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 method for the determination of a bisheteroarylpiperazine, non-nucleoside HIV-1 reverse transcriptase inhibitor, delavirdine, and its N-desisopropyl metabolite in human plasma, is described. Samples were deproteinized by addition of two parts of a solution of internal standard in acetonitrile (1 microgram/ml) to one part plasma. The supernatant was diluted with 10 mM phosphate buffer, pH 6.0, and injected onto the HPLC system. Fluorescence of the eluent was monitored with excitation at 302 nm and emission at 425 nm. Quantitation of delavirdine and its metabolite was achieved by comparing the peak-height ratio of each component relative to the internal standard to a through-the-origin linear regression curve determined from fortified plasma calibration standards. The assay was linear over the concentration range 0.02-17 microM for both delavirdine and its metabolite. The precision of the method, as expressed by the mean C.V. of the back-calculated, non-zero, standard concentrations, was +/- 4.4% for delavirdine and +/- 4.3% for the metabolite. The assay has been validated and utilized to analyze samples from human and animal pharmacokinetic studies.
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PMID:Simple, rapid and sensitive high-performance liquid chromatographic determination of delavirdine and its N-desisopropyl metabolite in human plasma. 755 Sep 87

Intrinsic protein fluorescence has been used to study dimerization of the HIV-1 reverse transcriptase (RT). We observed a 25% increase of the tryptophan fluorescence of the enzyme during dissociation of the subunits induced by the addition of acetonitrile. Upon reassociation of the separated subunits, the original fluorescence emission of the heterodimer is restored. A two-state transition model for the RT dimerization process in which the dimers are in equilibrium with folded monomers is proposed. The free energy of dissociation was determined to be 12.2 (+/- 0.2) kcal/mol. In the absence of Mg2+ ions a decrease of this value was observed, whereas the addition of a synthetic primer/template (18/36mer) results in an increase of dimer stability. Analyzing the effect of Mg2+ on the establishment of the binding equilibrium, a dramatic effect with a 100-fold acceleration of the association by the divalent ion was observed.
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PMID:Characterization of the dimerization process of HIV-1 reverse transcriptase heterodimer using intrinsic protein fluorescence. 768 95

The dissociation of dimeric reverse transcriptase (RT) of the human immunodeficiency virus (HIV) types 1 and 2 has been investigated using acetonitrile as a dissociating agent. The equilibrium transitions were monitored by combining different approaches (fluorescence spectroscopy, polymerase activity assay, and size-exclusion HPLC). The dissociation of RT induced a complete loss of polymerase activity and a 25% increase of the intrinsic fluorescence. It is fully reversible, and the midpoints of the equilibrium transition curves are dependent on the concentration of the enzyme used, suggesting a two-state transition model for the dissociation of RT in which dimers are in equilibrium with folded monomers. For both RTs, the heterodimeric form is more stable against dissociating agents and different pH than the corresponding homodimeric form. Moreover, heterodimeric HIV-2 RT exhibits a higher stability than HIV-1 RT, with a free energy of dissociation of 12.1 kcal/mol at pH 6.5 and 25 degrees C, instead of 10 kcal/mol for HIV-1 RT. The binding of a primer/template induces a marked conformational change in both RTs, shown by the lower accessibility of the tryptophans to quenchers and the increase in tryptophan heterogeneity, and stabilized the dimeric form of both RTs (10-100-fold). The central role of hydrophobic interactions in dimer formation has been revealed by the 30% increase of exposure of the tryptophan cluster to quenchers upon dissociation of RT and the binding of 4 equiv of 1-anilino-8-naphthalenesulfonate to the dissociated enzymes.
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PMID:Conformational stability of dimeric HIV-1 and HIV-2 reverse transcriptases. 884 59

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is a dimeric enzyme consisting of p66 and p51 subunits. The functional role of the p51 subunit remains elusive since all the catalytic functions appear to be executed through the p66 subunit. We report here that the p51 subunit, in addition to providing structural support to the p66 subunit, may be involved in facilitating the loading of the p66 subunit on to the template-primer (TP). This possibility is supported by following observations: (i) Upon binding to the TP, the p51 subunit can be dissociated by acetonitrile treatment and the template-primer-bound p66 monomer alone is capable of catalyzing DNA synthesis. (ii) Photo-cross-linking of template-primer to HIV-1 RT is abolished by dissociation of the p51 subunit prior to the TP binding but remains unaffected after the TP binding step. (iii) The p66-TP covalent complex selectively generated by UV irradiation and separated by gel electrophoresis can incorporate a single nucleotide in situ upon its renaturation in the gel. (iv) Treatment of HIV-1 RT with (tert-butyldimethylsilyl)spiroaminooxathioledioside (TSAO), an inhibitor that specifically binds to the beta7 beta8 loop of p51, destabilizes the heterodimeric enzyme, resulting in the subsequent loss of DNA binding.
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PMID:The p51 subunit of human immunodeficiency virus type 1 reverse transcriptase is essential in loading the p66 subunit on the template primer. 955 23

Two HPLC methods were developed: one for the quantitation of HBY 097 reverse transcriptase inhibitor and its metabolites M2 and M3 in human serum, and one for the quantitation of metabolite M5 in urine. The HPLC procedure for the quantitation of HBY 097 and its metabolites M2 and M3 in human serum involved protein precipitation with acetonitrile followed by automated on-line trace enrichment. The HPLC procedure for the analysis of metabolite M5 in urine involved enzymatic hydrolysis of urine with beta-glucuronidase to convert metabolite M5 (glucuronide of M3) to M3. Reverse phase chromatographic separation with gradient elution. UV detection at 335 nm, and internal standard were used to quantitate analytes in both procedures. The lower quantitation limits were 25 ng ml-1 for HBY 097 and metabolites M2 and M3 in serum, and 0.5 microgram ml-1 for the metabolite M5 in urine measured as metabolite M3 after hydrolysis. The HBY 097 and metabolite M3 concentrations were specific but metabolite M2 was semi-specific because the two diastereomers of M2 were not resolved by the present chromatographic procedure. Both procedures were applied to the quantitation of HBY 097 and its metabolites in serum and urine of HIV positive patients who were enrolled in a clinical study of drug safety and pharmacokinetics.
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PMID:Quantitative analysis of HBY 097 and its metabolites in human serum and urine by HPLC. 957 37

NSC 615985 (UC 84) has demonstrated anti-HIV activity in the NCI-AIDS antiviral screen and was under consideration as an anti-AIDS drug. The compound was subsequently shown to be a non-nucleoside reverse transcriptase inhibitor (NNRTI). An HPLC method was developed for the analysis of NSC 615985 in mouse, dog and human plasma; and was used to study its stability in plasma and blood as well as its absorption and metabolism in mice. The method involved precipitation of plasma protein with three volumes of methanol followed by HPLC analysis of the supernatant. The HPLC analysis was carried out on a reversed-phase Nova-Pak C18 column with a mobile phase of KH2PO4 (0.01 M; pH 4.8)-acetonitrile (52:48, v/v) at a flow rate of 1 ml min-1 and quantification with a UV detector set at 259 nm. The lower limit of quantitation was 0.05 microgram ml-1 in 1 ml of dog or human plasma or 0.1 microgram ml-1 in 0.5 ml of mouse plasma. NSC 615985 was more stable in dog and human plasma than in mouse plasma, and was less stable in blood than in plasma of the three species investigated. Following bolus intravenous (i.v.) administration at 10 mg kg-1 to male CDF1 mice, NSC 615985 elimination followed biexponential kinetics with half-lives of 1 and 7 min, and was extensively metabolized. NSC 615985 was very poorly absorbed following oral (PO) administration as a suspension in water or in 20% lipid emulsion (Liposyn II). Following bolus subcutaneous (s.c.) administration of [14C]NSC 615985 at 10 mg kg-1, relatively low concentrations of the parent compound were observed in three of 36 mice. One metabolite was tentatively identified in plasma of both the i.v.- and s.c.-treated animals as the sulfoxide of the parent compound. No parent compound was detected in the urine of NSC 615985 dosed mice. At least seven metabolites were present in urine; one metabolite (constituting 8-14% of urinary radioactivity) was tentatively identified as the carboxylic acid resulting from the hydrolysis of the isopropyl group from the parent compound. In summary, NSC 615985 was poorly absorbed following oral administration and extensively metabolized and eliminated following i.v. or s.c. administration. This unfavorable pharmacokinetic profile of NSC 615985 as well as its pattern of activity against NNRTI-resistant strains of HIV-1 precluded its progression to clinical trial; however, other members of the general chemical class are currently being evaluated by the NCI.
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PMID:Liquid chromatographic analysis in mouse, dog and human plasma; stability, absorption, metabolism and pharmacokinetics of the anti-HIV agent 2-chloro-5-(2-methyl-5,6-dihydro-1,4-oxathiin-3-yl carboxamido) isopropylbenzoate (NSC 615985, UC84). 960 23

Nevirapine is a non-nucleoside reverse transcriptase inhibitor for the treatment of HIV-1-infected patients. A simple and rapid high-performance liquid chromatographic method for the quantification of nevirapine in human plasma is described. Sample pretreatment consists of protein precipitation with acetonitrile. The analyte is separated from endogenous compounds by isocratic reversed-phase, ion-pair, high-performance liquid chromatography with ultraviolet detection at 282 nm. The method has been validated over the range of 52-10400 ng/ml using 250 microl of plasma. The assay was linear over this concentration range. Within- and between-day precisions were less than 4.5% for all quality control samples. The lower limit of quantitation was 52 ng/ml. Recovery of nevirapine from human plasma was 94.5%. This validated assay is suited for use in pharmacokinetic studies with nevirapine and can readily be used in a hospital laboratory for the monitoring of nevirapine concentrations.
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PMID:Rapid determination of nevirapine in human plasma by ion-pair reversed-phase high-performance liquid chromatography with ultraviolet detection. 974 55

Delavirdine is a novel non-nucleoside reverse transcriptase inhibitor for the treatment of HIV-1-infected patients. A simple and rapid high-performance liquid chromatographic method for the quantification of delavirdine in human plasma suitable for drug monitoring in patients is described. Sample pretreatment consists of protein precipitation with acetonitrile and subsequent evaporation of the extract to concentrate the analyte. The drug is separated from endogenous compounds by isocratic reversed-phase, high-performance liquid chromatography coupled with fluorescence detection. The optimal excitation and emission wavelengths are 300 and 425 nm, respectively. The method has been validated over the range of 50-50 000 ng/ml using only 200 microl of plasma samples. The assay is linear over this concentration range as indicated by the F-test for lack of fit. Within- and between-day precisions are less than 4.4% for all quality control samples. The lower limit of quanititation is 50 ng/ml. Recovery of delavirdine from human plasma is 93.8%. Delavirdine is stable under various conditions, for example 1 h at 60 degrees C and one week at 4 degrees C. This validated assay is suited for use in pharmacokinetic studies with delavirdine and can readily be implemented in the setting of a hospital laboratory for the monitoring of delavirdine concentrations.
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PMID:Rapid quantification of delavirdine, a novel non-nucleoside reverse transcriptase inhibitor, in human plasma using isocratic reversed-phase high-performance liquid chromatography with fluorescence detection. 1036 Apr 34

Efavirenz (EFV, DMP-266) is a new antiretroviral agent belonging to the class of nonnucleoside reverse transcriptase inhibitors. It has recently been approved by the Food and Drug Administration in management of human immunodeficiency virus (HIV). Preliminary pharmacokinetic studies on EFV in healthy volunteers show that the drug may influence the metabolism of protease inhibitors. For the determination of EFV in human plasma, a validated and specific reverse-phase high-performance liquid chromatography (HPLC) method, with UV detection, was developed. We used 100 microL plasma sample for a liquid-liquid extraction with diethyl ether after basification. The mobile phase was a mixture of acetonitrile and water, pumped at a flow rate of 1.2 mL/min. Ultraviolet detection was carried out at a wavelength of 247 nm. Retention times for EFV and internal standard (IS) were 5.3 and 4.5 minutes, respectively, and there was no chromatographic interference from other commonly administered drugs. The limit of detection was 100 ng/mL. The described assay is a rapid and accurate method for measurement of EFV in plasma: the easy preparation and small sample size makes this assay highly suitable for pharmacokinetic studies and routine clinical analysis in patients with HIV. In addition, the reproducibility of the method is only moderately increased by including IS, so analyzing without IS may be an alternative.
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PMID:High-performance liquid chromatography method for analyzing the antiretroviral agent efavirenz in human plasma. 1036 51

Efavirenz is a novel non-nucleoside reverse transcriptase inhibitor for the treatment of HIV-1-infected individuals. A simple and rapid high-performance liquid chromatographic method for the quantification of efavirenz in human plasma suitable for therapeutic drug monitoring in plasma is described. Sample pretreatment consists of protein precipitation with acetonitrile and subsequent evaporation of the extract to concentrate the analyte. The drug is separated from endogenous compounds by isocratic reversed-phase high-performance liquid chromatography with ultraviolet detection at 246 nm. The method has been validated over the range of 10 to 10,000 ng/ml using a volume of 250 microl of plasma. The assay is linear over this concentration range as indicated by the F-test for lack of fit. Within- and between-day precisions are less than 4.3% for all quality control samples. The lower limit of quantitation is 10 ng/ml and the recovery of efavirenz from human plasma is 106.4% (+/- 1.8%). Frequently co-administered drugs did not interfere with the described methodology. Efavirenz is stable under various relevant storage conditions, for example when stored for 24 h at room temperature. This validated assay is suited for use in pharmacokinetic studies with efavirenz and can readily be implemented in the setting of a hospital laboratory for the monitoring of efavirenz concentrations.
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PMID:Quantitative determination of efavirenz (DMP 266), a novel non-nucleoside reverse transcriptase inhibitor, in human plasma using isocratic reversed-phase high-performance liquid chromatography with ultraviolet detection. 1057 90


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