Cytochrome P450 (CYP) 2C8 is the principal enzyme responsible for the metabolism of the anti-cancer drug paclitaxel (Taxol). It is also the predominant P450 responsible for the metabolism of arachidonic acid to biologically active epoxyeicosatrienoic acids (EETs) in human liver and kidney. In this study, we describe two new CYP2C8 alleles containing coding changes: CYP2C8*2 has an Ile269Phe substitution in exon 5 and CYP2C8*3 includes both Arg139Lys and Lys399Arg amino acid substitutions in exons 3 and 8. CYP2C8*2 was found only in African-Americans, while CYP2C8*3 occurred primarily in Caucasians. Neither occurred in Asians. The frequency of the CYP2C8*2 allele was 0.18 in African-Americans, and that of CYP2C8*3 was 0.13 in Caucasians. CYP2C8*1 (wild-type), CYP2C8*2 and CYP2C8*3 cDNAs were expressed in Escherichia coli, and the ability of these enzymes to metabolize both paclitaxel and arachidonic acid was assessed. Recombinant CYP2C8*3 was defective in the metabolism of both substrates. The turnover number of CYP2C8*3 for paclitaxel was 15% of CYP2C8*1. CYP2C8*2 had a two-fold higher Km and two-fold lower intrinsic clearance for paclitaxel than CYP2C8*1. CYP2C8*3 was also markedly defective in the metabolism of arachidonic acid to 11,12- and 14,15-EET (turnover numbers 35-40% that of CYP2C8*1). Thus, CYP2C8*3 is defective in the metabolism of two important CYP2C8 substrates: the anticancer drug paclitaxel and the physiologically important compound arachidonic acid. This polymorphism has important clinical and physiological implications in individuals homozygous for this allele.

CYP2C9 is a clinically important enzyme, responsible for the metabolism of numerous clinically important therapeutic drugs. In the present study, we discovered 38 single nucleotide polymorphisms in CYP2C9 by resequencing of genomic DNA from 92 individuals from three different racial groups. Haplotype analysis predicted that there are at least 21 alleles of CYP2C9 in this group of individuals. Six new alleles were identified that contained coding changes: L19I (CYP2C9*7), R150H (CYP2C9*8), H251R (CYP2C9*9), E272G (CYP2C9*10), R335W(CYP2C9*11) and P489S (CYP2C9*12). When expressed in a bacterial cDNA expression system, several alleles exhibited altered catalytic activity. CYP2C9*11 appeared to be a putative poor metabolizer allele, exhibiting a three-fold increase in the Km and more than a two-fold decrease in the intrinsic clearance for tolbutamide. Examination of the crystal structure of human CYP2C9 reveals that R335 is located in the turn between the J and J' helices and forms a hydrogen-bonding ion pair with D341 from the J' helix. Abolishing this interaction in CYP2C9*11 individuals could destabilize the secondary structure and alter the substrate affinity. This new putative poor metabolizer (PM) allele was found in Africans. A second potentially PM allele CYP2C9*12 found in a racially unidentified sample also exhibited a modest decrease in the Vmax and the intrinsic clearance for tolbutamide in a recombinant system. Further clinical studies are needed to determine the effect of these new polymorphisms on the metabolism of CYP2C9 substrates.

ABSTRACT:The cytochrome P450 (P450) enzymes CYP2C8, CYP2C9, and CYP2J2 metabolize arachidonic acid to epoxyeicosatrienoic acids, which are known to be vital in regulation of vascular tone and cardiovascular homeostasis. Because there is limited information regarding the relative expression of these P450 enzymes in cardiovascular tissues, this study examined the expression of CYP2C8, CYP2C9, and CYP2J2 mRNA and protein in human heart, aorta, and coronary artery samples by real-time polymerase chain reaction, immunoblotting, and immunohistochemistry. CYP2J2 and CYP2C9 mRNA levels were highly variable in human hearts, whereas CYP2C8 mRNA was present in lower abundance. CYP2J2 mRNA was approximately 10 3 times higher than CYP2C9 or CYP2C8 in human heart. However, CYP2C9 mRNA was more abundant than CYP2J2 or CYP2C8 in one ischemic heart. In human aorta, mean CYP2C9 mRNA levels were ϳ50 times higher than that of CYP2J2 and 5-fold higher than that of CYP2C8. In human coronary artery, mean values for CYP2C9 mRNA were ϳ2-fold higher than that of CYP2J2 mRNA and 6-fold higher than that of CYP2C8 mRNA. Immunoblotting results show relatively high levels of CYP2J2 and CYP2C8 protein in human hearts, which was confirmed by immunohistochemistry. CYP2C9 protein was also detected at high levels in one ischemic heart by immunoblotting. CYP2C9 was present at higher levels than CYPJ2 in aorta and coronary artery, whereas CYP2C8 protein was below the limits of detection. The expression of CYP2J2 and CYP2C8 in human heart, and CYPC9 and CYP2J2 in aorta and coronary artery is consistent with a physiological role for these enzymes in these tissues.