Genetic variations in the genes encoding the cytochrome P450s (CYPs) are important determinants for interindividual differences in sensitivity to drugs and environmental chemicals as well as for the pathogenesis of several human diseases. In order to standardise the nomenclature of the rapidly increasing number of alleles described, a web page was established a few years ago. Here, we describe the present status of the web page and summarise the principles used for CYP allele nomenclature.

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  Orally administered drugs suffer from attack by metabolic enzymes not only in the liver, but also in the gastrointestine during the absorption process across the intestinal tissue. Although kinetic study on hepatic metabolism has been done well, the intestinal metabolism has not been well focused on compared with hepatic metabolism. In order to emphasize the role of intestinal metabolism in drug absorption and bioavailability, I have reviewed the experimental methods for intestinal absorption and metabolism, and the data analysis. Since Klippert et al. reported the prediction of intestinal first-pass effect of phenacetin in the rat from enzyme kinetic data in 1982, several reports have showed a good prediction, but others have not. Although intestinal absorption is an integrated process of transport (transporters) and metabolism (metabolic enzymes), most of the researchers missed the pathway of intestinal drug absorption and applied the kinetic model effective on only systemic metabolism to presystemic intestinal metabolism for their analysis of intestinal metabolism of orally administered drugs. A kinetic model, which incorporated factors of membrane transport, metabolic activity and protein binding, was structured to compare the equations in the reported models. In conclusion, we need more studies including kinetic modeling and experiments to understand the impact of intestinal metabolism on drug absorption. That knowledge must lead to the construction of ADME in silico (e-ADME).

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  (+)-Limonene is shown to cause renal toxicity in male rats, but not in female rats and other species of animals including mice, guinea pigs, rabbits, and dogs. We have previously shown that male-specific rat CYP2C11 (but not female-specific CYP2C12) is able to convert limonenes to carveols and perillyl alcohols (M. Miyazawa, M. Shindo, and T. Shimada: Chem. Res. Toxicol., 15, 15-20, 2002). Here, we investigated whether (+)- and (-)-limonene enantiomers are differentially metabolized by P450 enzymes in liver microsomes of mice, rats, guinea pigs, rabbits, dogs, monkeys, and humans. Limonene enantiomers were converted to respective carveols, perillyl alcohols, and carvones (oxidative metabolites of carveols) by liver microsomes of dogs, rabbits, and guinea pigs. Mice, rats, monkeys, and humans produced carveols and perilly alcohols, but not carvones. Reconstituted monooxygenase systems containing purified rabbit CYP1A2 and 2B4 and NADPH-P450 reductase were found to catalyze (+)-limonene to (+)-carveol, (+)-carvone, and (+)-perillyl alcohol, being more active with CYP2B4. When (+)-carveol and (+)-carvone were used as substrates, dogs, rabbits, and guinea pigs metabolized them to (+)-carvone and (+)-carveol, respectively. Again humans, monkeys, rats, and mice did not convert (+)-carveol to (+)-carvone, but metabolized (+)-carvone to (+)-carveol, with male rats having the highest rates. CYP2C enzymes were suggested to play major roles in metabolizing (+)-carveol to (+)-carvone and (+)-carvone to (+)-carveol by liver microsomes, since the activities were inhibited significantly by anti-human CYP2C9 antibodies in these animal species. Studies with recombinant P450 enzymes suggested that CYP2C9 and 2C19 in humans and CYP2C11 in untreated male rats were the major enzymes in metabolizing (+)-carvone. These results suggest that there are species-related differences in the metabolism of limonenes by P450 enzymes, particularly in the way from (+)-carveol to (+)-carvone. However, it remains unclear whether these differences in limonene metabolism by these animal species explain species-related differences in limonene-induced renal toxicity.

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  Hepatic microsomes from human liver catalyzed oxidation of the allyl aldehydes such as 11-oxo-Δ⁸-tetrahydrocannabinol and 9-anthraldehyde to the corresponding carboxylic acid metabolites. The oxygenation mechanism was confirmed by GC-MS that molecular oxygen was exclusively incorporated into Δ⁸-tetrahydrocannabinol-11-oic acid and 9-anthracene carboxylic acid formed under oxygen-18 gas. The microsomal aldehyde oxygenase (named MALDO) activities of 11-oxo-Δ⁸-tetrahydrocannabinol and 9-anthraldehyde were significantly inhibited by the antibody against CYP2C and CYP3A, respectively. MALDO activity for 11-oxo-Δ⁸-tetrahydrocannabinol was significantly inhibited by sulfaphenazole whereas that for 9-anthraldehyde was markedly inhibited by troleandomycin, but not by sulfaphenazole. CYP2C9 expressed in human B-lymphoblastoid cells catalyzed efficiently the MALDO activity for 11-oxo-Δ⁸-tetrahydrocannabinol (10.1 nmol/min/nmol P450), while the catalytic activities of other human CYPs expressed in the cells were lesser extents. In MALDO activity for 9-anthraldehyde, CYP3A4 expressed in the cells had the highest catalytic activity (7.72 nmol/min/nmol P450). These results indicate that CYP2C9 and CYP3A4 are major enzymes responsible for the MALDO activity in human liver for 11-oxo-Δ⁸-tetrahydrocannabinol and 9-anthraldehyde, respectively.

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  To investigate whether the free-drug theory is accurate in that only unbound drug is available for drug metabolism or enzyme inhibition. The effect of addition of rat liver cytosol to an in vitro system using human liver microsomes was examined by measuring the catalytic activities of CYP2C9 (tolbutamide and diclofenac) and CYP3A4 (terfenadine). And, the results were compared with those obtained when human serum albumin (HSA) was added to microsomes as far as unbound drug concentrations were concerned.
  After addition of rat liver cytosol, the unbound Km value (Km,u) for terfenadine metabolism by CYP3A4, and the unbound Ki value of miconazole (Ki,u) for CYP2C9 were smaller than for the controls. Addition of HSA resulted in smaller Km,u values for diclofenac and terfenadine metabolism by CYP2C9 and CYP3A4, respectively, and the Ki,u value for ketoconazole inhibition of CYP3A4 was also reduced. These results suggest protein-facilitated effects on drug metabolism and enzyme inhibition for both CYP2C9 and CYP3A4. However, no protein-facilitated drug metabolism was observed for tolbutamide in the presence of HSA or cytosol, or for diclofenac in the presence of cytosol. Protein-facilitated enzyme inhibition did not occur with miconazole in the presence of HSA or with ketoconazole in the presence of rat liver cytosol.
  Protein-facilitated metabolism and enzyme inhibition were observed for CYP2C9 and CYP3A4 in five cases but there was no obvious pattern of enzyme, substrate, or binding protein specificity. Further investigations are necessary to clarify the relevance of these results to in vivo observations.

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  The β₂-adrenergic receptor (B2AR) is a G protein-coupled cell surface receptor that is the key target for the β₂-agonist drugs used for bronchodelation in asthma and chromic obstructive pulmonary disease. To detect four SNPs with amino acid variations in the B2AR gene, we used the electronic microchip assay (NanoChip system), DHPLC and sequencing. Genomic DNA samples were obtained from the blood of 84 Japanese healthy volunteers. In sum, the agreement rates of the first data set with the final agreement data (allele calls) were 99.7% (328/329), 99.2% (243/245) and 96.7% (325/336). The percentages of no allele designation (ND) were 2.06% (7/336), 2.75% (7/252), and 0.00% (0/336) for the NanoChip system, DHPLC, and sequencing, respectively. As a result of SNP genotyping, we found three samples that might have a novel haplotype. Furthermore we identified the novel haplotype by a simple technique combining the NanoChip system and allele-specific PCR. These results indicated that NanoChip system was the useful method for clinical SNP genotyping and/or haplotyping because of its accuracy, simplicity and versatility.

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  Retinol-binding protein (RBP) is a specific transport protein which carries retinol in the circulation. RBP concentration in plasma and liver of rats following a large dose of acetaminophen (APAP) intraperitoneally was examined. The RBP concentration in plasma decreased significantly at 12 hr after the APAP administration, while the plasma albumin concentration was affected a little. Western blot and northern blot analyses showed marked changes in RBP but not in albumin. Thus, RBP was suggested to be more sensitive for the acute drug-induced hepatotoxicity than albumin. The decrease of RBP concentration in plasma was suggested to be caused by the dysfunction of RBP synthesis in the liver.

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  Nifedipine is one of drugs that have been suggested to undergo significant first-pass metabolism by cytochrome P450 (CYP) 3A in the intestine, based mainly on pharmacokinetic analyses of in vivo observations. To further substantiate this suggestion, we examined the metabolic extraction of nifedipine from the rat small intestine, using intestine perfused in situ by a single-pass technique and microsomes in vitro. When the intestinal lumen was perfused with nifedipine solution (30 μM) at the flow rate of 0.15 mL/min and steady-state was achieved, the fraction that disappeared from the intestinal lumen (F_a) and the fraction absorbed into the mesenteric venous blood (F_a,b) was 0.26 and 0.13, respectively. Thus, F_a,b was 50% smaller than F_a, indicating a significant extraction of nifedipine during passage through the intestinal mucosa. When ketoconazole (40 μM), a specific inhibitor of CYP3A, was added to the perfusion solution, F_a,b was increased to a level comparable with F_a, while F_a remained unchanged, suggesting the complete inhibition of metabolic extraction by CYP3A. A similar result was obtained for cyclosporin A (40 μM), another specific CYP3A inhibitor. In intestinal microsomes, the metabolic degradation of nifedipine (1 μM) was almost completely inhibited by ketoconazole (10 μM) and cyclosporin A (10 μM), consistent with the results in the perfused intestine. It was also found in intestinal microsomes that anti-rat CYP3A2 antibody can inhibit nifedipine metabolism completely. Thus, the present study demonstrates that nifedipine undergoes significant extraction during passage through the intestinal mucosa, and provides substantial evidence that CYP3A2 is responsible for that.

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  We measured the nicotine concentrations in tissues after a bolus i.v. administration of [³H]nicotine to rats to characterize the distribution profile of nicotine. The kidney showed the greatest distribution of nicotine compared to other tissues including liver, lung, heart, brain, and intestine. We also performed an HPLC assay for the determination of nicotine and its major metabolite, cotinine, and found that cotinine was negligible in the distribution of almost all tissues, except for the kidney and lung. In the kidney, cotinine was detected at a lower level than nicotine, while cotinine tended to be distributed in the lung compared to nicotine. [³H]Nicotine was accumulated in renal slices in a concentration dependent fashion, suggesting that the nicotine uptake in the renal tubules could be mediated by a specific transport system. Unlabeled nicotine, cotinine, and quinidine showed potent inhibitory effects on [³H]nicotine uptake by renal slices. In contrast, tetraethylammonium (TEA), cimetidine, and N¹-methylnicotinamide (NMN), which were substrates of renal organic cation transporters, had no effects on the uptake. These findings suggested that a specific transporter was involved in nicotine transport at the basolateral membranes of rat renal tubules, which could mediate the high accumulation of nicotine from blood into the kidney.

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  Eleven novel single nucleotide polymorphisms (SNPs) were found in the NR1I2 (PXR/SXR) gene from 205 Japanese subjects. The detected SNPs were as follows:
1) SNP, MPJ6_1I2001; GENE NAME, NR1I2; ACCESSION NUMBER, AF364606; LENGTH, 25 bases; 5′-TTTCTACCTCTAC/TTATTGAAAGGGC-3′
2) SNP, MPJ6_1I2004; GENE NAME, NR1I2; ACCESSION NUMBER, AF364606; LENGTH, 25 bases; 5′-AGGCCCAAATGTG/AAGTGATGCATAG-3′
3) SNP, MPJ6_1I2007; GENE NAME, NR1I2; ACCESSION NUMBER, AF364606; LENGTH, 25 bases; 5′-TGCCAGGCCTGCC/TGCCTGCGCAAGT-3′
4) SNP, MPJ6_1I2008; GENE NAME, NR1I2; ACCESSION NUMBER, AF364606; LENGTH, 25 bases; 5′-GAGTGAGCAGTGG/CGCGCGCGGGCGG-3′
5) SNP, MPJ6_1I2010; GENE NAME, NR1I2; ACCESSION NUMBER, AF364606; LENGTH, 25 bases; 5′-CAGAGGAGCAGCG/AGATGATGATCAG-3′
6) SNP, MPJ6_1I2011; GENE NAME, NR1I2; ACCESSION NUMBER, AF364606; LENGTH, 25 bases; 5′-CTGGAAGTGGCCA/GGGAGGTTCAAAG-3′
7) SNP, MPJ6_1I2013; GENE NAME, NR1I2; ACCESSION NUMBER, AF364606; LENGTH, 25 bases; 5′-TCTTCCTCTCGCC/TCCCAACTTCTGG-3′
8) SNP, MPJ6_1I2017; GENE NAME, NR1I2; ACCESSION NUMBER, AF364606; LENGTH, 25 bases; 5′-ATTGAATGCAATC/TGGCCCCAGCCTG-3′
9) SNP, MPJ6_1I2018; GENE NAME, NR1I2; ACCESSION NUMBER, AF364606; LENGTH, 25 bases; 5′-GGTGAGCACAGCA/GGGGGGTGAGGAC-3′
10) SNP, MPJ6_1I2019; GENE NAME, NR1I2; ACCESSION NUMBER, AF364606; LENGTH, 25 bases; 5′-GAGCTCCGCAGCA/GTCAATGCTCAGC-3′
11) SNP, MPJ6_1I2021; GENE NAME, NR1I2; ACCESSION NUMBER, AF364606; LENGTH, 25 bases; 5′-GGTGACACCTCCG/AAGAGGCAGCCAG-3′.
  The frequencies were 0.0293 for MPJ6_1I2021, 0.0073 for MPJ6_1I2011, and 0.0024 for the other 9 SNPs. All SNPs were found as heterozygous. Among these SNPs, MPJ6_1I2007, MPJ6_1I2010, MPJ6_1I2017 and MPJ6_1I2019 induce non-synonymous amino acid alterations (R98C, R148Q, R381W and I403V, respectively, in PAR1).

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  Twelve novel single nucleotide polymorphisms (SNPs) were found in the gene encoding the ATP-binding cassette transporter, P-glycoprotein, from 60 Japanese individuals who were administered the anti-antiarrythmic drug, amiodarone. The detected SNPs were as follows:
1) SNP, MPJ6_AB1017 (IVS6-109); GENE NAME, ABCB1; ACCESSION NUMBER, NT_017168; 2) SNP, MPJ6_AB1018 (IVS7+14); GENE NAME, ABCB1; ACCESSION NUMBER, NT_017168; 3) SNP, MPJ6_AB1021 (IVS9-44); GENE NAME, ABCB1; ACCESSION NUMBER, NT_017168; 4) SNP, MPJ6_AB1052 (IVS12+17); GENE NAME, ABCB1; ACCESSION NUMBER, NT_017168; 5) SNP, MPJ6_AB1029 (IVS15-69); GENE NAME, ABCB1; ACCESSION NUMBER, NT_017168; 6) SNP, MPJ6_AB1040 (IVS24+16); GENE NAME, ABCB1; ACCESSION NUMBER, NT_017168; 7) SNP, MPJ6_AB1053 (IVS27-189); GENE NAME, ABCB1; ACCESSION NUMBER, NT_017168; 8) SNP, MPJ6_AB1054 (IVS27-172); GENE NAME, ABCB1; ACCESSION NUMBER, NT_017168; 9) SNP, MPJ6_AB1048 (IVS27-167); GENE NAME, ABCB1; ACCESSION NUMBER, NT_017168; 10) SNP, MPJ6_AB1055 (IVS27-152); GENE NAME, ABCB1; ACCESSION NUMBER, NT_017168; 11) SNP, MPJ6_AB1049 (IVS27-119); GENE NAME, ABCB1; ACCESSION NUMBER, NT_017168; 12) SNP, MPJ6_AB1051 (at nucleotide 3751 (exon 28) from the A of the translation initiation codon); GENE NAME, ABCB1; ACCESSION NUMBER, NT_017168. Among these SNPs, only MPJ6_AB1051 resulted in an amino acid alteration, V1251I.

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