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John O Miners - One of the best experts on this subject based on the ideXlab platform.
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spironolactone and canrenone inhibit ugt2b7 catalyzed human liver and kidney microsomal aldosterone 18β Glucuronidation a potential drug interaction
Drug Metabolism and Disposition, 2010Co-Authors: Kathleen M. Knights, Kushari Bowalgaha, John O MinersAbstract:Elevated plasma concentrations of aldosterone (ALDO) are observed in patients treated with spironolactone. Because ALDO is eliminated via UGT2B7-catalyzed 18beta-Glucuronidation, this study aimed to determine whether spironolactone and its primary metabolites, canrenone and canrenoic acid, inhibit ALDO 18beta-Glucuronidation by recombinant UGT2B7 and by human liver (HLM) and human kidney cortical (HKCM) microsomes. Initial experiments characterized the effects of all three compounds on 4-methylumbelliferone and ALDO Glucuronidation by recombinant human UGT2B7. IC(50) values for spironolactone and canrenone ranged from 26 to 50 microM, whereas canrenoic acid was a weak inhibitor. Inhibitor constant (K(i)) values for spironolactone and canrenone inhibition of ALDO 18beta-Glucuronidation were subsequently determined with HLM, HKCM, and UGT2B7 as the enzyme sources. Spironolactone and canrenone were competitive inhibitors of ALDO 18beta-Glucuronidation by HLM, HKCM, and UGT2B7. Mean (+/-) K(i) values for spironolactone were 52 +/- 22 (HLM) and 34 +/- 4 microM (HKCM), and mean (+/-) K(i) values for canrenone were 41 +/- 19 (HLM) and 23 +/- 2 microM (HKCM). K(i) values for spironolactone and canrenone inhibition of ALDO 18beta-Glucuronidation by recombinant UGT2B7 were 23 and 11 microM, respectively. "Actual" K(i) values for spironolactone and canrenone inhibition of ALDO 18beta-Glucuronidation, which take into account the role of endogenous microsomal inhibitors, are predicted to be 3 to 5 and 2 to 4 microM, respectively. The data indicate that the elevated ALDO concentrations observed in patients treated with spironolactone may be due, at least in part, to a pharmacokinetic interaction, and spironolactone and canrenone should be considered to be potential inhibitors of the UGT2B7-mediated metabolic clearance of drugs in both liver and kidney.
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the prediction of drug Glucuronidation parameters in humans udp glucuronosyltransferase enzyme selective substrate and inhibitor probes for reaction phenotyping and in vitro in vivo extrapolation of drug clearance and drug drug interaction potential
Drug Metabolism Reviews, 2010Co-Authors: John O Miners, Peter I. Mackenzie, Kathleen M. KnightsAbstract:Major advances in the characterization of uridine diphosphate (UDP)-glucuronosyltransferase (UGT) enzyme substrate and inhibitor selectivities and the development of experimental paradigms to investigate xenobiotic Glucuronidation in vitro now permit the prediction of a range of drug-Glucuronidation parameters in humans. In particular, the availability of substrate and inhibitor “probes” for the major hepatic drug metabolizing UGTs together with batteries of recombinant enzymes allow the reaction phenotyping of drug Glucuronidation reactions. Additionally, in vitro experimental approaches and scaling strategies have been successfully applied to the quantitative prediction of in vivo clearance via Glucuronidation and drug-drug interaction potential.
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kinetic modeling of the interactions between 4 methylumbelliferone 1 naphthol and zidovudine Glucuronidation by udp glucuronosyltransferase 2b7 ugt2b7 provides evidence for multiple substrate binding and effector sites
Molecular Pharmacology, 2008Co-Authors: Verawan Uchaipichat, Brian J Houston, Peter I. Mackenzie, Andrew J Williams, Aleksandra Galetin, John O MinersAbstract:Interactions between the UGT2B7-catalyzed Glucuronidation of zidovudine (AZT), 4-methylumbelliferone (4MU), and 1-naphthol (1NP) were analyzed using multisite and empirical kinetic models to explore the existence of multiple substrate and effector binding sites within this important drug metabolizing enzyme. 4MU and 1NP Glucuronidation by UGT2B7 exhibit sigmoidal kinetics characteristic of homotropic cooperativity (autoactivation), which may be modeled assuming the existence of two equivalent, interacting substrate binding sites. In contrast, UGT2B7-catalyzed AZT Glucuronidation follows hyperbolic (Michaelis-Menten) kinetics. Although 4MU and 1NP decreased the binding affinity of AZT, the kinetics of AZT Glucuronidation changed from hyperbolic to sigmoidal in the presence of both modifiers. Data were well described by a generic two-substrate binding site model in which there is no interaction between the sites in the absence of 4MU or 1NP, but heterotropic cooperativity results from the binding of modifier. Inhibition of 4MU and 1NP Glucuronidation by AZT and interactions between 4MU and 1NP required more complex three-site models, where the modifier acts via a distinct effector site to alter either substrate binding affinity or Vmax without affecting the homotropic cooperativity characteristic of 4MU and 1NP Glucuronidation. It is noteworthy that 1NP inhibited 4MU Glucuronidation, whereas 4MU activated 1NP Glucuronidation. The results are consistent with the existence of two "catalytic" sites for each substrate within the UGT2B7 active site, along with multiple effector sites. The multiplicity of binding and effector sites results in complex kinetic interactions between UGT2B7 substrates, which potentially complicates inhibition screening studies.
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the albumin effect and drug Glucuronidation bovine serum albumin and fatty acid free human serum albumin enhance the Glucuronidation of udp glucuronosyltransferase ugt 1a9 substrates but not ugt1a1 and ugt1a6 activities
Drug Metabolism and Disposition, 2008Co-Authors: Andrew Rowland, Kathleen M. Knights, Peter I. Mackenzie, John O MinersAbstract:Bovine serum albumin (BSA) and fatty acid-free human serum albumin (HSAFAF) reduce the K m values for UGT2B7 substrates by sequestering inhibitory long-chain fatty acids released by incubations of human liver microsomes (HLM) and HEK293 cells expressing this enzyme. However, the scope of the “albumin effect” is unknown. In this investigation we characterized the effects of albumin on the kinetics of 4-methylumbelliferone (4MU) Glucuronidation by UDP-glucuronosyltransferase (UGT) 1A1, 1A6, and 1A9, and propofol (PRO) Glucuronidation by UGT1A9 and HLM. BSA and HSAFAF, but not human serum albumin, reduced the K m values for 4MU and PRO Glucuronidation by UGT1A9. For example, HSAFAF (2%) reduced the K m values for 4MU and PRO Glucuronidation from 13.4 to 2.9 and 41 to 7.2 μM, respectively. Similarly, HSAFAF (2%) reduced the K m for PRO Glucuronidation by HLM from 127 to 10.6 μM. Arachidonic, linoleic, and oleic acids and a mixture of these decreased the rates of 4MU and PRO Glucuronidation by UGT1A9. K m values for these reactions were increased 3- to 6-fold by the fatty acid mixture. Inhibition was reversed by the addition of BSA (2%). Extrapolation of kinetic constants for PRO Glucuronidation by HLM in the presence of HSAFAF predicted in vivo hepatic clearance within 15%. Fatty acids had no effect on 4MU Glucuronidation by UGT1A1 and UGT1A6 but, paradoxically, all forms of albumin altered the kinetic model for 4MU Glucuronidation by UGT1A6 (from Michaelis-Menten to two-site). Only BSA caused a similar effect on 4MU Glucuronidation by UGT1A1. It is concluded that BSA and HSAFAF reduce the K m values of only those enzymes inhibited by long-chain unsaturated fatty acids.
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the Glucuronidation of delta4 3 keto c19 and c21 hydroxysteroids by human liver microsomal and recombinant udp glucuronosyltransferases ugts 6alpha and 21 hydroxyprogesterone are selective substrates for ugt2b7
Drug Metabolism and Disposition, 2007Co-Authors: Kushari Bowalgaha, Kathleen M. Knights, Peter I. Mackenzie, David J Elliot, John O MinersAbstract:The stereo- and regioselective Glucuronidation of 10 Delta(4)-3-keto monohydroxylated androgens and pregnanes was investigated to identify UDP-glucuronosyltransferase (UGT) enzyme-selective substrates. Kinetic studies were performed using human liver microsomes (HLMs) and a panel of 12 recombinant human UGTs as the enzyme sources. Five of the steroids, which were hydroxylated in the 6beta-, 7alpha-, 11beta- or 17alpha-positions, were not glucuronidated by HLMs. Of the remaining compounds, comparative kinetic and inhibition studies indicated that 6alpha- and 21-hydroxyprogesterone (OHP) were glucuronidated selectively by human liver microsomal UGT2B7. 6alpha-OHP Glucuronidation by HLMs and UGT2B7 followed Michaelis-Menten kinetics, whereas 21-OHP Glucuronidation by these enzyme sources exhibited positive cooperativity. UGT2B7 was also identified as the enzyme responsible for the high-affinity component of human liver microsomal 11alpha-OHP Glucuronidation. In contrast, UGT2B15 and UGT2B17 were the major forms involved in human liver microsomal testosterone 17beta-Glucuronidation and the high-affinity component of 16alpha-OHP Glucuronidation. Activity of UGT1A subfamily enzymes toward the hepatically glucuronidated substrates was generally low, although UGT1A4 and UGT1A9 contribute to the low-affinity components of microsomal 16alpha- and 11alpha-OHP Glucuronidation, respectively. Interestingly, UGT1A10, which is expressed only in the gastrointestinal tract, exhibited activity toward most of the glucuronidated substrates. The results indicate that 6alpha- and 21-OHP may be used as selective "probes" for human liver microsomal UGT2B7 activity and, taken together, provide insights into the regio- and stereoselectivity of hydroxysteroid Glucuronidation by human UGTs.
Peter I. Mackenzie - One of the best experts on this subject based on the ideXlab platform.
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transcriptional regulation of human udp glucuronosyltransferase genes
Drug Metabolism Reviews, 2014Co-Authors: Robyn Meech, Ross A Mckinnon, Peter I. MackenzieAbstract:AbstractGlucuronidation is an important metabolic pathway for many small endogenous and exogenous lipophilic compounds, including bilirubin, steroid hormones, bile acids, carcinogens and therapeutic drugs. Glucuronidation is primarily catalyzed by the UDP-glucuronosyltransferase (UGT) 1A and two subfamilies, including nine functional UGT1A enzymes (1A1, 1A3–1A10) and 10 functional UGT2 enzymes (2A1, 2A2, 2A3, 2B4, 2B7, 2B10, 2B11, 2B15, 2B17 and 2B28). Most UGTs are expressed in the liver and this expression relates to the major role of hepatic Glucuronidation in systemic clearance of toxic lipophilic compounds. Hepatic Glucuronidation activity protects the body from chemical insults and governs the therapeutic efficacy of drugs that are inactivated by UGTs. UGT mRNAs have also been detected in over 20 extrahepatic tissues with a unique complement of UGT mRNAs seen in almost every tissue. This extrahepatic Glucuronidation activity helps to maintain homeostasis and hence regulates biological activity of en...
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the prediction of drug Glucuronidation parameters in humans udp glucuronosyltransferase enzyme selective substrate and inhibitor probes for reaction phenotyping and in vitro in vivo extrapolation of drug clearance and drug drug interaction potential
Drug Metabolism Reviews, 2010Co-Authors: John O Miners, Peter I. Mackenzie, Kathleen M. KnightsAbstract:Major advances in the characterization of uridine diphosphate (UDP)-glucuronosyltransferase (UGT) enzyme substrate and inhibitor selectivities and the development of experimental paradigms to investigate xenobiotic Glucuronidation in vitro now permit the prediction of a range of drug-Glucuronidation parameters in humans. In particular, the availability of substrate and inhibitor “probes” for the major hepatic drug metabolizing UGTs together with batteries of recombinant enzymes allow the reaction phenotyping of drug Glucuronidation reactions. Additionally, in vitro experimental approaches and scaling strategies have been successfully applied to the quantitative prediction of in vivo clearance via Glucuronidation and drug-drug interaction potential.
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kinetic modeling of the interactions between 4 methylumbelliferone 1 naphthol and zidovudine Glucuronidation by udp glucuronosyltransferase 2b7 ugt2b7 provides evidence for multiple substrate binding and effector sites
Molecular Pharmacology, 2008Co-Authors: Verawan Uchaipichat, Brian J Houston, Peter I. Mackenzie, Andrew J Williams, Aleksandra Galetin, John O MinersAbstract:Interactions between the UGT2B7-catalyzed Glucuronidation of zidovudine (AZT), 4-methylumbelliferone (4MU), and 1-naphthol (1NP) were analyzed using multisite and empirical kinetic models to explore the existence of multiple substrate and effector binding sites within this important drug metabolizing enzyme. 4MU and 1NP Glucuronidation by UGT2B7 exhibit sigmoidal kinetics characteristic of homotropic cooperativity (autoactivation), which may be modeled assuming the existence of two equivalent, interacting substrate binding sites. In contrast, UGT2B7-catalyzed AZT Glucuronidation follows hyperbolic (Michaelis-Menten) kinetics. Although 4MU and 1NP decreased the binding affinity of AZT, the kinetics of AZT Glucuronidation changed from hyperbolic to sigmoidal in the presence of both modifiers. Data were well described by a generic two-substrate binding site model in which there is no interaction between the sites in the absence of 4MU or 1NP, but heterotropic cooperativity results from the binding of modifier. Inhibition of 4MU and 1NP Glucuronidation by AZT and interactions between 4MU and 1NP required more complex three-site models, where the modifier acts via a distinct effector site to alter either substrate binding affinity or Vmax without affecting the homotropic cooperativity characteristic of 4MU and 1NP Glucuronidation. It is noteworthy that 1NP inhibited 4MU Glucuronidation, whereas 4MU activated 1NP Glucuronidation. The results are consistent with the existence of two "catalytic" sites for each substrate within the UGT2B7 active site, along with multiple effector sites. The multiplicity of binding and effector sites results in complex kinetic interactions between UGT2B7 substrates, which potentially complicates inhibition screening studies.
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the albumin effect and drug Glucuronidation bovine serum albumin and fatty acid free human serum albumin enhance the Glucuronidation of udp glucuronosyltransferase ugt 1a9 substrates but not ugt1a1 and ugt1a6 activities
Drug Metabolism and Disposition, 2008Co-Authors: Andrew Rowland, Kathleen M. Knights, Peter I. Mackenzie, John O MinersAbstract:Bovine serum albumin (BSA) and fatty acid-free human serum albumin (HSAFAF) reduce the K m values for UGT2B7 substrates by sequestering inhibitory long-chain fatty acids released by incubations of human liver microsomes (HLM) and HEK293 cells expressing this enzyme. However, the scope of the “albumin effect” is unknown. In this investigation we characterized the effects of albumin on the kinetics of 4-methylumbelliferone (4MU) Glucuronidation by UDP-glucuronosyltransferase (UGT) 1A1, 1A6, and 1A9, and propofol (PRO) Glucuronidation by UGT1A9 and HLM. BSA and HSAFAF, but not human serum albumin, reduced the K m values for 4MU and PRO Glucuronidation by UGT1A9. For example, HSAFAF (2%) reduced the K m values for 4MU and PRO Glucuronidation from 13.4 to 2.9 and 41 to 7.2 μM, respectively. Similarly, HSAFAF (2%) reduced the K m for PRO Glucuronidation by HLM from 127 to 10.6 μM. Arachidonic, linoleic, and oleic acids and a mixture of these decreased the rates of 4MU and PRO Glucuronidation by UGT1A9. K m values for these reactions were increased 3- to 6-fold by the fatty acid mixture. Inhibition was reversed by the addition of BSA (2%). Extrapolation of kinetic constants for PRO Glucuronidation by HLM in the presence of HSAFAF predicted in vivo hepatic clearance within 15%. Fatty acids had no effect on 4MU Glucuronidation by UGT1A1 and UGT1A6 but, paradoxically, all forms of albumin altered the kinetic model for 4MU Glucuronidation by UGT1A6 (from Michaelis-Menten to two-site). Only BSA caused a similar effect on 4MU Glucuronidation by UGT1A1. It is concluded that BSA and HSAFAF reduce the K m values of only those enzymes inhibited by long-chain unsaturated fatty acids.
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the Glucuronidation of delta4 3 keto c19 and c21 hydroxysteroids by human liver microsomal and recombinant udp glucuronosyltransferases ugts 6alpha and 21 hydroxyprogesterone are selective substrates for ugt2b7
Drug Metabolism and Disposition, 2007Co-Authors: Kushari Bowalgaha, Kathleen M. Knights, Peter I. Mackenzie, David J Elliot, John O MinersAbstract:The stereo- and regioselective Glucuronidation of 10 Delta(4)-3-keto monohydroxylated androgens and pregnanes was investigated to identify UDP-glucuronosyltransferase (UGT) enzyme-selective substrates. Kinetic studies were performed using human liver microsomes (HLMs) and a panel of 12 recombinant human UGTs as the enzyme sources. Five of the steroids, which were hydroxylated in the 6beta-, 7alpha-, 11beta- or 17alpha-positions, were not glucuronidated by HLMs. Of the remaining compounds, comparative kinetic and inhibition studies indicated that 6alpha- and 21-hydroxyprogesterone (OHP) were glucuronidated selectively by human liver microsomal UGT2B7. 6alpha-OHP Glucuronidation by HLMs and UGT2B7 followed Michaelis-Menten kinetics, whereas 21-OHP Glucuronidation by these enzyme sources exhibited positive cooperativity. UGT2B7 was also identified as the enzyme responsible for the high-affinity component of human liver microsomal 11alpha-OHP Glucuronidation. In contrast, UGT2B15 and UGT2B17 were the major forms involved in human liver microsomal testosterone 17beta-Glucuronidation and the high-affinity component of 16alpha-OHP Glucuronidation. Activity of UGT1A subfamily enzymes toward the hepatically glucuronidated substrates was generally low, although UGT1A4 and UGT1A9 contribute to the low-affinity components of microsomal 16alpha- and 11alpha-OHP Glucuronidation, respectively. Interestingly, UGT1A10, which is expressed only in the gastrointestinal tract, exhibited activity toward most of the glucuronidated substrates. The results indicate that 6alpha- and 21-OHP may be used as selective "probes" for human liver microsomal UGT2B7 activity and, taken together, provide insights into the regio- and stereoselectivity of hydroxysteroid Glucuronidation by human UGTs.
Kathleen M. Knights - One of the best experts on this subject based on the ideXlab platform.
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spironolactone and canrenone inhibit ugt2b7 catalyzed human liver and kidney microsomal aldosterone 18β Glucuronidation a potential drug interaction
Drug Metabolism and Disposition, 2010Co-Authors: Kathleen M. Knights, Kushari Bowalgaha, John O MinersAbstract:Elevated plasma concentrations of aldosterone (ALDO) are observed in patients treated with spironolactone. Because ALDO is eliminated via UGT2B7-catalyzed 18beta-Glucuronidation, this study aimed to determine whether spironolactone and its primary metabolites, canrenone and canrenoic acid, inhibit ALDO 18beta-Glucuronidation by recombinant UGT2B7 and by human liver (HLM) and human kidney cortical (HKCM) microsomes. Initial experiments characterized the effects of all three compounds on 4-methylumbelliferone and ALDO Glucuronidation by recombinant human UGT2B7. IC(50) values for spironolactone and canrenone ranged from 26 to 50 microM, whereas canrenoic acid was a weak inhibitor. Inhibitor constant (K(i)) values for spironolactone and canrenone inhibition of ALDO 18beta-Glucuronidation were subsequently determined with HLM, HKCM, and UGT2B7 as the enzyme sources. Spironolactone and canrenone were competitive inhibitors of ALDO 18beta-Glucuronidation by HLM, HKCM, and UGT2B7. Mean (+/-) K(i) values for spironolactone were 52 +/- 22 (HLM) and 34 +/- 4 microM (HKCM), and mean (+/-) K(i) values for canrenone were 41 +/- 19 (HLM) and 23 +/- 2 microM (HKCM). K(i) values for spironolactone and canrenone inhibition of ALDO 18beta-Glucuronidation by recombinant UGT2B7 were 23 and 11 microM, respectively. "Actual" K(i) values for spironolactone and canrenone inhibition of ALDO 18beta-Glucuronidation, which take into account the role of endogenous microsomal inhibitors, are predicted to be 3 to 5 and 2 to 4 microM, respectively. The data indicate that the elevated ALDO concentrations observed in patients treated with spironolactone may be due, at least in part, to a pharmacokinetic interaction, and spironolactone and canrenone should be considered to be potential inhibitors of the UGT2B7-mediated metabolic clearance of drugs in both liver and kidney.
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the prediction of drug Glucuronidation parameters in humans udp glucuronosyltransferase enzyme selective substrate and inhibitor probes for reaction phenotyping and in vitro in vivo extrapolation of drug clearance and drug drug interaction potential
Drug Metabolism Reviews, 2010Co-Authors: John O Miners, Peter I. Mackenzie, Kathleen M. KnightsAbstract:Major advances in the characterization of uridine diphosphate (UDP)-glucuronosyltransferase (UGT) enzyme substrate and inhibitor selectivities and the development of experimental paradigms to investigate xenobiotic Glucuronidation in vitro now permit the prediction of a range of drug-Glucuronidation parameters in humans. In particular, the availability of substrate and inhibitor “probes” for the major hepatic drug metabolizing UGTs together with batteries of recombinant enzymes allow the reaction phenotyping of drug Glucuronidation reactions. Additionally, in vitro experimental approaches and scaling strategies have been successfully applied to the quantitative prediction of in vivo clearance via Glucuronidation and drug-drug interaction potential.
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the albumin effect and drug Glucuronidation bovine serum albumin and fatty acid free human serum albumin enhance the Glucuronidation of udp glucuronosyltransferase ugt 1a9 substrates but not ugt1a1 and ugt1a6 activities
Drug Metabolism and Disposition, 2008Co-Authors: Andrew Rowland, Kathleen M. Knights, Peter I. Mackenzie, John O MinersAbstract:Bovine serum albumin (BSA) and fatty acid-free human serum albumin (HSAFAF) reduce the K m values for UGT2B7 substrates by sequestering inhibitory long-chain fatty acids released by incubations of human liver microsomes (HLM) and HEK293 cells expressing this enzyme. However, the scope of the “albumin effect” is unknown. In this investigation we characterized the effects of albumin on the kinetics of 4-methylumbelliferone (4MU) Glucuronidation by UDP-glucuronosyltransferase (UGT) 1A1, 1A6, and 1A9, and propofol (PRO) Glucuronidation by UGT1A9 and HLM. BSA and HSAFAF, but not human serum albumin, reduced the K m values for 4MU and PRO Glucuronidation by UGT1A9. For example, HSAFAF (2%) reduced the K m values for 4MU and PRO Glucuronidation from 13.4 to 2.9 and 41 to 7.2 μM, respectively. Similarly, HSAFAF (2%) reduced the K m for PRO Glucuronidation by HLM from 127 to 10.6 μM. Arachidonic, linoleic, and oleic acids and a mixture of these decreased the rates of 4MU and PRO Glucuronidation by UGT1A9. K m values for these reactions were increased 3- to 6-fold by the fatty acid mixture. Inhibition was reversed by the addition of BSA (2%). Extrapolation of kinetic constants for PRO Glucuronidation by HLM in the presence of HSAFAF predicted in vivo hepatic clearance within 15%. Fatty acids had no effect on 4MU Glucuronidation by UGT1A1 and UGT1A6 but, paradoxically, all forms of albumin altered the kinetic model for 4MU Glucuronidation by UGT1A6 (from Michaelis-Menten to two-site). Only BSA caused a similar effect on 4MU Glucuronidation by UGT1A1. It is concluded that BSA and HSAFAF reduce the K m values of only those enzymes inhibited by long-chain unsaturated fatty acids.
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the Glucuronidation of delta4 3 keto c19 and c21 hydroxysteroids by human liver microsomal and recombinant udp glucuronosyltransferases ugts 6alpha and 21 hydroxyprogesterone are selective substrates for ugt2b7
Drug Metabolism and Disposition, 2007Co-Authors: Kushari Bowalgaha, Kathleen M. Knights, Peter I. Mackenzie, David J Elliot, John O MinersAbstract:The stereo- and regioselective Glucuronidation of 10 Delta(4)-3-keto monohydroxylated androgens and pregnanes was investigated to identify UDP-glucuronosyltransferase (UGT) enzyme-selective substrates. Kinetic studies were performed using human liver microsomes (HLMs) and a panel of 12 recombinant human UGTs as the enzyme sources. Five of the steroids, which were hydroxylated in the 6beta-, 7alpha-, 11beta- or 17alpha-positions, were not glucuronidated by HLMs. Of the remaining compounds, comparative kinetic and inhibition studies indicated that 6alpha- and 21-hydroxyprogesterone (OHP) were glucuronidated selectively by human liver microsomal UGT2B7. 6alpha-OHP Glucuronidation by HLMs and UGT2B7 followed Michaelis-Menten kinetics, whereas 21-OHP Glucuronidation by these enzyme sources exhibited positive cooperativity. UGT2B7 was also identified as the enzyme responsible for the high-affinity component of human liver microsomal 11alpha-OHP Glucuronidation. In contrast, UGT2B15 and UGT2B17 were the major forms involved in human liver microsomal testosterone 17beta-Glucuronidation and the high-affinity component of 16alpha-OHP Glucuronidation. Activity of UGT1A subfamily enzymes toward the hepatically glucuronidated substrates was generally low, although UGT1A4 and UGT1A9 contribute to the low-affinity components of microsomal 16alpha- and 11alpha-OHP Glucuronidation, respectively. Interestingly, UGT1A10, which is expressed only in the gastrointestinal tract, exhibited activity toward most of the glucuronidated substrates. The results indicate that 6alpha- and 21-OHP may be used as selective "probes" for human liver microsomal UGT2B7 activity and, taken together, provide insights into the regio- and stereoselectivity of hydroxysteroid Glucuronidation by human UGTs.
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s naproxen and desmethylnaproxen Glucuronidation by human liver microsomes and recombinant human udp glucuronosyltransferases ugt role of ugt2b7 in the elimination of naproxen
British Journal of Clinical Pharmacology, 2005Co-Authors: Kushari Bowalgaha, Kathleen M. Knights, Peter I. Mackenzie, David J Elliot, Stellan Swedmark, John O MinersAbstract:Aims To characterize the kinetics of S-naproxen (‘naproxen’) acyl Glucuronidation and desmethylnaproxen acyl and phenolic Glucuronidation by human liver microsomes and identify the human UGT isoform(s) catalysing these reactions. Methods Naproxen and desmethylnaproxen Glucuronidation were investigated using microsomes from six and five livers, respectively. Human recombinant UGTs were screened for activity towards naproxen and desmethylnaproxen. Where significant activity was observed, kinetic parameters were determined. Naproxen and desmethylnaproxen glucuronides were measured by separate high-performance liquid chromatography methods. Results Naproxen acyl Glucuronidation by human liver microsomes followed biphasic kinetics. Mean apparent Km values (±SD, with 95% confidence interval in parentheses) for the high- and low-affinity components were 29 ± 13 µm (16, 43) and 473 ± 108 µm (359, 587), respectively. UGT 1A1, 1A3, 1A6, 1A7, 1A8, 1A9, 1A10 and 2B7 glucuronidated naproxen. UGT2B7 exhibited an apparent Km (72 µm) of the same order as the high-affinity human liver microsomal activity, which was inhibited by the UGT2B7 selective ‘probe’ fluconazole. Although data for desmethylnaproxen phenolic Glucuronidation by human liver microsomes were generally adequately fitted to either the single- or two-enzyme Michaelis–Menten equation, model fitting was inconclusive for desmethylnaproxen acyl Glucuronidation. UGT 1A1, 1A7, 1A9 and 1A10 catalysed both the phenolic and acyl Glucuronidation of desmethylnaproxen, while UGT 1A3, 1A6 and 2B7 formed only the acyl glucuronide. Atypical Glucuronidation kinetics were variably observed for naproxen and desmethylnaproxen Glucuronidation by the recombinant UGTs. Conclusion UGT2B7 is responsible for human hepatic naproxen acyl Glucuronidation, which is the primary elimination pathway for this drug.
Thomas R. Tephly - One of the best experts on this subject based on the ideXlab platform.
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genetic predisposition to the metabolism of irinotecan cpt 11 role of uridine diphosphate glucuronosyltransferase isoform 1a1 in the Glucuronidation of its active metabolite sn 38 in human liver microsomes
Journal of Clinical Investigation, 1998Co-Authors: Lalitha Iyer, Birgit L. Coffman, Mitchell D. Green, Thomas R. Tephly, Christopher D King, Peter F Whitington, Sandip K Roy, Mark J RatainAbstract:Irinotecan (CPT-11) is a promising antitumor agent, recently approved for use in patients with metastatic colorectal cancer. Its active metabolite, SN-38, is glucuronidated by hepatic uridine diphosphate glucuronosyltransferases (UGTs). The major dose-limiting toxicity of irinotecan therapy is diarrhea, which is believed to be secondary to the biliary excretion of SN-38, the extent of which is determined by SN-38 Glucuronidation. The purpose of this study was to identify the specific isoform of UGT involved in SN-38 Glucuronidation. In vitro Glucuronidation of SN-38 was screened in hepatic microsomes from normal rats (n = 4), normal humans (n = 25), Gunn rats (n = 3), and patients (n = 4) with Crigler-Najjar type I (CN-I) syndrome. A wide intersubject variability in in vitro SN-38 glucuronide formation rates was found in humans. Gunn rats and CN-I patients lacked SN-38 glucuronidating activity, indicating the role of UGT1 isoform in SN-38 Glucuronidation. A significant correlation was observed between SN-38 and bilirubin Glucuronidation (r = 0.89; P = 0.001), whereas there was a poor relationship between para-nitrophenol and SN-38 Glucuronidation (r = 0.08; P = 0.703). Intact SN-38 Glucuronidation was observed only in HK293 cells transfected with the UGT1A1 isozyme. These results demonstrate that UGT1A1 is the isoform responsible for SN-38 Glucuronidation. These findings indicate a genetic predisposition to the metabolism of irinotecan, suggesting that patients with low UGT1A1 activity, such as those with Gilbert's syndrome, may be at an increased risk for irinotecan toxicity.
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Glucuronidation of catechol estrogens by expressed human UDP-glucuronosyltransferases (UGTs) 1A1, 1A3, and 2B7.
Toxicological sciences : an official journal of the Society of Toxicology, 1998Co-Authors: Z Cheng, B Mojarrabi, C. D. King, Birgit L. Coffman, M.d. Green, Peter I. Mackenzie, Gladys R. Rios, Thomas R. TephlyAbstract:Catechol estrogens are major estrogen metabolites in mammals and are the most potent naturally occurring inhibitors of catecholamine metabolism. These estrogen compounds have been implicated in carcinogenic activity and the 4/2-hydroxyestradiol concentration has been shown to be elevated in neoplastic human mammary tissue compared to normal human breast tissue. Three human liver UDP-glucuronosyltransferases, UGT2B7, UGT1A1, and UGT1A3, have been shown to catalyze the Glucuronidation of catechol estrogens and lead to their enhanced elimination via urine or bile. The present study was designed to study the kinetic interaction of expressed human UGT2B7(Y) or (H), UGT1A1, and UGT1A3 toward 2- and 4-hydroxycatechol estrogens. cDNAs encoding UGT2B7(Y) or (H), UGT1A1, and UGT1A3 were expressed in HK293 cells, and cell homogenates or membrane preparations were used to determine their Glucuronidation ability. UGT2B7(Y) reacted with higher efficiency toward 4-hydroxyestrogenic catechols, whereas UGT1A1 and UGT1A3 showed higher activities toward 2-hydroxyestrogens. UGT2B7(H) catalyzed estrogen catechol Glucuronidation with efficiencies similar to UGT2B7(Y). Flunitrazepam (FNZ), a competitive inhibitor of morphine Glucuronidation in hepatic microsomes, competitively inhibited catechol estrogen Glucuronidation catalyzed by UGT2B7(Y), UGT1A1, and UGT1A3. Buprenorphine, an opioid substrate that reacts at high efficiency with each of these UGTs, was also studied. FNZ competitively inhibited buprenorphine Glucuronidation with UGT1A1 and UGT2B7 but had no inhibitory activity toward UGT1A3. This suggests that buprenorphine and 2-hydroxycatechol estrogens react with separate active sites of UGT1A3. A catecholamine, norepinephrine, did not inhibit UGT2B7(Y)-, UGT1A1-, and UGT1A3-catalyzed Glucuronidation of catechol estrogens. These results also suggest that drug-endobiotic interactions are possible in humans and may have implication in carcinogenesis.
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Genetic predisposition to the metabolism of irinotecan (CPT-11). Role of uridine diphosphate glucuronosyltransferase isoform 1A1 in the Glucuronidation of its active metabolite (SN-38) in human liver microsomes
1998Co-Authors: Lalitha Iyer, Birgit L. Coffman, Mitchell D. Green, Thomas R. Tephly, Christopher D King, Peter F Whitington, Ip K. Roy, Mark J RatainAbstract:Irinotecan (CPT-11) is a promising antitumor agent, recently approved for use in patients with metastatic colorectal cancer. Its active metabolite, SN-38, is glucuronidated by hepatic uridine diphosphate glucuronosyltransferases (UGTs). The major dose-limiting toxicity of irinotecan therapy is diarrhea, which is believed to be secondary to the biliary excretion of SN-38, the extent of which is determined by SN-38 Glucuronidation. The purpose of this study was to identify the specific isoform of UGT involved in SN-38 Glucuronidation. In vitro Glucuronidation of SN-38 was screened in hepatic microsomes from normal rats (n � 4), normal humans (n � 25), Gunn rats (n � 3), and patients (n � 4) with Crigler-Najjar type I (CN-I) syndrome. A wide intersubject variability in in vitro SN-38 glucuronide formation rates was found in humans. Gunn rats and CN-I patients lacked SN-38 glucuronidating activity, indicating the role of UGT1 isoform in SN-38 Glucuronidation. A significant correlation was observed between SN-38 and bilirubin Glucuronidation (r � 0.89; P � 0.001), whereas there was a poor relationship between para-nitrophenol and SN-38 Glucuronidation (r � 0.08; P � 0.703). Intact SN-38 Glucuronidation was observed only in HK293 cells transfected with the UGT1A1 isozyme. These results demonstrate that UGT1A1 is the isoform responsible for SN-38 Glucuronidation. These findings indicate a genetic predisposition to the metabolism of irinotecan, suggesting that patients with low UGT1A1 activity, such as those with Gilbert’s syndrome, may be at an increased risk for irinotecan toxicity. (J. Clin. Invest. 101:847
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the Glucuronidation of opioids other xenobiotics and androgens by human ugt2b7y 268 and ugt2b7h 268
Drug Metabolism and Disposition, 1998Co-Authors: Birgit L. Coffman, Gladys R. Rios, Christopher D King, Thomas R. TephlyAbstract:UGT2B7 has been cloned and expressed previously in COS cells and HK293 cells. Two forms have been identified: one with a tyrosine and one with a histidine at position 268. UGT2B7 has been shown to catalyze NSAIDs, catechol estrogens, and morphine-3- and -6-Glucuronidation. cDNAs for UGT2B7Y268 and H268 were cloned and stably expressed in HK 293 cells. Studies were designed to test each form for reactivity toward a number of opioid compounds, xenobiotics such as menthol, oxazepam, and propranolol, and androgens such as androsterone and testosterone using membrane preparations derived from HK 293 cells. Both UGT2B7Y and UGT2B7H are highly reactive with many opioids, menthol, androsterone, and (R)- and (S)-propranolol, and similar kinetic values were observed. UGT2B7Y and UGT2B7H react poorly with oxazepam and no difference in (R)- or (S)-Glucuronidation rate ratios was found. Thus, UGT2B7Y and H cannot account for the variability in the plasma or urine concentrations of these glucuronides in human populations. Our data suggest that UGT2B7 is a major isoform responsible for the Glucuronidation of androsterone. Neither UGT2B7Y nor H catalyzes the Glucuronidation of testosterone although each catalyzes the Glucuronidation of epitestosterone. UGT2B7 seems to be a major human isoform responsible for the Glucuronidation of opioids of the morphinan and oripavine class and is capable of catalyzing the Glucuronidation of both the 3- and 6-hydroxyl moieties on these molecules. Thus, UGT2B7 plays a major role in the conversion of morphine to morphine-6-glucuronide, the potent analgesic metabolite of morphine.
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Glucuronidation of amines and hydroxylated xenobiotics and endobiotics catalyzed by expressed human ugt1 4 protein
Drug Metabolism and Disposition, 1996Co-Authors: Mitchell D. Green, Thomas R. TephlyAbstract:Glucuronide conjugation of tertiary amine xenobiotics represents a unique and important metabolic pathway for these compounds in humans. In this study, we show that human UDP-glucuronosyltransferase 1.4 protein, stably expressed in human embryonic kidney 293 cells, catalyzes the N-Glucuronidation of primary, secondary, and tertiary amine substrates. In addition, the substrate specificity of the expressed enzyme toward many hydroxylated and carboxylic acid-containing compounds was examined. Of the hydroxylated compounds tested, only sapogenins gave Glucuronidation rates comparable with those observed for amine substrates. The apparent KM and Vmax values for sapogenins were such that the efficiency of Glucuronidation (Vmax/KM) for these compounds was higher than that determined for amine substrates. Human UDP-glucuronosyltranferase 1.4 also catalyzes the Glucuronidation of monoterpenoid alcohols and simple phenolic compounds. The enzyme kinetic values determined for these substrates suggested that this enzyme may have relatively limited significance for the conjugation of these classes of compounds. Of the endobiotics tested, androstanediol and progestins were glucuronidated at high rates by expressed human UDP-glucuronosyltransferase 1.4 protein. The Glucuronidation efficiency for 5alpha-pregnane-3beta,20alpha-diol was comparable with that determined for the sapogenins. Because UDP-glucuronosyltransferases are integral membrane proteins, the effects of different detergents on the catalytic activity of the expressed enzyme were determined. The results show that detergents (such as Lubrol PX, Emulgen 911, and Triton X-100) are inhibitory for the quaternary ammonium-linked Glucuronidation of chlorpromazine and imipramine catalyzed by expressed human UDP-glucuronosyltransferase 1.4. In contrast, CHAPS and nonanoyl-N-methylglucamide are less inhibitory toward the Glucuronidation of these compounds. The results suggest that human UDP-glucuronosyltransferase 1.4 may be an important enzyme for the detoxication of environmentally derived amines and sapogenins and for the conjugation of progestins.
Hidetaka Kamimura - One of the best experts on this subject based on the ideXlab platform.
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substrate dependent modulation of udp glucuronosyltransferase 1a1 ugt1a1 by propofol in recombinant human ugt1a1 and human liver microsomes
Basic & Clinical Pharmacology & Toxicology, 2007Co-Authors: Yuji Mano, Takashi Usui, Hidetaka KamimuraAbstract:Our previous study has shown that propofol, a probe substrate for human UDP-glucuronosyltransferase (UGT) 1A9, activated the Glucuronidation of 4-methylumbelliferone (4-MU) by recombinant UGT1A1 in a concentration-dependent manner. In the present study, we investigated the mechanism of activation, and whether the stimulatory effect occurs when another substrate is used with human liver microsomes. The Glucuronidation of 4-MU followed Michaelis-Menten kinetics with a K(m) value of 101 microM in the absence of propofol. In the presence of 200 microM propofol, a concentration that causes heterotopic activation of 4-MU Glucuronidation (4-MUG), the V(max) value increased to 1.5-fold, while the K(m) value decreased to 0.53-fold. In order to assess whether propofol activates UGT1A1 activity for a substrate other than 4-MU, the effect of propofol on oestradiol 3beta-Glucuronidation by recombinant UGT1A1 and in human liver microsomes was evaluated. In contrast to 4-MUG activity, propofol inhibited UGT1A1-catalysed oestradiol 3beta-Glucuronidation in recombinant UGT1A1 as well as in human liver microsomes with IC(50) values of 59 and 228 microM, respectively. In addition, a known UGT1A1 modulator, 17alpha-ethynyloestradiol, stimulated oestradiol 3beta-Glucuronidation slightly at a concentration of 5 microM, while it inhibited 4-MUG in recombinant UGT1A1 at all concentrations tested (5-100 microM). These findings indicate that the modulation of UGT1A1 by propofol is substrate-dependent, and thus care should be taken when extrapolating the stimulatory effects of drugs for one Glucuronidation substrate.
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identification of human udp glucuronosyltransferase responsible for the Glucuronidation of niflumic acid in human liver
Pharmaceutical Research, 2006Co-Authors: Yuji Mano, Takashi Usui, Hidetaka KamimuraAbstract:To assess the uridine diphosphate (UDP)-glucuronosyltransferase (UGT) isozymes involved in the Glucuronidation of niflumic acid in human liver. The Glucuronidation activity of niflumic acid was determined in liver microsomes and recombinant UGT isozymes by incubation of niflumic acid with UDP-glucuronic acid (UDPGA). Incubation of niflumic acid with liver microsomes and UDPGA produced one peak, which was identified as a glucuronide from mass spectrometric analysis. A study involving a panel of recombinant human UGT isozymes showed that Glucuronidation activity was highest in UGT1A1 among the isozymes investigated. The Glucuronidation in human liver microsomes (HLMs) followed Michaelis-Menten kinetics with a Km value of 16 μM, which is similar to that found with recombinant UGT1A1. The Glucuronidation activity of niflumic acid in microsomes from eight human livers significantly correlated with UGT1A1-catalyzed estradiol 3β-Glucuronidation activity (r=0.78, p<0.05). β-Estradiol inhibited niflumic acid Glucuronidation with an IC50 of 25 μM in HLMs, comparable to that for UGT1A1. These findings indicate that UGT1A1 is the main isozyme involved in the Glucuronidation of niflumic acid in the human liver.