In vitro inhibition of CYP2C9-mediated warfarin 7-hydroxylation by iguratimod: possible mechanism of iguratimod-warfarin interaction.

Iguratimod is a novel disease-modifying antirheumatic drug. A blue letter (safety advisory) for drug interaction between iguratimod and warfarin was issued by the Ministry of Health, Labour and Welfare of Japan in May 2013. Iguratimod may affect warfarin metabolism catalyzed by CYP. However, it is not clear whether iguratimod inhibits warfarin oxidation. This study was performed to investigate the effects of iguratimod on warfarin 7-hydroxylation with human liver microsomes (HLMs) and recombinant CYP enzymes. Iguratimod concentration-dependently inhibited R,S-warfarin 7-hydroxylase activity of HLMs with an IC50 value of 15.2 µM. The inhibitory effect was examined with S-warfarin and R-warfarin to determine which enantiomer was more potently inhibited by iguratimod. Iguratimod potently inhibited the S-warfarin 7-hydroxylase activity of HLMs with an IC50 value of 14.1 µM, but showed only slight inhibition of R-warfarin 7-hydroxylation. Furthermore, iguratimod inhibited the S-warfarin 7-hydroxylase activity of recombinant CYP2C9.1 (rCYP2C9.1) and rCYP2C9.3 in a concentration-dependent manner with IC50 values of 10.8 and 20.1 µM, respectively. Kinetic analysis of the inhibition of S-warfarin 7-hydroxylation by iguratimod indicated competitive-type inhibition for HLMs and rCYP2C9.1 but mixed-type inhibition for rCYP2C9.3. The Ki values for HLMs, rCYP2C9.1, and rCYP2C9.3 were 6.74, 4.23, and 14.2 µM, respectively. Iguratimod did not exert metabolism-dependent inhibition of S-warfarin 7-hydroxylation. These results indicated that iguratimod is a potent direct inhibitor of CYP2C9-mediated warfarin 7-hydroxylation and that its inhibitory effect on CYP2C9.1 was more sensitive than that on CYP2C9.3.

Functional characterization of wild-type and 49 CYP2D6 allelic variants for N-desmethyltamoxifen 4-hydroxylation activity.

Genetic variations in cytochrome P450 2D6 (CYP2D6) contribute to interindividual variability in the metabolism of clinically used drugs, e.g., tamoxifen. CYP2D6 is genetically polymorphic and is associated with large interindividual variations in therapeutic efficacy and drug toxicity. In this study, we performed an in vitro analysis of 50 allelic variants of CYP2D6 proteins. Wild-type CYP2D6.1 and 49 variants were transiently expressed in COS-7 cells, and the enzymatic activities of the CYP2D6 variants were characterized using N-desmethyltamoxifen as a substrate. The kinetic parameters K(m), V(max), and intrinsic clearance (V(max)/K(m)) of N-desmethyltamoxifen 4-hydroxylation were determined. Among the 50 CYP2D6 variants, the kinetic parameters for N-desmethyltamoxifen 4-hydroxylation were determined for 20 CYP2D6 variants. On the other hand, the kinetic parameters of 30 CYP2D6 variants could not be determined because the amount of metabolite produced was at or below the detection limit at the lower substrate concentrations. Among them, 8 variants, i.e., CYP2D6.2, .9, .26, .28, .32, .43, .45, and .70, showed decreased intrinsic clearance at <50% of CYP2D6.1. The comprehensive in vitro assessment of CYP2D6 variants provides novel insights into allele-specific activity towards tamoxifen and may be valuable when interpreting in vivo studies.

Functional characterization of 26 CYP2B6 allelic variants (CYP2B6.2-CYP2B6.28, except CYP2B6.22).

Cytochrome P450 2B6 (CYP2B6) is a potentially important enzyme for the metabolism of clinical drugs, and it exhibits genetic polymorphism. Thus far, 29 allelic variants of CYP2B6 (CYP2B6*1-CYP2B6*29) have been identified. This study aimed to investigate whether 26 of the variant alleles of CYP2B6 (CYP2B6*2-CYP2B6*21 and CYP2B6*23-CYP2B6*28) affect its kinetics in the metabolism of 7-ethoxy-4-trifluoromethylcoumarin (7-EFC) and selegiline. Wild-type CYP2B6.1 and the allelic variants were heterologously expressed in COS-7 cells. In-vitro kinetic analysis revealed that when compared with the wild-type protein CYP2B6.1, CYP2B6.10 and CYP2B6.14 exhibited significantly lower V(max)/K(m) values for selegiline N-demethylation. The kinetic parameters of CYP2B6.8, CYP2B6.11, CYP2B6.12, CYP2B6.13, CYP2B6.15, CYP2B6.18, CYP2B6.21, CYP2B6.24, and CYP2B6.28 could not be determined because these enzymes were inactive in the deethylation of 7-EFC and the N-demethylation/N-depropagylation of selegiline. These findings provide useful information for further genotype-phenotype studies on interindividual differences in the metabolism of CYP2B6 substrate drugs.

Functional characterization of 17 CYP2D6 allelic variants (CYP2D6.2, 10, 14A-B, 18, 27, 36, 39, 47-51, 53-55, and 57).

Cytochrome P450 2D6 (CYP2D6) is an enzyme of potential importance for the metabolism of drugs used clinically, and it exhibits genetic polymorphism with interindividual differences in metabolic activity. To date, 21 CYP2D6 allelic variants have been identified in the Japanese population. The aim of this study was to investigate the functional characterization of CYP2D6 variants identified in Japanese subjects. Wild-type CYP2D6 and its variants, namely, CYP2D6.2, CYP2D6.10, CYP2D6.14A, CYP2D6.14B, CYP2D6.18, CYP2D6.27, CYP2D6.36, CYP2D6.39, CYP2D6.47, CYP2D6.48, CYP2D6.49, CYP2D6.50, CYP2D6.51, CYP2D6.53, CYP2D6.54, CYP2D6.55, and CYP2D6.57 were transiently expressed in COS-7 cells, and enzymatic activities of the CYP2D6 variant proteins were characterized using bufuralol and dextromethorphan. Functional characterization of 17 CYP2D6 variants revealed an absence of enzyme activity in four (CYP2D6.14A, CYP2D6.36, CYP2D6.47, and CYP2D6.57), low activity in eight (CYP2D6.10, CYP2D6.14B, CYP2D6.18, CYP2D6.49, CYP2D6.50, CYP2D6.51, CYP2D6.54, and CYP2D6.55), and high activity in one (CYP2D6.53) compared with the wild type. Analysis of CYP2D6 variant proteins can be useful for predicting CYP2D6 phenotypes and could be applied to personalized drug therapy.

Competitive allele-specific short oligonucleotide hybridization (CASSOH) with enzyme-linked immunosorbent assay (ELISA) for the detection of pharmacogenetic single nucleotide polymorphisms (SNPs).

Individualization of drug therapy through genetic testing would maximize the effectiveness of medication and minimize its risks. Recent progress in genetic testing technologies has been remarkable, and they have been applied for the analysis of genetic polymorphisms that regulate drug responses. Clinical application of genetic information to individual health care requires simple and rapid identification of nucleotide changes in clinical settings. We previously reported a novel DNA diagnostic method for detecting single nucleotide polymorphisms (SNPs) using competitive allele-specific short oligonucleotide hybridization (CASSOH) with an immunochromatographic strip. We have developed the method further in order to incorporate an enzyme-linked immunosorbent assay (ELISA) into the final detection step; this enables multiple SNP detection. Special ELISA chips have been fabricated so that disposal of buffer waste is not required and handling procedures are minimized. This method (CASSOH-ELISA) has been successfully applied for the detection of clinically important SNPs in drug metabolism, such as N-acetyltransferase 2, NAT2*6 (590G>A) and NAT*7 (857G>A), and mitochondrial DNA (1555A>G). It would also facilitate point-of-care genetic testing for potentially diverse clinical applications.

Two novel single nucleotide polymorphisms (SNPs) of the CYP2D6 gene in Japanese individuals.

We analyzed all the exons and exon-intron junctions of the CYP2D6 gene from 286 Japanese individuals. We detected two novel single nucleotide polymorphisms (SNPs) 2556C>T in exon 5 (Thr261Ile) and 3835A>C in exon 8 (Lys404Gln). Both these SNPs showed a frequency of 0.002.