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Rat Liver Microsomes (rat + liver_microsome)
Selected AbstractsMechanisms underlying the inhibition of the cytochrome P450 system by copper ionsJOURNAL OF APPLIED TOXICOLOGY, Issue 8 2009M. E. Letelier Abstract Copper toxicity has been associated to the capacity of free copper ions to catalyze the production of superoxide anion and hydroxyl radical, reactive species that modify the structure and/or function of biomolecules. In addition, nonspecific Cu2+ -binding to thiol enzymes, which modifies their catalytic activities, has been reported. Cytochrome P450 (CYP450) monooxygenase is a thiol protein that binds substrates in the first and limiting step of CYP450 system catalytic cycle, necessary for the metabolism of lipophilic xenobiotics. Therefore, copper ions have the potential to oxidize and bind to cysteinyl residues of this monooxygenase, altering the CYP450 system activity. To test this postulate, we studied the effect of Cu2+ alone and Cu2+/ascorbate in rat liver microsomes, to independently evaluate its nonspecific binding and its pro-oxidant effects, respectively. We assessed these effects on the absorbance spectrum of the monooxygenase, as a measure of structural damage, and p -nitroanisole O -demethylating activity of CYP450 system, as a marker of functional impairment. Data obtained indicate that Cu2+ could both oxidize and bind to some amino acid residues of the CYP450 monooxygenase but not to its heme group. The differences observed between the effects of Cu2+ and Cu2+/ascorbate show that both mechanisms are involved in the catalytic activity inhibition of CYP450 system by copper ions. The significance of these findings on the pharmacokinetics and pharmacodynamics of drugs is discussed. Copyright © 2009 John Wiley & Sons, Ltd. [source] Lack of appreciable species differences in nonspecific microsomal bindingJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 8 2010Ying Zhang Abstract Species differences in microsomal binding were evaluated for 43 drug molecules in human, monkey, dog and rat liver microsomes, using a fixed concentration of microsomal protein. The dataset included 32 named drugs and 11 proprietary compounds encompassing a broad spectrum of physicochemical properties (11 acids, 24 bases, 8 neutral, c,log,D ,1 to 7, MW 200 to 700 and free fraction <0.001 to 1). Free fractions (fu,mic) in monkey, dog, rat and human microsomes were highly correlated, with linear regression correlation coefficients greater than 0.97. The average fold-difference in fu,mic between monkey, dog, or rat, and human was 1.6-, 1.3-, and 1.5-fold, respectively. Species differences in fu,mic were also assessed for a range of microsomal protein concentrations (0.2,2,mg/mL) for midazolam, clomipramine, astemizole, and tamoxifen, drugs with low to high microsomal binding. The mean fold species-difference in fu,mic for midazolam, clomipramine, astemizole, and tamoxifen was 1.1-, 1.2-, 1.3-, and 2.0-fold, respectively, and was independent of normalized microsomal protein concentration. For a fixed concentration of microsomal protein, greater than 76% and 90% of drugs examined in this study had preclinical species fu,mic within 1.5- and 2-fold, respectively, of experimentally measured human values. © 2010 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:3620,3627, 2010 [source] Allosteric kinetics of human carboxylesterase 1: Species differences and interindividual variability,JOURNAL OF PHARMACEUTICAL SCIENCES, Issue 12 2008Shiori Takahashi Abstract Esterified drugs such as imidapril, derapril, and oxybutynin hydrolyzed by carboxylesterase 1 (CES1) are extensively used in clinical practice. The kinetics using the CES1 substrates have not fully clarified, especially concerning species and tissue differences. In the present study, we performed the kinetic analyses in humans and rats in order to clarify these differences. The imidaprilat formation from imidapril exhibited sigmoidal kinetics in human liver microsomes (HLM) and cytosol (HLC) but Michaelis-Menten kinetics in rat liver microsomes and cytosol. The 2-cyclohexyl-2-phenylglycolic acid (CPGA) formation from oxybutynin were not detected in enzyme sources from rats, although HLM showed high activity. The kinetics were clarified to be different among species, tissues, and preparations. In individual HLM and HLC, there was large interindividual variability in imidaprilat (31- and 24-fold) and CPGA formations (15- and 9-fold). Imidaprilat formations exhibited Michaelis-Menten kinetics in HLM and HLC with high activity but sigmoidal kinetics in those with low activity. CPGA formations showed sigmoidal kinetics in high activity HLM but Michaelis-Menten kinetics in HLM with low activity. We revealed that the kinetics were different between individuals. These results could be useful for understanding interindividual variability and for the development of oral prodrugs. © 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 97:5434,5445, 2008 [source] Effect of herbal teas on hepatic drug metabolizing enzymes in ratsJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 10 2001Pius P. Maliakal We have investigated the effect of herbal teas (peppermint, chamomile and dandelion) on the activity of hepatic phase I and phase II metabolizing enzymes using rat liver microsomes. Female Wistar rats were divided into six groups (n = 5 each). Three groups had free access to a tea solution (2 %) while the control group had water. Two groups received either green tea extract (0.1 %) or aqueous caffeine solution (0.0625 %). After four weeks of pretreatment, different cytochrome P450 (CYP) isoforms and phase II enzyme activities were determined by incubation of liver microsomes or cytosol with appropriate substrates. Activity of CYP1A2 in the liver microsomes of rats receiving dandelion, peppermint or chamomile tea was significantly decreased (P < 0.05) to 15 %, 24 % and 39 % of the control value, respectively. CYP1A2 activity was significantly increased by pretreatment with caffeine solution. No alterations were observed in the activities of CYP2D and CYP3A in any group of the pretreated rats. Activity of CYP2E in rats receiving dandelion or peppermint tea was significantly lower than in the control group, 48 % and 60 % of the control, respectively. There was a dramatic increase (244 % of control) in the activity of phase II detoxifying enzyme UDP-glucuronosyl transferase in the dandelion tea-pretreated group. There was no change in the activity of glutathione-S-transferase. The results suggested that, like green and black teas, certain herbal teas can cause modulation of phase I and phase II drug metabolizing enzymes. [source] Metabolism of a novel antiangiogenic agent KR-31831 in rats using liquid chromatography-electrospray mass spectrometryJOURNAL OF SEPARATION SCIENCE, JSS, Issue 14 2005Hui-Hyun Kim Abstract KR-31831 ((2S,3R,4S)-4-(((1H-imidazol-2-yl)methyl)(4-chlorophenyl)amino)-6-amino-2-(dimethoxymethyl)-2-methyl-3,4-dihydro-2H-chromen-3-ol) is a novel antiangiogenic agent. In vitro and in vivo metabolism of KR-31831 in rats has been investigated using LC-MS and LC-MS/MS analysis. Incubation of rat liver microsomes and hepatocytes with KR-31831 produced three metabolites (M1,M3). M1, M2, and M3 were identified as N -((1H-imidazol-2-yl)methyl)-4-chlorobenzenamine, (2R,3R,4S)-4-(((1H-imidazol-2-yl)methyl)(4-chlorophenyl) amino)-6-amino-2-(hydroxymethyl)-2-methyl-3,4-dihydro-2H-chromen-3-ol, and N -((2S,3R,4S)-4-(((1H-imidazol-2-yl)methyl)(4-chlorophenyl)amino)-2-(dimethoxymethyl)-3-hydroxy-2-methyl-3,4-dihydro-2H-chromen-6-yl)acetamide, respectively, by co-chromatography with the authentic standards and by comparison with product ion spectra of the authentic standards. Those in vitro metabolites were also detected in bile, plasma, or urine samples after an intravenous administration of KR-31831 to rats. The metabolic routes for KR-31381 included the metabolism of acetal group to hydroxymethyl group (M2), N -dealkylation to M1, and N -acetylation at the 6-amino group (M3). [source] Inhibition of cytochrome P450 enzymes by rhein in rat liver microsomesPHYTOTHERAPY RESEARCH, Issue 2 2009Jing-cheng Tang Abstract Rhein, an active ingredient extensively found in plants such as Aloe, Cassitora L., rhubarb and so on, has been used for a long time in China. Pharmacological tests revealed that rhein not only had a strong antibacterial action, but also may be useful in cancer chemotherapy as a biochemical modulator. Its therapeutic action and toxicity is still the subject of considerable research. With microsome incubation assays in vitro and HPLC methods, the inhibition of rat liver CYP1A2, CYP2C9, CYP2D6, CYP2E1 and CYP3A enzymes by rhein were studied kinetically. The results showed the most inhibition of CYP2E1 by rhein (Ki = 10 µm, mixed); CYP3A and CYP2C9 were also inhibited by rhein, Ki = 30 µm (mixed) and Ki = 38 µm (mixed), respectively; rhein revealed some inhibition of CYP1A2 (Ki = 62 µm, uncompetitive) and CYP2D6 (Ki = 74 µm, mixed). Drug,drug interactions, especially cytochrome P450 (CYP)-mediated interactions, cause an enhancement or attenuation in the efficacy of co-administered drugs. Inhibition of the five major CYP enzymes observed for rhein suggested that changes in pharmacokinetics of co-administered drugs were likely to occur. Therefore, caution should be paid to the possible drug interaction of medicinal plants containing rhein and CYP substrates. Copyright © 2008 John Wiley & Sons, Ltd. [source] Antioxidant and antiglycation properties of total saponins extracted from traditional Chinese medicine used to treat diabetes mellitusPHYTOTHERAPY RESEARCH, Issue 2 2008Miaomiao Xi Abstract Eleven antidiabetic traditional Chinese medicine (TCM) extracts rich in saponins were examined for their antioxidant and antiglycation activities. The antioxidant activities of these extracts were evaluated by studying the inhibition of lipid peroxidation in rat liver microsomes induced by ascorbate/Fe2+, cumine hydroperoxide (CHP) or CCl4/reduced form of nicotinamide-adenine dinucleotide phosphate (NADPH). The antioxidant capacities were also evaluated by studying the scavenging of 2,2,-diphenyl-1-picrylhydrazyl (DPPH) free radical. The antiglycation activities of these extracts were evaluated by hemoglobin- , -gluconolactone (, -Glu) assay, bovine serum albumin (BSA)-glucose assay and N-acetyl-glycyl-lysine methyl ester (GK peptide)-ribose assay. Aralia taibaiensis outperformed other extracts in most of the assays except inhibition of early glycation products formation, where Acanthopanax senticosus showed higher activity. Aralia taibaiensis was particularly potent in inhibiting the late glycation and formation of advanced glycation end products (AGEs) on proteins. The antioxidant and antiglycation activities of most extracts were correlated with the saponin content. The results demonstrate that the antidiabetic activities of most extracts could be explained, at least in part, by their combined antioxidant and antiglycation properties. Copyright © 2007 John Wiley & Sons, Ltd. [source] Radiation protection of DNA and membrane in vitro by extract of Hemidesmus indicusPHYTOTHERAPY RESEARCH, Issue 5 2005T. K. Shetty Abstract Radioprotective effect of H. indicus root extract on lipid peroxidation in rat liver microsomes and plasmid DNA was examined. Hemidesmus indicus (HI) root extract was found to protect microsomal membranes as evident from reduction in lipid peroxidation values. The extract could also protect DNA from radiation induced strand breaks. Copyright © 2005 John Wiley & Sons, Ltd. [source] Protection of DNA and microsomal membranes in vitro by Glycyrrhiza glabra L. against gamma irradiationPHYTOTHERAPY RESEARCH, Issue 6 2002T. K. Shetty Abstract The radioprotective effect of the root extract of Glycyrrhiza glabra L on lipid peroxidation in rat liver microsomes and plasmid pBR322 DNA was investigated. The extract was found to protect microsomal membranes, as evident from reduction in lipid peroxidation, and could also protect plasmid DNA from radiation-induced strand breaks. Copyright © 2002 John Wiley & Sons, Ltd. [source] Structural elucidation of metabolites of ginkgolic acid in rat liver microsomes by ultra-performance liquid chromatography/electrospray ionization tandem mass spectrometry and hydrogen/deuterium exchangeRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 13 2009Z. H. Liu Ginkgolic acids have been shown to possess allergenic as well as genotoxic and cytotoxic properties. The question arises whether the metabolism of ginkgolic acids in the liver could decrease or increase their toxicity. In this study, the invitro metabolism of ginkgolic acid (15:1, GA), one component of ginkgo acids, was investigated as a model compound in Sprague-Dawley rat liver microsomes. The metabolites were analyzed by ultra-performance liquid chromatography coupled with photodiode array detector/negative-ion electrospray ionization tandem mass spectrometry (UPLC-PDA/ESI-MS/MS) and hydrogen/deuterium (H/D) exchange. The result showed that the benzene ring remained unchanged and the oxidations occurred at the side alkyl chain in rat liver microsomes. At least eight metabolites were found. Among them, six phase I metabolites were tentatively identified. This study might be useful for the investigation of toxicological mechanism of ginkgolic acids. Copyright © 2009 John Wiley & Sons, Ltd. [source] Metabolism of olaquindox in rat liver microsomes: structural elucidation of metabolites by high-performance liquid chromatography combined with ion trap/time-of-flight mass spectrometry,RAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 7 2008Zhaoying Liu Olaquindox (N -(2-hydroxyethyl)-3-methyl-2-quinoxalincarboxamide-1,4-dioxide) is a growth-promoting feed additive for food-producing animals. Its toxicity is closely related to the metabolism. The complete metabolic pathways of olaquindox are not revealed. To improve studies of the metabolism and toxicity of olaquindox, its biotransformation in rat liver microsomes and the structure of its metabolites using high-performance liquid chromatography combined with ion trap/time-of-flight mass spectrometry (LC/MS-ITTOF) were investigated. When olaquindox was incubated with an NADPH-generating system and rat liver microsomes, ten metabolites (M1,M10) were detected. The structures of these metabolites were identified from mass spectra and comparison of their changes in their accurate molecular masses and fragment ions with those of the parent drug. With the high resolution and good mass accuracy achieved by this technique, the elemental compositions of the metabolites and their fragment ions were exactly determined. The results indicate that the N,,,O group reduction is the main metabolic pathway of olaquindox metabolism in rat liver microsomes, because abundant 1-desolaquindox (M2), 4-desolaquindox (M1) and bisdesoxyolaquindox (M9) were produced during the incubation step. Seven other minor metabolites were revealed which were considered to be hydroxylation metabolites, based on the position of the quinoxaline ring or 3-methyl group and a carboxylic acid derivative on the side chain at position 2 of the quinoxaline ring. Among the identified metabolites, five new hydroxylated metabolites (M3,M7) were found for the first time in rat liver microsomes. This work will conduce to complete clarification of olaquindox metabolism, and improve the in vivo metabolism of olaquindox in food animals. Copyright © 2008 John Wiley & Sons, Ltd. [source] Rat Liver Microsomal Lipid Peroxidation Produced during the Oxidative Metabolism of Ethacrynic AcidBASIC AND CLINICAL PHARMACOLOGY & TOXICOLOGY, Issue 4 2001Kyoko Yamamoto Two oxidative metabolites of ethacrynic acid with dicarboxylic acid and hydroxylated ethyl group, respectively, were formed in the reaction mixture. The oxidative metabolism of ethacrynic acid was inhibited by cytochrome P450 inhibitors. The formation of TBARS was remarkably depressed by inhibitors like diethyldithiocarbamate and disulfiram. These results indicate that lipid peroxidation occurred in rat liver microsomes through the oxidative metabolism of ethacrynic acid. [source] A new metabolite of nodakenetin by rat liver microsomes and its quantification by RP-HPLC methodBIOMEDICAL CHROMATOGRAPHY, Issue 2 2010Peng Zhang Abstract The biotransformation of nodakenetin (NANI) by rat liver microsomes in vitro was investigated. Two major polar metabolites were produced by liver microsomes from phenobarbital-pretreated rats and detected by reversed-phase high-performance liquid chromatography (RP-HPLC) analysis. The chemical structures of two metabolites were firmly identified as 3,(R)-hydroxy-nodakenetin-3,-ol and 3,(S)-hydroxy-nodakenetin-3,-ol, respectively, on the basis of their 1H-NMR, MS and optical rotation analysis. The latter was a new compound. A sensitive, selective and simple RP-HPLC method has been developed for the simultaneous determination of NANI and its two major metabolites in rat liver microsomes. Chromatographic conditions comprise a C18 column, a mobile phase with MeOH-H2O (40 : 60, v/v), a total run time of 40 min, and ultraviolet absorbance detection at 330 nm. In the rat heat-inactivated liver microsomal supernatant, the lower limits of detection and quantification of metabolite I, metabolite II and NANI were 5.0, 2.0, 10.0 ng/mL and 20.0, 5.0, 50.0 ng/mL, respectively, and their calibration curves were linear over the concentration range 50,400, 20,120 and 150,24000 ng/mL, respectively. The results provided a firm basis for further evaluating the pharmacokinetics and clinical efficacy of NANI. Copyright © 2009 John Wiley & Sons, Ltd. [source] High-performance liquid chromatography assays for desmethoxyyangonin, methysticin, kavain and their microsomal metabolitesBIOMEDICAL CHROMATOGRAPHY, Issue 1 2009Shuang Fu Abstract Three novel, simple and reproducible high-performance liquid chromatography quantitative assays with UV detection were developed and validated for three major kavalactones,desmethoxyyangonin, methysticin and kavain,in rat liver microsomes using diazepam as an internal standard; liquid,liquid extraction was used for sample preparation and analysis was performed on a Shimadzu® 10A high-performance liquid chromatography system. The analysis was carried out in reversed-phase mode with a Luna® C18 column (150 × 2.00 mm, 3 µm) at 40°C. The limit of quantitation was 0.1 µg/mL using 0.25 mL of microsomal solution. The assays were linear over the range 0.1,10 µg/mL for desmethoxyyangonin, methysticin and kavain. Quality control samples exhibited good accuracy and precision with relative standard deviations lower than 15% and recoveries between 85 and 105%. The assays exhibited satisfactory performance with high sensitivity for quantifying desmethoxyyangonin, methysticin and kavain in rat liver microsomes and were successfully used to determine the three kavalactones and their microsomal metabolites. Copyright © 2008 John Wiley & Sons, Ltd. [source] In vitro metabolism of a new H+/K+ ATPase inhibitor DBM-819 in liver microsomes using HPLC and electrospray mass spectrometryBIOMEDICAL CHROMATOGRAPHY, Issue 8 2001Sung Jin Choi The metabolism of 1-(2-methyl-4-methoxyphenyl)-4-[(3-hydroxypropyl)amino]-6-methyl-2,3-dihydropyrrolo[3,2c]quinoline (DBM-819), a new H+/K+ ATPase inhibitor, has been studied by HPLC with spectrometric detection and on-line LC-electrospray mass spectrometry. In vitro incubation of DBM-819 with rat liver microsomes in the presence of NADPH resulted in the production of four metabolites (M1-4), whereas DBM-819 was oxidized to two metabolites, M2 and M4, by human liver microsomes. M2, M3 and M4 were identified as O-demethyl-DBM-819, 8-hydroxy-DBM-819 and N-dehydroxypropyl-DBM-819, respectively, based on LC/MS/MS analysis with authentic standards. M1 was tentatively identified as 1-(hydroxy-2-methyl-4-methoxyphenyl)-4-[(3-hydroxypropyl)amino]-6-methyl-2,3-dihydropyrrolo[3,2c]quinoline. Rat liver CYP1A1/2 catalyzed the oxidation of DBM-819 to 8-hydroxy-DBM-819 and N-dehydroxypropyl-DBM-819. Human CYP3A4 was a major isozyme for the formation of O-demethyl-DBM-819 as well as N-dehydroxypropyl-DBM-819. Copyright © 2001 John Wiley & Sons, Ltd. [source] Characterisation of the human liver in vitro metabolic pattern of artemisinin and auto-induction in the rat by use of nonlinear mixed effects modellingBIOPHARMACEUTICS AND DRUG DISPOSITION, Issue 2 2003Ulrika S.H. Svensson Abstract Aims: The aims of the study were to characterise the metabolic pattern of artemisinin in human and rat liver microsomes and to assess the magnitude of auto-induction in the rat. Methods: 14C-artemisinin was incubated with human liver microsomes and with liver microsomes from rats pretreated with oral artemisinin or placebo. The metabolic fate of 14C-artemisinin in microsomes from human B-lymphoblastoid cell lines transformed with CYP2A6, CYP2B6 and CYP3A4 was also investigated. The human liver microsome data and the rat liver microsomes data were analysed by nonlinear mixed effects modelling and naïve pooling using NONMEM, respectively. Results: Four metabolites were radiometrically detected in experiments with rat liver microsomes. The model that best described the data involved three primary metabolites of which one metabolite was further metabolised to a secondary metabolite. The formation of the four metabolites was induced 2.8, 7.2, 4.8 and 2.5-fold, respectively, in liver microsomes from rats pre-treated with artemisinin. Three metabolites were formed in human liver microsomes; having the same retention times as three of the metabolites formed in the rat. The final model consisted of two primary metabolites and a secondary metabolite with CYP2B6 and CYP2A6 influencing the formation rates of the major and minor primary metabolites, respectively. Conclusions: CYP2B6 and CYP2A6 activities described variability in the formation of the major and minor primary metabolites, respectively, in human liver microsomes. All artemisinin metabolic pathways in rat liver microsomes were induced in artemisinin pretreated animals. We suggest modelling as a method for the discrimination and detection of more complex metabolic patterns from in vitro metabolism rate data. Copyright © 2002 John Wiley & Sons, Ltd. [source] | |||||||||||||