Significant variations in the metabolism of drugs and environmental chemicals which are metabolized via the cytochrome P450 (CYP) enzymes exist between humans on an individual and population scale. The overall objectives of this work is to elucidate the molecular and metabolic basis of CYP related polymorphisms, characterization of the substrate-structure metabolism relationships, and the determination of critical protein structures of CYP enzymes that infer substrate specificity and metabolic activity. Methods to study the metabolism and kinetics of a number of CYP substrates inculding the anticancer drug taxol, the anticonvulsant drug mephenytoin, and the antihypertension drug nifidipine were developed in order to identify the polymorphisms, their functional significance, and the substrate recognition site (SRS) in CYP enzymes. The CYP3As are important in the metabolism of a wide range of drugs, steroids, and environmental chemicals. Variation in CYP3A activity has an important influence on the metabolism and the elimination of CYP3A substrates in humans. We previously have reported single nucleotide polymorphisms resulting in four CYP3A4 coding variants in three different racial groups. In the present study, using a bacterial cDNA expression system, we examined nifedipine metabolism by these recombinant allelic variants to determine whether any of these CYP3A4 allelic proteins are defective in metabolizing this drug. Metabolism of nifedipine by the L293P (CYP3A4*18), M445T (CYP3A4*3), and P467S (CYP3A4*19) recombinant allelic proteins was comparable to that of wild type CYP3A4*1. However, the 3A4 F189S (CYP3A4*17) allele exhibited >99% decrease in both Vmax and CLmax compared to CYP3A4*1. Out of 72 racially diverse individuals, CYP3A4*17 was identified in a single individual who was also homozygous for CYP3A5*3, indicating this SNP is part of one haplotype containing the CYP3A5*3 splice variant. Since most CYP3A substrates are metabolized by both CYP3A4 and CYP3A5 in humans, new specific PCR-RFLP genotyping procedures were developed to assess both the CYP3A4*17 allele and the major splice variant of CYP3A5 (CYP3A5*3). These new genetic tests will be useful in future clinical studies to study genotype/phenotype associations in vivo in individuals with this haplotype who would be predicted to metabolize CYP3A substrates poorly.
