Due to the importance of in vitro cytochrome P450 (P450) induction assay to assess the possible drug-drug interaction events, the recent US Food and Drug Administration draft guidance and European Medicines Agency guideline recommend to assess P450 induction using fresh or cryopreserved hepatocytes at mRNA level and/or enzyme activity level. Although cryopreserved hepatocytes are commercially available for P450 induction assays, feasibility and practicability of these hepatocytes have not been fully investigated. In this study, a total of 23 lots of human cryopreserved hepatocytes were treated with three typical inducers (omeprazole, phenobarbital, and rifampicin), and induction of CYP1A2, CYP2B6, and CYP3A4 enzyme activity was measured. In 8 of these 23 hepatocyte lots, induction of CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, and CYP3A4 mRNA was also analyzed. The results revealed that CYP1A2, CYP2B6, and CYP3A4 were induced (>2.0-fold) by omeprazole, phenobarbital, and rifampicin, respectively, in all the hepatocyte lots tested at enzyme activity level (23 lots) and mRNA level (8 lots). In contrast, of the 8 hepatocyte lots treated with rifampicin, CYP2C8 and CYP2C9 mRNA were not induced in 5 and 2 hepatocyte lots, respectively, and CYP2C19 mRNA was not induced in any of the 8 hepatocyte lots tested. These results suggest that induction of CYP1A2, CYP2B6, and CYP3A4 can be readily assessed, but evaluation for CYP2C mRNA induction might not be feasible, using commercially available human cryopreserved hepatocytes. IntroductionIn vitro P450 induction assays are one of the important experiments in drug discovery and development to assess the potential for drugdrug interaction. The recent Food and Drug Administration (FDA) draft guidance recommends to evaluate induction of CYP1A2, CYP2B6, CYP2C, and CYP3A4 using fresh or cryopreserved hepatocytes by measuring mRNA and/or enzyme activity level (www.fda.gov/downloads/ Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm292362. pdf). A similar recommendation has also been documented in European Medicines Agency (EMA) guidelines (www.ema.europa.eu/docs/en_GB/ document_library/Scientific_guideline/2012/07/WC500129606.pdf). One of the major changes from the previous FDA draft guidance is the use of P450 mRNA levels as an endpoint for evaluation of induction. P450 induction has been assessed by measuring enzyme activity; however, mRNA data are also essential to reliably detect test compounds as inducers, which are also mechanism-based inactivators.The FDA draft guidance indicates the use of fresh or cryopreserved hepatocytes in induction assays. Induction of various P450 mRNAs, including CYP1A2, CYP2B6, CYP2C, and CYP3A4 mRNA, has been largely demonstrated mostly using fresh hepatocytes (Raucy et al., 2002;Madan et al., 2003). In contrast, only a limited number of papers has reported such induction results using cryopreserved hepatocytes (Roymans et al., 2005;Hewitt et al., 2007). For P450 induction assays at pharmaceutical companies, the cryopreserved hepato...

1. Human cytochrome P450 (CYP) enzymes and esterases involved in the metabolism of mirabegron, a potent and selective human β(3)-adrenoceptor agonist intended for the treatment of overactive bladder, were identified in in vitro studies. 2. Incubations of mirabegron with recombinant human CYP enzymes showed significant metabolism of mirabegron by CYP2D6 and CYP3A4 only. Correlation analyses showed a significant correlation between mirabegron metabolism and testosterone 6β-hydroxylation (CYP3A4/5 marker activity). In inhibition studies using antiserum against CYP3A4, a strong inhibition (at maximum 80% inhibition) of the metabolism of mirabegron was observed, whereas the inhibitory effects of monoclonal antibodies against CYP2D6 were small (at maximum 10% inhibition). These findings suggest that CYP3A4 is the primary CYP enzyme responsible for in vitro oxidative metabolism of mirabegron, with a minor role of CYP2D6. 3. Mirabegron hydrolysis was catalyzed in human blood, plasma and butyrylcholinesterase (BChE) solution, but not in human liver microsomes, intestinal microsomes, liver S9, intestinal S9 and recombinant acetylcholinesterase solution. K(m) values of mirabegron hydrolysis in human blood, plasma and BChE solution were all similar (13.4-15.2 μM). The inhibition profiles in human blood and plasma were also similar to those in BChE solution, suggesting that mirabegron hydrolysis is catalyzed by BChE.

Heterotropic cooperativity of human cytochrome P450 (P450) 3A4/3A5 by the teratogen thalidomide was recently demonstrated by H. Yamazaki et al. (2013) using the model substrate midazolam in various in vitro and in vivo models. Chimeric mice with humanized liver also displayed enhanced midazolam clearance upon pre treatment with orally administered thalidomide, presumably because of human P450 3A induction. In the current study, we further investigated the regulation of human hepatic drug metabolizing enzymes. Thalidomide enhanced levels of P450 3A4 and 2B6 mRNA, protein expression, and/or oxidation activity in human hepatocytes, indirectly suggesting activation of upstream transcription factors involved in detoxication, e.g. the nuclear receptors pregnane X receptor (PXR) and constitutive androstane receptor (CAR). A key event after ligand binding is an alteration of nuclear receptor conformation and recruitment of co regulator proteins that alter chromatin accessibility of target genes. To investigate direct engagement and functional alteration of PXR and CAR by thalidomide, we utilized a peptide microarray with 154 co regulator derived nuclear receptor interaction motifs and co regulator and nuclear receptor boxes, which serves as a sensor for nuclear receptor conformation and activity status as a function of ligand. Thalidomide and its human proximate metabolite 5 hydroxythalidomide displayed significant modulation of co regulator interaction with PXR and CAR ligand binding domains, similar to established agonists for these receptors. These results collectively suggest that thalidomide acts as a ligand for PXR and CAR and causes enzyme induction leading to increased P450 enzyme activity. The possibilities of drug interactions during thalidomide therapy in humans require further evaluation.

ABSTRACT:Darexaban maleate is a novel oral direct factor Xa inhibitor, which is under development for the prevention of venous thromboembolism. Darexaban glucuronide was the major component in plasma after oral administration of darexaban to humans and is the pharmacologically active metabolite. In this study, we identified UDPglucuronosyltransferases (UGTs) responsible for darexaban glucuronidation in human liver microsomes (HLM) and human intestinal microsomes (HIM). In HLM, the K m value for darexaban glucuronidation was >250 M. In HIM, the reaction followed substrate inhibition kinetics, with a K m value of 27.3 M. Among recombinant human UGTs, UGT1A9 showed the highest intrinsic clearance for darexaban glucuronidation, followed by UGT1A8, -1A10, and -1A7. All other UGT isoforms were inactive toward darexaban. The K m value of recombinant UGT1A10 for darexaban glucuronidation (34.2 M) was comparable to that of HIM. Inhibition studies using typical UGT substrates suggested that darexaban glucuronidation in both HLM and HIM was mainly catalyzed by UGT1A8, -1A9, and -1A10. Fatty acid-free bovine serum albumin (2%) decreased the unbound K m for darexaban glucuronidation from 216 to 17.6 M in HLM and from 35.5 to 18.3 M in recombinant UGT1A9. Recent studies indicated that the mRNA expression level of UGT1A9 is extremely high among UGT1A7, -1A8, -1A9, and -1A10 in human liver, whereas that of UGT1A10 is highest in the intestine. Thus, the present results strongly suggest that darexaban glucuronidation is mainly catalyzed by UGT1A9 and UGT1A10 in human liver and intestine, respectively. In addition, UGT1A7, -1A8, and -1A9 play a minor role in human intestine.

The fraction of substrate metabolized (f ) can be used to estimate drug interactions and can be determined by comparison of the intrinsic clearances (CL ) of victim drugs obtained from inhibited and uninhibited hepatic enzymes Commercially available human liver microsomes were recently developed in which one cytochrome P450 (P450) isoform is selectively inactivated. These inactivated liver microsomes were used to evaluate the roles of P450 2C isoforms in the depletion and oxidation of probe substrates. Determination of CL with sets of control and P450 2C9-inactivated liver microsomes yielded f values of 0.69-1.0 for celecoxib, diclofenac and warfarin. Apparent minor contributions of P450 1A2/2C8/3A4 were seen in depletion assays, yielding ~1 for the sum of the f values. Selectively inactivated liver microsomes were thereby shown to be potentially useful for determining the in vitro f values for major P450 2C9 contributions to substrate oxidations. Metabolite formations from diclofenac and warfarin were suppressed by 62-84% by the replacement of control liver microsomes with P450 2C9-inactivated liver microsomes. R-, S- and racemic omeprazole and troglitazone oxidation activities by liver microsomes at multiple substrate concentrations were suppressed by 26-36% and 22-50%, respectively, when P450 2C19- and 2C8-inactivated liver microsomes were used in place of control liver microsomes. This study provides important information to help elucidate the different roles of P450 isoforms in metabolite formation at different substrate concentrations. The data obtained allow the fractions metabolized to be calculated for victim drugs.

Previously, we showed that the toxic susceptibility of male rats to an ultraviolet absorber, 2-(2'-hydroxy- 3',5'-di-tert-butylphenyl)benzotriazole (HDBB), was nearly 25 times higher than that of females. The present study aimed to clarify the mechanism of gender-related differences in HDBB toxicity. Male and female rats were given HDBB by gavage at 0.5, 2.5, or 12.5 mg/kg/day for 28 days, and plasma HDBB levels were measured at various time points by using liquid chromatography-tandem mass spectrometry. HDBB was rapidly absorbed and eliminated from the plasma in both sexes, and no sexual variations were found in the plasma levels. In the plasma, HDBB metabolites were not detected at any dose by the liquid chromatography-photodiode array detector. In an in vitro metabolic study using hepatic microsomes from male and female rats, HDBB was slightly metabolized, but no sexual differences were found in the residual HDBB ratio after a 60-minute incubation with an NADPH-generation system. Following 28-day HDBB administration, sexually different changes were found in cytochrome P450-dependent microsomal mixed-function oxidase activities in the liver. In males, 7-ethoxyresorufin O-deethylase activity decreased and lauric acid 12-hydroxylase activity increased at all doses. Decreases in aminopyrine N-demethylase activity and testosterone 2alpha- and 16alpha-hydroxylase activity were also found at 2.5 mg/kg and above in males. In females, the only significant change was increased lauric acid 12-hydroxylase activity at 12.5 mg/kg. These findings indicate that HDBB would have hepatic peroxisome proliferative activity, and the difference in susceptibility of male and female rats to this effect might lead to marked gender-related differences in HDBB toxicity.

Beraprost sodium (BPS), a chemically stable and orally active prostacyclin analogue used for the treatment of chronic occlusive disease and primary pulmonary hypertension, was investigated in terms of its drug-drug interaction mediated by cytochrome P450. In a metabolic enzyme characterization study using P450-expressing insect cell microsomes, beraprost (BP) was slightly metabolized in the presence of CYP2C8, but not metabolized by the other P450 isoforms (CYP1A1, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, and CYP4A11) at a concentration of 20 mM. These results suggest that none of the P450 isoforms is a major metabolic enzyme of BP. In a P450 induction study using human hepatocytes, BP did not induce any P450 isoform (CYP1A2, CYP2C9, CYP2C19, and CYP3A4) at concentrations of 1 100 mM. Furthermore, in a P450 inhibition study using human liver microsomes, BP did not inhibit any P450 isoform (CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4) at concentrations of 0.05 1 mM. Therefore it is concluded that BP is not involved in drug-drug interaction mediated by P450 isoforms.

Abstract:The aim of this study was to collect data on chronological changes in clinical laboratory tests, pathological examinations, and hepatic drug-metabolizing enzymes from Wistar Hannover rats at 8, 10, 19, and 32 weeks of age. The serum triglyceride concentration and the serum LDL cholesterol level were higher in males than in females at all ages. In contrast, serum total protein and creatinine concentrations and cholinesterase activity were lower in males than in females. In addition, sex differences were confirmed in pituitary weight and hepatic CYP3A2 and CYP2C11 activities. In conclusion, the general toxicological data noted in clinical laboratory tests, pathological examinations, and hepatic drug-metabolizing enzymes relating to chronological changes and sex differences may be useful in assessing drug-related toxicity in this strain. Key words: clinical laboratory test, hepatic drug-metabolizing enzyme, pathological examination sprague-dawley (sd) and F344 rats are commonly used for toxicity studies in japan [2], and the inbred F344/N rat substrain has been used for the National toxicity Program rodent toxicity and carcinogenicity bioassays for more than thirty years [10,19]. the F344/N rat is known to have high background incidences of certain types of tumor including testicular interstitial cell tumors and mononuclear cell leukemia [7], while typical spontaneous diseases in aged sd rats are chronic nephropathy and pituitary and mammary tumors [5,16]. therefore, the use of these strains in long-term toxicity studies should be carefully considered when planning study design and assessing toxicity.wistar Hannover rats are used widely in Europe [21], particularly for toxicity studies, because of their small body size, long survival rate, low spontaneous tumor incidence, and ease of handling. research with this strain has been limited in japan; however, its use in toxicity studies is expected to increase. in 2006, Petterino et al. reported clinical chemistry and hematology historical control data from preclinical toxicity studies using sd rats [14], but published historical data on wistar Hannover rats relating to a general toxicity study are scarce [21]. Furthermore, age-related changes and sex differences in serum chemistry values in sd rats have been reported [18]. therefore, collection of general toxico-