John Miners - Academia.edu (original) (raw)
Papers by John Miners
Clinical and Experimental Pharmacology and Physiology, Aug 1, 1982
Biochemical Pharmacology, Oct 1, 1984
Buthionine sulphoximine (BSO) is an inhibitor of gamma-glutamylcysteine synthetase (gamma-GCS) an... more Buthionine sulphoximine (BSO) is an inhibitor of gamma-glutamylcysteine synthetase (gamma-GCS) and, consequently lowers tissue glutathione (GSH) concentrations. In fed male C3H mice, liver and kidney GSH levels were depleted by BSO in a dose dependent manner with maximum effect (35% of initial levels) occurring with doses between 0.8 and 1.6 g/kg, i.p. At these doses maximum effects on gamma-GCS and GSH were observed 2-4 hr after BSO administration; initial gamma-GCS activity and GSH content were restored approximately 16 hr post BSO. BSO, either in vivo or in vitro, had no effect on hepatic microsomal cytochrome P-450 levels, a range of cytochrome P-450 dependent enzyme activities or p-nitrophenol glucuronyl transferase activity. Similarly, BSO had no effect on phenol sulphotransferase and two GSH-transferase activities in the 105,000 g supernatant fraction. BSO had no effect on the duration of hexobarbitone induced narcosis in mice. Consistent with specific inhibition of GSH synthesis, BSO pretreatment of mice decreased the proportion of a 50 mg/kg dose of paracetamol excreted in the urine as GSH-derived conjugates but did not affect paracetamol clearance through the glucuronidation or sulphation pathways. Since BSO does not affect cytochrome P-450 or conjugating enzyme activity, its use as a specific depletor of tissue GSH in the investigation of mechanisms of xenobiotic-induced toxicities is preferable to the standard GSH-depleting agents as these have other enzymic effects.
Biochemical Pharmacology, Jun 1, 1984
Clinical and Experimental Pharmacology and Physiology, Dec 1, 1981
Clinical and Experimental Pharmacology and Physiology, 1981
Improved predictions of in vivo hepatic clearance (CLH) from in vitro intrinsic clearance (CLint)... more Improved predictions of in vivo hepatic clearance (CLH) from in vitro intrinsic clearance (CLint) by bovine serum albumin (BSA) have been reported for drugs that were metabolized by certain cytochrome P450 (CYP) isoforms (i.e. CYP2C8 and CYP2C9) as well as certain UDP-glucuronosyltransferase (UGT) isoforms (i.e. UGT1A9, UGT2B4, and UGT2B7). BSA increases the activity of these enzymes by sequestering polyunsaturated fatty acids (PUFAs) that are released from membranes, especially when using human liver microsomes (HLM) as the enzyme source. The aims of this study were to characterize the effect of BSA on the kinetics of CYP1A2-mediated phenacetin O-deethylation and determine whether addition of BSA to incubation improves the prediction of in vivo hepatic clearance of phenacetin. The kinetics of phenacetin O-deethylation by HLM and E.coli-expressed CYP1A2 (rCYP1A2) were performed with and without BSA (2% w/v) supplementation. BSA increased CLint for phenacetin O-deethylation, due to a...
There is increasing evidence that drug glucuronidation reactions in vitro may exhibit atypical... more There is increasing evidence that drug glucuronidation reactions in vitro may exhibit atypical' or non-Michaelis-Menten kinetics. For example, 4-methylumbelliferone (4MU) and 1-naphthol (1NP) glucuronidation by UGT2B7 exhibit sigmoidal kinetics, characteristic of homotropic cooperativity. In contrast, zidovudine (AZT) glucuronidation follows Michaelis-Menten kinetics. This study investigated mutual interactions of the three commonly used UGT2B7 substrates, namely AZT, 4MU and 1NP, due to their distinctive kinetic properties. In addition to the empirical models (i.e., Michaelis-Menten and Hill equations), multisite kinetic models were employed to determine the Ki values and provide mechanistic insights into the interactions. AZT inhibited 4MU and 1NP glucuronidation by UGT2B7, with Ks (binding affinity constant) values of both substrates increasing with increasing AZT concentration. However, Vmax and the sigmoidal properties of the substrate showed no significant change, suggesti...
Methods in Enzymology, 1996
Publisher Summary This chapter describes the assays of omeprazole metabolism as a substrate probe... more Publisher Summary This chapter describes the assays of omeprazole metabolism as a substrate probe for human CYP isoforms. Omeprazole, a substituted benzimidazole inhibitor of the gastric proton pump H + , K + -ATPase, is essentially completely metabolized in vivo . The major metabolites detected in blood in vivo are omeprazole sulfone and hydroxyomeprazole. The major urinary metabolites identified are hydroxyomeprazole and its corresponding carboxylic acid, with neither omeprazole itself nor omeprazole sulfone being detected. Hydroxyomeprazole is converted into carboxylic acid by cytosolic alcohol and aldehyde dehydrogenases, and omeprazole sulfone is essentially completely further metabolized. Omeprazole is an effective substrate for both CYP2C19 and CYP3A4, but different metabolites are formed by the two isoforms. Hydroxyomeprazole and omeprazole sulfone show similar CYP specificity to the parent drug. Sulfone formation is mediated by CYP3A4, and the hydroxy formation is mediated mainly by CYP2C19.
Current protocols in toxicology / editorial board, Mahin D. Maines (editor-in-chief) ... [et al.], 2006
Cytochrome P4501A2 (CYP1A2) is responsible for the metabolism of a diverse range of clinically us... more Cytochrome P4501A2 (CYP1A2) is responsible for the metabolism of a diverse range of clinically used drugs and dietary and environmental chemicals (including many procarcinogens). CYP1A2 expression is influenced by numerous factors, and hence wide interindividual variability is a characteristic feature of this enzyme in humans. Phenacetin represents a convenient probe for the assessment of human CYP1A2 activity in vitro (hepatic microsomes and recombinant enzyme). It is a relatively high-turnover substrate that forms only one major primary metabolite, the O-deethylated derivative acetaminophen. Acetaminophen formation in incubations of phenacetin with a CYP1A2 source is readily measured by HPLC with UV detection. The assay has a low requirement for human liver microsomes or recombinant enzyme, and is both selective and sensitive without the requirement for a solvent extraction step. Overall assay reproducibility is excellent, with coefficients of variation <4%.
The Journal of pharmacology and experimental therapeutics, 2001
Interindividual variability in acetaminophen (APAP) glucuronidation may contribute to differences... more Interindividual variability in acetaminophen (APAP) glucuronidation may contribute to differences in susceptibility to APAP intoxication in humans. The purpose of this study was to identify the relevant UDP-glucuronosyltransferase (UGT) isoforms mediating APAP-UGT activity in human liver microsomes (HLMs). APAP-UGT activities and enzyme kinetics were determined using HLMs from 56 donors and nine recombinant human UGTs. Activities mediated by UGT1A1, UGT1A4, UGT1A9, and UGT2B7, and relative UGT1A6 protein content were quantified using 20 livers. More than 15-fold variation in liver microsomal APAP-UGT activities was observed with a distribution skewed toward lower activities. Although most UGTs could glucuronidate APAP, UGT1A1, UGT1A6, and UGT1A9 were most active. UGT1A6 was a relatively high-affinity (K(m) = 2.2 mM), low-capacity enzyme; UGT1A1 was intermediate in affinity (K(m) = 9.4 mM) and capacity; and UGT1A9 was a low-affinity (K(m) = 21 mM), high-capacity enzyme. K(m) values w...
In Silico Pharmacology, 2014
Molecular dynamics (MD) simulation is an emerging in silico technique with potential applications... more Molecular dynamics (MD) simulation is an emerging in silico technique with potential applications in diverse areas of pharmacology. Over the past three decades MD has evolved as an area of importance for understanding the atomic basis of complex phenomena such as molecular recognition, protein folding, and the transport of ions and small molecules across membranes. The application of MD simulations in isolation and in conjunction with experimental approaches have provided an increased understanding of protein structure-function relationships and demonstrated promise in drug discovery.
The analysis of membrane domain segregation and phase separation is of significant interest for t... more The analysis of membrane domain segregation and phase separation is of significant interest for the understanding of cell membranes. In this paper, we report a new approach to atomic force microscopy (AFM) of artificial membranes, using both functionalised AFM tips and "Force Volume" AFM imaging for the analysis of membrane phase separation. Simultaneous topology and mapping of interaction forces of binary component phospholipid bilayer membranes was performed yielding novel insights into membrane structure at unprecedented resolution.
Pharmacogenetics, 1997
Single nucleotide substitutions are known to result in a different amino acid at one of four site... more Single nucleotide substitutions are known to result in a different amino acid at one of four sites in cytochrome P4502C9 (CYP2C9) namely: residue 144: Arg/Cys; residue 358: Tyr/Cys; residue 359: Ile/Leu and residue 417: Gly/Asp. Polymerase chain reaction (PCR)-based amplification of the nucleotide fragments encompassing the four residues (144, 358-359 and 417) in 18 samples of human genomic DNA from a liver bank and one sample of DNA extracted from the blood of a known poor metabolizer of tolbutamide has been carried out. The products of PCR amplification were analysed by either allele-specific restriction endonucleases or probed with radioactively labelled allele-specific oligonucleotides in dot blot hybridizations. Fourteen individuals were homozygous for Arg144 and four were heterozygous Arg/Cys144. All individuals analysed were homozygous for Tyr358 (n = 17) and for Gly417 (n = 18). With the exception of one heterozygote the other 17 subjects were homozygous for Ile359. The genotype of the known poor metabolizer of tolbutamide was homozygous for Arg144, Leu359 and Gly417. The relative levels of expression of the Cys and Arg144 alleles was studied in the heterozygotes. A relative 5- to 10-fold greater expression of the Cys- over the Arg144 allele was noted in two heterozygotes. There was no apparent correlation of genotype to the hydroxylation of the known CYP2C9 substrates phenytoin, tolbutamide, torasemide and diclofenac. Apparent K(m) values for the cDNA-expressed Arg144/Ile359, Cys144/ Ile359 and Arg144/Leu359 variants towards tolbutamide were 91 microM, 62 microM and 229 microM, respectively. It is likely that functional changes occurring as a result of the Ile359Leu transition are responsible for the tolbutamide poor metabolizer phenotype.
Molecular Pharmacology, 2004
The UDP-glucuronosyltransferase (UGT) enzyme 'superfamily' contributes to the metabolism of a myr... more The UDP-glucuronosyltransferase (UGT) enzyme 'superfamily' contributes to the metabolism of a myriad of drugs, nondrug xenobiotic agents, and endogenous compounds. Although the individual UGT isoforms exhibit distinct but overlapping substrate selectivities, structural features of substrates that confer selectivity remain largely unknown. Using methods developed for pharmacophore fingerprinting combined with optimization and pattern recognition techniques, subsets of pharmacophores associated with the substrates and nonsubstrates of 12 human UGT isoforms were selected to generate predictive models of substrate selectivity and to elucidate the chemical and structural features associated with substrates and nonsubstrates. For all 12 UGT isoforms, the pharmacophore model generated showed predictive ability, as determined by a test set comprising 30% of the available data for each isoform.
Journal of Pharmacology and Experimental Therapeutics, 2010
Since COD (codeine) is eliminated primarily via glucuronidation, factors that alter COD glucuroni... more Since COD (codeine) is eliminated primarily via glucuronidation, factors that alter COD glucuronide formation potentially affect the proportion of the dose converted to the pharmacologically active metabolite morphine. Thus, in vitro-in vivo extrapolation (IV-IVE) approaches were utilized to identify potential drug-drug interactions arising from inhibition of COD glucuronidation in humans. Initial studies characterized the kinetics of COD 6-glucuronide (C6G) formation by human liver microsomes (HLM), and demonstrated an 88% reduction in K m (0.29 vs. 2.32 mM) for incubations performed in the presence of 2% bovine serum albumin (BSA). Of 13 recombinant UGT enzymes screened for COD glucuronidation activity, only UGT2B4 and UGT2B7 exhibited activity. The respective S 50 values (0.32 and 0.27 mM) generated in the presence of BSA were comparable to the mean K m observed in HLM. Known inhibitors of UGT2B7 activity in vitro or in vivo and drugs marketed as compound formulations with COD were investigated for inhibition of C6G formation by HLM. Inhibition screening identified potential interactions with dextropropoxyphene, fluconazole, ketoconazole and methadone. K i values generated for dextropropoxyphene (3.5 µM), fluconazole (202 µM), ketoconazole (0.66 µM) and methadone (0.32 µM) predicted 1.60-to 3.66-fold increases in the AUC ratio for COD in vivo. Whereas fluconazole and ketoconazole inhibited UGT2B4-and UGT2B7-catalyzed COD glucuronidation to a similar extent, inhibition by dextropropoxyphene and methadone resulted largely from an effect on UGT2B4. Interactions with dextropropoxyphene, fluconazole, ketoconazole and methadone potentially affect the intensity and duration of COD analgesia.
European Journal of Clinical Pharmacology, 1984
The effects of cimetidine (1 g/day) on theophylline disposition and metabolism were examined in s... more The effects of cimetidine (1 g/day) on theophylline disposition and metabolism were examined in smokers and non-smokers for single dose intravenous and chronic oral administration of theophylline. In the intravenous study the effect of cimetidine on plasma theophylline clearance was more marked in smokers (22.7% reduction) than in non-smokers (12.2% reduction). Similarly, in the multiple dose study the effect of cimetidine on theophylline clearance was greater in smokers (28.3% decrease) than in non-smokers (1I .3% decrease). The reduction in clearance was largely due to a reduction in metabolic clearances by 3-demethylation (C13 DM) and 1-demethylation (Cll DM) with no significant effect on clearance by 8-oxidation (C180x). There was a strong correlation between C13DM and CI1DM (r =0.98, p < 0.001) in both control and cimetidine study phases, whereas other coErelations between partial clearances were less marked and were not apparent during the cimetidine phase. The results are consistent with the view that 1-and 3-demethylation of theophylline are carried out by a common form of cytochrome P-450 which is selectively induced by cigarette smoking and preferentially inhibited by cimetidine.
Drug Metabolism Reviews, 2009
Major advances in the characterization of uridine diphosphate (UDP)-glucuronosyltransferase (UGT)... more 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 &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot;probes&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot; 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.
Drug Metabolism Reviews, 2010
The role of the kidney in drug and chemical disposition has traditionally focused on the excretio... more The role of the kidney in drug and chemical disposition has traditionally focused on the excretion of polar xenobiotics and metabolites. However, there is increasing evidence demonstrating that renal UGTs are integral to the &amp;amp;amp;amp;amp;amp;amp;quot;local&amp;amp;amp;amp;amp;amp;amp;quot; intrarenal, and, possibly, systemic, metabolic clearance of numerous drugs and nondrug xenobiotics, as well as to the maintenance of renal homeostasis through limiting the biological activity of endogenous renal mediators that control electrolyte balance and renal blood flow. The common involvement of UGT1A9 and UGT2B7 in the metabolism of both endogenous and exogenous compounds in kidney predicates significant renal drug-endobiotic interactions that may explain, in part, the adverse renal effects of some drugs.
Drug Metabolism and Disposition, 2005
Relatively few selective substrate and inhibitor probes have been identified for human UDP-glucur... more Relatively few selective substrate and inhibitor probes have been identified for human UDP-glucuronosyltransferases (UGT). This work investigated the selectivity of trifluoperazine (TFP), as a substrate, and amitriptyline, androsterone, canrenoic acid, hecogenin, phenylbutazone, quinidine, quinine and sulfinpyrazone, as inhibitors, for human UGTs. Selectivity was assessed using UGT 1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B7 and 2B15 expressed in HEK293 cells. TFP was confirmed as a highly selective substrate for UGT1A4. However, TFP bound extensively to both HEK293 lysate and to human liver microsomes in a concentration-dependent manner (fu inc 0.20-0.59). When corrected for non-specific binding, K m values for TFP glucuronidation were similar for both UGT1A4 (4.1 µM) and human liver microsomes (6.1 ± 1.2 µM) as the enzyme sources. Of the compounds screened as inhibitors, hecogenin alone was selective; significant inhibition was observed only for UGT1A4 (IC 50 1.5 µM). Using phenylbutazone and quinine as 'models', inhibition kinetics were variously described by competitive and noncompetitive mechanisms. Inhibition of UGT2B7 by quinidine was also investigated further since the effects of this compound on morphine pharmacokinetics (a known UGT2B7 substrate) have been ascribed to inhibition of P-glycoprotein. Quinidine inhibited human liver microsomal and recombinant UGT2B7 with respective K i values of 335 ± 128 µM and 186 µM. In conclusion, TFP and hecogenin represent selective substrate and inhibitor probes for UGT1A4, although the extensive non-selective binding of the former should be taken into account in kinetic studies. Amitriptyline, androsterone, canrenoic acid, hecogenin, phenylbutazone, quinidine, quinine and sulfinpyrazone are non-selective UGT inhibitors.
Clinical and Experimental Pharmacology and Physiology, Aug 1, 1982
Biochemical Pharmacology, Oct 1, 1984
Buthionine sulphoximine (BSO) is an inhibitor of gamma-glutamylcysteine synthetase (gamma-GCS) an... more Buthionine sulphoximine (BSO) is an inhibitor of gamma-glutamylcysteine synthetase (gamma-GCS) and, consequently lowers tissue glutathione (GSH) concentrations. In fed male C3H mice, liver and kidney GSH levels were depleted by BSO in a dose dependent manner with maximum effect (35% of initial levels) occurring with doses between 0.8 and 1.6 g/kg, i.p. At these doses maximum effects on gamma-GCS and GSH were observed 2-4 hr after BSO administration; initial gamma-GCS activity and GSH content were restored approximately 16 hr post BSO. BSO, either in vivo or in vitro, had no effect on hepatic microsomal cytochrome P-450 levels, a range of cytochrome P-450 dependent enzyme activities or p-nitrophenol glucuronyl transferase activity. Similarly, BSO had no effect on phenol sulphotransferase and two GSH-transferase activities in the 105,000 g supernatant fraction. BSO had no effect on the duration of hexobarbitone induced narcosis in mice. Consistent with specific inhibition of GSH synthesis, BSO pretreatment of mice decreased the proportion of a 50 mg/kg dose of paracetamol excreted in the urine as GSH-derived conjugates but did not affect paracetamol clearance through the glucuronidation or sulphation pathways. Since BSO does not affect cytochrome P-450 or conjugating enzyme activity, its use as a specific depletor of tissue GSH in the investigation of mechanisms of xenobiotic-induced toxicities is preferable to the standard GSH-depleting agents as these have other enzymic effects.
Biochemical Pharmacology, Jun 1, 1984
Clinical and Experimental Pharmacology and Physiology, Dec 1, 1981
Clinical and Experimental Pharmacology and Physiology, 1981
Improved predictions of in vivo hepatic clearance (CLH) from in vitro intrinsic clearance (CLint)... more Improved predictions of in vivo hepatic clearance (CLH) from in vitro intrinsic clearance (CLint) by bovine serum albumin (BSA) have been reported for drugs that were metabolized by certain cytochrome P450 (CYP) isoforms (i.e. CYP2C8 and CYP2C9) as well as certain UDP-glucuronosyltransferase (UGT) isoforms (i.e. UGT1A9, UGT2B4, and UGT2B7). BSA increases the activity of these enzymes by sequestering polyunsaturated fatty acids (PUFAs) that are released from membranes, especially when using human liver microsomes (HLM) as the enzyme source. The aims of this study were to characterize the effect of BSA on the kinetics of CYP1A2-mediated phenacetin O-deethylation and determine whether addition of BSA to incubation improves the prediction of in vivo hepatic clearance of phenacetin. The kinetics of phenacetin O-deethylation by HLM and E.coli-expressed CYP1A2 (rCYP1A2) were performed with and without BSA (2% w/v) supplementation. BSA increased CLint for phenacetin O-deethylation, due to a...
There is increasing evidence that drug glucuronidation reactions in vitro may exhibit atypical... more There is increasing evidence that drug glucuronidation reactions in vitro may exhibit atypical' or non-Michaelis-Menten kinetics. For example, 4-methylumbelliferone (4MU) and 1-naphthol (1NP) glucuronidation by UGT2B7 exhibit sigmoidal kinetics, characteristic of homotropic cooperativity. In contrast, zidovudine (AZT) glucuronidation follows Michaelis-Menten kinetics. This study investigated mutual interactions of the three commonly used UGT2B7 substrates, namely AZT, 4MU and 1NP, due to their distinctive kinetic properties. In addition to the empirical models (i.e., Michaelis-Menten and Hill equations), multisite kinetic models were employed to determine the Ki values and provide mechanistic insights into the interactions. AZT inhibited 4MU and 1NP glucuronidation by UGT2B7, with Ks (binding affinity constant) values of both substrates increasing with increasing AZT concentration. However, Vmax and the sigmoidal properties of the substrate showed no significant change, suggesti...
Methods in Enzymology, 1996
Publisher Summary This chapter describes the assays of omeprazole metabolism as a substrate probe... more Publisher Summary This chapter describes the assays of omeprazole metabolism as a substrate probe for human CYP isoforms. Omeprazole, a substituted benzimidazole inhibitor of the gastric proton pump H + , K + -ATPase, is essentially completely metabolized in vivo . The major metabolites detected in blood in vivo are omeprazole sulfone and hydroxyomeprazole. The major urinary metabolites identified are hydroxyomeprazole and its corresponding carboxylic acid, with neither omeprazole itself nor omeprazole sulfone being detected. Hydroxyomeprazole is converted into carboxylic acid by cytosolic alcohol and aldehyde dehydrogenases, and omeprazole sulfone is essentially completely further metabolized. Omeprazole is an effective substrate for both CYP2C19 and CYP3A4, but different metabolites are formed by the two isoforms. Hydroxyomeprazole and omeprazole sulfone show similar CYP specificity to the parent drug. Sulfone formation is mediated by CYP3A4, and the hydroxy formation is mediated mainly by CYP2C19.
Current protocols in toxicology / editorial board, Mahin D. Maines (editor-in-chief) ... [et al.], 2006
Cytochrome P4501A2 (CYP1A2) is responsible for the metabolism of a diverse range of clinically us... more Cytochrome P4501A2 (CYP1A2) is responsible for the metabolism of a diverse range of clinically used drugs and dietary and environmental chemicals (including many procarcinogens). CYP1A2 expression is influenced by numerous factors, and hence wide interindividual variability is a characteristic feature of this enzyme in humans. Phenacetin represents a convenient probe for the assessment of human CYP1A2 activity in vitro (hepatic microsomes and recombinant enzyme). It is a relatively high-turnover substrate that forms only one major primary metabolite, the O-deethylated derivative acetaminophen. Acetaminophen formation in incubations of phenacetin with a CYP1A2 source is readily measured by HPLC with UV detection. The assay has a low requirement for human liver microsomes or recombinant enzyme, and is both selective and sensitive without the requirement for a solvent extraction step. Overall assay reproducibility is excellent, with coefficients of variation <4%.
The Journal of pharmacology and experimental therapeutics, 2001
Interindividual variability in acetaminophen (APAP) glucuronidation may contribute to differences... more Interindividual variability in acetaminophen (APAP) glucuronidation may contribute to differences in susceptibility to APAP intoxication in humans. The purpose of this study was to identify the relevant UDP-glucuronosyltransferase (UGT) isoforms mediating APAP-UGT activity in human liver microsomes (HLMs). APAP-UGT activities and enzyme kinetics were determined using HLMs from 56 donors and nine recombinant human UGTs. Activities mediated by UGT1A1, UGT1A4, UGT1A9, and UGT2B7, and relative UGT1A6 protein content were quantified using 20 livers. More than 15-fold variation in liver microsomal APAP-UGT activities was observed with a distribution skewed toward lower activities. Although most UGTs could glucuronidate APAP, UGT1A1, UGT1A6, and UGT1A9 were most active. UGT1A6 was a relatively high-affinity (K(m) = 2.2 mM), low-capacity enzyme; UGT1A1 was intermediate in affinity (K(m) = 9.4 mM) and capacity; and UGT1A9 was a low-affinity (K(m) = 21 mM), high-capacity enzyme. K(m) values w...
In Silico Pharmacology, 2014
Molecular dynamics (MD) simulation is an emerging in silico technique with potential applications... more Molecular dynamics (MD) simulation is an emerging in silico technique with potential applications in diverse areas of pharmacology. Over the past three decades MD has evolved as an area of importance for understanding the atomic basis of complex phenomena such as molecular recognition, protein folding, and the transport of ions and small molecules across membranes. The application of MD simulations in isolation and in conjunction with experimental approaches have provided an increased understanding of protein structure-function relationships and demonstrated promise in drug discovery.
The analysis of membrane domain segregation and phase separation is of significant interest for t... more The analysis of membrane domain segregation and phase separation is of significant interest for the understanding of cell membranes. In this paper, we report a new approach to atomic force microscopy (AFM) of artificial membranes, using both functionalised AFM tips and "Force Volume" AFM imaging for the analysis of membrane phase separation. Simultaneous topology and mapping of interaction forces of binary component phospholipid bilayer membranes was performed yielding novel insights into membrane structure at unprecedented resolution.
Pharmacogenetics, 1997
Single nucleotide substitutions are known to result in a different amino acid at one of four site... more Single nucleotide substitutions are known to result in a different amino acid at one of four sites in cytochrome P4502C9 (CYP2C9) namely: residue 144: Arg/Cys; residue 358: Tyr/Cys; residue 359: Ile/Leu and residue 417: Gly/Asp. Polymerase chain reaction (PCR)-based amplification of the nucleotide fragments encompassing the four residues (144, 358-359 and 417) in 18 samples of human genomic DNA from a liver bank and one sample of DNA extracted from the blood of a known poor metabolizer of tolbutamide has been carried out. The products of PCR amplification were analysed by either allele-specific restriction endonucleases or probed with radioactively labelled allele-specific oligonucleotides in dot blot hybridizations. Fourteen individuals were homozygous for Arg144 and four were heterozygous Arg/Cys144. All individuals analysed were homozygous for Tyr358 (n = 17) and for Gly417 (n = 18). With the exception of one heterozygote the other 17 subjects were homozygous for Ile359. The genotype of the known poor metabolizer of tolbutamide was homozygous for Arg144, Leu359 and Gly417. The relative levels of expression of the Cys and Arg144 alleles was studied in the heterozygotes. A relative 5- to 10-fold greater expression of the Cys- over the Arg144 allele was noted in two heterozygotes. There was no apparent correlation of genotype to the hydroxylation of the known CYP2C9 substrates phenytoin, tolbutamide, torasemide and diclofenac. Apparent K(m) values for the cDNA-expressed Arg144/Ile359, Cys144/ Ile359 and Arg144/Leu359 variants towards tolbutamide were 91 microM, 62 microM and 229 microM, respectively. It is likely that functional changes occurring as a result of the Ile359Leu transition are responsible for the tolbutamide poor metabolizer phenotype.
Molecular Pharmacology, 2004
The UDP-glucuronosyltransferase (UGT) enzyme 'superfamily' contributes to the metabolism of a myr... more The UDP-glucuronosyltransferase (UGT) enzyme 'superfamily' contributes to the metabolism of a myriad of drugs, nondrug xenobiotic agents, and endogenous compounds. Although the individual UGT isoforms exhibit distinct but overlapping substrate selectivities, structural features of substrates that confer selectivity remain largely unknown. Using methods developed for pharmacophore fingerprinting combined with optimization and pattern recognition techniques, subsets of pharmacophores associated with the substrates and nonsubstrates of 12 human UGT isoforms were selected to generate predictive models of substrate selectivity and to elucidate the chemical and structural features associated with substrates and nonsubstrates. For all 12 UGT isoforms, the pharmacophore model generated showed predictive ability, as determined by a test set comprising 30% of the available data for each isoform.
Journal of Pharmacology and Experimental Therapeutics, 2010
Since COD (codeine) is eliminated primarily via glucuronidation, factors that alter COD glucuroni... more Since COD (codeine) is eliminated primarily via glucuronidation, factors that alter COD glucuronide formation potentially affect the proportion of the dose converted to the pharmacologically active metabolite morphine. Thus, in vitro-in vivo extrapolation (IV-IVE) approaches were utilized to identify potential drug-drug interactions arising from inhibition of COD glucuronidation in humans. Initial studies characterized the kinetics of COD 6-glucuronide (C6G) formation by human liver microsomes (HLM), and demonstrated an 88% reduction in K m (0.29 vs. 2.32 mM) for incubations performed in the presence of 2% bovine serum albumin (BSA). Of 13 recombinant UGT enzymes screened for COD glucuronidation activity, only UGT2B4 and UGT2B7 exhibited activity. The respective S 50 values (0.32 and 0.27 mM) generated in the presence of BSA were comparable to the mean K m observed in HLM. Known inhibitors of UGT2B7 activity in vitro or in vivo and drugs marketed as compound formulations with COD were investigated for inhibition of C6G formation by HLM. Inhibition screening identified potential interactions with dextropropoxyphene, fluconazole, ketoconazole and methadone. K i values generated for dextropropoxyphene (3.5 µM), fluconazole (202 µM), ketoconazole (0.66 µM) and methadone (0.32 µM) predicted 1.60-to 3.66-fold increases in the AUC ratio for COD in vivo. Whereas fluconazole and ketoconazole inhibited UGT2B4-and UGT2B7-catalyzed COD glucuronidation to a similar extent, inhibition by dextropropoxyphene and methadone resulted largely from an effect on UGT2B4. Interactions with dextropropoxyphene, fluconazole, ketoconazole and methadone potentially affect the intensity and duration of COD analgesia.
European Journal of Clinical Pharmacology, 1984
The effects of cimetidine (1 g/day) on theophylline disposition and metabolism were examined in s... more The effects of cimetidine (1 g/day) on theophylline disposition and metabolism were examined in smokers and non-smokers for single dose intravenous and chronic oral administration of theophylline. In the intravenous study the effect of cimetidine on plasma theophylline clearance was more marked in smokers (22.7% reduction) than in non-smokers (12.2% reduction). Similarly, in the multiple dose study the effect of cimetidine on theophylline clearance was greater in smokers (28.3% decrease) than in non-smokers (1I .3% decrease). The reduction in clearance was largely due to a reduction in metabolic clearances by 3-demethylation (C13 DM) and 1-demethylation (Cll DM) with no significant effect on clearance by 8-oxidation (C180x). There was a strong correlation between C13DM and CI1DM (r =0.98, p < 0.001) in both control and cimetidine study phases, whereas other coErelations between partial clearances were less marked and were not apparent during the cimetidine phase. The results are consistent with the view that 1-and 3-demethylation of theophylline are carried out by a common form of cytochrome P-450 which is selectively induced by cigarette smoking and preferentially inhibited by cimetidine.
Drug Metabolism Reviews, 2009
Major advances in the characterization of uridine diphosphate (UDP)-glucuronosyltransferase (UGT)... more 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 &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot;probes&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot; 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.
Drug Metabolism Reviews, 2010
The role of the kidney in drug and chemical disposition has traditionally focused on the excretio... more The role of the kidney in drug and chemical disposition has traditionally focused on the excretion of polar xenobiotics and metabolites. However, there is increasing evidence demonstrating that renal UGTs are integral to the &amp;amp;amp;amp;amp;amp;amp;quot;local&amp;amp;amp;amp;amp;amp;amp;quot; intrarenal, and, possibly, systemic, metabolic clearance of numerous drugs and nondrug xenobiotics, as well as to the maintenance of renal homeostasis through limiting the biological activity of endogenous renal mediators that control electrolyte balance and renal blood flow. The common involvement of UGT1A9 and UGT2B7 in the metabolism of both endogenous and exogenous compounds in kidney predicates significant renal drug-endobiotic interactions that may explain, in part, the adverse renal effects of some drugs.
Drug Metabolism and Disposition, 2005
Relatively few selective substrate and inhibitor probes have been identified for human UDP-glucur... more Relatively few selective substrate and inhibitor probes have been identified for human UDP-glucuronosyltransferases (UGT). This work investigated the selectivity of trifluoperazine (TFP), as a substrate, and amitriptyline, androsterone, canrenoic acid, hecogenin, phenylbutazone, quinidine, quinine and sulfinpyrazone, as inhibitors, for human UGTs. Selectivity was assessed using UGT 1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B7 and 2B15 expressed in HEK293 cells. TFP was confirmed as a highly selective substrate for UGT1A4. However, TFP bound extensively to both HEK293 lysate and to human liver microsomes in a concentration-dependent manner (fu inc 0.20-0.59). When corrected for non-specific binding, K m values for TFP glucuronidation were similar for both UGT1A4 (4.1 µM) and human liver microsomes (6.1 ± 1.2 µM) as the enzyme sources. Of the compounds screened as inhibitors, hecogenin alone was selective; significant inhibition was observed only for UGT1A4 (IC 50 1.5 µM). Using phenylbutazone and quinine as 'models', inhibition kinetics were variously described by competitive and noncompetitive mechanisms. Inhibition of UGT2B7 by quinidine was also investigated further since the effects of this compound on morphine pharmacokinetics (a known UGT2B7 substrate) have been ascribed to inhibition of P-glycoprotein. Quinidine inhibited human liver microsomal and recombinant UGT2B7 with respective K i values of 335 ± 128 µM and 186 µM. In conclusion, TFP and hecogenin represent selective substrate and inhibitor probes for UGT1A4, although the extensive non-selective binding of the former should be taken into account in kinetic studies. Amitriptyline, androsterone, canrenoic acid, hecogenin, phenylbutazone, quinidine, quinine and sulfinpyrazone are non-selective UGT inhibitors.