In vitro functional characterization of 37 CYP2C9 allelic isoforms found in Chinese Han population

Abstract

Aim: Cytochrome P450 2C9 (CYP2C9) is a polymorphic enzyme that is responsible for the metabolism of approximately 15% of clinically important drugs. The aim of this study was to assess the catalytic characteristics of 37 CYP2C9 allelic isoforms found in Chinese Han population on the metabolism of tolbutamide in vitro. Methods: The wild-type and 36 CYP2C9 variants were expressed in sf21 insect cells using a baculovirus-mediated expression system. Then the insect microsomes were prepared for assessing the metabolic characteristics of each variant toward the CYP2C9-specific drug substrate tolbutamide. Results: Of 36 allelic variants tested, the intrinsic clearance values of 2 allelic isoforms (CYP2C9.36 and CYP2C9.51) were much higher than the wild-type CYP2C9.1 protein, 3 allelic isoforms (CYP2C9.11, CYP2C9.56 and N418T) exhibited similar intrinsic clearance values as the wild-type enzyme, whereas the other 31 variants showed significantly reduced intrinsic clearance values, ranging from 0.08% to 66.88%, for tolbutamide. Conclusion: Our study provides the most comprehensive data concerning the enzymatic activity of the CYP2C9 variants that are present in the Chinese Han population, and our data suggest that most of the carriers of these alleles might be paid more attention when using CYP2C9 mediated drugs clinically.

In Vitro Functional Assessment of 22 Newly Identified CYP2D6 Allelic Variants in the Chinese Population

“…Four different cDNA expression systems have been successfully used for the in vitro drug metabolism studies, including bacteria [22], yeast [23], insect cells [24] and mammalian cells [13]. Compared with other expression systems, mammalian cells have adequate endogenous OR and cytochrome b 5 to support CYP activities and are much closer to the inherent environment of human proteins; thus, it is much more relevant to assess the potential effects of recombinant human CYP variant proteins in the mammalian cell system [21].…”

Identification and Functional Assessment of a New CYP2C9 Allelic Variant CYP2C9*59

“…Detailed information for all of the other primers is illustrated in Supplemental Table 1. The full-length sequence of each CYP2C9 allele was purified, digested, and cloned into the receptor vector pFastBacoxidoreductase (OR) to produce the ultimate dual-expression vector pFastBac-OR-CYP2C9, as described previously (Dai et al, 2013).…”

“…Then, the CYP2C9 variants and OR were simultaneously expressed at high levels in insect microsomes, according to the manufacturer's procedure. The determination and quantification of expressed proteins were performed via immunoblotting, as reported previously (Dai et al, 2013). Briefly, 2 mg of microsomes were used for the immunoblotting analysis using a rabbit polyclonal anti-CYP2C9 antibody (AbD Serotec, Oxford, United Kingdom) as the primary antibody at a dilution of 1:1000.…”

“…To gain insight into the metabolic characteristics of the novel CYP2C9 variant, the insect cell expression system was used to simultaneously express the recombinant CYP2C9 variants and the NADPH-cytochrome P450 OR according to the methods described previously (Dai et al, 2013). As shown in Fig.…”

“…Enzymatic Activity Assay Using Typical CYP2C9 Probe Substrates. To gain a better understanding of the enzymatic characteristics of the newly identified CYP2C9 variants, three different CYP2C9-specific probe substrates (tolbutamide, losartan, and diclofenac) were included in the in vitro experiments, as described previously (Dai et al, 2013;Luo et al, 2014;Wang et al, 2014). The reaction mixtures contained 4-20 pmol of CYP2C9 insect microsome, 8-40 pmol of cytochrome b5 (CYP2C9/b5 = 1:2), 50 mM (for losartan) or 100 mM (for tolbutamide and diclofenac) of Tris-HCl (pH 7.5), and a series of different concentrations of drugs (10-1000 mM for tolbutamide, 0.5-25 mM for losartan, and 1-100 mM for diclofenac).…”