Cytochrome P450 2A6 Phenotyping Using Dietary Caffeine Salivary Metabolite Ratios and Genotyping Using Blood on Storage Cards in Non-smoking Japanese Volunteers.

BACKGROUND: A simple method of genotyping and phenotyping cytochrome P450 2A6 (CYP2A6) was previously reported using individual blood samples and urinary caffeine metabolite ratios of 1,7-dimethyluric acid (17U) to 1-methylxanthine (1X). OBJECTIVE: Blood spotted onto storage cards and salivary caffeine metabolites were analyzed in 27 healthy non-smoking Japanese volunteers with no prior abstention from dietary caffeine intake. METHODS: 1,7-Dimethylxanthine (17X), 17U, 1X, and caffeine levels in spot saliva samples were determined in Japanese non-smokers by high-performance liquid chromatography under normal dietary caffeine consumption. RESULTS: 17U/17X ratios in saliva were almost constant over time, but those of 17U/1X were variable in two subjects tested before and 1-2.5 h after caffeine treatment (a cup of black tea). In seven subjects, 17U/17X ratios in saliva were highly correlated with those in plasma (r = 0.98, p < 0.01) and well correlated with those in urine samples (r = 0.78, p < 0.05). The average 17U/17X ratios, but not 17U/1X ratios, in saliva under dietary caffeine consumption obtained from subjects with CYP2A6*1/*4 (n=11) and CYP2A6*4/*4 (whole-gene deletion, n=2) genotypes were significantly lower than those from subjects with wild-type CYP2A6*1/*1 (n=14). Genotyping was done by a multiplex real-time polymerase chain reaction method using blood spotted onto storage cards. CONCLUSION: The present results suggest that the decreased CYP2A6 function associated with the whole-gene deletion genotype (determined using blood samples) could be detected using 17U/17X ratios, but not 17U/1X ratios, in spot saliva samples under normal dietary caffeine consumption in Japanese non-smokers, just as it could be detected using urinary 17U/1X ratios.

Mechanism-based inhibition of cytochrome P450 (CYP)2A6 by chalepensin in recombinant systems, in human liver microsomes and in mice in vivo.

BACKGROUND AND PURPOSE: Chalepensin is a pharmacologically active furanocoumarin compound found in rue, a medicinal herb. Here we have investigated the inhibitory effects of chalepensin on cytochrome P450 (CYP) 2A6 in vitro and in vivo. EXPERIMENTAL APPROACH: Mechanism-based inhibition was studied in vitro using human liver microsomes and bacterial membranes expressing genetic variants of human CYP2A6. Effects in vivo were studied in C57BL/6J mice. CYP2A6 activity was assayed as coumarin 7-hydroxylation (CH) using HPLC and fluorescence measurements. Metabolism of chalepensin was assessed with liquid chromatography/mass spectrometry (LC/MS). KEY RESULTS: CYP2A6.1, without pre-incubation with NADPH, was competitively inhibited by chalepensin. After pre-incubation with NADPH, inhibition by chalepensin was increased (IC(50) value decreased by 98%). This time-dependent inactivation (k(inact) 0.044 min(-1) ; K(I) 2.64 µM) caused the loss of spectrally detectable P450 content and was diminished by known inhibitors of CYP2A6, pilocarpine or tranylcypromine, and by glutathione conjugation. LC/MS analysis of chalepensin metabolites suggested an unstable epoxide intermediate was formed, identified as the corresponding dihydrodiol, which was then conjugated with glutathione. Compared with the wild-type CYP2A6.1, the isoforms CYP2A6.7 and CYP2A6.10 were less inhibited. In mouse liver microsomes, pre-incubation enhanced inhibition of CH activity. Oral administration of chalepensin to mice reduced hepatic CH activity ex vivo. CONCLUSIONS AND IMPLICATIONS: Chalepensin was a substrate and a mechanism-based inhibitor of human CYP2A6. Formation of an epoxide could be a key step in this inactivation. 'Poor metabolizers' carrying CYP2A6*7 or *10 may be less susceptible to inhibition by chalepensin. Given in vivo, chalepensin decreased CYP2A activity in mice.

Cytochrome P450-dependent drug oxidation activity of liver microsomes from Microminipigs, a possible new animal model for humans in non-clinical studies.

SUMMARY: Small minipigs (Bland name, Micromini Pig; registered as a novel variety of pig in the Japanese Ministry of Agriculture, Forestry and Fisheries) were developed with the aim of non-clinical pharmacological/toxicological use. They were principally mated with<10 kg body weight at 7 months-old resulting in good handling. Cytochrome P450 (P450)-and flavin-containing monooxygenases (FMO)-dependent drug oxidation activity of liver microsomes prepared from male Microminipigs (8 months-old) was compared with that for pooled dogs, monkeys, and humans. High P450 2D-dependent bufuralol 1'-hydroxylation and FMO-dependent benzydamine N-oxygenation activity was observed in liver microsomes from Microminipigs. Typical P450 1A, 2B, 2C, 2E, and 3A-dependent drug oxidation activity was also seen in Microminipigs. However, occasional differences might give undetected low P450 2A-dependent coumarin 7-hydroxylation in Microminipigs at 8-months-old, in contrast to liver microsomes from one 10-days-old Microminipis and commercially available pooled minipigs which had low but detectable coumarin 7-hydroxylation activity. The present results suggest that there is some overlap in Microminipig and human P450 substrate specificity. These findings should provide important information for greater understanding of drug metabolism in Microminipigs, as an experimental animal model for non-clinical use.

Complex mechanism underlying transcriptional control of the haplotyped flavin-containing monooxygenase 3 (FMO3) gene in Japanese: different regulation between mutations in 5'-upstream distal region and common element in proximal region.

We reported the human flavin-containing monooxygenase 3 (FMO3) haplotypes (Pharmacogenet. Genomics: 17, 827, 2007). The objective was to gain the insight into transcriptional regulation in a Japanese population. The wild-type FMO3 reporter plasmids carrying 5'-flanking sequence from the transcriptional initiation site of the FMO3 haplotype 1 (prepared from three individuals) showed higher luciferase activities in HepG2 cells than those from the FMO3 haplotypes 2 and 3, with the wild-type coding region. Several deletion mutants of the FMO3 haplotype 1 (extending from -5,167 to -1,764, numbered relative to the A of the ATG translational initiation codon) revealed that the region of -2,064 to -1,804 contained an important cis-acting element(s) for activation of the FMO3 gene expression. Putative hepatocyte nuclear factor-4 (HNF-4) binding site and CCAAT box, but not Yin Yang 1 element, could be responsible cis-acting elements of the FMO3 gene, by site-directed mutagenesis analysis. The unknown suppressive cis-element(s) at the 5'-upstream region from -2,064 might show genetic polymorphism, because the FMO3 haplotypes 2 and 3 had three and ten mutations, respectively. These results suggest that the putative HNF-4 binding site and CCAAT box could be responsible cis-acting elements of the FMO3 gene in Japanese.