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The relationship between the methylhydroxylation off tolbutamide and the 4-hydroxylation off phenytoin in human liver microsomes
1643 O.th.25.4 [
The relationship between the methylhydroxylation of tolbutamide and the 4-hydroxylafion of phenytoin in human fiver microsomes Veronese, M.E., Doecke *, C.J., McManus, M.E., Sansom *, L.N., Miners, J.O. and Birkett, D.J. Department of Clinical Pharmacology, Fiinders University of South Australia, Bedford Park, SA 5042 and * School of Pharmacy, South Australian Institute of Technology, Adelaide SA 5000, Australia
Genetic polymorphisms of cytochrome P450s have been suggested to contribute significantly to the marked inter-individual differences in elimination of both tolbutamide and phenytoin in man. To define what isozyme(s) of P450 are involved in the metabolism of these two commonly used drugs we have studied their metabolism in human liver microsomes. The oxidation of the tolyl methyl group of tolbutamide to form hydroxytolbutamide (OHTB) in human liver microsomes when analysed by Eadie-Hofstee plots is best described by a single enzyme component, which as consistent with the involvement of a single isozyme of cytochrome P450 (Miners et al., 1988). Recently it has been shown in a yeast expression system that this reaction can be mediated by human P45011C10 [P450~] (Brien et al., 1989). We have also shown that the kinetics of phenytoin 4-hydroxylation to 5-(4-hydroxyphenyl)-5-phenylhydantoin (HPPH) is also best described by a single enzyme component. In human liver microsomes from 18 subjects the mean tolbutamide hydroxylase activity was 272 + 88 pmol O H T B / m i n / m g protein, with a range of 155 to 430 pmol O H T B / m i n / m g protein. In these same human liver microsomes the mean phenytoin hydroxylase activity was 4.20 + 0.29 pmol H P P H / m i n / m g protein, with a range of 2.64 to 7.05 pmol H P P H / m i n / m g protein. A statistically significant correlation was obtained between these two activities (r - 0.852, p < 0.001). We then investigated these two activities with respect to inhibition by: (1) sulphaphenazole, a selective and potent inhibitor for the human tolbutamide cytochrome P450 isozyme, and (2) a polyclonal antibody raised against rabbit P45011C3 which is totally inhibitory to phenytoin hydroxylation in rabbit liver microsomes. Sulphaphenazole gave identical ICs0s (0.5/~M) for both tolbutamide and phenytoin hydroxylations. Both of these reactions were inhibited to a similar extent (65%) by anti-P4501IC3 IgG. To distinguish further the similarities in the isozymes hydroxylating these two drugs the kinetics of reciprocal inhibition was analysed by Lineweaver-Burke plots. Phenytoin was a competitive inhibitor of tolbutamide hydroxylation (Ki = 22.6 + 5.6 /tM, n--3), this being simiiar to the Km for phenytoin •*J,.,*,,,,J,,-~,-t"' . . . . . , .... a ;n" "*'o~o,,,.....hum~..n _...t;,,,'rs(u-,.,,..~..-_ .....°~ ,; .,.-L....'~ 1 p~.~). Tolbutamide was also a competitive inhibitor of phenytoin hydroxylation (Ki -- 114.4 + 16.7 pM, n -- 2), this being similar to the Km for tolbutamide hydroxylase (Kin = 87.8 + 7.7 tiM, n = 3). The agreement between the Km and Ki values indicate that tolbutamide and phenytoin act as competitive inhibitors of the hydroxylations of each other. These data suggest that the methylhydroxylation of tolbutamide and the 4-hydroxylation of phenytoin, the major pathways for the elimination of these drugs, are mediated by the same isozyme(s) of cytochrome P450. Studies are being undertaken to determine whether this isozyme is subject to genetic polymorphism in the Australian population. References Brien, W.R. et al., 1989, Biochemistry 28, 4993. Miners, J.O. et al., 1988, Biochem. Pharmacol. 37, 1137.
The relationship between the methylhydroxylation of tolbutamide and the 4-hydroxylafion of phenytoin in human fiver microsomes Veronese, M.E., Doecke *, C.J., McManus, M.E., Sansom *, L.N., Miners, J.O. and Birkett, D.J. Department of Clinical Pharmacology, Fiinders University of South Australia, Bedford Park, SA 5042 and * School of Pharmacy, South Australian Institute of Technology, Adelaide SA 5000, Australia
Genetic polymorphisms of cytochrome P450s have been suggested to contribute significantly to the marked inter-individual differences in elimination of both tolbutamide and phenytoin in man. To define what isozyme(s) of P450 are involved in the metabolism of these two commonly used drugs we have studied their metabolism in human liver microsomes. The oxidation of the tolyl methyl group of tolbutamide to form hydroxytolbutamide (OHTB) in human liver microsomes when analysed by Eadie-Hofstee plots is best described by a single enzyme component, which as consistent with the involvement of a single isozyme of cytochrome P450 (Miners et al., 1988). Recently it has been shown in a yeast expression system that this reaction can be mediated by human P45011C10 [P450~] (Brien et al., 1989). We have also shown that the kinetics of phenytoin 4-hydroxylation to 5-(4-hydroxyphenyl)-5-phenylhydantoin (HPPH) is also best described by a single enzyme component. In human liver microsomes from 18 subjects the mean tolbutamide hydroxylase activity was 272 + 88 pmol O H T B / m i n / m g protein, with a range of 155 to 430 pmol O H T B / m i n / m g protein. In these same human liver microsomes the mean phenytoin hydroxylase activity was 4.20 + 0.29 pmol H P P H / m i n / m g protein, with a range of 2.64 to 7.05 pmol H P P H / m i n / m g protein. A statistically significant correlation was obtained between these two activities (r - 0.852, p < 0.001). We then investigated these two activities with respect to inhibition by: (1) sulphaphenazole, a selective and potent inhibitor for the human tolbutamide cytochrome P450 isozyme, and (2) a polyclonal antibody raised against rabbit P45011C3 which is totally inhibitory to phenytoin hydroxylation in rabbit liver microsomes. Sulphaphenazole gave identical ICs0s (0.5/~M) for both tolbutamide and phenytoin hydroxylations. Both of these reactions were inhibited to a similar extent (65%) by anti-P4501IC3 IgG. To distinguish further the similarities in the isozymes hydroxylating these two drugs the kinetics of reciprocal inhibition was analysed by Lineweaver-Burke plots. Phenytoin was a competitive inhibitor of tolbutamide hydroxylation (Ki = 22.6 + 5.6 /tM, n--3), this being simiiar to the Km for phenytoin •*J,.,*,,,,J,,-~,-t"' . . . . . , .... a ;n" "*'o~o,,,.....hum~..n _...t;,,,'rs(u-,.,,..~..-_ .....°~ ,; .,.-L....'~ 1 p~.~). Tolbutamide was also a competitive inhibitor of phenytoin hydroxylation (Ki -- 114.4 + 16.7 pM, n -- 2), this being similar to the Km for tolbutamide hydroxylase (Kin = 87.8 + 7.7 tiM, n = 3). The agreement between the Km and Ki values indicate that tolbutamide and phenytoin act as competitive inhibitors of the hydroxylations of each other. These data suggest that the methylhydroxylation of tolbutamide and the 4-hydroxylation of phenytoin, the major pathways for the elimination of these drugs, are mediated by the same isozyme(s) of cytochrome P450. Studies are being undertaken to determine whether this isozyme is subject to genetic polymorphism in the Australian population. References Brien, W.R. et al., 1989, Biochemistry 28, 4993. Miners, J.O. et al., 1988, Biochem. Pharmacol. 37, 1137.