Search

金沢大学附属病院薬剤部, We have noticed that the trough level of blood concentration of cyclosporin A (CyA) tends to be lower in patients receiving long-term oral levothyroxine (LTX) than in patients not receiving LTX. We confirmed this clinical observation in experiments using Wistar rats orally given LTX (8 microg/kg) or saline (control) for 3 weeks, followed by CyA (10 mg/kg). The LTX treatment had little effect on the blood concentrations of CyA after i.v. administration, whereas they were decreased significantly after p.o. administration. After p.o. administration, the value of the area under the blood concentration-time curve from 0 to 24 hr and the bioavailability of CyA in the LTX group were decreased to only about one-fifth and a quarter of those in the control group, respectively. After treatment with LTX, the expression levels of mdr1a, mdr1b and CYP3A2 mRNAs in the duodenum were markedly increased to about twice the control, but in jejunum, ileum and liver the expression levels were little changed. These findings suggest that the absorption of CyA, which occurs mainly from the upper intestine, is reduced as a result of efflux transport via P-glycoprotein induced by LTX. In conclusion, careful monitoring of CyA levels is required in the event of LTX administration to patients receiving immunotherapy with CyA.

(+)-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.

Levels and catalytic activities of cytochrome P450 (P450) enzymes involved in the oxidation of drugs and carcinogens were determined in human adult lungs and fetal livers and compared with those in microsomes from adult livers. P450s immunoreactive with anti-human P450 1A1 and anti-human P450 3A antibodies were detected in fetal liver microsomes by immunoblotting analysis, and P450s related to P450 1A1, 2A6, 2C9, 2E 1, and 3A4 were determined in adult lung microsomes; all of these P450 enzymes were detected in much higher amounts in adult liver microsomes except that P450 1A2 was only the 1A subfamily of P450 found in adults. Drug oxidation activities with several substrates were determined in these microsomes, and we found that none of the activities were higher in microsomes of adult lungs and fetal livers than in adult livers. Activation of procarcinogens by P450 enzymes was also examined and it was found that activities with (+)- and (-)-enantiomers of 7, 8-dihydroxy-7, 8-dihydrobenzo[a]pyrene were higher in fetal liver microsomes than adult lung or liver microsomes. Anti-human P450 1A1 and 1A2 and a-naphthoflavone, known to inhibit P450 IA-related activities, did not affect these procarcinogen activation in fetal liver microsomes. Fetal liver microsomes catalyzed activation of aflatoxin B1 and sterigmatocystin, although activation of carcinogenic arylamines was much lower in microsomes of fetal livers and adult lungs than of adult livers. These results suggest that in human fetal livers at least two P450 enzymes, namely P450 IA-related enzyme and P450 3A7, are actually expressed and these enzymes are involved in the activation of the (+)- and (-)-enantiomers of 7, 8-dihydroxy-7, 8-dihydrobenzo[a]pyrene and the carcinogenic mycotoxins, respectively. In adult human lungs, several P450 enzymes are expressed, though the precise roles of these enzymes in the oxidation of xenobiotics were not determined due to the low level of expression of these P450s.

A new P450 responsible for mutagenic activation of 3-methoxy-4-aminoazobenzene (3-MeO-AAB) which is a potent procarcinogen was purified from renal microsomes of male mice using an index of umu gene expression. The purified P450 had high bioactivation toward 2-aminofluorene (2-AF), 2-aminoanthracene (2-AA), and 3, 3'-dichlorobenzidine (DCB) as well as 3-MeO-AAB. The antibody against this P450 completely inhibited mutagenic activation of 3-MeO-AAB, 2-AF, 2-AA, and DCB by mouse renal microsomes. With immunoblotting, this form was present abundantly in renal microsomes of male mice but not in those of female mice. This P450 was also present in mouse pulmonary and bladder microsomes but not in hepatic microsomes. The NH2-terminal amino acid sequence analysis indicated that this form belonged to the CYP4B subfamily. Thus, mouse kidney cDNA library was screened with rat CYP4B1 probe. The cDNA-deduced amino acid sequence of isolated cDNA consisted of 511 amino acids and the NH2-terminal amino acid sequence of the purified renal P450 agreed with the cDNA-deduced amino acid sequence, indicating that renal P450 purified in this study is mouse CYP4B1. Human pulmonary and renal cDNA library were screened with mouse CYP4B1 cDNA probe. Two full-length cDNAs were isolated and they had two or three amino acid changes to human CYP4B1 reported previously.

Twenty one types of genetic polymorphisms in CYP2D6 gene were determined in liver DNA of Japanese and Caucasians and compared these CYP2D6 genotypes with CYP2D6 protein levels and bufuralol 1'- and 6-hydroxylation activities in liver microsomes of these human samples. We detected 11 types of CYP2D6 genetic polymorphisms and classified these humans into 17 genotypes; 7 types were found in the Japanese and 13 types in the Caucasian. CYP2D6* 10B, but not CYP2D6*10A, was the most frequent in mutation at 34.6% in the Japanese, whereas in Caucasians, CYP2D6 polymorphisms including CYP2D6*4A, *4D, *4E, *4L *3, *9, and *M12 (frequencies at 6.8, 3.4, 4.5, 9.1, 2.3, 2.3, 4.5%) respectively, were detected. A Caucasian having homozygous CYP2D6*3/*3 had a protein with slower gel mobility (immunoblotting with anti-CYP2D6 antibody) and a very low activity for bufuralol 1'-hydroxylation. Five Caucasian samples with CYP2D6*4A/*4A, *4A/*4L, or *4D/*4L had no measurable CYP2D6 protein and very low bufuralol 1'-hydroxylation activities. Seven Japanese subjects with CYP2D6*10B/*IOB had CYP2D6 protein at levels of -20% of those present in humans with CYP2D6*1 and *2 and catalyzed bufuralol 1'-hydroxylation at low rates. These results support the view that CYP2D6*3, *4A, *4D, and *4L are major genotypes in producing poor metabolizer phenotypes in CYP2D6 in Caucasians, whereas CYP2D6*10B is a major causes in decreasing CYP2D6 protein expression and catalytic activities in Japanese. We also determine three types of CYP2E1 genetic polymorphisms, namely RsaI/PstI-, DraI-, and MspI-types, and compared these genotypes with levels of CYP2E1 and activities of 7-ethoxycoumarin O-deethylation and chlorzoxazone 6-hydroxylation in liver microsomes from these human samples. The results obtained collectively indicated that RsaI/PstI-, DraI-, and MspI-types of CYP2E1 polymorphisms may not cause significant alterations in protein expression and enzyme catalytic activities of CYP2E1 enzyme in human livers.

The roles of human P450 enzymes in the oxidation of xenobiotics have been shown to be determined by a) the ratio of Vmax to Km of individual P450 forms and b) the levels of expression of individual P450 forms In liver microsomes of human samples. We examined which P450 enzymes are most important in the 4-deethylation of 7-ethoxycoumarin and 6-hydroxylation of chlorzoxazone, two model reactions suggested to be catalyzed by several P450 enzymes, in liver microsomes of different human samples. Kinetic parameters for these two reactions were determined in 14 forms of recombinant human P450 expressed in baculovirus systems. Genetic polymorphisms In P450 genes are also important in understanding basis of different susceptibilities of individuals towards numerous xenobiotic chemicals. We examined the frequency distribution of CYP2A6 genetic polymorphisms in liver DNA of 39 Japanese and 43 Caucasians in the newly-developed two-step PCR method with modifications and the differences in CYP2A6-dependent catalytic activities were determined and compared in liver microsomes of individuals genotyped for CYP2A6 gene.

Omeprazole 5-hydroxylation and sulfoxidation activities were determined in liver microsomes of different humans whose levels of individual forms of cytochrome P450 (P450 or CYP) varied. Omeprazole 5-hydroxylation by liver microsomes of a human sample which contained relatively high levels of CYP3A4 and low levels of CYP2C19 were inhibited very significantly by ketoconazole and anti-CYP3A4 antibodies, while a human sample having high in CYP2C19 and low in CYP3A4 was found to be sensitive towards fluvoxamine and anti-CYP2C9 antibodies. Both recombinant human CYP2C19 and CYP3A4 enzymes had activities for omeprazole 5-hydroxylation, with low Km and high Vmax values for the former enzyme and high Km and low Vmax values for the CYP3A4. Omeprazole 5-hydroxylation activities of different human samples were found to be related to predicted values calculated from the kinetic parameters of recombinant enzymes and the levels of liver microsomal P450 enzymes. When recombinant human CYP2C19 and CYP3A4 were mixed at levels found in different human samples, relatively similar profiles of omeprazole oxidation by the recombinant and microsomal enzyme systems were determined. These results suggest that both CYP2C19 and CYP3A4 are involved in the 5-oxidation of omeprazole (at a substrate concentration of 10 μM) in human liver microsomes and that contributions of these P450 enzymes depend on the compositions of CYP2C19 and CYP3A4 in liver. Different contributions of CYP2C19 and CYP3A4 In the oxidation of testosterone and progesterone by human liver microsomes are also reported.