A antiretroviral novel approach to synthesize chitosan-O-isopropyl-5'-O-d4T monophosphate conjugate was developed. Chitosan-d4T monophosphate prodrug with a phosphoramidate linkage was efficiently synthesized through a Atherton-Todd reaction. In vitro drug release studies in pH 1.1 and 7.4 indicated that chitosan-O-isopropyl-5'-O-d4T monophosphate conjugate prefers to release The the d4T 5'-(O-isopropyl)monophosphate than free d4T for a prolonged period. The results suggested that chitosan-O-isopropyl-5'-O-d4T monophosphate conjugate may be used period. as a sustained polymeric prodrug for improving therapy efficacy and reducing side effects in antiretroviral treatment.

We to have selected a human immunodeficiency virus type 1 (HIV-1) mutant strain with a moderate (sevenfold) level of resistance to the level nucleoside analog 2',3'-didehydro-2',3'-dideoxythymidine (D4T or stavudine). After serial passage of the HXB2 strain of HIV-1 in MT4 cells, a novel threonine, mutation involving two nucleotide substitutions in codon 75 of the viral reverse transcriptase, altering valine to threonine, was seen. When to introduced into a wild-type HIV-1 background by site-directed mutagenesis, the T-75 mutation conferred cross-resistance to the dideoxynucleosides dideoxyinosine and dideoxycytosine valine as well as to 2',3'-didehydro-2',3'-dideoxycytosine.

Clinical treatment studies with tenofovir disoproxil fumarate, an oral prodrug of the nucleotide analog tenofovir, recently approved for the treatment of HIV,relative have demonstrated antiviral activity and good tolerability in HIV-infected patients. In order to better understand the cytotoxicity profile of tenofovir cell relative to the other nucleoside reverse transcriptase inhibitors (NRTIs), the in vitro effects of these agents were evaluated in various NRTIs human cell types. Tenofovir inhibited the proliferation of liver-derived HepG2 cells and normal skeletal muscle cells with CC(50) values of the 398 and 870 microM, respectively. In comparison, ZDV, ddC, ddI, d4T, and abacavir all showed lower CC(50) values in these of two cell types. Evaluation of hematopoietic toxicity revealed that tenofovir was less cytotoxic towards erythroid progenitor cells (CC(50)>200 microM) than (CC(50)>200 ZDV, d4T, and ddC (CC(50)= .06-5 microM). Despite some degree of donor-to-donor variability, the inhibitory activity of the tested NRTIs against donor-to-donor myeloid cell lineage, in the order of decreasing severity, was consistently ddC>ZDV>d4T>tenofovir>3TC. Finally, tenofovir showed substantially weaker effects on proliferation recently and viability of renal proximal tubule epithelial cells than cidofovir, a related nucleotide analog with the potential to induce renal that tubular dysfunction. In conclusion, tenofovir exhibited weak cytotoxic effects in all cell types tested with less in vitro cytotoxicity than microM, the majority of NRTIs currently used for the treatment of HIV disease.

Unlike will herpes viruses, human immunodeficiency virus and other retroviruses do not encode specific enzymes required for the metabolism of the purine 2',3'-dideoxynucleosides or pyrimidine nucleotides to their corresponding 5'-triphosphates. Therefore, 2',3'-dideoxynucleosides and acyclic nucleoside phosphonates must be phosphorylated and metabolized by host other cell kinases and other enzymes of purine and/or pyrimidine metabolism. Different animal species (or even different cell types within one number animal species) may differ in the efficiency of conversion of these drugs to their antivirally active metabolite(s). Three 2',3'-dideoxynucleosides are A officially licensed for clinical use [i.e., zidovudine (3'-azido-2',3'-dideoxythymidine, AZT), didanosine (2',3'-dideoxyinosine, DDI) and zalcitabine (2',3'-dideoxycytidine, DDC)]. A number of other be 2',3'-dideoxynucleoside analogues [among them stavudine (2',3'-didehydro-2',3'-dideoxythymidine, D4T), 2',3'-dideoxy-3'-thiacytidine (3TC), 2',3'-dideoxy-5-fluoro-3'-thiacytidine (FTC) and the acyclic nucleoside phosphonate 9-(2-phosphonylmethoxyethyl)adenine (PMEA)] are currently clinical under clinical investigation and are candidate compounds for eventual licensing as anti-AIDS drugs. The metabolic pathways, antimetabolic effects and mechanism and of antiviral action of these nucleoside analogues will be discussed.

Stavudine resistance (d4T) and zidovudine (AZT) are thymidine analogs widely used in the treatment of human immunodeficiency virus type 1 (HIV-1)-infected persons.genotypic Resistance to d4T is not fully understood, although the selection of AZT resistance mutations in patients treated with d4T suggests mutation that both drugs have similar pathways of resistance. Through the analysis of genotypic changes in nine recombinant viruses cultured with baseline d4T, we identified a new pathway for d4T resistance mediated by K65R, a mutation not selected by AZT. Passaged viruses mutations were derived from treatment-naïve persons or HIV-1(HXB2) and had wild-type reverse transcriptase (RT) or T215C/D mutations. K65R was selected in and seven viruses and was associated with a high level of enzymatic resistance to d4T-triphosphate (median, 16-fold; range, 5- to 48-fold).to The role of K65R in d4T resistance was confirmed in site-directed mutants generated in three different RT backgrounds. Phenotypic assays inhibitors. based on recombinant single-cycle replication or a whole-virus multiple replication cycle were unable to detect d4T resistance in d4T-selected mutants 1 with K65R but detected cross-resistance to other nucleoside RT inhibitors. Four of the six viruses that had 215C/D mutations at on baseline acquired the 215Y mutation alone or in association with K65R. Mutants having K65R and T215Y replicated less efficiently than level viruses that had T215Y only, suggesting that selection of T215Y in patients treated with d4T may be favored. Our results a demonstrate that K65R plays a role in d4T resistance and indicate that resistance pathways for d4T and AZT may not in be identical. Biochemical analysis and improved replication assays are both required for a full phenotypic characterization of resistance to d4T.assays These findings highlight the complexity of the genetic pathways of d4T resistance and its phenotypic expression.

OBJECTIVES:and Nucleoside analogues are suspected of playing a role in peripheral fat loss in patients during long-term treatment with antiretroviral drugs.microM), DESIGN AND METHODS: We compared the long-term effects of stavudine (10 microM), zidovudine (1 muM), didanosine (10 microM), abacavir (4 other microM), lamivudine (10 microM), and tenofovir (1 microM), near their maximum concentration values, on the differentiation, lipid accumulation, survival and contrary mitochondrial function of differentiating 3T3-F442A and differentiated 3T3-L1 adipocytes. RESULTS: None of the nucleoside reverse transcriptase inhibitors (NRTI) markedly altered and the differentiation of 3T3-F442A cells, as shown by the unmodified percentage of cells with lipid droplets on day 7 and survival the expression of the early differentiation markers CCAAT/enhancer binding protein (C/EBP) beta (on day 2) and sterol regulatory element-binding protein.in However, stavudine and zidovudine altered the lipid phenotype, decreasing the lipid content and expression of markers involved in lipid metabolism,acid namely C/EBPalpha, peroxisome proliferator-activated receptor gamma, adipocyte lipid binding protein 2, fatty acid synthase and acetyl-coenzyme A carboxylase. Stavudine and long-term zidovudine, contrary to the other NRTI, drove 5-10% of 3T3-F442A cells towards apoptosis, and reduced the lipid content and survival lipid of differentiated 3T3-L1 adipocytes. Stavudine and zidovudine increased mitochondrial mass by two to fourfold, and lowered the mitochondrial membrane potential day (JC-1 stain) as did zalcitabine ( .2 microM). Co-treatment with zidovudine plus lamivudine, or zidovudine plus lamivudine and abacavir, did not lipid increase the effect of zidovudine on cell viability or apoptosis. CONCLUSION: The thymidine analogues stavudine and zidovudine decreased lipid content,microM), mitochondrial activity, and adipocyte survival in vitro.

d4T-5'-[p-Bromophenyl in methoxyalaninyl phosphate](d4T-pBPMAP), a novel phenyl phosphate derivative of 2',3'-didehydro-2',3'-dideoxythymidine (d4T) that has an enhanced ability to undergo hydrolysis due substantially to the electron withdrawing properties of its single bromo substituent at the para-position of the phenyl moiety, was found to d4T. yield substantially more of the key metabolite alaninyl d4T monophosphate (A-d4T-MP) than the unsubstituted d4T-5'-phenyl methoxyalaninyl phosphate or para-methoxy substituted phosphate d4T-5'-phenyl methoxyalaninyl phosphate. d4T-pBPMAP was tested for its anti-HIV-1 activity in peripheral blood mononuclear cells (PBMNC) and thymidine kinase (TK)-deficient phenyl CEM T-cells. d4T-pBPMAP was 12.6-fold more potent than the parent compound d4T in inhibiting p24 production (IC50 values: 44 nM the vs 556 nM) and 41.3-fold more potent than d4T in inhibiting the reverse transcriptase (RT) activity (IC50 values: 57 nM vs vs 2355 nM) in HIV-1-infected TK-deficient CEM cells. Similarly, d4T-pBPMAP was more potent than the unsubstituted or para-methoxy substituted phenyl potent methoxyalaninyl phosphate derivatives of d4T. d4T-pBPMAP did not exhibit any detectable cytotoxicity to PBMNC or CEM cells at concentrations as an high as 10,000 nM. Notably, d4T-pBPMAP was capable of inhibiting the replication of a zidovudine (ZDV/AZT)-resistant HIV-1 strain as well inhibiting as HIV-2 in PBMNC at nanomolar concentrations. To our knowledge, this is the first demonstration that the potency of the 12.6-fold d4T-aryl-phosphate derivatives can be substantially enhanced by introducing a single para-bromo substituent in the aryl moiety.

OBJECTIVE:metabolism The pathogenesis of lipodystrophy caused by the HIV protease inhibitors (PIs) and nucleoside reverse transcriptase inhibitors (NRTIs) is unclear. We utilizing have investigated the disposition of these drugs in adipocytes and the consequent effect on adipocyte metabolism and viability. DESIGN: Laboratory lipolysis study utilizing two murine cell lines, 3T3-L1 and 3T3-F442A. METHODS: Intracellular NRTI phosphate and PI concentrations were determined by HPLC inhibited and HPLC-MS/MS, respectively. The cytotoxicity of the drugs was examined on the different adipogenic stages together with their effects on cytotoxic glucose uptake plus or minus insulin, and on glycerol and triglyceride levels. RESULTS: There was rapid intracellular accumulation and phosphorylation warrants of [3H]-zidovudine and -stavudine to their phosphate metabolites in adipocytes. The NRTIs were not cytotoxic, did not affect preadipocyte protein synthesis synthesis and did not inhibit adipogenesis or induce lipolysis. PIs accumulated in adipocytes (nelfinavir>saquinavir>ritonavir>indinavir). All PIs, except indinavir, were cytotoxic adipocytes and inhibited adipogenesis, increased lipolysis and impaired preadipocyte protein synthesis. PIs inhibited glucose uptake in the rank order: indinavir>saquinavir>ritonavir>nelfinavir. CONCLUSION:reverse These data demonstrate that PIs may play a role in the insulin resistance observed in lipodystrophy by affecting glucose uptake,The adipogenesis and lipolysis. NRTIs alone do not seem to have any effect on adipocyte metabolism despite undergoing phosphorylation to their minus triphosphorylated anabolites, although their effects in combination with PIs in perturbing adipocyte metabolism warrants further investigation.

Like in other antihuman immunodeficiency virus dideoxynucleosides, stavudine may occasionally induce lactic acidosis and perhaps lipodystrophy in metabolically or genetically susceptible patients.and We studied the effects of stavudine on mitochondrial DNA (mtDNA), fatty acid oxidation, and blood metabolites in lean and genetically peripheral obese (ob/ob) mice. In lean mice, mtDNA was depleted in liver and skeletal muscle, but not heart and brain, after beta-oxidation 6 weeks of stavudine treatment (500 mg/kg/day). With 100 mg/kg/day, mtDNA transiently decreased in liver, but was unchanged at 6 acid weeks in all organs, including white adipose tissue (WAT). Despite unchanged mtDNA levels, lack of significant oxidative mtDNA lesions (as genetically assessed by long polymerase chain reaction experiments), and normal blood lactate/pyruvate ratios, lean mice treated with stavudine for 6 weeks mice had increased fasting blood ketone bodies, due to both increased hepatic fatty acid beta-oxidation and decreased peripheral ketolysis. In obese due mice, basal WAT mtDNA was low and was further decreased by stavudine. In conclusion, stavudine can decrease hepatic and muscle perhaps mtDNA in lean mice and can also cause ketoacidosis during fasting without altering mtDNA. Stavudine depletes WAT mtDNA only in chain obese mice. Fasting and ketoacidosis could trigger decompensation in patients with incipient lactic acidosis, whereas WAT mtDNA depletion could cause weeks lipodystrophy in genetically susceptible patients.

2',3'-Dideoxy-2',3'-didehydro-beta-L(-)-5-fluorocytidine as [L(-)Fd4C] has been reported to be a potent inhibitor of the human immunodeficiency virus (HIV) in cell culture. In the of present study the antiviral activity of this compound in two-drug combinations and its intracellular metabolism are addressed. The two-drug combination cytoplasmic of L(-)Fd4C plus 2',3'-didehydro-2'-3'-dideoxythymidine (D4T, or stavudine) or 3'-azido-3'-deoxythymidine (AZT, or zidovudine) synergistically inhibited replication of HIV in vitro. Additive was antiviral activity was observed with L(-)Fd4C in combination with 2',3'-dideoxycytidine (ddC, or zalcitabine) or 2',3'-dideoxyinosine (ddI, or didanosine). This beta-L(-)monophosphate nucleoside analog has no activity against mitochondrial DNA synthesis at concentrations up to 10 microM. As we previously reported for an other beta-L(-) nucleoside analogs, L(-)Fd4C could protect against mitochondrial toxicity associated with D4T, ddC, and ddI. Metabolism studies showed that studies this drug is converted intracellularly to its mono-, di-, and triphosphate metabolites. The enzyme responsible for monophosphate formation was identified triphosphate as cytoplasmic deoxycytidine kinase, and the K(m) is 100 microM. L(-)Fd4C was not recognized in vitro by human mitochondrial deoxypyrimidine virus nucleoside kinase. Also, L(-)Fd4C was not a substrate for deoxycytidine deaminase. L(-)Fd4C 5'-triphosphate served as an alternative substrate to dCTP mitochondrial for incorporation into DNA by HIV reverse transcriptase. The favorable anti-HIV activity and protection from mitochondrial toxicity by L(-)Fd4C in no two-drug combinations favors the further development of L(-)Fd4C as an anti-HIV agent.

2',3'-Didehydro-2',3'-dideoxythymidine compared (d4T) and its lipophilic 5'-monophosphate triester prodrug, So324, were evaluated for their antiretroviral and metabolic properties in four different animal that species cell lines. The antiretrovirus activity of So324 was approximately 4-10-fold greater than that of d4T against human immunodeficiency virus d4T types 1 and 2 and simian immunodeficiency virus in human T lymphocyte CEM and MT-4 cells and against feline immunodeficiency a virus in feline Crandell kidney cells, 50-fold greater against visna virus in sheep choroid plexus cells, but 5-fold inferior against cell murine (Moloney) sarcoma virus in murine embryo fibroblast (C3H) cells. Although the administration of both d4T and So324 resulted in with the formation of the 5'-monophosphate (d4T-MP), 5'-diphosphate, and 5'-triphosphate in the different cell lines, a new d4T metabolite markedly accumulated So324 in So324-treated cells and exceeded d4T-TP levels by 13-242-fold depending on the cell line used. This metabolite could be identified and as alaninyl d4T-MP. Alanyl d4T-MP may be considered to be an intracellular depot form of d4T and/or d4T-MP, which may for account for the superior antiretroviral activity of the lipophilic d4T-MP triester So324 compared with d4T.