Shigeru Ohmori

Cytochrome P450-dependent drug oxidation activities in liver microsomes of various animal species including rats, guinea pigs, dogs, monkeys, and humans

Abstract Levels of cytochrome P450 (P450 or CYP) proteins immunoreactive to antibodies raised against human CYP1A2, 2A6, 2C9, 2E1, and 3A4, monkey CYP2B17, and rat CYP2D1 were determined in liver microsomes of rats, guinea pigs, dogs, monkeys, and humans. We also examined several drug oxidation activities catalyzed by liver microsomes of these animal species using eleven P450 substrates such as phenacetin, coumarin, pentoxyresorufin, phenytoin, S-mephenytoin, bufuralol, aniline, benzphetamine, ethylmorphine, erythromycin, and nifedipine; the activities were compared with the levels of individual P450 enzymes. Monkey liver P450 proteins were found to have relatively similar immunochemical properties by immunoblotting analysis to the human enzymes, which belong to the same P450 gene families. Mean catalytic activities (on basis of mg microsomal protein) of P450-dependent drug oxidations with eleven substrates were higher in liver microsomes of monkeys than of humans, except that humans showed much higher activities for aniline p-hydroxylation than those catalyzed by monkeys. However, when the catalytic activities of liver microsomes of monkeys and humans were compared on the basis of nmol of P450, both species gave relatively similar rates towards the oxidation of phenacetin, coumarin, pentoxyresorufin, phenytoin, mephenytoin, benzphetamine, ethylmorphine, erythromycin, and nifedipine, while the aniline p-hydroxylation was higher and bufuralol 1′-hydroxylation was lower in humans than monkeys. On the other hand, the immunochemical properties of P450 proteins and the activities of P450-dependent drug oxidation reactions in dogs, guinea pigs, and rats were somewhat different from those of monkeys and humans; the differences in these animal species varied with the P450 enzymes examined and the substrates used. The results presented in this study provide useful information towards species-related differences in susceptibilities of various animal species regarding actions and toxicities of drugs and xenobiotic chemicals.

Differential catalytic properties in metabolism of endogenous and exogenous substrates among CYP3A enzymes expressed in COS-7 cells

The catalytic properties of CYP3A7 in the metabolism of endogenous and exogenous substrates were compared with those of CYP3A4 and CYP3A5 using COS-7 expressing enzymes. The highest activities of dehydroepiandrosterone (DHEA) and dehydroepiandrosterone 3-sulfate (DHEA-S) 16alpha-hydroxylase were observed in COS-7 cells expressing CYP3A7. In contrast, the activity of testosterone 6beta-hydroxylase of CYP3A7 expressed in COS-7 cells was much less than that of CYP3A4 expressed in COS-7 cells. The rate of carbamazepine 10, 11-epoxidation was the greatest in COS-7 cells expressing CYP3A4, followed by CYP3A5 and CYP3A7. On the other hand, the formation of reductive metabolite of zonisamide was the highest in COS-7 cells expressing CYP3A4, followed by CYP3A7 and CYP3A5. Furthermore, the addition of triazolam resulted in a decrease in 6beta-hydroxylation catalyzed by CYP3A7, but not by CYP3A4, whereas the pretreatment of microsomes with triacetyloleandomycin (TAO) resulted in a decrease in the reaction catalyzed by CYP3A4, but not by CYP3A7. Together with these results, it was suggested that CYP3A7 exerts differential catalytic properties not only in metabolism of endogenous substrates but also in drug metabolism compared to CYP3A4 and CYP3A5.

Interaction of drugs and Chinese herbs: pharmacokinetic changes of tolbutamide and diazepam caused by extract of Angelica dahurica.

The inhibitory effects of Angelica dahurica root extract on rat liver microsomal cytochrome P450 and drug‐drug interactions were studied.

Prediction of drug-drug interactions of zonisamide metabolism in humans from in vitro data

Objective: The purposes of this study were to identify the P450 enzyme (CYP) responsible for zonisamide metabolism in humans by using expressed human CYPs and to predict drug interaction of zonisamide in vivo from in vitro data. Methods: Ten expressed human CYPs and human liver microsomes were used in the experiments for the identification of enzymes responsible for zonisamide metabolism and for the prediction of drug-drug interactions of zonisamide metabolism in humans from in vitro data, respectively. Two-sulfamoylacetyl phenol, a reductive metabolite of zonisamide, was measured by the HPLC method. Results: From the experiments using ten expressed human CYPs, CYP2C19, CYP3A4 and CYP3A5 were shown to be capable of catalyzing zonisamide reduction. However, an intrinsic clearance, Vmax/kM, of CYP3A4 was much higher than those of CYP2C19 and CYP3A5. From the point of view of enzyme amount in human liver CYPs isoform and their intrinsic clearance, it was suggested that CYP3A4 is mainly responsible for zonisamide metabolism in human CYPs. Zonisamide metabolism in human liver microsomes was markedly inhibited by cyclosporin A, dihydroergotamine, ketoconazole, itraconazole, miconazole and triazolam. We estimated the possibility and degree of change of zonisamide clearance in vivo in clinical dose range from in vitro inhibition constant of other drugs against zonisamide metabolism (Ki) and unbound inhibitor concentration in blood (Iu) in clinical usage. Clearance of zonisamide was maximally estimated to decrease by 31%, 23% and 17% of the clearance without inhibitors i.e. ketoconazole, cyclospolin A and miconazole, respectively. Fluconazole and carbamazepine are estimated to decrease by 5–6% of the clearance of zonisamide. On the other hand, there may be lack of interaction of zonisamide metabolism by dihydroergotamine, itraconazole and triazolam in clinical dose range. Conclusion: We demonstrated that: (1) zonisamide is metabolized by recombinant CYP3A4, CYP2C19 and CYP3A5, (2) the metabolism is inhibited to a variable extent by known CYP3A4/5 substrates and/or inhibitors in human liver microsomes, and (3) in vitro-in vivo predictive calculations suggest that several compounds demonstrating CYP3A4-affinity might cause in vivo drug-drug interactions with zonisamide.

Histological and functional analysis of vascular smooth muscle cells in a novel culture system with honeycomb-like structure

Vascular smooth muscle cells (SMCs) undergo phenotype change with the development of atherosclerosis. The phenotype changes of SMCs have been observed in various culture conditions, such as collagen-coated dishes. Here, we report the morphological and functional features of SMCs in a novel culture system using type I-collagen in a characteristic three-dimensional structure designated as honeycombs. The number of ribosome and mitochondria in SMCs cultured in honeycombs was one half or third of those cultured on collagen-coated plastic plates. DNA and protein synthesis of SMCs cultured in honeycombs were less than 1 and 30-40%, respectively, of those cultured on plastic plates. In addition, PDGF-BB did not increase the amount of DNA synthesis in SMCs in honeycombs. SMCs in honeycombs were shown to express several proteins, which are known to express in SMCs in medial layers of arteries. Particularly, caldesmon heavy chain was expressed in SMCs cultured in honeycombs, whereas not in those on plastic plates. Although focal adhesion kinase (FAK) was clearly detected in SMCs in honeycomb, the phosphotyrosine content of focal adhesion kin ase decreased in the process of culture. Immunoblot analysis showed dear different expression of ERK1 and ERK2 of mitogen-activated protein kinase in SMCs. SMCs in honeycombs expressed ERK2, more abundantly compared to ERK1, whereas SMCs in plates show the same levels of expressions for both proteins. Thus, the histological and functional feature of SMCs in the novel culture system is different from SMCs in plastic plates. The three-dimensional culture system described here may be indicating that cultured SMCs are able to express different proteins responding to the surrounding structures.

Immunochemical characterization and toxcological significance of P-450HFLb purified from human fetal livers

Immunochemical properties of P-450HFLb purified from human fetal livers were investigated. P-450HFLb cross-reacted with antibodies to rat P-4501A1 but not with antibodies to CYP2A6, CYP2C9, CYP3A7 (P-450HFLa) and rat CYP2B1. In addition, P-450HFLb also cross-reacted with both monospecific antibodies to rat CYP1A1 and CYP1A2. However, P-450HFLb was shown to be an immunochemically distinct form of cytochrome P-450 from P-450PA (human CYP1A2). Immunoblot analysis of human fetal livers with the antibodies to P-450HFLb showed that P-450HFLb was expressed in all fetal livers studied although there appeared to be individual differences in the amounts of P-450HFLb expressed in fetal livers. The formation of mutagens from IQ (but not from AFB1) in fetal liver homogenates was inhibited by the antibodies to P-450HFLb in a dose dependent manner. These results suggest that P-450HFLb may be a form of human cytochrome P-450 classified into CYP1 gene family, and that the cytochrome P-450 is, in part, responsible for the mutagenic activation of IQ in human fetal livers as well as CYP3A7 (P-450HFLa).

Enantioselectivity of bunitrolol 4-hydroxylation is reversed by the change of an amino acid residue from valine to methionine at position 374 of cytochrome P450-2D6.

The enantioselectivity of 4-hydroxylation of bunitrolol (BTL), a beta-adrenoceptor blocking drug, was studied in microsomes from human liver, human hepatoma (Hep G2) cells expressing CYP2D6, and lymphoblastoid cells expressing CYP2D6. Kinetics in human liver microsomes showed that the Vmax value for (+)-BTL was 2.1-fold that of (-)-BTL, and that the Km value for (+)-BTL was lower than that for the (-)-antipode, resulting in the intrinsic clearance (Vmax/Km) of (+)-BTL being 2.1-fold over its (-)-antipode. CYP2D6 (CYP2D6-met) expressed in Hep G2 cells had a methionine residue at position 373 of the amino acid sequence and a rat-type N-terminal peptide (MELLNGTGLWSM) instead of the human-type (MGLEALVPLAVIV), and showed enantioselectivity of [(+)-BTL < (-)-BTL] for the rate of BTL 4-hydroxylation. In contrast, enantioselectivity [(+)-BTL > (-)-BTL] for Hep G2-CYP2D6 (CYP2D6-val) with a human-type N-terminal peptide that had a valine residue at 374, which corresponds to the methionine of the CYP2D6-met variant, was the same as that for human liver microsomes. We further confirmed that CYP2D6-met and CYP2D6-val expressed in human lymphoblastoid cells, both of which have methionine and valine, respectively, at position 374 and a human-type N-terminal peptide, exhibited the same enantioselectivities as those obtained from CYP2D6-met and CYP2D6-val expressed in the Hep G2 cell system. These results indicate that the amino acid at 374 of CYP2D6 is one of the key factors influencing the enantioselectivity of BTL 4-hydroxylation.

Intestinal first-pass metabolism of eperisone in the rat.

AbstractPurpose. The purpose of this study was to clarify quantitatively the contribution of the intestine to the first-pass metabolism of eperisone in rats. Methods. The systemic availabilities of eperisone were estimated by administering the drug into the duodenum, portal vein, and femoral vein in rats in vivo. The first-pass metabolism of eperisone was confirmed in the perfused rat small intestine in situ. Metabolism of eperisone to an ω-1-hydroxylated metabolite (HMO), the first step of eperisone metabolism, was studied using rat intestinal microsomes in vitro. Results. The bioavailabilities in the intestine were 0.176 and 0.0879 at administration rates of 100 and 25 mg/h/kg, respectively, whereas those in the liver were 0.532 and 0.486, respectively. In the intestinal perfusion experiment, the appearance clearance to the portal vein from the intestinal lumen was much lower than the elimination clearance from the intestinal lumen, resulting in high metabolic clearance of eperisone in the small intestine. Eperisone was biotransformed to HMO by rat intestinal microsomes, and this was inhibited by α-naphthoflavone and an anti-rat CYP1A antibody. Conclusions. Those data strongly suggest that eperisone may be metabolized to HMO by CYP1A in rat intestinal microsomes during the first-pass through the epithelium of the small intestine.

Species differences of testosterone 16-hydroxylases in liver microsomes of guinea pig, rat and dog

1. In hepatic microsomes, remarkable species differences in the activity of testosterone 16-hydroxylase was observed in guinea pig, dog, and rat. The activity of testosterone 16 beta-hydroxylase was higher than that of 16 alpha-hydroxylase in guinea pig, whereas 16 alpha-hydroxylated testosterone was predominant as the metabolite in dog and rat. 2. Since P4502B isoenzyme has been shown to be a catalyst for testosterone 16-hydroxylations, we compared the catalytic properties of the P4502B subfamily (P450GP-1, P450b and P450PBD-2) purified from liver microsomes of guinea pig, dog, and rat, respectively. P450GP-1, P450b and P450PBD-2 showed different stereoselectivities for hydroxylation of testosterone at the 16-position. 3. P450GP-1, P450b and P450PBD-2 together comprised 47, < 0.1 and 23% of total P450 in liver microsomes of untreated guinea pig, rat and dog, respectively, indicating that the amounts of the P4502B isoenzyme in untreated animals were clearly different in these three animal species. Both 16 alpha- and 16 beta-hydroxylations of testosterone in liver microsomes of phenobarbital-treated guinea pig, rat and dog were inhibited by anti-P450GP-1, anti-P450b and anti-P450PBD-2 antibodies, respectively. 4. These and other results indicate that the species difference observed in testosterone 16-hydroxylation may be, in part, due to differences in the amounts of P450 of the P4502B subfamily, and their stereoselectivities for 16-hydroxylation.

Species difference in enantioselectivity for the oxidation of propranolol by cytochrome P450 2D enzymes.

We examined and compared enantioselectivity in the oxidation of propranolol (PL) by liver microsomes from humans and Japanese monkeys (Macaca fuscata). PL was oxidized at the naphthalene ring to 4-hydroxypropranolol, 5-hydroxypropranolol and side chain N-desisopropylpropranolol by human liver microsomes with enantioselectivity of [R(+)>S(-)] in PL oxidation rates at substrate concentrations of 10 microM and 1 mM. In contrast, reversed enantioselectivity [R(+)<S(-)] in PL 5-hydroxylation and N-desalkylation rates at the same substrate concentrations was observed in monkey liver microsomes, although the selectivity was the same for PL 4-hydroxylation between the two species. All oxidation reactions of the PL enantiomers in human liver microsomes showed biphasic kinetics, i.e. the reactions could be expressed as the summation of a low-K(m) phase and a high-K(m) phase. Inhibition studies using antibodies and characterization of CYP2D6 enzymes expressed in insect cells or human lymphoblastoid cells indicated that the enantioselectivity of PL oxidation, especially the ring 4- and 5-hydroxylations reflected the properties of CYP2D6 in human liver microsomes. In monkey liver microsomes, all of the oxidation reactions of S(-)-PL showed biphasic kinetics, whereas ring 4- and 5-hydroxylations were monophasic and side chain N-desisopropylation was biphasic for R(+)-PL. Similarly, from the results of inhibition studies using antibodies and inhibitors of cytochrome P450 (P450), it appears that the reversed selectivity [R(+)<S(-)] of PL oxidation rates is catalyzed by CYP2D enzyme(s) in monkey liver at low substrate concentrations. These results indicate that different properties of P450s belonging to the 2D subfamily cause the reversed enantioselectivity between human and monkey liver microsomes.