A single amino acid-substituted mutant protein, CYP2D6 (G42R) was expressed in Saccharomyces cerevisiae and its enzymatic properties were compared with those of other single (P34S, R296C and S486T) and double amino acid-substituted mutant proteins (P34S/S486T and R296C/S486T) expressed in yeast cells, all of which were known to occur in the CYP2D6 gene as single nucleotide polymorphisms. The protein levels of G42R, P34S and P34S/S486T in microsomal fractions and their oxidation capacities towards debrisoquine as a prototypic substrate and bunitrolol as a chiral substrate were different from those of wild-type CYP2D6, while the R296C, S486T and R296C/S486T behaved similarly to the wild-type in these indices. The CYP contents both in yeast microsomal and in whole cell fractions indicated that some part of G42R protein was localized in the endoplasmic reticulum membrane fraction, whereas most of G42R protein was in some subcellular fractions other than endoplasmic reticulum. In kinetic analysis, the G42R substitution increased apparent Km and decreased Vmax for debrisoquine 4-hydroxylation, while it increased both Km and Vmax for bunitrolol 4-hydroxylation. The P34S substitution did not drastically change Km but decreased Vmax for debrisoquine 4-hydroxylation, whereas Km was increased and Vmax unchanged or decreased for bunitrolol 4-hydroxylation by P34S substitution. These results suggest that the G42R substitution causes a change in the CYP2D6 conformation, which may be different from the change produced by the P34S substitution.

Bufuralol (BF), a nonselective beta-adrenoceptor blocking agent, has a chiral center in its molecule, yielding the enantiomers 1'R-BF and 1'S-BF. beta-Adrenoceptor blocking potency is much higher in 1'S-BF than in 1'R-BF. One of the metabolic pathways of BF is 1"-hydroxylation of an ethyl group attached at the aromatic 7-position forming a carbinol metabolite (1"-hydroxybufuralol, 1"-OH-BF), and further oxidation (or dehydrogenation) produces a ketone metabolite (1-oxobufuralol, 1"-Oxo-BF). Both 1"-OH-BF and 1"-Oxo-BF are known to have beta-adrenoceptor blocking activities comparable to or higher than those of the parent drug. The 1"-hydroxylation introduces another chiral center into the BF molecule and four 1"-OH-BF diastereomers are formed from BF racemate in mammals, including humans, making elucidation of the metabolic profiles complicated. HPLC methods employing derivatization, reversed phase, or chiral columns have been developed to efficiently separate the four 1"-OH-BF diastereomers formed from BF enantiomers or racemate. Accumulated in vitro experimental results revealed that 1'R-BF is a much more preferential substrate than 1'S-BR for BF 1"-hydroxylation in human liver microsomes. Kinetic studies using recombinant human cytochrome P450 (CYP) enzymes indicate that CYP2D6 serves as a major BF 1"-hydroxylase and that CYP1A2 and CYP2C19 also contribute to BF 1"-hydroxylation in human livers. This mini-review summarizes the knowledge reported so far on the pharmacology of BF and its metabolites and the profiles of BF metabolism, especially focusing on the stereoselectivity in the oxidation of BF mainly in human livers and recombinant CYP enzymes.

A new HPLC method was developed using a chiral column to efficiently separate four 1ЈЈ-hydroxybufuralol (1ЈЈ-OH-BF) diastereomers that are major metabolites of bufuralol (BF). Employing this method, we examined diastereomer selectivity in the formation of 1ЈЈ-OH-BF from BF racemate or enantiomers in four individual samples of human liver microsomes. Three different human liver microsomes showed a selectivity of 1ЈЈR-OH Ͻ 1ЈЈS-OH for BF enantiomers, which was similar to that of recombinant CYP2D6 expressed in insect cell microsomes, whereas one human liver microsomal fraction yielded a selectivity of 1ЈЈR-OH Ͼ 1ЈЈS-OH for BF enantiomers, which was similar to those of recombinant CYP2C19 expressed in insect cell microsomes. Recombinant CYP1A2 and CYP3A4 showed a selectivity similar to that of CYP2D6, but their BF 1ЈЈ-hydroxylase activities were much lower than those of CYP2D6. In inhibition studies, quinidine, a known CYP2D6 inhibitor, markedly inhibited BF 1ЈЈ-hydroxylation in the fractions of human liver microsomes that showed the CYP2D6-type selectivity. Furthermore, omeprazole, a known CYP2C19 inhibitor, efficiently suppressed the formation of 1ЈЈ-OH-BF diastereomers from BF in the microsomal fraction that showed the CYP2C19-type selectivity. From these results, we concluded that the diastereomer selectivity in the formation of 1ЈЈ-OH-BF from BF differs between CYP2D6 and CYP2C19, both of which can be determinant enzymes in the diastereoselective 1ЈЈ-hydroxylation of BF in human liver microsomes.Cytochrome P450 2D6 (CYP2D6) is known to be one of the major drug-metabolizing CYP enzymes in human livers and is responsible for the major metabolic pathways of more than 60 clinically used medicines that are often prescribed (Gonzalez, 1996;Meyer and Zanger, 1997). Bufuralol (BF; Fig. 1), a ␤-adrenoceptor blocking agent, is a typical substrate of CYP2D6. As shown in Fig. 1, BF has an asymmetric carbon at the side chain, yielding enantiomers, 1ЈR-BF (R-BF) and 1ЈS-BF (S-BF). The major metabolic pathway of BF is 1ЈЈ-hydroxylation of the ethyl group at the 7-position of its aromatic ring by CYP2D6. The 1ЈЈ-hydroxylation produces a new chiral center in the BF molecule, thereby theoretically yielding four diastereomers [1ЈЈR-OH-1ЈR-BF (1ЈЈR-OH-R-BF), Fig. 1). 1ЈЈS-OH-1ЈR-BF (1ЈЈS-OH-R-BF), 1ЈЈR-OH-1ЈS-BF (1ЈЈR-OH-S-BF) and 1ЈЈS-OH-1ЈS-BF (1ЈЈS-OH-S-BF)] of 1ЈЈ-hydroxybufurarol (1ЈЈ-OH-BF) from racemic BF (seeFor studies on the stereoselectivity in the oxidative metabolism of BF enantiomers by mammalian liver microsomes, Weerawarna et al. (1991) developed an HPLC method employing phenylethylurea derivatives of diastereomeric 1ЈЈ-OH-BFs and demonstrated that rat liver microsomes favored the formation of 1ЈЈR-OH metabolite over 1ЈЈS-OH metabolite from both R-BF and S-BF, whereas human liver microsomes favor the formation of 1ЈЈS-OH metabolite over 1ЈЈR-OH metabolite from the same substrates. Therefore, between rats and humans the stereoselectivity in the formation of 1ЈЈ-OH-BF diastereomers was shown to be reversed. ...

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