Metabolism of 2,5-bis(trifluoromethyl)-7-benzyloxy-4-trifluoromethyl- coumarin by human hepatic CYP isoforms : evidence for selectivity towards CYP3A4
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1. The metabolism of 2,5-bis(trifluoromethyl)-7-benzyloxy-4-trifluoromethylcoumarin (BFBFC) to 7-hydroxy-4-trifluoromethylcoumarin (HFC) was studied in human liver microsomes and in cDNA-expressed human liver CYP isoforms. For purposes of comparison, some limited studies were also performed with 7-benzyloxyquinoline (7BQ).
2. Initial interactive docking studies with a homology model of human CYP3A4 indicated that BFBFC was likely to be a selective substrate for CYP3A4 with a relatively high binding affinity, due to the presence of several key hydrogen bonds with active site amino acid residues.
3. Kinetic analysis of NADPH-dependent BFBFC metabolism to HFC in three preparations of pooled human liver microsomes revealed mean (±SEM) K<sub>m</sub> and V<sub>max</sub> = 4.6 ± 0.3 μM and 20.0 ± 3.8 pmol/min/mg protein, respectively.
4. The metabolism of BFBFC to HFC was determined in a characterized bank of 24 individual human liver microsomal preparations employing a BFBFC substrate concentration of 10 μM (i.e. around twice K<sub>m</sub>). Good correlations (r<sup>2</sup> = 0.736-0.904) were observed between BFBFC metabolism and markers of CYP3A isoforms.
5. While 10 μM BFBFC was metabolized to HFC by cDNA-expressed CYP3A4, little or no metabolism was observed with cDNA-expressed CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP2E1.
6. The metabolism of 10 μM BFBFC in human liver microsomes was markedly inhibited by 5-50 μM troleandomycin and 0.2-5 μM ketoconazole, but stimulated by 0.2-10 μM α-naphthoflavone. The metabolism of 10 μM BFBFC in human liver microsomes was also markedly inhibited by an antibody to CYP3A4.
7. Kinetic analysis of NADPH-dependent 7BQ metabolism to 7-hydroxyquinoline (7HQ) in human liver microsomes revealed K<sub>m</sub> and V<sub>max</sub> = 70 μM and 3.39 nmol/min/mg protein, respectively.
8. While 80 μM 7BQ was metabolized to 7HQ by cDNA-expressed CYP3A4, only low rates of metabolism were observed with cDNA-expressed CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP2E1.
9. In summary, by correlation analysis, the use of cDNA-expressed CYP isoforms, chemical inhibition and inhibitory antibodies, BFBFC metabolism in human liver microsomes appears to be primarily catalysed by CYP3A4. BFBFC may be a useful fluorescent probe substrate for human hepatic CYP3A4, but compared with 7BQ has only a low rate of metabolism in human liver microsomes.
2. Initial interactive docking studies with a homology model of human CYP3A4 indicated that BFBFC was likely to be a selective substrate for CYP3A4 with a relatively high binding affinity, due to the presence of several key hydrogen bonds with active site amino acid residues.
3. Kinetic analysis of NADPH-dependent BFBFC metabolism to HFC in three preparations of pooled human liver microsomes revealed mean (±SEM) K<sub>m</sub> and V<sub>max</sub> = 4.6 ± 0.3 μM and 20.0 ± 3.8 pmol/min/mg protein, respectively.
4. The metabolism of BFBFC to HFC was determined in a characterized bank of 24 individual human liver microsomal preparations employing a BFBFC substrate concentration of 10 μM (i.e. around twice K<sub>m</sub>). Good correlations (r<sup>2</sup> = 0.736-0.904) were observed between BFBFC metabolism and markers of CYP3A isoforms.
5. While 10 μM BFBFC was metabolized to HFC by cDNA-expressed CYP3A4, little or no metabolism was observed with cDNA-expressed CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP2E1.
6. The metabolism of 10 μM BFBFC in human liver microsomes was markedly inhibited by 5-50 μM troleandomycin and 0.2-5 μM ketoconazole, but stimulated by 0.2-10 μM α-naphthoflavone. The metabolism of 10 μM BFBFC in human liver microsomes was also markedly inhibited by an antibody to CYP3A4.
7. Kinetic analysis of NADPH-dependent 7BQ metabolism to 7-hydroxyquinoline (7HQ) in human liver microsomes revealed K<sub>m</sub> and V<sub>max</sub> = 70 μM and 3.39 nmol/min/mg protein, respectively.
8. While 80 μM 7BQ was metabolized to 7HQ by cDNA-expressed CYP3A4, only low rates of metabolism were observed with cDNA-expressed CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP2E1.
9. In summary, by correlation analysis, the use of cDNA-expressed CYP isoforms, chemical inhibition and inhibitory antibodies, BFBFC metabolism in human liver microsomes appears to be primarily catalysed by CYP3A4. BFBFC may be a useful fluorescent probe substrate for human hepatic CYP3A4, but compared with 7BQ has only a low rate of metabolism in human liver microsomes.
Topics
2,5 bis(trifluoromethyl) 7 benzyloxy 4 trifluoromethylcoumarin7 benzyloxyquinoline7 hydroxy 4 trifluoromethylcoumarin7 hydroxyquinolineAlpha naphthoflavoneComplementary DNACoumarin derivativeCytochrome P450 1A2Cytochrome P450 2A6Cytochrome P450 2B6Cytochrome P450 2C19Cytochrome p450 2c8Cytochrome P450 2C9Cytochrome P450 2D6Cytochrome P450 2E1Cytochrome P450 3A4Fluorescent dyeKetoconazolel 871420Quinoline derivativeQuinolinol derivativeReduced nicotinamide adenine dinucleotide phosphateTroleandomycinUnclassified drugBinding affinityControlled studyDrug metabolismEnzyme active siteEnzyme kineticsEnzyme specificityEnzyme substrate complexHumanHuman cellHydrogen bondInhibition kineticsLiver microsomeCoumarinsCytochrome P-450 Enzyme SystemDNA, ComplementaryDose-Response Relationship, DrugHepatocytesHumansKineticsLiverMicrosomes, LiverMixed Function OxygenasesModels, ChemicalModels, MolecularPhenotypeProtein BindingProtein IsoformsQuinolinesSubstrate SpecificityTime Factors
TNO Identifier
72303
Source
Xenobiotica, 31(4), pp. 187-204.
Pages
187-204
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