido-ddA and 3'-azido-ddG retain activity against viruses containing K65R, L74V, or M184V (IC50 change of <2.0-fold) and against those containing three or more thymidine analog mutations (IC50 change of <3.5-fold). In addition, 3'-azido-ddG does not exhibit cytotoxicity in primary lymphocytes or epithelial or T-cell lines and does not decrease the mitochondrial DNA content of HepG2 cells. Furthermore, 3'-azido-ddG is efficiently phosphorylated to 3'-azido-ddGTP in human lymphocytes, with an intracellular half-life of the nucleoside triphosphate of 9 h. The present data suggest that additional preclinical studies are warranted to assess the potential of ADPNs for treatment of HIV-1 infection.

Antiviral and cellular metabolism interactions between Dexelvucitabine and lamivudine.

Studies on cellular drug interactions with antiretroviral agents prior to clinical trials are critical to detect possible drug interactions. Herein, we demonstrated that two 2'-deoxycytidine antiretroviral agents, dexelvucitabine (known as beta-d-2',3'-didehydro-2',3'-dideoxy-5-fluorocytidine, DFC, d-d4FC, or RVT) and lamivudine (3TC), combined in primary human peripheral blood mononuclear (PBM) cells infected with human immunodeficiency virus 1 strain LAI (HIV-1(LAI)), resulted in additive-to-synergistic effects. The cellular metabolism of DFC and 3TC was studied in human T-cell lymphoma (CEM) and in primary human PBM cells to determine whether this combination caused any reduction in active nucleoside triphosphate (NTP) levels, which could decrease with their antiviral potency. Competition studies were conducted by coincubation of either radiolabeled DFC with different concentrations of 3TC or radiolabeled 3TC with different concentrations of DFC. Coincubation of radiolabeled 3TC with DFC at concentrations up to 33.3 microM did not cause any marked reduction in 3TC-triphosphate (TP) or any 3TC metabolites. However, a reduction in the level of DFC metabolites was noted at high concentrations of 3TC with radiolabeled DFC. DFC-TP levels in CEM and primary human PBM cells decreased by 88% and 94%, respectively, when high concentrations of 3TC (33.3 and 100 microM) were added, which may influence the effectiveness of DFC-5'-TP on the HIV-1 polymerase. The NTP levels remained well above the median (50%) inhibitory concentration for HIV-1 reverse transcriptase. These results suggest that both beta-d- and beta-l-2'-deoxycytidine analogs, DFC and 3TC, respectively, substrates of 2'-deoxycytidine kinase, could be used in a combined therapeutic modality. However, it may be necessary to decrease the dose of 3TC for this combination to prove effective.

Inhibition of hepatitis C replicon RNA synthesis by beta-D-2'-deoxy-2'-fluoro-2'-C-methylcytidine: a specific inhibitor of hepatitis C virus replication.

beta-D-2'-Deoxy-2'-fluoro-2'-C-methylcytidine (PSI-6130) is a cytidine analogue with potent and selective anti-hepatitis C virus (HCV) activity in the subgenomic HCV replicon assay, 90% effective concentration (EC90)=4.6 +/- 2.0 microM. The spectrum of activity and cytotoxicity profile of PSI-6130 was evaluated against a diverse panel of viruses and cell types, and against two additional HCV-1b replicons. The S282T mutation, which confers resistance to 2'-C-methyl adenosine and other 2'-methylated nucleosides, showed only a 6.5-fold increase in EC90. When assayed for activity against bovine diarrhoea virus (BVDV), which is typically used as a surrogate assay to identify compounds active against HCV, PSI-6130 showed no anti-BVDV activity. Weak antiviral activity was noted against other flaviviruses, including West Nile virus, Dengue type 2, and yellow fever virus. These results indicate that PSI-6130 is a specific inhibitor of HCV. PSI-6130 showed little or no cytotoxicity against various cell types, including human peripheral blood mononuclear and human bone marrow progenitor cells. No mitochondrial toxicity was observed with PSI-6130. The reduced activity against the RdRp S282T mutant suggests that PSI-6130 is an inhibitor of replicon RNA synthesis. Finally, the no-effect dose for mice treated intraperitoneally with PSI-6130 for six consecutive days was > or =100 mg/kg per day.

Tissue-specific differences of p53 inhibition by Mdm2 and Mdm4.

The function of the p53 tumor suppressor to inhibit proliferation or initiate apoptosis is often abrogated in tumor cells. Mdm2 and its homolog, Mdm4, are critical inhibitors of p53 that are often overexpressed in human tumors. In mice, loss of Mdm2 or Mdm4 leads to embryonic lethal phenotypes that are completely rescued by concomitant loss of p53. To examine the role of Mdm2 and Mdm4 in a temporal and tissue-specific manner and to determine the relationships of these inhibitors to each other, we generated conditional alleles. We deleted Mdm2 and Mdm4 in cardiomyocytes, since proliferation and apoptosis are important processes in heart development. Mice lacking Mdm2 in the heart were embryonic lethal and showed defects at the time recombination occurred. A critical number of cardiomyocytes were lost by embryonic day 13.5, resulting in heart failure. This phenotype was completely rescued by deletion of p53. Mice lacking Mdm4 in the heart were born at the correct ratio and appeared to be normal. Our studies provide the first direct evidence that Mdm2 can function in the absence of Mdm4 to regulate p53 activity in a tissue-specific manner. Moreover, Mdm4 cannot compensate for the loss of Mdm2 in heart development.

Pharmacology and pharmacokinetics of the antiviral agent beta-D-2',3'-dideoxy-3'-oxa-5-fluorocytidine in cells and rhesus monkeys.

Beta-D-2',3'-dideoxy-3'-oxa-5-fluorocytidine (D-FDOC) is an effective inhibitor of human immunodeficiency virus 1 (HIV-1) and HIV-2, simian immunodeficiency virus, and hepatitis B virus (HBV) in vitro. The purpose of this study was to evaluate the intracellular metabolism of d-FDOC in human hepatoma (HepG2), human T-cell lymphoma (CEM), and primary human peripheral blood mononuclear (PBM) cells by using tritiated compound. By 24 h, the levels of D-FDOC-triphosphate (D-FDOC-TP) were 2.8 +/- 0.4, 6.7 +/- 2.3, and 2.0 +/- 0.1 pmol/10(6) cells in HepG2, CEM, and primary human PBM cells, respectively. Intracellular D-FDOC-TP concentrations remained greater than the 50% inhibitory concentration for HIV-1 reverse transcriptase for up to 24 h after removal of the drug from cell cultures. In addition to d-FDOC-monophosphate (D-FDOC-MP), -diphosphate (D-FDOC-DP), and -TP, D-FDOC-DP-ethanolamine and d-FDOC-DP-choline were detected in all cell extracts as major intracellular metabolites. D-FDOC was not a substrate for Escherichia coli thymidine phosphorylase. No toxicity was observed in mice given D-FDOC intraperitoneally for 6 days up to a dose of 100 mg/kg per day. Pharmacokinetic studies in rhesus monkeys indicated that D-FDOC has a t(1/2) of 2.1 h in plasma and an oral bioavailability of 38%. The nucleoside was excreted unchanged primary in the urine, and no metabolites were detected in plasma or urine. These results suggest that further safety and pharmacological studies are warranted to assess the potential of this nucleoside for the treatment of HIV- and HBV-infected individuals.

Emergence of a novel mutation in the FLLA region of hepatitis B virus during lamivudine therapy.

The emergence of resistance to lamivudine has been one of the major stumbling blocks to successful treatment and control of hepatitis B virus (HBV) infections. The major mechanism of resistance has been attributed to the alteration in the YMDD motif of the HBV polymerase due to an amino acid change of rtM204 to V/I and an accompanying rtL180M conversion. A novel mutation pattern in a patient having clinical breakthrough under lamivudine therapy was discovered. The mutant had a rtL180C/M204I genotype and was detected after 2 years of therapy with lamivudine. To characterize this novel variant, site-directed mutagenesis was performed using a vector construct containing the HBV genome. Transient transfection studies in human hepatoma cells with HBV carrying the new mutant demonstrated that the rtL180C/M204I mutant was resistant to lamivudine up to 10 microM. The resistance profile was comparable to that of the previously reported rtL180 M/M204I-containing virus. These observations were further confirmed by generation of stable cultures transfected with the mutant virus.

N4-acyl-modified D-2',3'-dideoxy-5-fluorocytidine nucleoside analogues with improved antiviral activity.

A series of 2',3'-dideoxy (D2) and 2',3'-didehydro-2',3'-dideoxy (D4) 5-fluorocytosine nucleosides modified with substituted benzoyl, heteroaromatic carbonyl, cycloalkylcarbonyl and alkanoyl at the N4-position were synthesized and evaluated for anti-human immunodeficiency virus type 1 (HIV-1) and anti-hepatitis B virus (HBV) activity in vitro. For most D2-nucleosides, N4-substitutions improved the anti-HIV-1 activity markedly without increasing the cytotoxicity. In the D4-nucleosides series, some of the substituents at the N4-position enhanced the anti-HIV-1 activity with a modest increase in the cytotoxicity. The most potent and selective N4-modified nucleoside for the D2-series was N4-p-iodobenzoyl-D2FC, which had a 46-fold increase in anti-HIV-1 potency in MT-2 cells compared to the parent nucleoside D-D2FC. In the D4-series, N4-p-bromobenzoyl-D4FC was 12-fold more potent in MT-2 cells compared to the parent nucleoside D-D4FC. All eight N4-p-halobenzoyl-substituted D2- and D4-nucleosides evaluated against HBV in HepAD38 cells demonstrated equal or greater potency than the two parental compounds, D-D2FC and D-D4FC. The N4-modification especially in the D2-nucleoside series containing the N4-nicotinoyl, o-nitrobenzoyl and n-butyryl showed a significant reduction in mitochondrial toxicity relative to the parent nucleoside analogue. Although the 5'-triphosphate of the parent compound (D-D4FC-TP) was formed from the N4-acyl-D4FC analogues in different cells, the levels of the 5'-triphosphate nucleotide did not correlate with the cell-derived 90% effective antiviral concentrations (EC90), suggesting that a direct interaction of the triphosphates of these N4-acyl nucleosides was involved in the antiviral activity.