Kazuhide Iwasaki

Metabolism of FK506, a potent immunosuppressive agent, by cytochrome P450 3A enzymes in rat, dog and human liver microsomes

The oxidative metabolism of FK506 by liver microsomes and purified cytochrome P450 (P450) enzymes from rats, dogs and humans was studied. The major metabolite formed by liver microsomes from all species was 13-demethylated FK506, named M-I. In adult rats, liver microsomal metabolic activity toward FK506 was higher in males than in females and was stimulated by treatment with P450 3A inducers such as dexamethasone and phenobarbital. In a reconstituted monooxygenase system containing various forms of purified P450 3A enzymes, rat P450 3A2, dog P450 DPB-1 (a form of the P450 3A family) and human P450 3A4 catalyzed FK506 oxidation efficiently in the presence of cytochrome b5, a mixture of phospholipids (dilauroylphosphatidylcholine, dioleoylphosphatidylcholine and phosphatidylserine), and sodium cholate. Rat P450 2C6 and 2D1 and human P450 2CMP also metabolized FK506, with significant lower activity than the P450 3A enzymes, and other rat P450 1A, 2A, 2B, 2C and 2E families including C11 did not show catalytic activities for FK506. Anti-P450 3A2 and anti-P450 3A4 antibodies strongly inhibited FK506 oxidation catalyzed by rat and human liver microsomes, respectively. The formation rate of M-I correlated well with testosterone 2 beta- and 6 beta-hydroxylase activities in rat liver microsomes and with immunoquantified P450 3A4 content, nifedipine oxidase activity, and testosterone 6 beta-hydroxylase activity in human liver microsomes. These in vitro findings indicate that the P450 3A enzymes in liver microsomes from various species of animals, including human, play a major role in the first step oxidation of FK506.

Distribution and protein binding of FK506, a potent immunosuppressive macrolide lactone, in human blood and its uptake by erythrocytes

Abstract— The distribution of FK506 in the blood was estimated in‐vitro. At a drug level of 5 ng mL−1, FK506 mainly distributed in erythrocytes (95–98%) in dog, monkey and human blood, and its distribution was affected by drug concentration, temperature, and haematocrit values. In erythrocytes most of FK506 was distributed in cytoplasmic components and was bound strongly to a protein having a molecular weight of 10–11 kDa. The molecular weight of this protein agrees with FK506‐binding protein found in various cells. Greater than 98·8% of FK506 was bound to the plasma proteins in all species studied. FK506 bound to various plasma proteins such as lipoproteins, globulins, α1‐acid glycoprotein and albumin.

EXPRESSION OF HUMAN CYTOCHROMES P450 IN CHIMERIC MICE WITH HUMANIZED LIVER

Recently, a chimeric mouse line in which the liver could be replaced by more than 80% with human hepatocytes was established in Japan. Because the chimeric mouse produces human albumin (hAlb), replacement by human hepatocytes could be estimated by the hAlb concentration in the blood of chimeric mice. In this study, we investigated human major cytochrome P450 (P450) in the livers of chimeric mice by mRNA, protein, and enzyme activity using real-time polymerase chain reaction, Western blot analysis, and high-performance liquid chromatography, respectively. Chimeric mice with humanized liver generated using hepatocytes from a Japanese and white donor were used. Human P450 mRNAs were expressed in the liver of chimeric mice, and major human P450 proteins such as CYP1A2, CYP2C9, and CYP3A4 were detected. The expression of P450 mRNA and protein was correlated with the hAlb concentration in the blood. The enzyme activities such as diclofenac 4′-hydroxylase activity, dexamethasone 6-hydroxylase activity, and coumarin 7-hydroxylase activity, activities that are specific to human P450 but not to murine P450, were increased in a hAlb concentration-dependent manner. The chimeric mice with nearly 90% replacement by human hepatocytes demonstrated almost the same protein contents of human P450s and drug-metabolizing enzyme activity as those of the donor. It was confirmed that genomic DNA from the livers of the chimeric mice and that from the liver of the donor exhibited the same genotype. In conclusion, the chimeric mice exhibited a similarly efficient capacity of drug metabolism as humans, suggesting that they could be a useful animal model for drug development.

Macaque cytochromes P450: nomenclature, transcript, gene, genomic structure, and function

Monkeys, especially macaques, including cynomolgus (Macaca fascicularis) and rhesus monkeys (Macaca mulatta), are frequently used in drug metabolism studies due to their evolutionary closeness to humans. Recently, numerous cytochrome P450 (P450 or CYP) cDNAs have been identified and characterized in cynomolgus and rhesus monkeys and were named by the P450 Nomenclature Committee. However, recent advances in genome analysis of cynomolgus and rhesus monkeys revealed that some monkey P450s are apparently orthologous to human P450s and thus need to be renamed corresponding to their human orthologs. In this review, we focus on the P450s identified in cynomolgus and rhesus monkeys and present an overview of the identity and functional characteristics of each P450 cDNA in the CYP1-4 families. Information on the Japanese monkey (Macaca fuscata), African green monkey (Cercopithecus aethiops), and marmoset (Callithrix jacchus), primate species used in some drug metabolism studies, are also included. We compared the genomic structure of the macaque P450 genes to those of human and rat P450 genes in the CYP1-4 families. Based on sequence identity, phylogeny, and genomic organization of monkey P450s, we determined orthologous relationships of monkey P450s and, in this article, propose a revised nomenclature: CYP2B17/CYP2B30 to CYP2B6, CYP2C20/CYP2C74 to CYP2C8, CYP2C43/CYP2C83 to CYP2C9, CYP2C75 to CYP2C19, CYP2F6 to CYP2F1, CYP3A8/CYP3A21/CYP3A64 to CYP3A4, CYP3A66 to CYP3A5, and CYP4F45 to CYP4F2. The information presented in this review is expected to promote a better understanding of monkey P450 genes through comparative genomics and thereby make it more feasible to use monkeys in drug metabolism studies.

Evidence for the involvement of cytochrome P-450 in tiaramide N-oxide reduction

Abstract Tiaramide N-oxide, a major metabolite of tiaramide, is reduced anaerobically to tiaramide by rat liver microsomes. The reaction requires NADPH and is inhibited by oxygen and carbon monoxide. Both phenobarbital and 3-methylcholanthrene treatments induced the reductase activity with increasing cytochrome P-450 content. Tiaramide N-oxide produced a pronounced spectral change with reduced cytochrome P-450 and the difference spectrum showed a peak of absorbance at 442 nm. These findings provide evidence in support of an essential role for cytochrome P-450 in the process of the N-oxide reduction.

Induction of cytochrome P-448 activity as exemplified by the O-deethylation of ethoxyresorufin: Effects of dose, sex, tissue and animal species

The effects of tissue, sex, animal species and dose on the induction of cytochrome P-448 activity by various inducing agents were investigated using O-ethoxyresorufin as a model substrate. The liver was by far more effective in catalysing the O-deethylation of ethoxyresorufin (EROD) than the lung and kidney. The extent of induction was also highest in the liver, with the exception of benzo(a)pyrene and 3-methylcholanthrene where inducibility was more pronounced in the kidney. The benzo(a)pyrene-induced hepatic EROD activity in the rat decayed to reach control levels four days after a single administration. Rat hepatic EROD activity was induced in both sexes but tended to be higher in the male. Marked species differences in the inducibility of hepatic EROD activity by various chemicals was observed, the rat being always more responsive when compared to the hamster or mouse. The induction of rat hepatic EROD activity by benzo(a)pyrene, 2-acetylaminofluorene and safrole was dose-dependent, maximum induction being achieved with single doses of 5, 2 and 5 mg/kg, respectively.

Identification of cytochrome P450 enzymes involved in the metabolism of zotepine, an antipsychotic drug, in human liver microsomes.

1. Studies using human liver microsomes and recombinant human cytochrome P450 (P450) enzymes and flavin-containing monooxygenase (FMO) were performed to identify the enzymes responsible for the formation of zotepine metabolites in man. 2. Human liver microsomes produced four metabolites and a tentative order of importance was: norzotepine, 3-hydroxyzotepine, zotepine S-oxide and 2-hydroxyzotepine. Zotepine N-oxide was also detected, but it could not be quantified. 3. The rates of formation of the major metabolite, norzotepine, and zotepine S-oxide (at a substrate concentration of 20 microM) were significantly correlated with the testosterone 6beta-hydroxylase activities and CYP3A4 contents of the 12 different human liver microsomal samples. Inhibition studies with P450 enzyme selective inhibitors and anti-rat CYP3A2 antibodies also indicated a predominant role of CYP3A4 in the formation of norzotepine and zotepine S-oxide. Furafylline and sulphaphenazole inhibited the N-demethylation of zotepine by up to approximately 30%. 4. Correlation and inhibition data for the 2- and 3-hydroxylation of zotepine were consistent with the predominant role of CYP1A2 and 2D6 in the formation of these metabolites, respectively. 5. Recombinant CYP1A1, 1A2, 2B6, 2C19, 3A4 and 3A5 efficiently catalysed N-demethylation of zotepine. CYP1A1, 1A2, 2B6 and 3A4 were also active for S-oxidation. CYP1A2 and 2D6*1-Val374 efficiently produced 2-hydroxyzotepine and 3-hydroxyzotepine, respectively. Recombinant human FMO3 did not catalyse zotepine S-oxidation. 6. These results suggest that both the N-demethylation and S-oxidation of zotepine are mediated mainly by CYP3A4, and that CYP1A2 and 2D6 play an important role in the 2- and 3-hydroxylation of zotepine, respectively.

Species and sex differences of testosterone and nifedipine oxidation in liver microsomes of rat, dog and monkey

1. Species and sex differences in testosterone hydroxylation and nifedipine oxidation in liver microsomes from rat, dog and monkey have been investigated. 2. The formation of 2 alpha-, 2 beta-, 6 beta-, and 16 alpha-hydroxytestosterone and androstenedione in the male rat was higher than that in the female rat. Microsomes prepared from the male rat oxidized nifedipine about eight times faster than did those from the female rat. In contrast, marked sex-related differences were not seen in the dog and monkey. 3. Nifedipine oxidase activity in rat, dog and monkey correlated significantly with the activities for both testosterone 2 beta-hydroxylation and 6 beta-hydroxylation, suggesting the involvement of P4503A isozymes in these reactions. The ratios of formation of the 2 beta- to 6 beta-hydroxytestosterone in male rat and monkey were 0.17 and 0.18 respectively, whereas that in dog was 0.46. The corresponding activity ratios catalysed by P450DPB-1, a P4503A isoform purified from dog liver microsomes, was 0.36. 4. The formation of 16 beta-hydroxytestosterone was higher than that of the 16 alpha-hydrolated metabolite in liver microsomes from monkey, whereas 16 alpha-hydroxytestosterone was the predominant metabolite in the rat and dog, indicating species differences in stereoselectivity at the 16-position.

Macaque CYP2C76 Encodes Cytochrome P450 Enzyme Not Orthologous to Any Human Isozymes

Cynomolgus monkey is used in the study of drug metabolism and toxicity due to its evolutionary closeness to human as compared with other non-human primate species. However, it has become certain that drug metabolism in monkeys is different than in humans. Such species differences have not been fully investigated at a molecular level largely due to the scarcity of information on drug-metabolizing enzyme genes. In cynomolgus monkey, we have identified cDNAs for 21 kinds of cytochromes P450 (CYPs), among which CYP2C76 does not correspond to any human CYP isozymes and is partly responsible for the difference in pitavastatin metabolism between cynomolgus monkey and human. In cynomolgus monkey CYP2C76, we identified numerous genetic variants including a null genotype. Heterozygotes for this null genotype are expected to be poor metabolizers in CYP2C76-mediated drug metabolism. To provide new clues to CYP2C76 function, here, we have taken advantage of sequence information that has been recently deposited to public databases to assess the presence of CYP2C76 orthologs in primate species. In this assessment, we found the CYP2C76 cDNA sequence in rhesus monkey, and a gene sequence highly homologous to cynomolgus monkey CYP2C76 in the marmoset and orangutan genomes, raising the possibility that CYP2C76 could also play a role in these primate species. This review paper gives an overview of CYP2C76 from isolation to molecular characterization, and its implication in drug metabolism.

Urinary metabolite profile of tiaramide in man and in some animal species

1. The metabolism of tiaramide, 4-[(5-chloro-2-oxo-3(2H)-benzothiazolyl)acetyl]-1-piperazineethanol, was studied in healthy male volunteers and experimental animals. 2. Tiaramide was extensively metabolized in human with only 1.5% excreted unchanged. 3. Urinary metabolites were identified by FD, CI and EI mass spectral comparison with authentic standards. The major urinary metabolites in human were 4-[(5-chloro-2-oxo-3(2H)-benzothiazolyl)acetyl]-1-piperazineacetic acid (TRAA), 4-[(5-chloro-2-oxo-3(2H)-benzothiazolyl)acetyl]-1-piperazineacetic acid 1-oxide (TRAO) and the O-glucuronide of tiaramide. 4. TRAO, a new metabolite identified in human urine, was also present in mouse, rat, guinea-pig and monkey, but in smaller amounts than for human. 5. Sex differences in the excretion of sulphate of tiaramide were noted only in the rat.