Sally Coulter

Flinders Medical Centre, Tarndarnya, South Australia, Australia

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Publications (6)15.56 Total impact

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    ABSTRACT: Aims To investigate inhibition of recombinant CYP2C8 by: (i) prototypic CYP isoform selective inhibitors (ii) imidazole/triazole antifungal agents (known inhibitors of CYP), and (iii) certain CYP3A substrates (given the apparent overlapping substrate specificity of CYP2C8 and CYP3A).Methods CYP2C8 and NADPH-cytochrome P450 oxidoreductase were coexpressed in Spodoptera frugiperda (Sf21) cells using the baculovirus expression system. CYP isoform selective inhibitors, imidazole/triazole antifungal agents and CYP3A substrates were screened for their inhibitory effects on CYP2C8-catalysed torsemide tolylmethylhydroxylation and, where appropriate, the kinetics of inhibition were characterized. The conversion of torsemide to its tolylmethylhydroxy metabolite was measured using an h.p.l.c. procedure.Results At concentrations of the CYP inhibitor ‘probes’ employed for isoform selectivity, only diethyldithiocarbamate and ketoconazole inhibited CYP2C8 by > 10%. Ketoconazole, at an added concentration of 10 µm, inhibited CYP2C8 by 89%. Another imidazole, clotrimazole, also potently inhibited CYP2C8. Ketoconazole and clotrimazole were both noncompetitive inhibitors of CYP2C8 with apparent Ki values of 2.5 µm. The CYP3A substrates amitriptyline, quinine, terfenadine and triazolam caused near complete inhibition (82–91% of control activity) of CYP2C8 at concentrations five-fold higher than the known CYP3A Km. Kinetic studies with selected CYP3A substrates demonstrated that most inhibited CYP2C8 noncompetitively. Apparent Ki values for midazolam, quinine, terfenadine and triazolam ranged from 5 to 25 µm.Conclusions Inhibition of CYP2C8 occurred at concentrations of ketoconazole and diethyldithiocarbamate normally employed for selective inhibition of CYP3A and CYP2E1, respectively. Some CYP3A substrates have the capacity to inhibit CYP2C8 activity and this may have implications for inhibitory drug interactions in vivo.
    British Journal of Clinical Pharmacology 01/2008; 50(6):573-580. · 3.69 Impact Factor
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    ABSTRACT: To investigate inhibition of recombinant CYP2C8 by: (i) prototypic CYP isoform selective inhibitors (ii) imidazole/triazole antifungal agents (known inhibitors of CYP), and (iii) certain CYP3A substrates (given the apparent overlapping substrate specificity of CYP2C8 and CYP3A). CYP2C8 and NADPH-cytochrome P450 oxidoreductase were coexpressed in Spodoptera frugiperda (Sf21) cells using the baculovirus expression system. CYP isoform selective inhibitors, imidazole/triazole antifungal agents and CYP3A substrates were screened for their inhibitory effects on CYP2C8-catalysed torsemide tolylmethylhydroxylation and, where appropriate, the kinetics of inhibition were characterized. The conversion of torsemide to its tolylmethylhydroxy metabolite was measured using an h.p.l.c. procedure. At concentrations of the CYP inhibitor 'probes' employed for isoform selectivity, only diethyldithiocarbamate and ketoconazole inhibited CYP2C8 by > 10%. Ketoconazole, at an added concentration of 10 microM, inhibited CYP2C8 by 89%. Another imidazole, clotrimazole, also potently inhibited CYP2C8. Ketoconazole and clotrimazole were both noncompetitive inhibitors of CYP2C8 with apparent Ki values of 2.5 microM. The CYP3A substrates amitriptyline, quinine, terfenadine and triazolam caused near complete inhibition (82-91% of control activity) of CYP2C8 at concentrations five-fold higher than the known CYP3A Km. Kinetic studies with selected CYP3A substrates demonstrated that most inhibited CYP2C8 noncompetitively. Apparent Ki values for midazolam, quinine, terfenadine and triazolam ranged from 5 to 25 microM. Inhibition of CYP2C8 occurred at concentrations of ketoconazole and diethyldithiocarbamate normally employed for selective inhibition of CYP3A and CYP2E1, respectively. Some CYP3A substrates have the capacity to inhibit CYP2C8 activity and this may have implications for inhibitory drug interactions in vivo.
    British Journal of Clinical Pharmacology 01/2001; 50(6):573-80. · 3.69 Impact Factor
  • Pharmacogenetics 05/2000; 10(3):267-70.
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    ABSTRACT: 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.
    Pharmacogenetics 03/1997; 7(1):51-8.
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    ABSTRACT: 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 IIe359. 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 Km values for the cDNA-expressed Arg144/Ile359, Cys144/ Ile359 and Arg144/Leu359 variants towards tolbutamide were 91[micro]M, 62[micro]M and 229[micro]M, respectively. It is likely that functional changes occurring as a result of the Ile359Leu transition are responsible for the tolbutamide poor metabolizer phenotype. (C) Lippincott-Raven Publishers.
    Pharmacogenetics and Genomics 01/1997; 7(1). · 3.61 Impact Factor
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    ABSTRACT: A preliminary report implicated cytochrome P450 (CYP) 2C9 in the human liver microsomal O-demethylation of S-naproxen, suggesting that this pathway may be suitable for investigation of human hepatic CYP2C9 in vitro. Kinetic and inhibitor studies with human liver microsomes and confirmatory investigations with cDNA-expressed enzymes were undertaken here to define the role of CYP2C9 and other isoforms in the O-demethylation of R- and S-naproxen. All studies utilised a newly developed sensitive and specific HPLC assay that measured the respective O-desmethyl metabolites of R- and S-naproxen in incubations of human liver microsomes and in COS cell lysates. Microsomal R- and S-naproxen O-demethylation kinetics followed Michaelis-Menten kinetics, with respective mean apparent Km values of 123 μM and 143 μM. Sulfaphenazole, a specific inhibitor of CYP2C9, reduced the microsomal O-demethylation of R-and S-naproxen by 43% and 47%, respectively, and the CYP1A2 inhibitor furafylline decreased R- and S-naproxen O-demethylation by 38% and 28%, respectively. R,S-Mephenytoin was a weak inhibitor of R- and S-naproxen O-demethylation, but other CYP isoform specific inhibitors (e.g., coumarin, diethyldithiocarbamate, quinidine, troleandomycin) had little or no effect on these reactions. cDNA-expressed CYP2C9 and CYP1A2 were both shown to O-demethylate R- and S-naproxen. Apparent Km values (92–156 μM) for the reactions catalysed by the recombinant enzymes were similar to those observed for human liver microsomal R- and S-naproxen O-demethylation. The data demonstrate that CYP2C9 and CYP1A2 together account for the majority of human liver R- and S-naproxen O-demethylation, precluding the use of either R- or S-naproxen as a CYP isoform-specific substrate in vitro and in vivo.
    Biochemical Pharmacology 05/1996; · 4.58 Impact Factor