Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A high-performance liquid chromatographic method for the quantitative determination of the HIV reverse transcriptase inhibitor lamivudine ((-)-2'-deoxy-3'-thiacytidine, 3TC, Epivir) in human plasma, saliva and cerebrospinal fluid is described. Lamivudine was extracted from samples using silica extraction columns prior to reversed-phase high-performance liquid chromatography with ultraviolet detection at 270 nm. The method has been validated over the range of 10 (lower limit of quantitation) to 5000 ng/ml using a 0.5-ml sample volume. Between-day and within-day precisions ranged from 3.5 to 9.0%. The assay has been used for the quantitative analysis of lamivudine in plasma and cerebrospinal fluid of HIV-1 infected patients.
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PMID:Quantitative determination of (-)-2'-deoxy-3'-thiacytidine (lamivudine) in human plasma, saliva and cerebrospinal fluid by high-performance liquid chromatography with ultraviolet detection. 974 54

Lamivudine (3TC), the negative enantiomer of 2'-deoxy-3'-thiacytidine, is a dideoxynucleoside analogue used in combination with other agents in the treatment of human immunodeficiency virus type 1 (HIV-1) infection and as monotherapy in the treatment of hepatitis B virus (HBV) infection. Lamivudine undergoes anabolic phosphorylation by intracellular kinases to form lamivudine 5'-triphosphate, the active anabolite which prevents HIV-1 and HBV replication by competitively inhibiting viral reverse transcriptase and terminating proviral DNA chain extension. The pharmacokinetics of lamivudine are similar in patients with HIV-1 or HBV infection, and healthy volunteers. The drug is rapidly absorbed after oral administration, with maximum serum concentrations usually attained 0.5 to 1.5 hours after the dose. The absolute bioavailability is approximately 82 and 68% in adults and children, respectively. Lamivudine systemic exposure, as measured by the area under the serum drug concentration-time curve (AUC), is not altered when it is administered with food. Lamivudine is widely distributed into total body fluid, the mean apparent volume of distribution (Vd) being approximately 1.3 L/kg following intravenous administration. In pregnant women, lamivudine concentrations in maternal serum, amniotic fluid, umbilical cord and neonatal serum are comparable, indicating that the drug diffuses freely across the placenta. In postpartum women lamivudine is secreted into breast milk. The concentration of lamivudine in cerebrospinal fluid (CSF) is low to modest, being 4 to 8% of serum concentrations in adults and 9 to 17% of serum concentrations in children measured at 2 to 4 hours after the dose. In patients with normal renal function, about 5% of the parent compound is metabolised to the trans-sulphoxide metabolite, which is pharmacologically inactive. In patients with renal impairment, the amount of trans-sulphoxide metabolite recovered in the urine increases, presumably as a function of the decreased lamivudine elimination. As approximately 70% of an oral dose is eliminated renally as unchanged drug, the dose needs to be reduced in patients with renal insufficiency. Hepatic impairment does not affect the pharmacokinetics of lamivudine. Systemic clearance following single intravenous doses averages 20 to 25 L/h (approximately 0.3 L/h/kg). The dominant elimination half-life of lamivudine is approximately 5 to 7 hours, and the in vitro intracellular half-life of its active 5'-triphosphate anabolite is 10.5 to 15.5 hours and 17 to 19 hours in HIV-1 and HBV cell lines, respectively. Drug interaction studies have shown that trimethoprim increases the AUC and decreases the renal clearance of lamivudine, although lamivudine does not affect the disposition of trimethoprim. Other studies have demonstrated no significant interaction between lamivudine and zidovudine or between lamivudine and interferon-alpha-2b. There is limited potential for drug-drug interactions with compounds that are metabolised and/or highly protein bound.
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PMID:Clinical pharmacokinetics of lamivudine. 998 42

Human immunodeficiency virus (HIV) type 1 (HIV-1) variants were selected for resistance to the (+) and (-) enantiomers of a novel nucleoside analogue, 2'-deoxy-3'-oxa-4'-thio-5-fluorocytidine (dOTFC), by use of the infectious molecular clone HIV HXB2D and the human T-cell line MT-4. The dOTFC-resistant variants that were selected were 10-fold less sensitive than wild-type virus, and cloning and sequencing of the complete reverse transcriptase (RT)-coding region identified the mutation M184V. Studies with mutated recombinant HXB2D virus confirmed the importance of the M184V mutation in conferring resistance to (-)dOTFC in MT-4 cells, although no difference in sensitivity was observed in primary cells. The M184V substitution also displayed decreased susceptibility to (+)dOTFC. Selection with (+)dOTFC also produced variants which were 10-fold more resistant than the wild type, and a novel mutation, D67G, was identified following cloning and sequencing of the RT genes. The D67G mutation was introduced into HXB2D by site-directed mutagenesis, and the data obtained confirmed the importance of this mutation in conferring resistance to both (+)dOTFC and (-)dOTFC. Mutated recombinant molecular clone HXB2D-D67G was further selected with (+)dOTFC, and three of six clones sequenced contained both the D67G and M184V mutations, while the other three of the six clones contained only the D67G mutation. Clinical isolates of HIV-1 which are (-) 2'-deoxy-3'-thiacytidine-resistant also displayed resistance to both (+)dOTFC and (-)dOTFC.
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PMID:Selection and characterization of human immunodeficiency virus type 1 variants resistant to the (+) and (-) enantiomers of 2'-deoxy-3'-oxa-4'-thio-5-fluorocytidine. 1077 Jul 40

Several first-generation nucleoside analogues have been tested against chronic hepatitis B virus (HBV) infection, but trials were unsuccessful or accompanied by toxicity. Recently, oral second-generation nucleoside analogues have been developed that have potent activity against HBV. The best-studied compound so far is lamivudine ((-)2'-deoxy-3'-thiacytidine; 3TC). Lamivudine is an inhibitor of reverse transcriptase (RT) activity and is in clinical use in human immunodeficiency virus (HIV)-infected individuals. As several studies on the use of lamivudine for hepatitis B show, the development of resistance in the viral polymerase under lamivudine treatment, however, causes a significant clinical problem. All other drugs in advanced clinical development for HBV are nucleosides; cross-resistance is therefore expected in most cases. The history of HIV treatment demonstrates that new classes of drugs, the protease inhibitors and non-nucleosidic inhibitors of RT, allowed for a longer-term clinical benefit when used in combination with nucleoside analogues. The development of non-nucleosidic compounds with different modes of action therefore appears very important for the treatment of chronic hepatitis B as well.
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PMID:Development of resistance and perspectives for future therapies against hepatitis B infections: lessons to be learned from HIV. 1088 29

The patterns of resistance-conferring mutations that are selected in HIV-1 reverse transcriptase (RT) by the racemates of 2'-dideoxy-3'-oxa-4'-thiocytidine (+/-)dOTC and its fluorinated derivative (+/-)dOTFC were characterized. Genotypic and phenotypic analyses of HIV-1 clinical isolates and HXB2D variants selected with (+/-)dOTC and (+/-)dOTFC were performed in primary cells and in the MT-2 T cell line. HIV-1 variants selected with (+/-)dOTC or (+/-)dOTFC displayed fivefold decreased susceptibility to the respective compounds. A substitution of methionine to valine was identified at position 184 (M184V) in variants selected with (+/-)dOTC. In contrast, a mutation of lysine to arginine at position 65 (K65R) was found in variants selected with (+/-)dOTFC. These patterns of selected mutations differ from those seen with the individual enantiomers. Studies with mutated recombinant HXB2D-M184V and -K65R confirmed that these mutations are important for phenotypic resistance in MT-2 cells. Clinical isolates that display resistance to (-)2'-deoxy-3'-thiacytidine (3TC) also showed cross-resistance to (+/-)dOTC and (+/-)dOTFC. These studies demonstrate that similar genotypes may be selected by the dOTC and dOTFC compounds to those with the structurally related drug 3TC.
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PMID:Selection of resistance-conferring mutations in HIV-1 by the nucleoside reverse transcriptase inhibitors (+/-)dOTC and (+/-)dOTFC. 1122 93

Lamivudine, the negative enantiomer of 2'-deoxy-3'-thiacytidine, is a dideoxynucleoside analog that prevents hepatitis B virus (HBV) DNA synthesis by competitively inhibiting the viral reverse transcriptase and DNA polymerase stages of HBV replication and by terminating proviral DNA chain extension. A dose-ranging study established that once daily oral lamivudine 3 mg/kg up to a maximum of 100 mg/day has the optimum efficacy and tolerability profile for achieving a maximal reduction in serum HBV DNA levels in children aged 2 to 12 years and adolescents aged 13 to 17 years with chronic HBV infection and active viral replication (chronic hepatitis B). Significantly more children and adolescents with chronic hepatitis B receiving lamivudine demonstrated a virologic response (undetectable serum hepatitis Be antigen and undetectable HBV DNA level) [23 vs 13%; p = 0.04] and/or biochemical response (55 vs 12%; p < 0.001) compared with placebo in a large, randomized, double-blind, 52-week phase III study. Despite the emergence of YMDD-variant HBV in 19% of lamivudine-treated children and adolescents, serum alanine aminotransferase and HBV DNA levels remained below baseline in these patients. Oral lamivudine is generally well tolerated by children and adolescents with chronic hepatitis B, with a similar tolerability profile to placebo at the recommended once daily dosage of 3 mg/kg up to a maximum of 100 mg/day.
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PMID:Lamivudine: in children and adolescents with chronic hepatitis B virus infection. 1226 43

The methionine-to-valine mutation in codon 184 (M184V) in reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) or simian immunodeficiency virus (SIV) confers resistance to (-)-2'-deoxy-3'-thiacytidine (3TC; lamivudine) and increased sensitivity to 9-[2-(phosphonomethoxy)propyl]adenine (PMPA; tenofovir). We have used the SIV model to evaluate the effect of the M184V mutation on the emergence of resistance to the combination of 3TC plus PMPA. A site-directed mutant of SIVmac239 containing M184V (SIVmac239-184V) was used to select for resistance to both 3TC and PMPA by serial passage in the presence of increasing concentrations of both drugs. Under these selection conditions, the M184V mutation reverted in the majority of the selections. Variants resistant to both drugs were found to have the lysine-to-arginine mutation at codon 65 (K65R), which has previously been associated with resistance to PMPA in both SIV and HIV. Similarly, in rhesus macaques infected with SIVmac239-184V for 46 weeks and treated daily with (-)-2'-deoxy-5-fluoro-3'-thiacytidine [(-)-FTC], there was no reversion of M184V, but this mutation reverted to 184 M in all three animals within 24 weeks of treatment with (-)-FTC and PMPA. Although the addition of PMPA to the (-)-FTC therapy induced a decrease in virus loads in plasma, these loads eventually returned to pre-PMPA levels in each case. All animals receiving this combination developed the K65R mutation. These results demonstrate that the combination of PMPA with 3TC or (-)-FTC selects for the K65R mutation and against the M184V mutation in SIV RT.
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PMID:Reversion of the M184V mutation in simian immunodeficiency virus reverse transcriptase is selected by tenofovir, even in the presence of lamivudine. 1250 28

Antiretroviral therapy for the human immunodeficiency virus-1 (HIV-1) typically includes two nucleoside reverse transcriptase inhibitors (NRTIs). 3'-Azido-3'-deoxythymidine (AZT, Zidovudine) plus 2'-deoxy-3'-thiacytidine (3TC, Lamivudine) is a combination that is used frequently. The NRTIs are mutagenic nucleoside analogs that become incorporated into DNA and terminate replication. We therefore hypothesized that exposure to this class of drug may alter cell cycle parameters. We used flow cytometry to examine the cell cycle in human epithelioid carcinoma (HeLa) cells exposed to AZT and 3TC alone, as well as a series of AZT/3TC dose combinations: (A) 125.0 microM AZT/12.5 microM 3TC; (B) 250.0 microM AZT/25.0 microM 3TC; and (C) 500 microM AZT/50 microM 3TC. At 24 h, at all doses, there was a good cell viability (>/=68%), and incorporation of AZT into nuclear DNA. Using flow cytometry, a dose-related increase in the percentage of cells in S phase, from 9.5% with no drug, to 36.0% with dose C, was observed in cells exposed for 24 h (P = 0.001, ANOVA). A concomitant decrease in the percentage of cells in G(1) phase, from 82.6% with no drug to 58.5% with dose C, was observed in cells exposed for 24 h (P = 0.017, ANOVA). A similar S phase arrest was seen in cells exposed to 125, 250 and 500 microM AZT alone, but there was no S phase alteration with 50 microM 3TC alone, suggesting that AZT is responsible for the accumulation of cells in S phase. To elucidate the accumulation of cells in S phase and explore the cell cycle gene expression changes induced by AZT and 3TC, we used c-DNA microarray, Cell Cycle Super Array and real-time PCR. There was a strong upregulation of the DNA damage-inducible transcript 3 (DDIT3 or GADD153) in NRTI-exposed cells. In addition, AZT induced an upregulation of cyclin D1 accompanied by a downregulation of the cyclin D1-associated inhibitors P18 and P57, and the G(1)-S check point gene P21, the net effect of which would be to foster a cell progression into S phase. Cyclin A2 was down-regulated in cells exposed to AZT, suggesting a block in S-G(2)-M progression that would also be consistent with the accumulation of cells in S phase. Overall, the study demonstrates that AZT, but not 3TC, causes an arrest of cells in S phase with a consistent alteration in the expression of several cell cycle genes.
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PMID:Zidovudine induces S-phase arrest and cell cycle gene expression changes in human cells. 1578 90

Glutathione S-transferase (GST) theta 1 (GSTT1) has been regarded as one of the key enzymes involved in phase II reactions because of its unique substrate specificity. In this study, we generated mice with the disrupted Gstt1 gene (Gstt1-null mice) by gene targeting and analyzed the metabolic properties in cytosolic and in vivo studies. The resulting Gstt1-null mice failed to express the Gstt1 mRNA and GSTT1 protein by reverse transcriptase-polymerase chain reaction analysis and two-dimensional fluorescence difference gel electrophoresis/mass spectrometry analysis, respectively. However, the Gstt1-null mice appeared to be normal and were fertile. In an enzymatic study using cytosolic samples from the liver and kidney, GST activity toward 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP), dichloromethane (DCM), and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) was markedly lower in Gstt1-null mice than in the wild-type controls, despite there being no difference in GST activity toward 1-choloro-2,4-dinitrobenzene between Gstt1-null mice and the wild-type controls. Gstt1-null mice had GST activity of only 8.7 to 42.1% of the wild-type controls to EPNP, less than 2.2% of the wild-type controls to DCM, and 13.2 to 23.9% of the wild-type controls to BCNU. Plasma BCNU concentrations after a single i.p. administration of BCNU to Gstt1-null mice were significantly higher, and there was a larger area under the curve(5-60) min (male, 2.30 times; female, 2.28 times, versus the wild-type controls) based on the results. In conclusion, Gstt1-null mice would be useful as an animal model of humans with the GSTT1-null genotype.
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PMID:Generation and functional characterization of mice with a disrupted glutathione S-transferase, theta 1 gene. 1782 37


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