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)

We found previously that long-chain fatty acids could inhibit eukaryotic DNA polymerase activities in vitro [1,2]. The purpose of the present study was to investigate the mode of this inhibition in greater detail. Among the C18 to C24 fatty acids examined, the strongest inhibitor was a C24 fatty acid, nervonic acid (NA), and the weakest was a C18 fatty acid, linoleic acid (LA). We analyzed the inhibitory effect of these two fatty acids and their modes of action. For DNA polymerase beta (pol. beta), NA acted by competing with both the substrate- and template-primer, but for DNA polymerase alpha (pol. alpha) or human immunodeficiency virus type 1 reverse transcriptase (HIV-1 reverse transcriptase or HIV-RT), NA acted non-competitively. NA-binding to pol. beta could be stopped with a non-ionic detergent, but the binding to pol. alpha or HIV-RT could not. The inhibition mode of LA showed the same characteristics, except that the minimum inhibitory dose of the longer chain was much lower. We also tested the effects of NA and LA using pol. beta and its proteolytic fragments, as described by Kumar et al. [3,4]. Both of the fatty acids were found to bind to the 8 kDa DNA-binding domain fragment, and to suppress binding to the template-primer DNA. We found that 10,000 times more of either fatty acid was required for it to bind to the 31 kDa catalytic domain or inhibit the DNA polymerase activity. The possible modes of inhibition by these long-chain fatty acids are discussed, based on the present findings.
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PMID:The inhibitory action of fatty acids on DNA polymerase beta. 936 79

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

As part of an on-going study on the suitability of a formal therapeutic drug monitoring (TDM) of antiviral drugs for improving the management of HIV infection, a high-performance liquid chromatography method has been developed to quantify simultaneously in plasma five HIV protease inhibitors (PIs) (i.e., indinavir, amprenavir, saquinavir, ritonavir, nelfinavir) and the novel non-nucleoside reverse transcriptase inhibitor efavirenz. After viral inactivation by heat (60 degrees C for 60 min), plasma (600 microl), with clozapine added as internal standard, is diluted 1:1 with phosphate buffer, pH 7 and subjected to a solid-phase extraction on a C18 cartridge. Matrix components are eliminated with 2 x 500 microl of a solution of 0.1% H3PO4 neutralised with NaOH to pH 7. PIs and efavirenz are eluted with 3 x 500 microl MeOH. The resulting eluate is evaporated under nitrogen at room temperature and is reconstituted in 100 microl 50% MeOH. A 40-microl volume is subjected to HPLC analysis onto a Nucleosil 100, 5 microm C18 AB column, using a gradient elution of MeCN and phosphate buffer adjusted to pH 5.15 and containing 0.02% sodium heptanesulfonate: 15:85 at 0 min-->30:70 at 2 min-->32:68 at 8 min-->42:58 at 18 min-->46:54 at 34 min, followed by column cleaning with MeCN-buffer, pH 5.15 (90:10), onto which 0.3% AcOH is added. Clozapine, indinavir, amprenavir, saquinavir, ritonavir, efavirenz and nelfinavir are detected by UV at 201 nm at a retention time of 8.2, 13.0, 16.3, 21.5, 26.5, 28.7 and 31.9 min, respectively. The total run time for a single analysis is 47 min, including the washing-out and reequilibration steps. The calibration curves are linear over the range 100-10,000 ng/ml. The absolute recovery of PIs/efavirenz is always higher than 88%. The method is precise with mean inter-day relative standard deviations within 2.5-9.8% and accurate (range of inter-day deviations -4.6 to +4.3%). The in vitro stability of plasma spiked with PIs/efavirenz at 750, 3000 and 9000 ng/ml has been studied at room temperature, -20 degrees C and +60 degrees C. The method has been validated and is currently applied to the monitoring of PIs and efavirenz in HIV patients. This HPLC assay may help clinicians confronted to questionable compliance, side effects or treatment failure in elucidating whether patients are exposed to adequate circulating drug levels. The availability of such an assay represents an essential step in elucidating the utility of a formal TDM for the optimal follow-up of HIV patients.
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PMID:Simultaneous determination of the HIV protease inhibitors indinavir, amprenavir, saquinavir, ritonavir, nelfinavir and the non-nucleoside reverse transcriptase inhibitor efavirenz by high-performance liquid chromatography after solid-phase extraction. 1079 93

A sensitive and selective liquid chromatographic assay has been developed for the determination of the five protease inhibitors currently approved by the Food and Drug Administration (FDA) (amprenavir, indinavir, nelfinavir, ritonavir, and saquinavir) in a single run. Pretreatment of a 1-mL plasma sample spiked with internal standard was made by a solid-phase extraction procedure using a polymeric reversed-phase sorbent. Liquid chromatography was performed using a narrowbore C18 reversed-phase column and gradient elution. A double ultraviolet detection at 265 nm (amprenavir) and at 210 nm (indinavir, nelfinavir, ritonavir, saquinavir and internal standard) was used. Calibration curves were linear in the range 25-10000 ng/mL and the assay has been validated over the range 25-5000 ng/mL. Average accuracy at four concentrations was in the range of 100.5-104.2% and 96.9-100.5% for within-day and between-day, respectively. The coefficients of variation were less than 10%. Mean absolute recoveries varied from 85.4% (ritonavir) to 98.8% (saquinavir). No metabolite of the protease inhibitors was found to coelute with the drugs of interest or with the internal standard. At this time, among the tested drugs, especially all the presently licensed nucleoside and nonnucleoside reverse transcriptase inhibitors that can be used in combination with the protease inhibitors, none was found to interfere with the assay. This method is now in use in the authors' laboratory for the therapeutic monitoring of the HIV-protease inhibitors.
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PMID:Simultaneous determination of the five HIV-protease inhibitors: amprenavir, indinavir, nelfinavir, ritonavir, and saquinavir in human plasma by solid-phase extraction and column liquid chromatography. 1094 89

A new high-performance liquid chromatography (HPLC) with UV detection assay was developed for the simultaneous determination of protease inhibitors (PIs), nucleoside and non-nucleoside reverse transcriptase inhibitors (NRTIs, NNRTIs) using a single 1-ml plasma samples. A solid-liquid extraction procedure without internal standard was coupled with two separate reversed-phase HPLC systems; one for the determination of amprenavir, efavirenz, indinavir, nelfinavir, ritonavir, saquinavir (run time=32 min) and one for the determination of abacavir, didanosine, lamivudine, stavudine, nevirapine, zidovudine (run time=40 min). The first requires a mobile phase containing sodium phosphate buffer+ion pair-acetonitrile (50:50, v/v) through a C18 Symmetry column (250x4.6 mm I.D., 5 microm particle size), using variable wavelengths (241, 254 and 261 nm). The second system requires three mobile phases (potassium phosphate buffer+ion pair-acetonitrile) for different elution through a C18 Symmetry Shield column (250x4.6 mm I.D., 5 microm), using a single wavelength (260 nm). Peak-areas are linear; correlation coefficients are better than 0.998 for all compounds, with both inter- and intra-day relative standard deviations lower than 12%. Extraction recoveries are higher than 93% for PIs and NNRTIs and higher than 70% for NRTIs. The method is specific and sensitive and was used to determine trough and peak levels of antiretroviral drugs in HIV infected patients under various combinations of RTIs and PIs.
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PMID:Determination of twelve antiretroviral agents in human plasma sample using reversed-phase high-performance liquid chromatography. 1099 10

Nevirapine is an antiretroviral agent belonging to the class of non-nucleoside reverse transcriptase inhibitors. We describe a fast, simple isocratic reversed-phase high-performance liquid chromatography method with a 30-mm long column for assaying nevirapine in human serum. After deproteinization of 200 microl serum samples with 50% trichloroacetic acid, the supernatant was injected into a reversed-phase C18 column, using 10 mM phosphate buffer (pH 5)-acetonitrile (82:18, v/v) as the mobile phase. Peak detection was performed at 240 nm. Nevirapine retention time was 2 min. The method was validated over 0.1-10 microg/ml and the assay was linear over this concentration range (r2>0.998). Within- and between-day precisions were less than 5.4%. The lower limit of quantification was 0.1 microg/ml. Nevirapine in human serum samples was stable for 2 days at 20-25 degrees C, 15 days at 4 degrees C and 3 months at -20 degrees C.
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PMID:Simple and rapid determination of nevirapine in human serum by reversed-phase high-performance liquid chromatography. 1123 94

An analytical technique using liquid chromatography (LC) coupled with electrospray-mass spectrometry (ESI--MS) has been developed for the simultaneous determination of five protease inhibitors (PIs): saquinavir, indinavir, ritonavir, nelfinavir, and amprenavir; and three non-nucleoside reverse transcriptase inhibitors (NNRTIs): nevirapine, delavirdine, and efavirenz, in human plasma. This assay allows the elution and identification of these drugs in a single run (10 minutes) using a linear gradient with water and acetonitrile. The procedure involves liquid--liquid extraction. High-performance liquid chromatography (HPLC) separation was achieved on a C18 reversed-phase column, with a linear gradient elution followed by mass spectrometry detection. The calibration curves, obtained by automatic process peak area integration, show a good linearity in a range of concentrations between 20 and 10,000 ng/mL (40--10,000 ng/mL for efavirenz). The limit of detection was approximately 10 ng/mL for seven drugs (25 ng/mL for efavirenz). The coefficients of variation (CV) were always less than 15% for both intraday and interday precision for each compound. The recovery of the eight drugs ranged from 88.5% to 100%. This novel LC/ESI--MS assay provides an excellent method for simultaneous quantitative monitoring of different components of the highly active antiretroviral treatments (HAARTs) in patients treated simultaneously with PIs and NNRTIs, and it has been successfully applied to therapeutic drug monitoring and pharmacokinetic studies.
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PMID:Antiretrovirals: simultaneous determination of five protease inhibitors and three nonnucleoside transcriptase inhibitors in human plasma by a rapid high-performance liquid chromatography--mass spectrometry assay. 1147 20

A single HPLC assay was developed for therapeutic drug monitoring of 5 HIV protease inhibitors (indinavir, amprenavir, saquinavir, ritonavir, nelfinavir) and a non-nucleoside reverse transcriptase inhibitor (nevirapine) in human plasma. After liquid-liquid extraction in a mixture ethyl acetate-hexane, compounds are separated on a C18 column with a gradient elution of solvent A [acetonitrile and 0.025 M tetramethylammonium perchlorate in 0.2% aqueous trifluoroacetic acid (55:45 (v/v))] and solvent B [methanol and 0.025 M tetramethylammonium perchlorate in 0.2% aqueous trifluoroacetic acid (55:45 (v/v))]. The compounds are detected at various wavelengths: 320 nm (nevirapine), 259 nm (indinavir), 254 nm (amprenavir, nelfinavir, saquinavir) and 239 nm (ritonavir). The intra-day and inter-day precision and accuracy are lower than 15%. The limits of quantitation are 0.05 mg/l (amprenavir), 0.2 mg/l (indinavir, saquinavir, nelfinavir) and 0.4 mg/l (ritonavir, nevirapine). This method which allows to estimate simultaneously plasma levels of protease inhibitors and nevirapine can be used for therapeutic drug monitoring.
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PMID:High-performance liquid chromatographic assay to determine the plasma levels of HIV-protease inhibitors (amprenavir, indinavir, nelfinavir, ritonavir and saquinavir) and the non-nucleoside reverse transcriptase inhibitor (nevirapine) after liquid-liquid extraction. 1148 21

The glycolipid galactosyldiacylglycerol (GDG), containing C16:0 and C18:1 fatty acids, was isolated from the sea alga Petalonia bingbamiae as a potent inhibitor of the activities of mammalian DNA polymerase alpha (pol. alpha). GDG, however, had no effect on pol. alpha from a fish or a higher plant. The inhibition of pol. alpha by GDG was dose-dependent with an IC50 value of 54 microM. The compound did not influence the activities of other replicative DNA polymerases such as mammalian pol. delta, or repair-related enzymes such as mammalian pol. beta. GDG also did not influence the activities of prokaryotic DNA polymerases such as the Klenow fragment of DNA polymerase I, T4 DNA polymerase, Taq DNA polymerase, DNA polymerases from the higher plant, cauliflower, or DNA metabolic enzymes such as calf thymus terminal deoxynucleotidyl transferase, human immunodeficiency virus type 1 reverse transcriptase and deoxyribonuclease 1. Kinetic analysis of the compound showed that pol. alpha was non-competitively inhibited with respect to both the DNA template and the nucleotide substrate. In this study, we demonstrated the structure-function relationship in the selective inhibition of pol. alpha by the glycolipid group.
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PMID:Galactosyldiacylglycerol, a mammalian DNA polymerase alpha-specific inhibitor from a sea alga, Petalonia bingbamiae. 1155 81

A reversed-phase high-performance liquid chromatography method for the simultaneous quantitative determination of the currently available HIV protease inhibitors amprenavir, indinavir, lopinavir, nelfinavir, ritonavir, saquinavir, the active nelfinavir metabolite M8, and the nonnucleoside reverse transcriptase inhibitor nevirapine in human plasma is described. The method involved liquid-liquid extraction from plasma, followed by high-performance liquid chromatography with an OmniSpher 5 C18 column and ultraviolet detection set at a wavelength of 215 nm for the protease inhibitors and 280 nm for nevirapine. The runtime was 25 minutes. The assay has been validated over the concentration range of 0.05 to 30 mg/L for indinavir, nelfinavir, ritonavir, and saquinavir, 0.07 to 30 mg/L for amprenavir and lopinavir, and 0.05 to 15 mg/L for M8 and nevirapine. This method proved to be simple, accurate, and precise and is useful for the therapeutic drug monitoring of protease inhibitors and the nonnucleoside reverse transcriptase inhibitor nevirapine on a routine basis.
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PMID:Simultaneous determination of the HIV drugs indinavir, amprenavir, saquinavir, ritonavir, lopinavir, nelfinavir, the nelfinavir hydroxymetabolite M8, and nevirapine in human plasma by reversed-phase high-performance liquid chromatography. 1276 71


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