Open AccessJournal Article
Validation of Bupropion Hydroxylation as a Selective Marker of Human Cytochrome P450 2B6 Catalytic Activity
Stephanie R. Faucette,Roy L. Hawke,Edward L. LeCluyse,Stacy S. Shord,Bingfang Yan,Ronald M. Laethem,Celeste Lindley +6 more
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TLDR
Results demonstrate selectivity of BUP hydroxylation for CYP2B6 at 500 microM BUP, thereby validating its use as a diagnostic in vitro marker of CYP 2B6 catalytic activity.Abstract:
The purpose of this study was to establish bupropion (BUP) hydroxylation as a selective in vitro marker of cytochrome P450 (CYP) 2B6 catalytic activity. Among a panel of 16 human liver microsomes (HLMs), BUP hydroxylase activity varied 80-fold when assayed at 500 microM substrate and significantly correlated with CYP2B6 blotting density (r(2) = 0.99) and S-mephenytoin N-demethylase activity (r(2) = 0.98). Kinetic analysis of BUP hydroxylation was performed in a subset of seven HLMs representative of the 80-fold range in activity. Sigmoidal kinetics suggestive of allosteric activation was observed in five HLMs exhibiting low or high activity; the mean apparent K(m) for BUP hydroxylation in these HLMs (130 microM) was similar to the K(m) for cDNA-expressed CYP2B6 (156 microM). Nonsaturable, biphasic kinetics was observed in two HLMs exhibiting low activity. Among a panel of cDNA-expressed P450 isoforms, CYP2B6 and CYP2E1 demonstrated the highest rates of BUP hydroxylation at 12 mM BUP (7.0 and 2.4 pmol/min/pmol of P450, respectively). The relative contributions of CYP2B6 and CYP2E1 to BUP hydroxylation were estimated by using immunoinhibitory monoclonal antibodies (MAB) to these enzymes. MAB-2B6 produced 88% maximum inhibition of BUP hydroxylation when assayed at 12 mM BUP in a high activity HLM, whereas MAB-2E1 produced 81% maximum inhibition in a low activity HLM. However, negligible inhibition by MAB-2E1 was observed when low and high activity HLMs were assayed at 500 microM BUP. These results demonstrate selectivity of BUP hydroxylation for CYP2B6 at 500 microM BUP, thereby validating its use as a diagnostic in vitro marker of CYP2B6 catalytic activity.read more
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Cytochrome P450 enzymes in drug metabolism: Regulation of gene expression, enzyme activities, and impact of genetic variation
TL;DR: Recent progress on drug metabolism activity profiles, interindividual variability and regulation of expression, and the functional and clinical impact of genetic variation in drug metabolizing P450s are reviewed.
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Microscale culture of human liver cells for drug development
TL;DR: This work presents a miniaturized, multiwell culture system for human liver cells with optimized microscale architecture that maintains phenotypic functions for several weeks and demonstrates utility through assessment of gene expression profiles, phase I/II metabolism, canalicular transport, secretion of liver-specific products and susceptibility to hepatotoxins.
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Summary of information on human cyp enzymes: human p450 metabolism data
TL;DR: This chapter is an update of the data on substrates, reactions, inducers, and inhibitors of human CYP enzymes published previously by Rendic and DiCarlo, now covering selection of the literature through 2001 in the reference section.
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Primary Hepatocytes: Current Understanding of the Regulation of Metabolic Enzymes and Transporter Proteins, and Pharmaceutical Practice for the Use of Hepatocytes in Metabolism, Enzyme Induction, Transporter, Clearance, and Hepatotoxicity Studies
Nicola J. Hewitt,Maria Jose Gomez Lechon,J. Brian Houston,David Hallifax,Hayley S. Brown,Patrick Maurel,Patrick Maurel,J. Gerry Kenna,Lena Gustavsson,Christina Lohmann,Christian Skonberg,André Guillouzo,Gregor Tuschl,Albert P. Li,Edward L. LeCluyse,Geny M. M. Groothuis,Jan G. Hengstler +16 more
TL;DR: This review brings you up-to-date with the hepatocyte research on in vitro–in vivo correlations of metabolism and clearance, the function and regulation of hepatic transporters and models used to elucidate their role in drug clearance, mechanisms and examples of idiosyncratic and intrinsic hepatotoxicity.
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Polymorphism of human cytochrome P450 enzymes and its clinical impact
TL;DR: Current pharmacogenetic knowledge on important human drug-metabolizing cytochrome P450s (CYPs) is highlighted to understand the large interindividual variability in drug clearance and responses in clinical practice and to improve the efficacy and safety of both prospective and currently available drugs.
References
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Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.
TL;DR: A method has been devised for the electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets that results in quantitative transfer of ribosomal proteins from gels containing urea.
Journal Article
Interindividual variations in human liver cytochrome P-450 enzymes involved in the oxidation of drugs, carcinogens and toxic chemicals: studies with liver microsomes of 30 Japanese and 30 Caucasians.
TL;DR: The results presented in this study provide useful information for the study of drug biotransformation in humans and for the basis of drug toxicities, carcinogenesis and teratogenesis.
Journal ArticleDOI
Activation of CYP3A4: evidence for the simultaneous binding of two substrates in a cytochrome P450 active site.
Magang Shou,James Grogan,J. A. Mancewicz,Kristopher W. Krausz,Frank J. Gonzalez,Harry V. Gelboin,Kenneth R. Korzekwa +6 more
TL;DR: Kinetic analyses of these two substrates show that 7,8-benzoflavone increases the Vmax of phenanthrene metabolism without changing the Km and that Phenanthrene decreases the V max of 7, 8-benZ oflavone metabolism without increasing the KM, providing the first evidence that two different molecules can be simultaneously bound to the same P450 active site.
Journal Article
Development of a Substrate-Activity Based Approach To Identify the Major Human Liver P-450 Catalysts of Cyclophosphamide and Ifosfamide Activation Based on cDNA-Expressed Activities and Liver Microsomal P-450 Profiles
TL;DR: The significance of human liver CYP2B6 for the activation of the clinically important cancer chemotherapeutic prodrug CPA is established as well as the presence of 2B6 protein at a readily detectable level in all but one of 17 livers.
Journal ArticleDOI
Effects of freezing, thawing, and storing human liver microsomes on cytochrome P450 activity
Robin Pearce,C J McIntyre,Ajay Madan,U Sanzgiri,Alison J. Draper,Peter Bullock,D C Cook,L A Burton,J Latham,Cheryl Nevins,Andrew Parkinson +10 more
TL;DR: The results suggest that the current methods for storing and processing human liver are well suited to preserving microsomal P450 enzyme activity, and that long-term storage of human liver microsomes at -80 degrees C is well suited.