Defective Activity of Recombinant Cytochromes P450 3A4.2 and 3A4.16 in Oxidation of Midazolam, Nifedipine, and Testosterone

Abstract

ABSTRACT:Cytochrome P4503A4 (CYP3A4) is the most abundant cytochrome P450 in adult human liver and small intestine and oxidizes numerous clinically, physiologically, and toxicologically important compounds. The metabolic activity of CYP3A4 in patients varies at least 10-fold in vivo, and CYP3A4 genetic variants are considered one of the causes of individual differences. The cDNAs for the CYP3A4*2 (S222P), *7 (G56D), *16 (T185S), and *18 (L293P) mutant alleles, found in high frequencies in Caucasians or Asians, were constructed by site-directed mutagenesis and expressed in an Escherichia coli expression system. Midazolam (MDZ), testosterone (TST), and nifedipine (NIF) were used to assess the catalytic activities of the CYP3A4 wild type (CYP3A4.1) and its variants. The catalytic activities of CYP3A4.2 and CYP3A4.16 were reduced (lower V max and increased K m relative to CYP3A4.1) for all substrates. The CYP3A4.7 showed lower V max values for MDZ and NIF (60 and 84%, respectively) and a higher K m (2-fold) for TST but not for MDZ or NIF. Although CYP3A4.18 showed low V max values for MDZ, NIF, and TST (88, 72, and 80% of CYP3A4.1, respectively), no significant differences were identified in the ratio V max/Km . In summary, CYP3A4.2 and CYP3A4.16 exhibited significantly lower activity for MDZ, TST, and NIF oxidations than CYP3A4.1. Therefore, drugs metabolized by only CYP3A should be carefully administered to patients with these alleles.The CYP3A subfamily of cytochrome P450 enzymes comprises approximately 30 to 60% of total cytochrome P450 in human liver (Shimada et al., 1994). The CYP3A enzymes are responsible for the metabolism of more than 50% of clinically used drugs and also some steroids and environmental chemicals (Li et al., 1995;Evans and Relling, 1999;Guengerich, 1999). Four human CYP3A enzymes-CYP3A4, CYP3A5, CYP3A7, and CYP3A43-have been identified, and CYP3A4 is regarded as the most dominant CYP3A enzyme in the liver and small intestine (Wrighton et al., 1990;Shimada et al., 1994;Hustert et al., 2001;Koch et al., 2002). It has been reported that CYP3A4 expression shows large interindividual variation (Guengerich, 1999;Ozdemir et al., 2000;Lin et al., 2002) and that these variations can lead to different responses to human drugs that are substrates for CYP3A4. Because the expression of CYP3A4 and CYP3A5 can be induced by pregnane X receptor, constitutive androstane receptor, and glucuronide receptor ligands, both environmental and genetic factors can influence the CYP3A activity; however, the genetic contribution has been estimated to be larger (Ozdemir et al., 2000), suggesting that polymorphisms in CYP3A4 may predict CYP3A phenotype, at least in part. Genetic analysis is needed to understand interindividual variability. Extensive studies searching allelic variants in the coding regions of CYP3A4 have been done, and currently 20 different CYP3A4 variant proteins have been described, some of them representing proteins of either decreased or increased activity. Those polymorphisms are reported on the Hum...

Comparison of the Inhibitory Profiles of Itraconazole and Cimetidine in Cytochrome P450 3A4 Genetic Variants

“…Consistent with our previous report (Miyazaki et al, 2008), the K m values for TST 6␤-hydroxylation were increased in CYP3A4.7 and 16, decreased in CYP3A4.18, and comparable in CYP3A4.2 (in comparison with CYP3A4.1). The observed influences of genetic variation on the enzyme catalytic efficiency (V max /K m ) were also consistent with previous in vitro findings (Sata et al, 2000;Eiselt et al, 2001;Lamba et al, 2002;Murayama et al, 2002;FukushimaUesaka et al, 2004).…”

“…The observed influences of genetic variation on the enzyme catalytic efficiency (V max /K m ) were also consistent with previous in vitro findings (Sata et al, 2000;Eiselt et al, 2001;Lamba et al, 2002;Murayama et al, 2002;FukushimaUesaka et al, 2004). The absolute K m and V max values in the present study were not identical to those in our previous report (Miyazaki et al, 2008). Although the cause of this discrepancy remains uncertain, minor modification of the experimental condition, such as the composition of reaction buffer to solubilize both substrate and inhibitor in our present study, may have affected the absolute value of V max /K m .…”

“…Concentrations of 6 ␤-OHT were measured by an HPLC-UV method reported previously (Miyazaki et al, 2008). In brief, the HPLC system consisted of a pump (LC-10AD; Shimadzu, Kyoto, Japan), a UV detector (SPD-10AV spectrophotometer; Shimadzu), and an octadecylsilane column (Cosmosil, 5C18-AR-2, 4.6 ϫ 150 mm; Nacalai Tesque, Kyoto, Japan).…”

“…The membrane fractions for CYP3A4.1 (wild type), 2(S222P, 7(G56D, 16(T185S))), and 18(L293P) were prepared as described previously (Miyazaki et al, 2008). All other chemicals and reagents were of analytical or high-performance liquid chromatography (HPLC) grade and were obtained from commercial sources.…”

“…In our previous study, we compared the enzymatic activities of major CYP3A4 variants from alleles with relatively higher allelic frequency, such as CYP3A4*1 (wild type),*2 (2.7% in whites) (Sata et al, 2000), *7 (1.4 -3% in white) (Eiselt et al, 2001;Lamba et al, 2002), *16 (1.4 -5% in Japanese) (Lamba et al, 2002;FukushimaUesaka et al, 2004), and *18 (1.3-2.8% in Japanese) (FukushimaUesaka et al, 2004) by using Escherichia coli expression systems (Miyazaki et al, 2008). CYP3A4.2 and CYP3A4.16 exhibited lower catalytic activity for 6␤-hydroxylation of testosterone (TST), a probe substrate of CYP3A4, than CYP3A4.1 (wild type), whereas CYP3A4.18 had a higher activity, and CYP3A4.7 had comparable activity.…”