The decline in bone mineral density that occurs after longterm treatment with some antiepileptic drugs is thought to be mediated by increased vitamin D 3 metabolism. In this study, we show that the inducible enzyme CYP3A4 is a major source of oxidative metabolism of 1␣,25-dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ] in human liver and small intestine and could contribute to this adverse effect. Heterologously-expressed CYP3A4 catalyzed the 23-and 24-hydroxylation of 1,25(OH) 2 D 3 . No human microsomal cytochrome P450 enzyme tested, other than CYP3A5, supported these reactions. CYP3A4 exhibited opposite product stereochemical preference compared with that of CYP24A1, a known 1,25(OH) 2 D 3 hydroxylase. The three major metabolites generated by CYP3A4 were 1,23R,25(OH) 3 D 3 , 1,24S,25(OH) 3 D 3 , and 1,23S,25(OH) 3 D 3 . Although the metabolic clearance of CYP3A4 was less than that of CYP24A1, comparison of metabolite profiles and experiments using CYP3A-specific inhibitors indicated that CYP3A4 was the dominant source of 1,25(OH) 2 D 3 23-and 24-hydroxylase activity in both human small intestine and liver. Consistent with this observation, analysis of mRNA isolated from human intestine and liver (including samples from donors treated with phenytoin) revealed a general absence of CYP24A1 mRNA. In addition, expression of CYP3A4 mRNA in a panel of duodenal samples was significantly correlated with the mRNA level of a known vitamin D receptor gene target, calbindin-D9K. These and other data suggest that induction of CYP3A4-dependent 1,25(OH) 2 D 3 metabolism by antiepileptic drugs and other PXR ligands may diminish intestinal effects of the hormone and contribute to osteomalacia.

The purpose of the study was to elucidate human intestinal cytochrome P450 isoform(s) involved in the metabolism of an antihistamine, ebastine, having two major pathways of hydroxylation and N-dealkylation. The ebastine dealkylase in human intestinal microsomes was CYP3A4, based on the inhibition studies with antibodies against CYP1A, CYP2A, CYP2C, CYP2D, CYP2E, and CYP3A isoforms and their selective inhibitors. However, ebastine hydroxylase could not be identified. We then examined the inhibitory effects of anti-CYP4F antibody and 17-octadecynoic acid, an inhibitor of the CYP4 family, on ebastine hydroxylation in intestinal microsomes, since CYP4F was recently found to be the predominant ebastine hydroxylase in monkey intestine; and a novel CYP4F isoform (CYP4F12), also capable of hydroxylating ebastine, was found to exist in human intestine. However, the inhibitory effects were only partial (about 20%) and thus it was thought that, although human CYP4F was involved in ebastine hydroxylation, another predominant enzyme exists. Further screening showed that the hydroxylation was inhibited by arachidonic acid. CYP2J2 was selected as a candidate expressed in the intestine and closely related to arachidonic acid metabolism. The catalytic activity of recombinant CYP2J2 was much higher than that of CYP4F12. Anti-CYP2J antibody inhibited the hydroxylation to about 70% in human intestinal microsomes. These results demonstrate that CYP2J2 is the predominant ebastine hydroxylase in human intestinal microsomes. Thus, the present paper for the first time indicates that, in human intestinal microsomes, both CYP2J and CYP4F subfamilies not only metabolize endogenous substrates but also are involved in the drug metabolism. Ebastine is a potent nonsedative H 1 -receptor antagonist (Fig. 1), and after oral administration to experimental animals and humans, the agent is almost completely metabolized to the pharmacologically active principle, the carboxylated metabolite (carebastine), and other inactive metabolites Matsuda et al., 1994;Yamaguchi et al., 1994). Carebastine alone was the major metabolite detectable in the blood. Our previous in situ studies using rats indicated that the small intestine extensively converted the orally given ebastine to carebastine via hydroxylated ebastine and the dealkylated metabolite (Fujii et al., 1997). Therefore, it seemed that small intestine plays an important role in the first-pass metabolism of this drug, and the enzymes responsible for its metabolism exist there.We reported that ebastine was primarily metabolized by human liver microsomes to two metabolites, hydroxy-and desalkyl-ebastine (Hashizume et al., 1998). N-Dealkylation to desalkyl-ebastine was mediated by CYP3A4, whereas hydroxylation to hydroxy-ebastine, the most important intermediate metabolite yielding carebastine, was mediated by unidentified P450(s) other than CYP3A4. Our recent studies revealed that two novel CYP4F isoforms (P450 MI-2 and CYP4F12) obtained from monkey and human small intestine, respectively, were ...

ABSTRACT:Prediction of idiosyncratic drug-induced liver injury (DILI) is difficult, and the underlying mechanisms are not fully understood. However, many drugs causing DILI are considered to form reactive metabolites and covalently bind to cellular macromolecules in the liver. The objective of this study was to clarify whether the risk of idiosyncratic DILI can be estimated by comparing in vitro covalent binding (CB) levels among 12 positive compounds (acetaminophen, alpidem, bromfenac, carbamazepine, diclofenac, flutamide, imipramine, nefazodone, tacrine, ticlopidine, tienilic acid, and troglitazone) for DILI and 12 negative compounds (acetylsalicylic acid, caffeine, dexamethasone, losartan, ibuprofen, paroxetine, pioglitazone, rosiglitazone, sertraline, theophylline, venlafaxine, and zolpidem). After incubation with human liver microsomes in the presence of NADPH, there was a large overlap in the distribution of CB amounts between the positive and negative groups. On addition of UDP-glucuronic acid (UDPGA) as a cofactor for glucuronidation, the CB levels of bromfenac and diclofenac were increased. With addition of nucleophilic glutathione (GSH), values for most compounds were decreased. However, separation of the two groups on the basis of CB could not be improved by UDPGA or GSH. Furthermore, CB with human hepatocytes also failed to discriminate positive from negative compounds. Therefore, the CB amount alone is not sufficient for risk assessment of DILI. In contrast, when the CB amount was multiplied by the maximum daily dose, which may reflect maximum hepatic exposure, the two groups did become discriminated. Taken together, our findings suggest that the combination of CB amount and daily dose can estimate the risk of idiosyncratic DILI.

Vitamin D 3 is critical for the regulation of calcium and phosphate homeostasis. In some individuals, mineral homeostasis can be disrupted by long-term therapy with certain antiepileptic drugs and the antimicrobial agent rifampin, resulting in druginduced osteomalacia, which is attributed to vitamin D deficiency. We now report a novel CYP3A4-dependent pathway, the 4-hydroxylation of 25-hydroxyvitamin D 3 (25OHD 3 ), the induction of which may contribute to drug-induced vitamin D deficiency.

The current results indicated that an extrastriatal spreading of microglial activation reflects one of PD pathophysiology occurring at an early stage.

25-Hydroxyvitamin D3 (25OHD3) is used as a clinical biomarker for assessment of vitamin D status. Blood levels of 25OHD3 represent a balance between its formation rate and clearance by several oxidative and conjugative processes. In the present study, the identity of human uridine 5'-diphosphoglucuronyltransferases (UGTs) capable of catalyzing the 25OHD3 glucuronidation reaction was investigated. Two isozymes, UGT1A4 and UGT1A3, were identified as the principal catalysts of 25OHD3 glucuronidation in human liver. Three 25OHD3 monoglucuronides (25OHD3-25-glucuronide, 25OHD3-3-glucuronide, and 5,6-trans-25OHD3-25-glucuronide) were generated by recombinant UGT1A4/UGT1A3, human liver microsomes, and human hepatocytes. The kinetics of 25OHD3 glucuronide formation in all systems tested conformed to the Michaelis-Menten model. An association between the UGT1A4*3 (Leu48Val) gene polymorphism with the rates of glucuronide formation was also investigated using human liver microsomes isolated from 80 genotyped livers. A variant allele dose effect was observed: the homozygous UGT1A4*3 livers (GG) had the highest glucuronidation activity, whereas the wild type (TT) had the lowest activity. Induction of UGT1A4 and UGT1A3 gene expression was also determined in human hepatocytes treated with pregnane X receptor/constitutive androstane receptor agonists, such as rifampin, carbamazepine, and phenobarbital. Although UGT mRNA levels were increased significantly by all of the known pregnane X receptor/constitutive androstane receptor agonists tested, rifampin, the most potent of the inducers, significantly induced total 25OHD3 glucuronide formation activity in human hepatocytes measured after 2, but not 4 and 24 hours, of incubation. Finally, the presence of 25OHD3-3-glucuronide in both human plasma and bile was confirmed, suggesting that the glucuronidation pathway might be physiologically relevant and contribute to vitamin D homeostasis in humans.

Metabolites of arachidonic acid produced by P450 are interesting substances with prominent physiological functions. To elucidate the physiological function of P450, it is necessary to identify a specific P450 in a particular tissue or organ and to characterize its catalytic activities. In this study, the expression of CYP2A1, 2B1, 2C23, 2J3, and 4F1 was investigated in liver, lung, kidney, spleen, heart, brain, and testis of rats by RT-PCR. Furthermore, arachidonic acid metabolism was investigated using the rat P450s described above and human CYP2A6, 2B6, 2C9, 2C18, 2C19, 2J2, and 4F2. Among the rat P450s, CYP2B1 and 2C23 efficiently produced EETs and CYP4F1 produced 19/20-HETE in abundace. CYP2B1 was specifically expressed in the lung. CYP2C23 was detected in all tissues used in this study. CYP4F1 was expressed in the kidney as well as in the liver. Among the human P450s, CYP2C9 and 2C19 efficiently produced EETs. CYP4F2 produced 19/20-HETE. The catalytic properties of rat CYP2C23 were similar to those of human CYP2C9 and 2C19. The catalytic properties of CYP4F isoforms were also similar between humans and rats. A systematic analysis of P450 expression in various tissues and of its catalytic property may provide valuable information on the physiological roles of P450s in each tissue.

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