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Genetic polymorphisms contribute to inter-individual variability in the metabolism of multiple clinical drugs, including warfarin, thiopurines, primaquine, and aminoglycosides. A rapid and sensitive clinical assessment of various genome biomarkers is, therefore, required to predict the individual responsiveness of each patient to these drugs. In this study, we developed a novel genotyping method for the detection of nine pharmacogene variants that are important in the prediction of drug efficiency and toxicity. This genotyping method uses competitive allele-specific PCR and a single-stranded tag hybridization chromatographic printed-array strip (STH-PAS) that can unambiguously determine the presence or absence of the gene variant by displaying visible blue lines on the chromatographic printed-array strip. Notably, the results of our STH-PAS method were in 100% agreement with those obtained using standard Sanger sequencing and KASP assay genotyping methods for CYP4F2 gene deletion. Moreover, the results were obtained within 90 min, including the PCR amplification and signal detection processes. The sensitive and rapid nature of this novel method make it ideal for clinical genetic testing to predict drug efficacy and toxicity, and in doing so will aid in the development of individualized medicine and better patient care.

Cytochrome P450 2C8 (CYP2C8) is one of the enzymes primarily responsible for the metabolism of many drugs, including paclitaxel and amodiaquine. CYP2C8 genetic variants contribute to interindividual variations in the therapeutic efficacy and toxicity of paclitaxel. Although it is difficult to investigate the enzymatic function of most CYP2C8 variants in vivo, this can be investigated in vitro using recombinant CYP2C8 protein variants. The present study used paclitaxel to evaluate 6α-hydroxylase activity and amodiaquine for the N-deethylase activity of wild-type and 11 CYP2C8 variants resulting in amino acid substitutions in vitro. The wild-type and variant CYP2C8 proteins were heterologously expressed in COS-7 cells. Paclitaxel 6α-hydroxylation and amodiaquine N-deethylation activities were determined by measuring the concentrations of 6α-hydroxypaclitaxel and N-desethylamodiaquine, respectively, and the kinetic parameters were calculated. Compared to the wild-type enzyme (CYP2C8.1), CYP2C8.11 and CYP2C8.14 showed little or no activity with either substrate. In addition, the intrinsic clearance values of CYP2C8.8 and CYP2C8.13 for paclitaxel were 68% and 67% that of CYP2C8.1, respectively. In contrast, the CLint values of CYP2C8.2 and CYP2C8.12 were 1.4 and 1.9 times higher than that of CYP2C8.1. These comprehensive findings could inform for further genotype-phenotype studies on interindividual differences in CYP2C8-mediated drug metabolism.

Genetic variations in cytochrome P450 4A11 (CYP4A11) contributes to inter-individual variability in the metabolism of fatty acids such as arachidonic acid. CYP4A11 metabolizes arachidonic acid to 20-hydroxyeicosatetraenoic acid (20-HETE), which is important for the regulation of blood pressure. Polymorphisms in CYP4A11 are associated with susceptibility to hypertension. In this study, we evaluated the in vitro ω-hydroxylation of arachidonic acid by 10 CYP4A11 allelic variants, which cause amino acid substitutions in the encoded proteins. CYP4A11 variants were heterologously expressed in COS-7 cells and the kinetic parameters of arachidonic acid ω-hydroxylation were estimated. Among 10 CYP4A11 variants, 5 (CYP4A11-v1, CYP4A11-v2, CYP4A11-v3, CYP4A11-v4, and CYP4A11-v7) showed no or markedly lower activity compared to wild-type CYP4A11. This functional analysis of CYP4A11 variants could provide useful information for the effective prevention and treatment of hypertension.

Genetic variations in cytochrome P450 2C9 (CYP2C9) contribute to interindividual variability in the metabolism of clinically used drugs such as warfarin and tolbutamide. We functionally characterized 32 types of allelic variant CYP2C9 proteins. Recombinant CYP2C9 proteins generated using a heterologous expression system are useful for comparing functional changes in CYP2C9 variant proteins expressed from low-frequency alleles. Wild-type CYP2C9 and its 31 variants were found to be transiently expressed in COS-7 cells, and the enzymatic activity of the CYP2C9 variants was characterized using S-warfarin as a representative substrate. Among the 32 types of CYP2C9 allelic variants tested, CYP2C9.18, CYP2C9.21, CYP2C9.24, CYP2C9.26, CYP2C9.33 and CYP2C9.35 exhibited no enzyme activity, and 12 types showed significantly decreased enzyme activity. In vitro analysis of CYP2C9 variant proteins should be useful for predicting CYP2C9 phenotypes and for application to personalized drug therapy.

We devised a new method of determining the temperatures of materials with their electron-energy distributions. The Fermi–Dirac distribution convoluted with a linear combination of Gaussian and Lorentzian distributions was fitted to the photoelectron spectrum measured for the Au(110) single-crystal surface at liquid N2-cooled temperature. The fitting successfully determined the surface-local thermodynamic temperature and the energy resolution simultaneously from the photoelectron spectrum, without any preliminary results of other measurements. The determined thermodynamic temperature was 99 ± 2.1 K, which was in good agreement with the reference temperature of 98.5 ± 0.5 K measured using a silicon diode sensor attached to the sample holder.

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

Genetic variations in cytochrome P450 2C19 (CYP2C19) contribute to interindividual variability in the metabolism of therapeutic agents such as clopidogrel. Polymorphisms in CYP2C19 are associated with large interindividual variations in the therapeutic efficacy of clopidogrel. This study evaluated the in vitro oxidation of clopidogrel by 21 CYP2C19 variants harboring amino acid substitutions. These CYP2C19 variants were heterologously expressed in COS-7 cells, and the kinetic parameters of clopidogrel 2-oxidation were estimated. Among the 21 CYP2C19 variants, 12 (that is, CYP2C19.5A, CYP2C19.5B, CYP2C19.6, CYP2C19.8, CYP2C19.9, CYP2C19.10, CYP2C19.14, CYP2C19.16, CYP2C19.19, CYP2C19.22, CYP2C19.24 and CYP2C19.25) showed no or markedly low activity compared with the wild-type protein CYP2C19.1B. This comprehensive in vitro assessment provided insights into the specific metabolic activities of CYP2C19 proteins encoded by variant alleles, and this may to be valuable when interpreting the results of in vivo studies.