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Tsutomu Shimada - One of the best experts on this subject based on the ideXlab platform.

  • characterization of liver microsomal 7 ethoxycoumarin o deethylation and Chlorzoxazone 6 hydroxylation activities in japanese and caucasian subjects genotyped for cyp2e1 gene
    Archives of Toxicology, 2000
    Co-Authors: Kiyoshi Inoue, Hiroshi Yamazaki, Tsutomu Shimada
    Abstract:

    To determine whether the CYP2E1 genetic polymorphisms cause alterations in protein expression and enzyme catalytic activities, three CYP2E1 genetic polymorphisms, namely RsaI/PstI, DraI, and MspI types, were determined in liver genomic DNA isolated from 39 Japanese and 45 Caucasians. These genotypes were compared with levels of CYP2E1 and activities of 7-ethoxycoumarin O-deethylation and Chlorzoxazone 6-hydroxylation in liver microsomes from these human samples. In combination of three types of CYP2E1 polymorphisms, it was classified into seven genotypes in the Japanese population and four in the Caucasian population. The incidence in the occurrence of RsaI/PstI polymorphism or DraI polymorphism was 0.24 and 0.29 for Japanese, and 0.01 and 0.02 for Caucasians. Ethnic difference was also noted in the MspI polymorphism in which frequencies in Japanese and Caucasian populations were 0.15 and 0.02, respectively. Studies with liver microsomes showed that there were no significant differences in the levels of expression of CYP2E1 protein between wild-type (group A) and other 6 genotypes (B, C, D, E, F, and G) in Japanese and other three genotypes (B, D, and F) in Caucasians. Catalytic activities for 7-ethoxycoumarin O-deethylation and Chlorzoxazone 6-hydroxylation by liver microsomes were also found to be less significantly affected by mutations in the CYP2E1 gene in human samples examined in this study. These results support the view that RsaI/PstI, DraI, and MspI types of CYP2E1 genetic polymorphisms may not cause significant alterations in protein expression and enzyme catalytic activities of CYP2E1 enzyme in human livers.

  • prediction of human liver microsomal oxidations of 7 ethoxycoumarin and Chlorzoxazone with kinetic parameters of recombinant cytochrome p 450 enzymes
    Drug Metabolism and Disposition, 1999
    Co-Authors: Tsutomu Shimada, Fujiko Tsumura, Hiroshi Yamazaki
    Abstract:

    Different roles of individual forms of human cytochrome P-450 (CYP) in the oxidation of 7-ethoxycoumarin and Chlorzoxazone were investigated in liver microsomes of different human samples, and the microsomal activities thus obtained were predicted with kinetic parameters obtained from cDNA-derived recombinant CYP enzymes in microsomes of Trichoplusia ni cells. Of 14 forms of recombinant CYP examined, CYP1A1 had the highest activities ( V max / K m ratio) in catalyzing 7-ethoxycoumarin O -deethylation followed by CYP1A2, 2E1, 2A6, and 2B6, although CYP1A1 has been shown to be an extrahepatic enzyme. With these kinetic parameters (excluding CYP1A1) we found that CYP1A2 and 2E1 were the major enzymes catalyzing 7-ethoxycoumarin; the contributions of these two forms were dependent on the contents of these CYPs in liver microsomes of different humans. Similarly, Chlorzoxazone 6-hydroxylation activities of liver microsomes were predicted with kinetic parameters of recombinant human CYP enzymes and it was found that CYP3A4 as well as CYP1A2 and 2E1 were involved in Chlorzoxazone hydroxylation, depending on the contents of these CYP forms in the livers. Recombinant CYP2A6 and 2B6 and CYP2D6 had considerable roles ( V max / K m ratio) for 7-ethoxycoumarin O -deethylation and Chlorzoxazone 6-hydroxylation, respectively; however, these CYP forms had relatively minor roles in the reactions, probably due to low expression in human livers. These results support the view that the roles of individual CYP enzymes in the oxidation of xenobiotic chemicals in human liver microsomes could be predicted by kinetic parameters of individual CYP enzymes and by the levels of each of the CYP enzymes in liver microsomes of human samples.

  • Prediction of human liver microsomal oxidations of 7-ethoxycoumarin and Chlorzoxazone using kinetic parameters of recombinant cytochrome P450 enzymes. Drug Metab Dispos 27:1274–1280.
    1999
    Co-Authors: Tsutomu Shimada, Fujiko Tsumura, Hiroshi Yamazaki
    Abstract:

    This paper is available online at http://www.dmd.org ABSTRACT: Different roles of individual forms of human cytochrome P-450 (CYP) in the oxidation of 7-ethoxycoumarin and Chlorzoxazone were investigated in liver microsomes of different human samples, and the microsomal activities thus obtained were predicted with kinetic parameters obtained from cDNA-derived recombinant CYP enzymes in microsomes of Trichoplusia ni cells. Of 14 forms of recombinant CYP examined, CYP1A1 had the highest activities (V max /K m ratio) in catalyzing 7-ethoxycoumarin O-deethylation followed by CYP1A2, 2E1, 2A6, and 2B6, although CYP1A1 has been shown to be an extrahepatic enzyme. With these kinetic parameters (excluding CYP1A1) we found that CYP1A2 and 2E1 were the major enzymes catalyzing 7-ethoxycoumarin; the contributions of these two forms were dependent on the contents of these CYPs in liver microsomes of different humans. Similarly, Chlorzoxazone 6-hydroxylation activities of liver microsomes were predicted with kinetic parameters of recombinant human CYP enzymes and it was found that CYP3A4 as well as CYP1A2 and 2E1 were involved in Chlorzoxazone hydroxylation, depending on the contents of these CYP forms in the livers. Recombinant CYP2A6 and 2B6 and CYP2D6 had considerable roles (V max /K m ratio) for 7-ethoxycoumarin O-deethylation and Chlorzoxazone 6-hydroxylation, respectively; however, these CYP forms had relatively minor roles in the reactions, probably due to low expression in human livers. These results support the view that the roles of individual CYP enzymes in the oxidation of xenobiotic chemicals in human liver microsomes could be predicted by kinetic parameters of individual CYP enzymes and by the levels of each of the CYP enzymes in liver microsomes of human samples. Multiple forms of cytochrome P-450 (CYP) 2 exist in liver microsomes and these CYP forms play important roles in the oxidation of structurally diverse xenobiotic chemicals such as drugs, toxic chemicals, and carcinogens as well as endobiotic chemicals, including steroids, fatty acids, fat-soluble vitamins, and prostaglandins Several studies have reported that two or more CYP enzymes are able to oxidize xenobiotic and endobiotic chemicals at the same position of the molecules with different affinities (i.e., V max /K m ratio) in human liver microsomes 1274 predicted with kinetic parameters of recombinant enzymes and the levels of liver microsomal CYP2C19 and 3A4 enzymes Materials and Methods Chemicals. 7-Ethoxycoumarin was obtained from Aldrich Chemical Co. (Milwaukee, WI) and 7-hydroxycoumarin from Katayama Chemical Co. (Osaka, Japan). Chlorzoxazone and its 6-hydroxylated metabolite were donated by Dr. F. P. Guengerich of Vanderbilt University. Other reagents and chemicals used in this study were obtained from sources as described previously or were of highest qualities commercially available Enzyme Preparation. Human liver samples were obtained from organ donors or patients undergoing liver resection as described previously Recombinant CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 3A4, 3A5, and 4A11 expressed in microsomes of T. ni cells infected with a baculovirus containing human CYP and NADPH-CYP reductase cDNA inserts were obtained from Gentest Co. Enzyme Assays. 7-Ethoxycoumarin O-deethylation activities by CYP enzymes were determined by HPLC as described Incubation mixtures (final volume of 0.20 ml) for Chlorzoxazone 6-hydroxylation were the same as for the assay of 7-ethoxycoumarin Odeethylation, except that substrate was replaced by Chlorzoxazone. Reactions were carried out at 37°C for 10 min and terminated by adding a mixture of 1.5 ml of CH 2 Cl 2 and 25 l of 43% H 3 PO 4 . The organic layer, after collecting by centrifugation, was evaporated to dryness under nitrogen atmosphere and the materials were dissolved in 200 l of 27% CH 3 CN containing 0.5% H 3 PO 4 . Product formation was determined by HPLC with a 4.6 ϫ 150 mm Nucleosil octylsilyl (C 8 ) reversed phase column (Chemco Scientific, Osaka, Japan). CYP contents were estimated spectrally by the original method Statistical Analysis. Kinetic parameters for 7-ethoxycoumarin O-deethylation and Chlorzoxazone 6-hydroxylation by human CYP enzymes were estimated with a computer program (KaleidaGraph; Synergy Software, Reading, PA) designed for nonlinear regression analysis. Prediction of 7-Ethoxycoumarin O-Deethylation and Chlorzoxazone 6-Hydroxylation by Human Liver Microsomes Based on Kinetic Parameters of Recombinant CYP Enzymes. To define the potential roles of individual forms of CYP enzymes in the oxidation of 7-ethoxycoumarin and Chlorzoxazone by different human samples, we calculated the predicted activities of human liver microsomal 7-ethoxycoumarin O-deethylation and Chlorzoxazone 6-hydroxylation activities with kinetic parameters of recombinant human CYP enzymes and estimated contents of these CYP proteins by immunoblotting. The equation used for the prediction of expected activities was obtained from the previous method Results Kinetic Analysis of 7-Ethoxycoumarin O-Deethylation and Chlorzoxazone 6-Hydroxylation by 14 Forms of Recombinant Human CYPs. Kinetic parameters for 7-ethoxycoumarin O-deethylation and Chlorzoxazone 6-hydroxylation by 14 forms of recombinant human CYP enzymes (in microsomes of T. ni cells) were determined as described in Materials and Methods K m values for 7-ethoxycoumarin O-deethylation were the lowest in CYP1A1 followed by CYP1B1, CYP1A2, CYP2A6, CYP2E1, and CYP2C9 Of 14 forms of CYP examined, CYP2E1, 1A1, 2D6, 1A2, and 3A4 catalyzed Chlorzoxazone 6-hydroxylation at varying rates, whereas other enzymes, including CYP1B1, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 3A5, and 4A11 gave very low or undetectable rates for the hydroxylation Prediction of 7-Ethoxycoumarin O-Deethylation and Chlorzoxazone 6-Hydroxylation by Human Liver Microsomes with Kinetic Parameters of Recombinant CYP Enzymes. We examined the 7-ethoxycoumarin O-deethylation and Chlorzoxazone 6-hydroxylation activities catalyzed by human liver microsomes with the kinetic parameters of recombinant human CYP enzymes described above and the estimated contents of immunochemically determined CYPs in human liver microsomes The levels of individual forms of CYP, including CYP1A2, CYP2A6, CYP2B6, CYP2C, CYP2D6, CYP2E1, and CYP3A in liver microsomes of 16 Japanese and 8 Caucasian samples were determined by Western immunoblotting analysi

  • reconstitution of recombinant cytochrome p450 2c10 2c9 and comparison with cytochrome p450 3a4 and other forms effects of cytochrome p450 p450 and cytochrome p450 b5 interactions
    Archives of Biochemistry and Biophysics, 1997
    Co-Authors: Hiroshi Yamazaki, Elizabeth M J Gillam, F P Guengerich, Misook Dong, William W Johnson, Tsutomu Shimada
    Abstract:

    Tolbutamide methyl hydroxylation and S-warfarin 7-hydroxylation activities were reconstituted in systems containing recombinant human cytochrome P450 (P450 or CYP) 2C10(2C9) and the optimal conditions for the systems were compared with those of bufuralol 1'-hydroxylation by CYP1A1, theophylline 8-hydroxylation by CYP1A2, bufuralol 1'-hydroxylation by CYP2D6, Chlorzoxazone 6-hydroxylation by CYP2E1, and testosterone 6 beta-hydroxylation by CYP3A4. CYP2C10 required cytochrome b5 (b5) for optimal rates of tolbutamide and S-warfarin oxidations and b5 could be replaced by apo-b5; apo-b5 and b5 effects on the reconstituted systems have already been reported in systems containing CYP3A4 for the oxidation of testosterone and nifedipine and for the rapid reduction of CYP3A4 by NADPH-P450 reductase (H. Yamazaki et al., 1996, J. Biol. Chem. 271, 27438-27444). Stopped-flow studies, however, suggested that apo-b5 as well as b5 did not cause stimulation of the reduction of CYP2C10 by NADPH-P450 reductase, while the reduction rates were dependent on the substrates in reconstituted systems. Chlorzoxazone 6-hydroxylation by CYP2E1 was stimulated by b5, but not by apo-b5, in reconstituted systems. Neither apo- nor holo-b5 increased bufuralol 1'-hydroxylation activity by CYP1A1 or 2D6 or theophylline 8-hydroxylation by CYP1A2. Interestingly, we found that testosterone 6 beta-hydroxylation by CYP3A4 was stimulated by CYP1A2 (and also by a modified form in which the first 36 residues of the native human protein were removed) and CYP1A1 as well as by b5, and such stimulations were not seen when other P450 proteins (e.g., CYP2C10, 2D6, or 2E1) were added to the reconstituted systems. In contrast, substrate oxidations by CYP2C10 and CYP2E1 were not stimulated by other P450 proteins. The present results suggest that there are differences in optimal conditions for reconstitution of substrate oxidations by various forms of human P450 enzymes, and in some P450-catalyzed reactions protein-protein interactions between P450 and b5 and other P450 proteins are very important in some oxidations catalyzed by CYP2C10, 2E1, and 3A4.

  • requirements for cytochrome b5 in the oxidation of 7 ethoxycoumarin Chlorzoxazone aniline and n nitrosodimethylamine by recombinant cytochrome p450 2e1 and by human liver microsomes
    Biochemical Pharmacology, 1996
    Co-Authors: Hiroshi Yamazaki, Elizabeth M J Gillam, L C Bell, M Nakano, Tsutomu Shimada
    Abstract:

    NADH-dependent 7-ethoxycoumarin O-deethylation activities could be reconstituted in systems containing cytochrome b(5) (b(5)), NADH-b(5) reductase, and bacterial recombinant P450 2E1 in 100 mM potassium phosphate buffer (pH 7.4) containing a synthetic phospholipid mixture and cholate. Replacement of NADH-b(5) reductase with NADPH-P450 reductase yielded a 4-fold increase in 7-ethoxycoumarin O-deethylation activity, and further stimulation (similar to 1.5-fold) could be obtained when NADPH was used as an electron donor. Removal of b(5) from the NADH- and NADPH-supported systems caused a 90% loss of 7-ethoxycoumarin O-deethylation activities in the presence of NADPH-P450 reductase, but resulted in complete loss of the activities in the absence of NADPH-P450 reductase. K-m values were increased and V-max values were decreased for 7-ethoxycoumarin O-deethylation when b(5) was omitted from the NADPH-supported P450 2E1-reconstituted systems. Requirements for b(5) in P450 2E1 systems were also observed in Chlorzoxazone 6-hydroxylation, aniline p-hydroxylation, and N-nitrosodimethylamine N-demethylation. In human liver microsomes, NADH-dependent 7-ethoxycoumarin O-deethylation, Chlorzoxazone 6-hydroxylation, aniline P-hydroxylation, and N-nitrosodimethylamine N-demethylation activities were found to be about 55, 41, 33, and 50%, respectively, of those catalyzed by NADPH-supported systems. Anti-rat NADPH-P450 reductase immunoglobulin G inhibited 7-ethoxycoumarin O-deethylation activity catalyzed by human liver microsomes more strongly in NADPH- than NADH-supported reactions, while anti-human b(5) immunoglobulin G inhibited microsomal activities in both NADH- and NADPH-supported systems to similar extents. These results suggest that b(5) is an essential component in P450 2E1-catalyzed oxidations of several substrates used, that about 10% of the activities occur via P450 2E1 reduction by NADPH-P450 reductase in the absence of b(5), and that the NADH-supported system contributes, in part, to some reactions catalyzed by P450 2E1 in human liver microsomes.

Hiroshi Yamazaki - One of the best experts on this subject based on the ideXlab platform.

  • characterization of liver microsomal 7 ethoxycoumarin o deethylation and Chlorzoxazone 6 hydroxylation activities in japanese and caucasian subjects genotyped for cyp2e1 gene
    Archives of Toxicology, 2000
    Co-Authors: Kiyoshi Inoue, Hiroshi Yamazaki, Tsutomu Shimada
    Abstract:

    To determine whether the CYP2E1 genetic polymorphisms cause alterations in protein expression and enzyme catalytic activities, three CYP2E1 genetic polymorphisms, namely RsaI/PstI, DraI, and MspI types, were determined in liver genomic DNA isolated from 39 Japanese and 45 Caucasians. These genotypes were compared with levels of CYP2E1 and activities of 7-ethoxycoumarin O-deethylation and Chlorzoxazone 6-hydroxylation in liver microsomes from these human samples. In combination of three types of CYP2E1 polymorphisms, it was classified into seven genotypes in the Japanese population and four in the Caucasian population. The incidence in the occurrence of RsaI/PstI polymorphism or DraI polymorphism was 0.24 and 0.29 for Japanese, and 0.01 and 0.02 for Caucasians. Ethnic difference was also noted in the MspI polymorphism in which frequencies in Japanese and Caucasian populations were 0.15 and 0.02, respectively. Studies with liver microsomes showed that there were no significant differences in the levels of expression of CYP2E1 protein between wild-type (group A) and other 6 genotypes (B, C, D, E, F, and G) in Japanese and other three genotypes (B, D, and F) in Caucasians. Catalytic activities for 7-ethoxycoumarin O-deethylation and Chlorzoxazone 6-hydroxylation by liver microsomes were also found to be less significantly affected by mutations in the CYP2E1 gene in human samples examined in this study. These results support the view that RsaI/PstI, DraI, and MspI types of CYP2E1 genetic polymorphisms may not cause significant alterations in protein expression and enzyme catalytic activities of CYP2E1 enzyme in human livers.

  • prediction of human liver microsomal oxidations of 7 ethoxycoumarin and Chlorzoxazone with kinetic parameters of recombinant cytochrome p 450 enzymes
    Drug Metabolism and Disposition, 1999
    Co-Authors: Tsutomu Shimada, Fujiko Tsumura, Hiroshi Yamazaki
    Abstract:

    Different roles of individual forms of human cytochrome P-450 (CYP) in the oxidation of 7-ethoxycoumarin and Chlorzoxazone were investigated in liver microsomes of different human samples, and the microsomal activities thus obtained were predicted with kinetic parameters obtained from cDNA-derived recombinant CYP enzymes in microsomes of Trichoplusia ni cells. Of 14 forms of recombinant CYP examined, CYP1A1 had the highest activities ( V max / K m ratio) in catalyzing 7-ethoxycoumarin O -deethylation followed by CYP1A2, 2E1, 2A6, and 2B6, although CYP1A1 has been shown to be an extrahepatic enzyme. With these kinetic parameters (excluding CYP1A1) we found that CYP1A2 and 2E1 were the major enzymes catalyzing 7-ethoxycoumarin; the contributions of these two forms were dependent on the contents of these CYPs in liver microsomes of different humans. Similarly, Chlorzoxazone 6-hydroxylation activities of liver microsomes were predicted with kinetic parameters of recombinant human CYP enzymes and it was found that CYP3A4 as well as CYP1A2 and 2E1 were involved in Chlorzoxazone hydroxylation, depending on the contents of these CYP forms in the livers. Recombinant CYP2A6 and 2B6 and CYP2D6 had considerable roles ( V max / K m ratio) for 7-ethoxycoumarin O -deethylation and Chlorzoxazone 6-hydroxylation, respectively; however, these CYP forms had relatively minor roles in the reactions, probably due to low expression in human livers. These results support the view that the roles of individual CYP enzymes in the oxidation of xenobiotic chemicals in human liver microsomes could be predicted by kinetic parameters of individual CYP enzymes and by the levels of each of the CYP enzymes in liver microsomes of human samples.

  • Prediction of human liver microsomal oxidations of 7-ethoxycoumarin and Chlorzoxazone using kinetic parameters of recombinant cytochrome P450 enzymes. Drug Metab Dispos 27:1274–1280.
    1999
    Co-Authors: Tsutomu Shimada, Fujiko Tsumura, Hiroshi Yamazaki
    Abstract:

    This paper is available online at http://www.dmd.org ABSTRACT: Different roles of individual forms of human cytochrome P-450 (CYP) in the oxidation of 7-ethoxycoumarin and Chlorzoxazone were investigated in liver microsomes of different human samples, and the microsomal activities thus obtained were predicted with kinetic parameters obtained from cDNA-derived recombinant CYP enzymes in microsomes of Trichoplusia ni cells. Of 14 forms of recombinant CYP examined, CYP1A1 had the highest activities (V max /K m ratio) in catalyzing 7-ethoxycoumarin O-deethylation followed by CYP1A2, 2E1, 2A6, and 2B6, although CYP1A1 has been shown to be an extrahepatic enzyme. With these kinetic parameters (excluding CYP1A1) we found that CYP1A2 and 2E1 were the major enzymes catalyzing 7-ethoxycoumarin; the contributions of these two forms were dependent on the contents of these CYPs in liver microsomes of different humans. Similarly, Chlorzoxazone 6-hydroxylation activities of liver microsomes were predicted with kinetic parameters of recombinant human CYP enzymes and it was found that CYP3A4 as well as CYP1A2 and 2E1 were involved in Chlorzoxazone hydroxylation, depending on the contents of these CYP forms in the livers. Recombinant CYP2A6 and 2B6 and CYP2D6 had considerable roles (V max /K m ratio) for 7-ethoxycoumarin O-deethylation and Chlorzoxazone 6-hydroxylation, respectively; however, these CYP forms had relatively minor roles in the reactions, probably due to low expression in human livers. These results support the view that the roles of individual CYP enzymes in the oxidation of xenobiotic chemicals in human liver microsomes could be predicted by kinetic parameters of individual CYP enzymes and by the levels of each of the CYP enzymes in liver microsomes of human samples. Multiple forms of cytochrome P-450 (CYP) 2 exist in liver microsomes and these CYP forms play important roles in the oxidation of structurally diverse xenobiotic chemicals such as drugs, toxic chemicals, and carcinogens as well as endobiotic chemicals, including steroids, fatty acids, fat-soluble vitamins, and prostaglandins Several studies have reported that two or more CYP enzymes are able to oxidize xenobiotic and endobiotic chemicals at the same position of the molecules with different affinities (i.e., V max /K m ratio) in human liver microsomes 1274 predicted with kinetic parameters of recombinant enzymes and the levels of liver microsomal CYP2C19 and 3A4 enzymes Materials and Methods Chemicals. 7-Ethoxycoumarin was obtained from Aldrich Chemical Co. (Milwaukee, WI) and 7-hydroxycoumarin from Katayama Chemical Co. (Osaka, Japan). Chlorzoxazone and its 6-hydroxylated metabolite were donated by Dr. F. P. Guengerich of Vanderbilt University. Other reagents and chemicals used in this study were obtained from sources as described previously or were of highest qualities commercially available Enzyme Preparation. Human liver samples were obtained from organ donors or patients undergoing liver resection as described previously Recombinant CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 3A4, 3A5, and 4A11 expressed in microsomes of T. ni cells infected with a baculovirus containing human CYP and NADPH-CYP reductase cDNA inserts were obtained from Gentest Co. Enzyme Assays. 7-Ethoxycoumarin O-deethylation activities by CYP enzymes were determined by HPLC as described Incubation mixtures (final volume of 0.20 ml) for Chlorzoxazone 6-hydroxylation were the same as for the assay of 7-ethoxycoumarin Odeethylation, except that substrate was replaced by Chlorzoxazone. Reactions were carried out at 37°C for 10 min and terminated by adding a mixture of 1.5 ml of CH 2 Cl 2 and 25 l of 43% H 3 PO 4 . The organic layer, after collecting by centrifugation, was evaporated to dryness under nitrogen atmosphere and the materials were dissolved in 200 l of 27% CH 3 CN containing 0.5% H 3 PO 4 . Product formation was determined by HPLC with a 4.6 ϫ 150 mm Nucleosil octylsilyl (C 8 ) reversed phase column (Chemco Scientific, Osaka, Japan). CYP contents were estimated spectrally by the original method Statistical Analysis. Kinetic parameters for 7-ethoxycoumarin O-deethylation and Chlorzoxazone 6-hydroxylation by human CYP enzymes were estimated with a computer program (KaleidaGraph; Synergy Software, Reading, PA) designed for nonlinear regression analysis. Prediction of 7-Ethoxycoumarin O-Deethylation and Chlorzoxazone 6-Hydroxylation by Human Liver Microsomes Based on Kinetic Parameters of Recombinant CYP Enzymes. To define the potential roles of individual forms of CYP enzymes in the oxidation of 7-ethoxycoumarin and Chlorzoxazone by different human samples, we calculated the predicted activities of human liver microsomal 7-ethoxycoumarin O-deethylation and Chlorzoxazone 6-hydroxylation activities with kinetic parameters of recombinant human CYP enzymes and estimated contents of these CYP proteins by immunoblotting. The equation used for the prediction of expected activities was obtained from the previous method Results Kinetic Analysis of 7-Ethoxycoumarin O-Deethylation and Chlorzoxazone 6-Hydroxylation by 14 Forms of Recombinant Human CYPs. Kinetic parameters for 7-ethoxycoumarin O-deethylation and Chlorzoxazone 6-hydroxylation by 14 forms of recombinant human CYP enzymes (in microsomes of T. ni cells) were determined as described in Materials and Methods K m values for 7-ethoxycoumarin O-deethylation were the lowest in CYP1A1 followed by CYP1B1, CYP1A2, CYP2A6, CYP2E1, and CYP2C9 Of 14 forms of CYP examined, CYP2E1, 1A1, 2D6, 1A2, and 3A4 catalyzed Chlorzoxazone 6-hydroxylation at varying rates, whereas other enzymes, including CYP1B1, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 3A5, and 4A11 gave very low or undetectable rates for the hydroxylation Prediction of 7-Ethoxycoumarin O-Deethylation and Chlorzoxazone 6-Hydroxylation by Human Liver Microsomes with Kinetic Parameters of Recombinant CYP Enzymes. We examined the 7-ethoxycoumarin O-deethylation and Chlorzoxazone 6-hydroxylation activities catalyzed by human liver microsomes with the kinetic parameters of recombinant human CYP enzymes described above and the estimated contents of immunochemically determined CYPs in human liver microsomes The levels of individual forms of CYP, including CYP1A2, CYP2A6, CYP2B6, CYP2C, CYP2D6, CYP2E1, and CYP3A in liver microsomes of 16 Japanese and 8 Caucasian samples were determined by Western immunoblotting analysi

  • reconstitution of recombinant cytochrome p450 2c10 2c9 and comparison with cytochrome p450 3a4 and other forms effects of cytochrome p450 p450 and cytochrome p450 b5 interactions
    Archives of Biochemistry and Biophysics, 1997
    Co-Authors: Hiroshi Yamazaki, Elizabeth M J Gillam, F P Guengerich, Misook Dong, William W Johnson, Tsutomu Shimada
    Abstract:

    Tolbutamide methyl hydroxylation and S-warfarin 7-hydroxylation activities were reconstituted in systems containing recombinant human cytochrome P450 (P450 or CYP) 2C10(2C9) and the optimal conditions for the systems were compared with those of bufuralol 1'-hydroxylation by CYP1A1, theophylline 8-hydroxylation by CYP1A2, bufuralol 1'-hydroxylation by CYP2D6, Chlorzoxazone 6-hydroxylation by CYP2E1, and testosterone 6 beta-hydroxylation by CYP3A4. CYP2C10 required cytochrome b5 (b5) for optimal rates of tolbutamide and S-warfarin oxidations and b5 could be replaced by apo-b5; apo-b5 and b5 effects on the reconstituted systems have already been reported in systems containing CYP3A4 for the oxidation of testosterone and nifedipine and for the rapid reduction of CYP3A4 by NADPH-P450 reductase (H. Yamazaki et al., 1996, J. Biol. Chem. 271, 27438-27444). Stopped-flow studies, however, suggested that apo-b5 as well as b5 did not cause stimulation of the reduction of CYP2C10 by NADPH-P450 reductase, while the reduction rates were dependent on the substrates in reconstituted systems. Chlorzoxazone 6-hydroxylation by CYP2E1 was stimulated by b5, but not by apo-b5, in reconstituted systems. Neither apo- nor holo-b5 increased bufuralol 1'-hydroxylation activity by CYP1A1 or 2D6 or theophylline 8-hydroxylation by CYP1A2. Interestingly, we found that testosterone 6 beta-hydroxylation by CYP3A4 was stimulated by CYP1A2 (and also by a modified form in which the first 36 residues of the native human protein were removed) and CYP1A1 as well as by b5, and such stimulations were not seen when other P450 proteins (e.g., CYP2C10, 2D6, or 2E1) were added to the reconstituted systems. In contrast, substrate oxidations by CYP2C10 and CYP2E1 were not stimulated by other P450 proteins. The present results suggest that there are differences in optimal conditions for reconstitution of substrate oxidations by various forms of human P450 enzymes, and in some P450-catalyzed reactions protein-protein interactions between P450 and b5 and other P450 proteins are very important in some oxidations catalyzed by CYP2C10, 2E1, and 3A4.

  • requirements for cytochrome b5 in the oxidation of 7 ethoxycoumarin Chlorzoxazone aniline and n nitrosodimethylamine by recombinant cytochrome p450 2e1 and by human liver microsomes
    Biochemical Pharmacology, 1996
    Co-Authors: Hiroshi Yamazaki, Elizabeth M J Gillam, L C Bell, M Nakano, Tsutomu Shimada
    Abstract:

    NADH-dependent 7-ethoxycoumarin O-deethylation activities could be reconstituted in systems containing cytochrome b(5) (b(5)), NADH-b(5) reductase, and bacterial recombinant P450 2E1 in 100 mM potassium phosphate buffer (pH 7.4) containing a synthetic phospholipid mixture and cholate. Replacement of NADH-b(5) reductase with NADPH-P450 reductase yielded a 4-fold increase in 7-ethoxycoumarin O-deethylation activity, and further stimulation (similar to 1.5-fold) could be obtained when NADPH was used as an electron donor. Removal of b(5) from the NADH- and NADPH-supported systems caused a 90% loss of 7-ethoxycoumarin O-deethylation activities in the presence of NADPH-P450 reductase, but resulted in complete loss of the activities in the absence of NADPH-P450 reductase. K-m values were increased and V-max values were decreased for 7-ethoxycoumarin O-deethylation when b(5) was omitted from the NADPH-supported P450 2E1-reconstituted systems. Requirements for b(5) in P450 2E1 systems were also observed in Chlorzoxazone 6-hydroxylation, aniline p-hydroxylation, and N-nitrosodimethylamine N-demethylation. In human liver microsomes, NADH-dependent 7-ethoxycoumarin O-deethylation, Chlorzoxazone 6-hydroxylation, aniline P-hydroxylation, and N-nitrosodimethylamine N-demethylation activities were found to be about 55, 41, 33, and 50%, respectively, of those catalyzed by NADPH-supported systems. Anti-rat NADPH-P450 reductase immunoglobulin G inhibited 7-ethoxycoumarin O-deethylation activity catalyzed by human liver microsomes more strongly in NADPH- than NADH-supported reactions, while anti-human b(5) immunoglobulin G inhibited microsomal activities in both NADH- and NADPH-supported systems to similar extents. These results suggest that b(5) is an essential component in P450 2E1-catalyzed oxidations of several substrates used, that about 10% of the activities occur via P450 2E1 reduction by NADPH-P450 reductase in the absence of b(5), and that the NADH-supported system contributes, in part, to some reactions catalyzed by P450 2E1 in human liver microsomes.

Khaled M Houamed - One of the best experts on this subject based on the ideXlab platform.

  • modulation of recombinant small conductance ca 2 activated k channels by the muscle relaxant Chlorzoxazone and structurally related compounds
    Journal of Pharmacology and Experimental Therapeutics, 2001
    Co-Authors: Ying-jun Cao, J C Dreixler, J D Roizen, M T Roberts, Khaled M Houamed
    Abstract:

    Using the patch clamp technique we investigated the effects of the centrally acting muscle relaxant Chlorzoxazone and three structurally related compounds, 1-ethyl-2-benzimidazolinone (1-EBIO), zoxazolamine, and 1,3-dihydro-1-[2-hydroxy-5-(triflu oromethyl)phenyl]-5-(trifluoromethyl)-2 H -benzimidazol-2-one (NS 1619) on recombinant rat brain SK2 channels (rSK2 channels) expressed in HEK293 mammalian cells. SK channels are small conductance K+ channels normally activated by a rise in intracellular Ca2+ concentration; they modulate the electrical excitability in neurons and neuroendocrine cells. When applied externally, Chlorzoxazone, 1-EBIO, and zoxazolamine activated rSK2 channel currents in cells dialyzed with a nominally Ca2+-free intracellular solution. The activation was reversible, reproducible, and depended on the chemical structure and concentration. The order of potency was 1-EBIO > Chlorzoxazone > zoxazolamine. Activation of rSK2 channels by Chlorzoxazone, 1-EBIO, and zoxazolamine declined at higher drug concentrations. Zoxazolamine, when applied in combination with Chlorzoxazone or 1-EBIO, partially inhibited the rSK2 channel current responses, suggesting a partial-agonist mode of action. 1-EBIO failed to activate rSK2 channel currents when applied to excised inside-out membrane patches exposed to a Ca2+-free intracellular solution. In contrast, 1-EBIO activated rSK2 currents in a concentration-dependent manner when coapplied to the patches with a solution containing 20 nM free Ca2+. NS 1619 did not activate rSK2 channel currents; it inhibited rSK2 channel currents activated by the other three test compounds or by high intracellular Ca2+. We conclude that Chlorzoxazone and its derivatives act through a common mechanism to modulate rSK2 channels, and SK channel modulation in the brain may partly underlie the clinical effects of Chlorzoxazone.

  • Modulation of Recombinant Small-Conductance Ca21-Activated K1 Channels by the Muscle Relaxant Chlorzoxazone and Structurally Related Compounds
    2000
    Co-Authors: Ying-jun Cao, J C Dreixler, J D Roizen, M T Roberts, Khaled M Houamed
    Abstract:

    Using the patch clamp technique we investigated the effects of the centrally acting muscle relaxant Chlorzoxazone and three structurally related compounds, 1-ethyl-2-benzimidazolinone (1-EBIO), zoxazolamine, and 1,3-dihydro-1-[2-hydroxy-5-(triflu oromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2-one (NS 1619) on recombinant rat brain SK2 channels (rSK2 chan-nels) expressed in HEK293 mammalian cells. SK channels are small conductance K1 channels normally activated by a rise in intracellular Ca21 concentration; they modulate the electrical excitability in neurons and neuroendocrine cells. When applied externally, Chlorzoxazone, 1-EBIO, and zoxazolamine activated rSK2 channel currents in cells dialyzed with a nominally Ca21-free intracellular solution. The activation was reversible, repro-ducible, and depended on the chemical structure and concen

Elizabeth M J Gillam - One of the best experts on this subject based on the ideXlab platform.

  • reconstitution of recombinant cytochrome p450 2c10 2c9 and comparison with cytochrome p450 3a4 and other forms effects of cytochrome p450 p450 and cytochrome p450 b5 interactions
    Archives of Biochemistry and Biophysics, 1997
    Co-Authors: Hiroshi Yamazaki, Elizabeth M J Gillam, F P Guengerich, Misook Dong, William W Johnson, Tsutomu Shimada
    Abstract:

    Tolbutamide methyl hydroxylation and S-warfarin 7-hydroxylation activities were reconstituted in systems containing recombinant human cytochrome P450 (P450 or CYP) 2C10(2C9) and the optimal conditions for the systems were compared with those of bufuralol 1'-hydroxylation by CYP1A1, theophylline 8-hydroxylation by CYP1A2, bufuralol 1'-hydroxylation by CYP2D6, Chlorzoxazone 6-hydroxylation by CYP2E1, and testosterone 6 beta-hydroxylation by CYP3A4. CYP2C10 required cytochrome b5 (b5) for optimal rates of tolbutamide and S-warfarin oxidations and b5 could be replaced by apo-b5; apo-b5 and b5 effects on the reconstituted systems have already been reported in systems containing CYP3A4 for the oxidation of testosterone and nifedipine and for the rapid reduction of CYP3A4 by NADPH-P450 reductase (H. Yamazaki et al., 1996, J. Biol. Chem. 271, 27438-27444). Stopped-flow studies, however, suggested that apo-b5 as well as b5 did not cause stimulation of the reduction of CYP2C10 by NADPH-P450 reductase, while the reduction rates were dependent on the substrates in reconstituted systems. Chlorzoxazone 6-hydroxylation by CYP2E1 was stimulated by b5, but not by apo-b5, in reconstituted systems. Neither apo- nor holo-b5 increased bufuralol 1'-hydroxylation activity by CYP1A1 or 2D6 or theophylline 8-hydroxylation by CYP1A2. Interestingly, we found that testosterone 6 beta-hydroxylation by CYP3A4 was stimulated by CYP1A2 (and also by a modified form in which the first 36 residues of the native human protein were removed) and CYP1A1 as well as by b5, and such stimulations were not seen when other P450 proteins (e.g., CYP2C10, 2D6, or 2E1) were added to the reconstituted systems. In contrast, substrate oxidations by CYP2C10 and CYP2E1 were not stimulated by other P450 proteins. The present results suggest that there are differences in optimal conditions for reconstitution of substrate oxidations by various forms of human P450 enzymes, and in some P450-catalyzed reactions protein-protein interactions between P450 and b5 and other P450 proteins are very important in some oxidations catalyzed by CYP2C10, 2E1, and 3A4.

  • requirements for cytochrome b5 in the oxidation of 7 ethoxycoumarin Chlorzoxazone aniline and n nitrosodimethylamine by recombinant cytochrome p450 2e1 and by human liver microsomes
    Biochemical Pharmacology, 1996
    Co-Authors: Hiroshi Yamazaki, Elizabeth M J Gillam, L C Bell, M Nakano, Tsutomu Shimada
    Abstract:

    NADH-dependent 7-ethoxycoumarin O-deethylation activities could be reconstituted in systems containing cytochrome b(5) (b(5)), NADH-b(5) reductase, and bacterial recombinant P450 2E1 in 100 mM potassium phosphate buffer (pH 7.4) containing a synthetic phospholipid mixture and cholate. Replacement of NADH-b(5) reductase with NADPH-P450 reductase yielded a 4-fold increase in 7-ethoxycoumarin O-deethylation activity, and further stimulation (similar to 1.5-fold) could be obtained when NADPH was used as an electron donor. Removal of b(5) from the NADH- and NADPH-supported systems caused a 90% loss of 7-ethoxycoumarin O-deethylation activities in the presence of NADPH-P450 reductase, but resulted in complete loss of the activities in the absence of NADPH-P450 reductase. K-m values were increased and V-max values were decreased for 7-ethoxycoumarin O-deethylation when b(5) was omitted from the NADPH-supported P450 2E1-reconstituted systems. Requirements for b(5) in P450 2E1 systems were also observed in Chlorzoxazone 6-hydroxylation, aniline p-hydroxylation, and N-nitrosodimethylamine N-demethylation. In human liver microsomes, NADH-dependent 7-ethoxycoumarin O-deethylation, Chlorzoxazone 6-hydroxylation, aniline P-hydroxylation, and N-nitrosodimethylamine N-demethylation activities were found to be about 55, 41, 33, and 50%, respectively, of those catalyzed by NADPH-supported systems. Anti-rat NADPH-P450 reductase immunoglobulin G inhibited 7-ethoxycoumarin O-deethylation activity catalyzed by human liver microsomes more strongly in NADPH- than NADH-supported reactions, while anti-human b(5) immunoglobulin G inhibited microsomal activities in both NADH- and NADPH-supported systems to similar extents. These results suggest that b(5) is an essential component in P450 2E1-catalyzed oxidations of several substrates used, that about 10% of the activities occur via P450 2E1 reduction by NADPH-P450 reductase in the absence of b(5), and that the NADH-supported system contributes, in part, to some reactions catalyzed by P450 2E1 in human liver microsomes.

Mark Helfand - One of the best experts on this subject based on the ideXlab platform.

  • Comparative efficacy and safety of skeletal muscle relaxants for spasticity and musculoskeletal conditions: a systematic review
    J Pain Symptom Manage, 2004
    Co-Authors: Roger Chou, Kim Peterson, Mark Helfand
    Abstract:

    Skeletal muscle relaxants are a heterogeneous group of medications used to treat two different types of underlying conditions: spasticity from upper motor neuron syndromes and muscular pain or spasms from peripheral musculoskeletal conditions. Although widely used for these indications, there appear to be gaps in our understanding of the comparative efficacy and safety of different skeletal muscle relaxants. This systematic review summarizes and assesses the evidence for the comparative efficacy and safety of skeletal muscle relaxants for spasticity and musculoskeletal conditions. Randomized trials (for comparative efficacy and adverse events) and observational studies (for adverse events only) that included oral medications classified as skeletal muscle relaxants by the FDA were sought using electronic databases, reference lists, and pharmaceutical company submissions. Searches were performed through January 2003. The validity of each included study was assessed using a data abstraction form and predefined criteria. An overall grade was allocated for the body of evidence for each key question. A total of 101 randomized trials were included in this review. No randomized trial was rated good quality, and there was little evidence of rigorous adverse event assessment in included trials or observational studies. There is fair evidence that baclofen, tizanidine, and dantrolene are effective compared to placebo in patients with spasticity (primarily multiple sclerosis). There is fair evidence that baclofen and tizanidine are roughly equivalent for efficacy in patients with spasticity, but insufficient evidence to determine the efficacy of dantrolene compared to baclofen or tizanidine. There is fair evidence that although the overall rate of adverse effects between tizanidine and baclofen is similar, tizanidine is associated with more dry mouth and baclofen with more weakness. There is fair evidence that cyclobenzaprine, carisoprodol, orphenadrine, and tizanidine are effective compared to placebo in patients with musculoskeletal conditions (primarily acute back or neck pain). Cyclobenzaprine has been evaluated in the most clinical trials and has consistently been found to be effective. There is very limited or inconsistent data regarding the effectiveness of metaxalone, methocarbamol, Chlorzoxazone, baclofen, or dantrolene compared to placebo in patients with musculoskeletal conditions. There is insufficient evidence to determine the relative efficacy or safety of cyclobenzaprine, carisoprodol, orphenadrine, tizanidine, metaxalone, methocarbamol, and Chlorzoxazone. Dantrolene, and to a lesser degree Chlorzoxazone, have been associated with rare serious hepatotoxicity.