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F P Guengerich - One of the best experts on this subject based on the ideXlab platform.
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kinetic deuterium isotope effects for 7 alkoxycoumarin o dealkylation reactions catalyzed by human cytochromes p450 and in liver microsomes rate limiting c h bond breaking in cytochrome p450 1a2 substrate oxidation
FEBS Journal, 2006Co-Authors: Keonhee Kim, Donghyun Kim, Chulho Yun, Emre M Isin, F P GuengerichAbstract:7-Ethoxy (OEt) coumarin has been used as a model substrate in many cytochrome P450 (P450) studies, including the use of kinetic isotope effects to probe facets of P450 kinetics. P450s 1A2 and 2E1 are known to be the major catalysts of 7-OEt coumarin O-deethylation in human liver microsomes. Human P450 1A2 also catalyzed 3-hydroxylation of 7-methoxy (OMe) coumarin at appreciable rates but P450 2E1 did not. Intramolecular kinetic isotope effects were used as estimates of the intrinsic kinetic deuterium isotope effects for both 7-OMe and 7-OEt coumarin dealkylation reactions. The apparent intrinsic isotope effect for P450 1A2 (9.4 for O-Demethylation, 6.1 for O-deethylation) showed little attenuation in other competitive and noncompetitive experiments. With P450 2E1, the intrinsic isotope effect (9.6 for O-Demethylation, 6.1 for O-deethylation) was attenuated in the noncompetitive intermolecular experiments. High noncompetitive intermolecular kinetic isotope effects were seen for 7-OEt coumarin O-deethylation in a baculovirus-based microsomal system and five samples of human liver microsomes (7.3-8.1 for O-deethylation), consistent with the view that P450 1A2 is the most efficient P450 catalyzing this reaction in human liver microsomes and indicating that the C-H bond-breaking step makes a major contribution to the rate of this P450 (1A2) reaction. Thus, the rate-limiting step appears to be the chemistry of the breaking of this bond by the activated iron-oxygen complex, as opposed to steps involved in the generation of the reactive complex. The conclusion about the rate-limiting step applies to all of the systems studied with this model P450 1A2 reaction including human liver microsomes, the most physiologically relevant.
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evidence for a 1 electron oxidation mechanism in n dealkylation of n n dialkylanilines by cytochrome p450 2b1 kinetic hydrogen isotope effects linear free energy relationships comparisons with horseradish peroxidase and studies with oxygen surrogates
Journal of Biological Chemistry, 1996Co-Authors: F P Guengerich, Chulho Yun, Timothy L MacdonaldAbstract:Many enzymes catalyze N-dealkylations of alkylamines, including cytochrome P450 (P450) and peroxidase enzymes. Peroxidases, exemplified by horseradish peroxidase (HRP), are generally accepted to catalyze N-dealkylations via 1-electron transfer processes. Several lines of evidence also support a 1-electron mechanism for many P450 reactions, although this view has been questioned in light of reported trends for kinetic hydrogen isotope effects for N-Demethylation with a series of 4-substituted N,N-dimethylanilines. No continuous trend for an increase of isotope effects with the electronic parameters of para-substitution was seen for the P450 2B1-catalyzed reactions in this study. The larger value seen with the 4-nitro derivative is consistent with a shift in mechanism due to either a reversible electron transfer step preceding deprotonation or to a hydrogen atom abstraction mechanism. With HRP, the trend is to lower isotope effects with para electron-withdrawing substituents, due to an apparent shift in rate-limiting steps. Biomimetic model high-valent porphyrins showed reduction rates with variously 4-substituted N,N-dialkylanilines that were consistent with a positively charged intermediate; such relationships were not seen for anisole O-Demethylation with P450 2B1. In contrast to the case with the NADPH-supported P450 reactions, high deuterium isotope effects ( approximately 7) were seen in the N-dealkylations supported by the oxygen surrogate iodosylbenzene. With iodosylbenzene, colored aminium radicals were observed in the oxidations of aminopyrine, N,N-dimethyl-4-aminothioanisole, and 4-methoxy-N,N-dimethylaniline. With the latter compound, a substantial intermolecular deuterium isotope effect was observed for N-Demethylation. In the N-dealkylation of N-ethyl,N-methylaniline by P450 2B1 (NADPH-supported), the ratio of N-Demethylation to N-deethylation was 16. Although it is probably possible for P450s to catalyze amine N-dealkylations via hydrogen atom abstraction when such a course is electronically or sterically favored, we interpret the evidence to favor a 1-electron pathway with N,N-dialkylamines with P450 2B1 as well as HRP and several biomimetic models.
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evidence for a 1 electron oxidation mechanism in n dealkylation of n n dialkylanilines by cytochrome p450 2b1 kinetic hydrogen isotope effects linear free energy relationships comparisons with horseradish peroxidase and studies with oxygen surrogates
Journal of Biological Chemistry, 1996Co-Authors: F P Guengerich, Chulho Yun, Timothy L MacdonaldAbstract:Many enzymes catalyze N-dealkylations of alkylamines, including cytochrome P450 (P450) and peroxidase enzymes. Peroxidases, exemplified by horseradish peroxidase (HRP), are generally accepted to catalyze N-dealkylations via 1-electron transfer processes. Several lines of evidence also support a 1-electron mechanism for many P450 reactions, although this view has been questioned in light of reported trends for kinetic hydrogen isotope effects for N-Demethylation with a series of 4-substituted N,N-dimethylanilines. No continuous trend for an increase of isotope effects with the electronic parameters of para-substitution was seen for the P450 2B1-catalyzed reactions in this study. The larger value seen with the 4-nitro derivative is consistent with a shift in mechanism due to either a reversible electron transfer step preceding deprotonation or to a hydrogen atom abstraction mechanism. With HRP, the trend is to lower isotope effects with para electron-withdrawing substituents, due to an apparent shift in rate-limiting steps. Biomimetic model high-valent porphyrins showed reduction rates with variously 4-substituted N,N-dialkylanilines that were consistent with a positively charged intermediate; such relationships were not seen for anisole O-Demethylation with P450 2B1. In contrast to the case with the NADPH-supported P450 reactions, high deuterium isotope effects (∼7) were seen in the N-dealkylations supported by the oxygen surrogate iodosylbenzene. With iodosylbenzene, colored aminium radicals were observed in the oxidations of aminopyrine, N,N-dimethyl-4-aminothioanisole, and 4-methoxy-N,N-dimethylaniline. With the latter compound, a substantial intermolecular deuterium isotope effect was observed for N-Demethylation. In the N-dealkylation of N-ethyl,N-methylaniline by P450 2B1 (NADPH-supported), the ratio of N-Demethylation to N-deethylation was 16. Although it is probably possible for P450s to catalyze amine N-dealkylations via hydrogen atom abstraction when such a course is electronically or sterically favored, we interpret the evidence to favor a 1-electron pathway with N,N-dialkylamines with P450 2B1 as well as HRP and several biomimetic models.
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Expression of Cytochrome-P450-3A5 in Escherichia-Coli - Effects of 5' Modification, Purification, Spectral Characterization, Reconstitution Conditions, and Catalytic Activities (Vol 317, Pg 374, 1995)
'Elsevier BV', 1995Co-Authors: Gillam Emj, Reilly Peb, Zy Guo, Yf Ueng, Yamazaki H, Wd Hooper, F P GuengerichAbstract:Cytochrome P450 (P450) 3A5 is a human enzyme with 85% amino acid sequence identity to the more predominantly expressed P450 3A4 and has been reported to have overlapping catalytic specificity. The 5'-terminus of a P450 3A5 cDNA was modified for optimal expression in Escherichia coli using the vector pCW, by aligning the MALLLAVFL N-terminal sequence of recombinant bovine P450 17A (H. J. Barnes, M. P. Arlotto, and M. R. Waterman, (1991) Proc. Natl. Acad. Sci. USA 88, 5597-5601) to the 3A5 cDNA. Two constructs were made, differing by their identity with the modified 3A4 N-terminal sequence (E. M. J. Gillam, T. Baba, B-R. Rim, S. Ohmori, and F. P. Guengerich, (1993) Arch. Biochem. Biophys. 305, 123-131). The first modified sequence (3A5#1) was identical to recombinant P450 3A4 up to codon 15, the 3A5 sequence being introduced thereafter. In the other (3A5#2), the successful 3A4 N-terminal nucleotide sequence was attached to codon 30. The yield was greater than fourfold higher in the first construct [up to 260 nmol (liter culture)(-1)]. The recombinant P450 3A5 (construct 1) was purified to electrophoretic homogeneity using a variation of a three-step procedure developed previously for P450 3A4, with an overall yield of similar to 40%. Purified P450 3A5 was active in nifedipine oxidation, testosterone 6 beta-hydroxylation, aflatoxin 3 alpha-hydroxylation and 8,9-epoxidation, N-ethylmorphine N-Demethylation, erythromycin N-Demethylation, and d-benzphetamine N-Demethylation. The reconstitution of nifedipine oxidation, testosterone 6 beta-hydroxylation, and the aflatoxin oxidation activities showed dependence upon the presence of cytochrome b(5), divalent cations, phospholipid mixtures, glutathione, and cholate similar to that previously found for purified P450 3A4. However, rates of the N-Demethylations of N-ethylmorphine, erythromycin, and d-benzphetamine were as high or higher for P450 3A5 than P450 3A4 and were not particularly dependent upon modifications of reconstitution systems. (C) 1995 Academic Press, Inc
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induction of cytochrome p450 dependent monooxygenases in hamster tissues by fasting
Toxicology and Applied Pharmacology, 1993Co-Authors: Tzuuhuei Ueng, Taliang Chen, Yungfang Ueng, S S Park, M Iwasaki, F P GuengerichAbstract:Abstract The effects of fasting on liver, kidney, and lung monooxygenases were studied using hamsters starved for 4 days. Fasting treatment increased microsomal cytochrome P450 content and NADPH-cytochrome P450 reductase activity in kidney and lung. The treatment caused significant increases of aniline hydroxylation, N-nitrosodimethylamine Demethylation, and 7-ethoxycoumarin 0-deethylation activities in the liver, kidney, and lung. Fasting caused a threefold increase of benzphetamine N-Demethylation activity in lung and a 25% increase in liver and had no effect in kidney. Benzo[a]pyrene hydroxylation activities in the fasted hamster liver, kidney, and lung were higher, lower, and similar to the controls, respectively. Gel electrophoresis of tissue microsomes from control and fasted hamsters revealed that fasting enhanced the intensity of protein band(s) in the P450 molecular weight region. Immunoblotting of the microsomal proteins showed that fasting induced a protein cross-reactive with rabbit antibody raised against human P450 2E1 in hamster liver, kidney, and lung. Immunoblotting analysis using mouse monoclonal antibody 2-66-3 raised against rat P450 2B1 revealed that fasting induced an immunorelated protein preferentially in hamster lung, with minimal effects on liver and kidney. Protein blots probed with mouse monoclonal antibody 1-12-3 indicated that fasting induced a protein related to P450 1A1 in hamster liver, kidney, and lung. These results demonstrate that fasting causes a variety of inductive effects on the enzyme components and catalytic activities of monooxygenase systems as well as on the P450s 2E, 2B, and 1A in the hamster tissues.
Timothy L Macdonald - One of the best experts on this subject based on the ideXlab platform.
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evidence for a 1 electron oxidation mechanism in n dealkylation of n n dialkylanilines by cytochrome p450 2b1 kinetic hydrogen isotope effects linear free energy relationships comparisons with horseradish peroxidase and studies with oxygen surrogates
Journal of Biological Chemistry, 1996Co-Authors: F P Guengerich, Chulho Yun, Timothy L MacdonaldAbstract:Many enzymes catalyze N-dealkylations of alkylamines, including cytochrome P450 (P450) and peroxidase enzymes. Peroxidases, exemplified by horseradish peroxidase (HRP), are generally accepted to catalyze N-dealkylations via 1-electron transfer processes. Several lines of evidence also support a 1-electron mechanism for many P450 reactions, although this view has been questioned in light of reported trends for kinetic hydrogen isotope effects for N-Demethylation with a series of 4-substituted N,N-dimethylanilines. No continuous trend for an increase of isotope effects with the electronic parameters of para-substitution was seen for the P450 2B1-catalyzed reactions in this study. The larger value seen with the 4-nitro derivative is consistent with a shift in mechanism due to either a reversible electron transfer step preceding deprotonation or to a hydrogen atom abstraction mechanism. With HRP, the trend is to lower isotope effects with para electron-withdrawing substituents, due to an apparent shift in rate-limiting steps. Biomimetic model high-valent porphyrins showed reduction rates with variously 4-substituted N,N-dialkylanilines that were consistent with a positively charged intermediate; such relationships were not seen for anisole O-Demethylation with P450 2B1. In contrast to the case with the NADPH-supported P450 reactions, high deuterium isotope effects ( approximately 7) were seen in the N-dealkylations supported by the oxygen surrogate iodosylbenzene. With iodosylbenzene, colored aminium radicals were observed in the oxidations of aminopyrine, N,N-dimethyl-4-aminothioanisole, and 4-methoxy-N,N-dimethylaniline. With the latter compound, a substantial intermolecular deuterium isotope effect was observed for N-Demethylation. In the N-dealkylation of N-ethyl,N-methylaniline by P450 2B1 (NADPH-supported), the ratio of N-Demethylation to N-deethylation was 16. Although it is probably possible for P450s to catalyze amine N-dealkylations via hydrogen atom abstraction when such a course is electronically or sterically favored, we interpret the evidence to favor a 1-electron pathway with N,N-dialkylamines with P450 2B1 as well as HRP and several biomimetic models.
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evidence for a 1 electron oxidation mechanism in n dealkylation of n n dialkylanilines by cytochrome p450 2b1 kinetic hydrogen isotope effects linear free energy relationships comparisons with horseradish peroxidase and studies with oxygen surrogates
Journal of Biological Chemistry, 1996Co-Authors: F P Guengerich, Chulho Yun, Timothy L MacdonaldAbstract:Many enzymes catalyze N-dealkylations of alkylamines, including cytochrome P450 (P450) and peroxidase enzymes. Peroxidases, exemplified by horseradish peroxidase (HRP), are generally accepted to catalyze N-dealkylations via 1-electron transfer processes. Several lines of evidence also support a 1-electron mechanism for many P450 reactions, although this view has been questioned in light of reported trends for kinetic hydrogen isotope effects for N-Demethylation with a series of 4-substituted N,N-dimethylanilines. No continuous trend for an increase of isotope effects with the electronic parameters of para-substitution was seen for the P450 2B1-catalyzed reactions in this study. The larger value seen with the 4-nitro derivative is consistent with a shift in mechanism due to either a reversible electron transfer step preceding deprotonation or to a hydrogen atom abstraction mechanism. With HRP, the trend is to lower isotope effects with para electron-withdrawing substituents, due to an apparent shift in rate-limiting steps. Biomimetic model high-valent porphyrins showed reduction rates with variously 4-substituted N,N-dialkylanilines that were consistent with a positively charged intermediate; such relationships were not seen for anisole O-Demethylation with P450 2B1. In contrast to the case with the NADPH-supported P450 reactions, high deuterium isotope effects (∼7) were seen in the N-dealkylations supported by the oxygen surrogate iodosylbenzene. With iodosylbenzene, colored aminium radicals were observed in the oxidations of aminopyrine, N,N-dimethyl-4-aminothioanisole, and 4-methoxy-N,N-dimethylaniline. With the latter compound, a substantial intermolecular deuterium isotope effect was observed for N-Demethylation. In the N-dealkylation of N-ethyl,N-methylaniline by P450 2B1 (NADPH-supported), the ratio of N-Demethylation to N-deethylation was 16. Although it is probably possible for P450s to catalyze amine N-dealkylations via hydrogen atom abstraction when such a course is electronically or sterically favored, we interpret the evidence to favor a 1-electron pathway with N,N-dialkylamines with P450 2B1 as well as HRP and several biomimetic models.
Chulho Yun - One of the best experts on this subject based on the ideXlab platform.
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kinetic deuterium isotope effects for 7 alkoxycoumarin o dealkylation reactions catalyzed by human cytochromes p450 and in liver microsomes rate limiting c h bond breaking in cytochrome p450 1a2 substrate oxidation
FEBS Journal, 2006Co-Authors: Keonhee Kim, Donghyun Kim, Chulho Yun, Emre M Isin, F P GuengerichAbstract:7-Ethoxy (OEt) coumarin has been used as a model substrate in many cytochrome P450 (P450) studies, including the use of kinetic isotope effects to probe facets of P450 kinetics. P450s 1A2 and 2E1 are known to be the major catalysts of 7-OEt coumarin O-deethylation in human liver microsomes. Human P450 1A2 also catalyzed 3-hydroxylation of 7-methoxy (OMe) coumarin at appreciable rates but P450 2E1 did not. Intramolecular kinetic isotope effects were used as estimates of the intrinsic kinetic deuterium isotope effects for both 7-OMe and 7-OEt coumarin dealkylation reactions. The apparent intrinsic isotope effect for P450 1A2 (9.4 for O-Demethylation, 6.1 for O-deethylation) showed little attenuation in other competitive and noncompetitive experiments. With P450 2E1, the intrinsic isotope effect (9.6 for O-Demethylation, 6.1 for O-deethylation) was attenuated in the noncompetitive intermolecular experiments. High noncompetitive intermolecular kinetic isotope effects were seen for 7-OEt coumarin O-deethylation in a baculovirus-based microsomal system and five samples of human liver microsomes (7.3-8.1 for O-deethylation), consistent with the view that P450 1A2 is the most efficient P450 catalyzing this reaction in human liver microsomes and indicating that the C-H bond-breaking step makes a major contribution to the rate of this P450 (1A2) reaction. Thus, the rate-limiting step appears to be the chemistry of the breaking of this bond by the activated iron-oxygen complex, as opposed to steps involved in the generation of the reactive complex. The conclusion about the rate-limiting step applies to all of the systems studied with this model P450 1A2 reaction including human liver microsomes, the most physiologically relevant.
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evidence for a 1 electron oxidation mechanism in n dealkylation of n n dialkylanilines by cytochrome p450 2b1 kinetic hydrogen isotope effects linear free energy relationships comparisons with horseradish peroxidase and studies with oxygen surrogates
Journal of Biological Chemistry, 1996Co-Authors: F P Guengerich, Chulho Yun, Timothy L MacdonaldAbstract:Many enzymes catalyze N-dealkylations of alkylamines, including cytochrome P450 (P450) and peroxidase enzymes. Peroxidases, exemplified by horseradish peroxidase (HRP), are generally accepted to catalyze N-dealkylations via 1-electron transfer processes. Several lines of evidence also support a 1-electron mechanism for many P450 reactions, although this view has been questioned in light of reported trends for kinetic hydrogen isotope effects for N-Demethylation with a series of 4-substituted N,N-dimethylanilines. No continuous trend for an increase of isotope effects with the electronic parameters of para-substitution was seen for the P450 2B1-catalyzed reactions in this study. The larger value seen with the 4-nitro derivative is consistent with a shift in mechanism due to either a reversible electron transfer step preceding deprotonation or to a hydrogen atom abstraction mechanism. With HRP, the trend is to lower isotope effects with para electron-withdrawing substituents, due to an apparent shift in rate-limiting steps. Biomimetic model high-valent porphyrins showed reduction rates with variously 4-substituted N,N-dialkylanilines that were consistent with a positively charged intermediate; such relationships were not seen for anisole O-Demethylation with P450 2B1. In contrast to the case with the NADPH-supported P450 reactions, high deuterium isotope effects ( approximately 7) were seen in the N-dealkylations supported by the oxygen surrogate iodosylbenzene. With iodosylbenzene, colored aminium radicals were observed in the oxidations of aminopyrine, N,N-dimethyl-4-aminothioanisole, and 4-methoxy-N,N-dimethylaniline. With the latter compound, a substantial intermolecular deuterium isotope effect was observed for N-Demethylation. In the N-dealkylation of N-ethyl,N-methylaniline by P450 2B1 (NADPH-supported), the ratio of N-Demethylation to N-deethylation was 16. Although it is probably possible for P450s to catalyze amine N-dealkylations via hydrogen atom abstraction when such a course is electronically or sterically favored, we interpret the evidence to favor a 1-electron pathway with N,N-dialkylamines with P450 2B1 as well as HRP and several biomimetic models.
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evidence for a 1 electron oxidation mechanism in n dealkylation of n n dialkylanilines by cytochrome p450 2b1 kinetic hydrogen isotope effects linear free energy relationships comparisons with horseradish peroxidase and studies with oxygen surrogates
Journal of Biological Chemistry, 1996Co-Authors: F P Guengerich, Chulho Yun, Timothy L MacdonaldAbstract:Many enzymes catalyze N-dealkylations of alkylamines, including cytochrome P450 (P450) and peroxidase enzymes. Peroxidases, exemplified by horseradish peroxidase (HRP), are generally accepted to catalyze N-dealkylations via 1-electron transfer processes. Several lines of evidence also support a 1-electron mechanism for many P450 reactions, although this view has been questioned in light of reported trends for kinetic hydrogen isotope effects for N-Demethylation with a series of 4-substituted N,N-dimethylanilines. No continuous trend for an increase of isotope effects with the electronic parameters of para-substitution was seen for the P450 2B1-catalyzed reactions in this study. The larger value seen with the 4-nitro derivative is consistent with a shift in mechanism due to either a reversible electron transfer step preceding deprotonation or to a hydrogen atom abstraction mechanism. With HRP, the trend is to lower isotope effects with para electron-withdrawing substituents, due to an apparent shift in rate-limiting steps. Biomimetic model high-valent porphyrins showed reduction rates with variously 4-substituted N,N-dialkylanilines that were consistent with a positively charged intermediate; such relationships were not seen for anisole O-Demethylation with P450 2B1. In contrast to the case with the NADPH-supported P450 reactions, high deuterium isotope effects (∼7) were seen in the N-dealkylations supported by the oxygen surrogate iodosylbenzene. With iodosylbenzene, colored aminium radicals were observed in the oxidations of aminopyrine, N,N-dimethyl-4-aminothioanisole, and 4-methoxy-N,N-dimethylaniline. With the latter compound, a substantial intermolecular deuterium isotope effect was observed for N-Demethylation. In the N-dealkylation of N-ethyl,N-methylaniline by P450 2B1 (NADPH-supported), the ratio of N-Demethylation to N-deethylation was 16. Although it is probably possible for P450s to catalyze amine N-dealkylations via hydrogen atom abstraction when such a course is electronically or sterically favored, we interpret the evidence to favor a 1-electron pathway with N,N-dialkylamines with P450 2B1 as well as HRP and several biomimetic models.
Drew Fackett - One of the best experts on this subject based on the ideXlab platform.
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a comparison of aroclor 1254 induced and uninduced rat liver microsomes to human liver microsomes in phenytoin o deethylation coumarin 7 hydroxylation tolbutamide 4 hydroxylation s mephenytoin 4 hydroxylation chloroxazone 6 hydroxylation and testoste
Chemico-Biological Interactions, 2001Co-Authors: Judy Easterbrook, Drew Fackett, Albert P LiAbstract:Abstract Aroclor 1254-induced rat liver homogenate supernatant (liver S-9) is routinely used as an exogenous metabolic activation system for the evaluation of mutagenicity of xenobiotics. The purpose of this study is to evaluate whether results obtained with Aroclor 1254-induced liver microsomes would be relevant to human. Aroclor 1254-induced and uninduced rat liver microsomes were compared to human liver microsomes in the metabolism of substrates which are known to be selectively metabolized by the major human cytochrome P450 (CYP) isoforms. The activities studied and the major CYP isoforms involved were as follows: phenacetin O -deethylation (CYP1A2); coumarin 7-hydroxylation, (CYP2A6); tolbutamide 4-hydroxylation (CYP2C9), S- mephenytoin 4′-hydroxylation (CYP2C19); dextromethorphan O -Demethylation (CYP2D6); chloroxazone 6-hydroxylation (CYP2E1); and testosterone 6β-hydroxylation (CYP3A4). We found that both induced and uninduced rat liver microsomes were active in all the pathways studied with the exception of coumarin 7-hydroxylation. Coumarin 7-hydroxylation was observed with human liver microsomes but not the rat liver microsomes. Aroclor-1254 was found to induce all activities measured, with the exception of coumarin 7-hydroxylation. Dextromethorphan O -deethylation activity was higher in the rat liver microsomes than the human liver microsomes. Testosterone 6β-hydroxylation activity was found to be similar between the human liver microsomes and the induced rat liver microsomes. Our results suggest that experimental data obtained with Aroclor 1254-induced rat liver microsomes may not always be relevant to human.
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a comparison of aroclor 1254 induced and uninduced rat liver microsomes to human liver microsomes in phenytoin o deethylation coumarin 7 hydroxylation tolbutamide 4 hydroxylation s mephenytoin 4 hydroxylation chloroxazone 6 hydroxylation and testoste
Chemico-Biological Interactions, 2001Co-Authors: Judy Easterbrook, Drew FackettAbstract:Aroclor 1254-induced rat liver homogenate supernatant (liver S-9) is routinely used as an exogenous metabolic activation system for the evaluation of mutagenicity of xenobiotics. The purpose of this study is to evaluate whether results obtained with Aroclor 1254-induced liver microsomes would be relevant to human. Aroclor 1254-induced and uninduced rat liver microsomes were compared to human liver microsomes in the metabolism of substrates which are known to be selectively metabolized by the major human cytochrome P450 (CYP) isoforms. The activities studied and the major CYP isoforms involved were as follows: phenacetin O-deethylation (CYP1A2); coumarin 7-hydroxylation, (CYP2A6); tolbutamide 4-hydroxylation (CYP2C9), S-mephenytoin 4'-hydroxylation (CYP2C19); dextromethorphan O-Demethylation (CYP2D6); chloroxazone 6-hydroxylation (CYP2E1); and testosterone 6beta-hydroxylation (CYP3A4). We found that both induced and uninduced rat liver microsomes were active in all the pathways studied with the exception of coumarin 7-hydroxylation. Coumarin 7-hydroxylation was observed with human liver microsomes but not the rat liver microsomes. Aroclor-1254 was found to induce all activities measured, with the exception of coumarin 7-hydroxylation. Dextromethorphan O-deethylation activity was higher in the rat liver microsomes than the human liver microsomes. Testosterone 6beta-hydroxylation activity was found to be similar between the human liver microsomes and the induced rat liver microsomes. Our results suggest that experimental data obtained with Aroclor 1254-induced rat liver microsomes may not always be relevant to human.
Lisa L. Von Moltke - One of the best experts on this subject based on the ideXlab platform.
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inhibition of human cytochrome p450 isoforms by nonnucleoside reverse transcriptase inhibitors
The Journal of Clinical Pharmacology, 2001Co-Authors: Brian W. Granda, Lisa L. Von Moltke, David J. Greenblatt, Gina M. GiancarloAbstract:The capacity of three clinically available nonnucleoside reverse transcriptase inhibitors (NNRTIs) to inhibit the activity of human cytochromes P450 (CYPs) was studied in vitro using human liver microsomes. Delavirdine, nevirapine, and efavirenz produced negligible inhibition of phenacetin O-deethylation (CYP1A2) or dextromethorphan O-Demethylation (CYP2D6). Nevirapine did not inhibit hydroxylation of tolbutamide (CYP2C9) or S-mephenytoin (CYP2C19), but these CYP isoforms were importantly inhibited by delavirdine and efavirenz. This indicates the likelihood of significantly impaired clearance of CYP2C substrate drugs (such as phenytoin, tolbutamide, and warfarin) upon initial exposure to these two NNRTIs. Delavirdine and efavirenz (but not nevirapine) also were strong inhibitors of CYP3A, consistent with clinical hazards of initial cotreatment with either of these drugs and substrates of CYP3A. The in vitro microsomal model provides relevant predictive data on probable drug interactions with NNRTIs when the mechanism is inhibition of CYP-mediated drug biotransformation. However, the model does not incorporate interactions attributable to enzyme induction.
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Inhibition of human cytochrome P450 isoforms in vitro by zafirlukast.
Biopharmaceutics & drug disposition, 1999Co-Authors: Richard I. Shader, Brian W. Granda, Lisa L. Von Moltke, Gina M. Giancarlo, David J. GreenblattAbstract:Zafirlukast is a cysteinyl leukotriene antagonist used to treat allergic and exercise-induced asthma. This in vitro study used human liver microsomes to evaluate the inhibitory activity of zafirlukast versus six human cytochrome P450 (CYP) isoforms. Zafirlukast (0-250 microM) was co-incubated with fixed concentrations of index substrates. Zafirlukast inhibited the hydroxylation of tolbutamide (CYP2C9; mean IC(50)=7.0 microM), triazolam (CYP3A; IC(50)=20.9 microM) and S-mephenytoin (CYP2C19; IC(50)=32.7 microM), and was a less potent inhibitor of phenacetin O-deethylation (CYP1A2; IC(50)=56 microM) and dextromethorphan O-Demethylation (CYP2D6; IC(50)=116 microM). Zafirlukast produced negligible inhibition of CYP2E1. In vitro inhibition of CYP2C9 by zafirlukast is consistent with clinical studies showing impaired clearance of S-warfarin and enhanced anti-thrombotic effects, although the in vitro IC(50) value is higher than the usual range of clinically relevant plasma concentrations. Zafirlukast deserves further clinical study as an inhibitor of other CYP2C9 substrates such as nonsteroidal anti-inflammatory agents, tolbutamide, phenytoin and mestranol. Clinically important inhibition by zafirlukast of other CYP isoforms is not established.
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protease inhibitors as inhibitors of human cytochromes p450 high risk associated with ritonavir
The Journal of Clinical Pharmacology, 1998Co-Authors: Lisa L. Von Moltke, Jerold S Harmatz, Brian W. Granda, David J. Greenblatt, Jeffrey M Grassi, Su Xiang Duan, Steven M Fogelman, Johanna P Daily, Richard I. ShaderAbstract:Four protease inhibitor antiviral agents (ritonavir, indinavir, nelfinavir, saquinavir) were evaluated as in vitro inhibitors of the activity of six human cytochromes using an in vitro model based on human liver microsomes. Ritonavir was a highly potent inhibitor of P450-3A activity (triazolam hydroxylation), having inhibitory potency slightly less than ketoconazole. Indinavir was also a potent 3A inhibitor, while nelfinavir and saquinavir were less potent. Ritonavir had high inhibition potency against cytochrome P450-2C9 (tolbutamide hydroxylation), -2C19 (S-mephenytoin hydroxylation), and -2D6 (dextromethorphan O-Demethylation and desipramine hydroxylation), while the other protease inhibitors had one or more orders of magnitude lower inhibitory activity against these reactions. None of the protease inhibitors had important inhibitory potency against P450-1A2 (phenacetin O-deethylation) or -2E1 (chlorzoxazone hydroxylation). Thus, among available protease inhibitors, ritonavir carries the highest risk of incurring drug interactions due to inhibition of cytochrome P450 activity.