System for the regulation of in vivo drug concentration ; Analyzes of genetic polymorphisms of drug metabolizing enzymes

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(1) The debrisoquine/sparteine drug oxidation polymorphism is due to mutant or null alleles of CYP2D6 causing an incapability to metabolize a large number of therapeutically-important drugs and some environmental toxicants. In this study, a group of Japanese subjects were phenotyped for CYP2D6 activity by determination of urine metabolic ratios of sparteine. Genomic DNA from a PM,having a high metabolic ratio(31.6), was subjected to sequence analysis. Sequence analysis of exon and exon-intron junction in CYP2D6 from the PM revealed nine base-insertion in exon 9. The nine base-insertion enhances the hydrophobicity at the carboxy-terminal region of the protein, and was demonstrated to inherit from mother of PM to PM as examined by PCR (polymerase chain reaction)-RFLP.(2) CYP1A2 is responsible for the metabolic activation of various carcinogens. In human, interindividual differences of CYP1A2 activities have been regarded as determinants of individual cancer susceptibility. The probit ana … More lyzes of non-smokers (n=147) and smokers (n=58) , using data from in vivo caffeine test in Japanese population, suggested that the CYP1A2 activity was not normally distributed and appeared bimodal. The bimodal probit plot suggested the existence of poor and extensive phenotypes. The percentage of individuals with the poor phenotype in Japanese was 14.1%. Induction of CYP1A2 by cigarette smoking was confirmed by the higher molar ratio observed in smokers (p<0.0001) . Family study in eight pedigrees suggested that the poor phenotype of CYP1A2 inherited as an autosomal recessive trait. Although no differences of nucleotide sequence were observed in exons, exon-intron junctions and 5-flanking regions (up to-2.6kb) of CYP1A2 gene between each phenotypes. This is the first report in which the CYP1A2 phenotype and a genetic polymorphism in the CYP1A2 gene were comparably investigated.(3) N-Acetyltransferase (NAT) was phenotyped by the urinary molar ratio of AFMU/1-methylxanthine in the caffeine test. PCR-RFLP method was applied to determine the NAT mutations causing slow N-acetylation. In vivo metabolic ratio (AFMU/1X) of wild-type was 1.46, whereas hetero-type showed 0.972, p=0.007. Homo-mutated type showed the value of 0.131 (p=0.001). The genotype of NAT2 completely correlated well with the phenotype but did not correlate with metabolic ratio due to CYP1A2.(4) We sequenced CYP2C18 and CYP2C9 cDNAs from PMs and EMs. No difference between PM and EM was observed. The hepatic expression level of CYP2C18 mRNA was examined in 20 human liver subjects by reverse transcriptase-PCR method. Individual variations in hepatic expression levels of CYP2C18 mRNA were over 10-fold. A good correlation (r=-0.758 ; p<0.02) was observed between the hepatic levels of CYP2C18 mRNA and R/S ratios of mephenytoin 4, -hydroxylation. Less