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Lamotrigine is a phenyltriazine anticonvulsant used to treat epilepsy and bipolar disorder, with species-dependent metabolic profiles. In this study, we investigated the metabolism of lamotrigine in chimeric NOG-TKm30 mice transplanted with human hepatocytes (humanised-liver mice).Substantial lamotrigine N2-glucuronidation activities were observed in the liver microsomes from humanised-liver mice, humans, marmosets, and rabbits, compared to those from monkeys, minipigs, guinea pigs, rats, and mice. Lamotrigine N2-glucuronidation activities in the liver microsomes from humanised-liver mice were dose-dependently inhibited by hecogenin, a specific inhibitor of the human UGT1A4.The major metabolite in the hepatocytes from humanised-liver mice and humans was lamotrigine N2-glucuronide, whereas that in mouse hepatocytes was lamotrigine N2-oxide. After a single oral dose of lamotrigine (10 mg/kg), the plasma levels of N2-glucuronide, N5-glucuronide, and N2-methyl were higher in humanised-liver mice compared to that in NOG-TKm30 mice. Lamotrigine N2-glucuronide was the most abundant metabolite in the urine in humanised-liver mice, similar to that reported in humans; whereas, lamotrigine N2-oxide was predominantly excreted in the urine in NOG-TKm30 mouse.These results suggest that humanised-liver mice may be a suitable animal model for studying the UGT1A4 mediated-lamotrigine metabolism.

Common marmosets (

Plasma concentrations of acetaminophen, its glucuronide and sulfate conjugates, and cysteinyl acetaminophen were experimentally determined after oral administrations of 10 mg/kg in humanised-liver mice, control mice, rats, common marmosets, cynomolgus monkeys, and minipigs; the results were compared with reported human pharmacokinetic data. Among the animals tested, only rats predominantly converted acetaminophen to sulfate conjugates, rather than glucuronide conjugates. In contrast, the values of area under the plasma concentration curves of acetaminophen, its glucuronide and sulfate conjugates, and cysteinyl acetaminophen after oral administration of acetaminophen in marmosets and minipigs were consistent with those reported in humans under the present conditions. Physiologically based pharmacokinetic (PBPK) models (consisting of the gut, liver, and central compartments) for acetaminophen and its primary metabolite could reproduce and estimate, respectively, the plasma and hepatic concentrations of acetaminophen in experimental animals and humans after single virtual oral doses. The values of area under the curves of hepatic concentrations of acetaminophen estimated using PBPK models were correlated with the measured levels of cysteinyl acetaminophen (a deactivated metabolite) in plasma fractions in these species. Consequently, using simple PBPK models and plasma data to predict hepatic chemical concentrations after oral doses could be helpful as an indicator of

1. In the cynomolgus macaque, UDP-glucuronosyltransferases (UGTs) 1As have similar molecular and enzymatic characteristics to those of their human orthologs. However, genetic polymorphisms in major cynomolgus

1. 2′-, 3′-, and 4′-Methoxyflavones (MeFs) were incubated with nine forms of recombinant human cytochrome P450 (P450 or CYP) enzymes in the presence of an NADPH-generating system and the products formed were analyzed with LC-MS/MS methods. 2. CYP1B1.1 and 1B1.3 were highly active in demethylating 4′MeF to form 4′-hydroxyflavone (rate of 5.0 nmol/min/nmol P450) and further to 3′,4′-dihydroxyflavone (rates of 2.1 and 0.66 nmol/min/nmol P450, respectively). 3′MeF was found to be oxidized by P450s to m/z 239 (M-14) products (presumably 3′-hydroxyflavone) and then to 3′,4′-dihydroxyflavone. P450s also catalyzed oxidation of 2′MeF to m/z 239 (M-14) and m/z 255 (M-14, M-14 +16) products, presumably mono- and di-hydroxylated products, respectively. 3. At least two types of ring oxidation products having m/z 269 fragments were formed, although at slower rates than the formation of mono- and di-hydroxylated products, on incubation of these MeFs with P450s; one type was products oxidized at the C-ring, having m/z 121 fragments, and the other one was the products oxidized at the A-ring (having m/z 137 fragments). 4. Molecular docking analysis indicated the preference of interaction of O-methoxy moiety of methoxyflavones in the active site of CYP1A2. 5. These results suggest that 2′-, 3′-, and 4′-methoxyflavones are principally demethylated by human P450s to form mono- and di-hydroxyflavones and that direct oxidation occurs in these MeFs to form mono-hydroxylated products, oxidized at the A- or B-ring of MeF.

Pharmacokinetics and metabolism of recently launched antidyslipidemic drug pemafibrate ((2R)-2-[3-({1,3-benzoxazol-2-yl[3-(4-methoxyphenoxy)propyl]amino}methyl)phenoxy]butanoic acid) was investigated in chimeric mice with humanized liver in the present study.The plasma unbound fractions of [

1. Desloratadine is an antiallergic drug with species-dependent metabolic profiles in mice, rats, monkeys and humans. We investigated whether humanized-liver mice could reproduce the reported human-specific

1. Flavin-containing monooxygenase 3 (FMO3) in humans is polymorphic in several ethnic groups, including Caucasians, Africans and Asians. Some

The evaluation of drug-mediated cytochrome P450 (P450) induction using human hepatocytes is important for predicting drug interactions. In this study, we prepared hepatocytes from chimeric mice with humanised livers (Hu-Liver mice) and evaluated the expression and inducibility of P450s in these hepatocytes. Up to 95% of the Hu-Liver cells stained positive for human leukocyte antigen and the mean viability exceeded 85% (n = 10). Monolayer-cultured Hu-Liver cells displayed a similar morphology to cultures of the corresponding human hepatocytes used as transplantation donors. The mRNA expression levels in Hu-Liver cells of 16 P450 forms belonging to P450 subfamilies 1-4 correlated well with the expression levels of the same enzymes in human hepatocytes. The variations in individual P450 mRNA levels between Hu-Liver cells and the corresponding human hepatocytes were within five-fold for 13 P450 forms. The production of 6β-hydroxytestosterone in Hu-Liver cells was significantly increased (p .05) following treatment with the CYP3A inducer, rifampicin. Hu-Liver cells have characteristics similar to those of human hepatocytes in terms of mRNA expression levels and the inducibility of the various P450 forms. Thus, Hu-Liver cells can potentially be used for in vitro drug-mediated induction assays of human hepatic P450s.

Albumin has reportedly enhanced cytochrome P450 (P450)-mediated drug oxidation rates in human liver microsomes. Consequently, measurements of clearances and fractions metabolized could vary depending on the experimental albumin concentrations used. In this study, the oxidation rates of diclofenac and warfarin by human liver microsomes were significantly enhanced in the presence of 0.10% (w/v) bovine serum albumin, whereas those of tolbutamide and phenytoin required 1.0% and 2.0% of albumin for significant enhancement. Values of the fractions metabolized by P450 2C9 for four substrates did not markedly change in the presence of albumin at the above-mentioned concentrations. The oxidation rates of bupropion, omeprazole, chlorzoxazone and phenacetin in human liver microsomes were reportedly enhanced by 0.5%, 1%, 2% and 2% of albumin, respectively. Analysis of reported intrinsic clearance values and suitable albumin concentrations for the currently analyzed substrates and the reported substrates revealed an inverse correlation, with warfarin as an outlier. Suitable albumin concentrations were multivariately correlated with physicochemical properties, that is, the plasma unbound fractions, octanol-water partition coefficient and acid dissociation constant (r = 0.98, p

1. A potentially useful animal model for preclinical studies is the common marmoset (Callithrix jacchus). In this study, using reverse-transcription polymerase chain reaction from marmoset livers, we identified a novel cytochrome P450 (P450) 2F1 cDNA with an open reading frame of 1473 bp. 2. High sequence identities of 92-94% with primate P450 2 F amino acid sequences were indicated by deduced amino acid sequences of P450 2F1 cDNA. Phylogenetic analysis indicates that marmoset P450 2F1 is more congruent with primate P450 2 F forms than those of other species such as rodents. 3. Among five tissue types examined, abundant expression of marmoset P450 2F1 mRNA and P450 2F1 protein in lungs was shown. Cynomolgus monkey P450 2F1 mRNA was abundantly expressed in lungs as well as testes and ovaries in 10 tissue types. 4. Similar to those of humans and cynomolgus monkeys, marmoset P450 2F1 heterologously expressed in Escherichia coli membranes efficiently catalyzed 7-ethoxycoumarin O-deethylation and biphenyl hydroxylation, however unlike human P450 2F1, marmoset P450 2F1 exhibited hydroxylation activity toward coumarin and chlorzoxazone. 5. These findings indicated that P450 2F1 enzyme expressed in marmoset lungs and also catalyzed metabolism of xenobiotics, suggesting the importance of P450 2 F-dependent drug metabolism in marmoset lungs.

1. The pharmacokinetics were investigated for human cytochrome P450 probes after single intravenous and oral administrations of 0.20 and 1.0 mg/kg, respectively, of caffeine, warfarin, omeprazole, metoprolol and midazolam to aged (10-14 years old, n = 4) or rifampicin-treated/young (3 years old, n = 3) male common marmosets all genotyped as heterozygous for a cytochrome P450 2C19 variant. 2. Slopes of the plasma concentration-time curves after intravenous administration of warfarin and midazolam were slightly, but significantly (two-way analysis of variance), decreased in aged marmosets compared with young marmosets. The mean hepatic clearances determined by in silico fitting for individual pharmacokinetic models of warfarin and midazolam in the aged group were, respectively, 23% and 56% smaller than those for the young group. 3. Significantly enhanced plasma clearances of caffeine, warfarin, omeprazole and midazolam were evident in young marmosets pretreated with rifampicin (25 mg/kg daily for 4 days). Two- to three-fold increases in hepatic intrinsic clearance values were observed in the individual pharmacokinetic models. 4. The in vivo dispositions of multiple simultaneously administered drugs in old, young and P450-enzyme-induced marmosets were elucidated. The results suggest that common marmosets could be experimental models for aged, induced or polymorphic P450 enzymes in P450-dependent drug metabolism studies.

1. Roles of human cytochrome P450 (P450) 3A4 in oxidation of an antihistaminic drug terfenadine have been previously investigated in association with terfenadine-ketoconazole interaction. Several antihistamine drugs have been recently identified as substrates for multiple P450 enzymes. In this study, overall roles of P450 3A4, 2J2, and 4F12 enzymes in terfenadine t-butyl hydroxylation were investigated in small intestines and livers from humans, marmosets, and/or cynomolgus monkeys. 2. Human liver microsomes and liver and small intestine microsomes from marmosets and cynomolgus monkeys effectively mediated terfenadine t-butyl hydroxylation. Ketoconazole and N-hydroxy-N'-(4-butyl-2-methylphenyl)-formamidine (a P450 4A/F inhibitor) almost completely and moderately inhibited these activities, respectively, in human liver microsomes; however, these chemicals did not show substantially suppression in marmoset liver. Anti-human P450 3A and 4F antibodies showed the roughly supportive inhibitory effects. 3. Recombinant P450 3A4/90 and 4F12 showed high terfenadine t-butyl hydroxylation activities with substrate inhibition constants of 84-144 μM (under 26-76 μM of Km values), in similar manners to liver and intestine microsomes. 4. These results suggest that human and marmoset P450 3A4/90 and 4F12 in livers or small intestines played important roles in terfenadine t-butyl hydroxylation. Marmosets could be a model for humans during first pass extraction of terfenadine and related substrates.

1. A cDNA encoding novel cytochrome P450 (P450) 4A enzyme was cloned from marmoset livers by reverse transcription (RT)-polymerase chain reaction (PCR) based on the marmoset genome sequences. The amino acid sequence deduced from P450 4A11 cDNA contained consensus sequences of six substrate recognition sites and one heme-binding domain. 2. Marmoset P450 4A11, highly identical (85-88%) to cynomolgus monkey and human P450 4A enzymes, was grouped into the same cluster as cynomolgus monkey and human P450 4A enzymes by phylogenetic analysis. 3. The tissue distribution analyses by real-time RT PCR and immunoblotting demonstrated that marmoset P450 4A11 mRNA and proteins were expressed in kidneys and livers. Marmoset P450 4A11 enzyme heterologously expressed in Escherichia coli preferentially catalyzed the ω-hydroxylation of arachidonic acid and lauric acid, similar to cynomolgus monkey and human P450 4A11 enzymes. However, lauric acid ω-hydroxylation activity of marmoset P450 4A11 was low compared with those of marmoset liver microsomes. 4. These results indicated that novel marmoset P450 4A11 was also a fatty acid ω-hydroxylase expressed in kidneys and livers, with the same regioselectivity (at ω-position of fatty acid) as cynomolgus monkey and human P450 4A enzymes.

1. The partial glucokinase activator N,N-dimethyl-5-((2-methyl-6-((5-methylpyrazin-2-yl)carbamoyl)benzofuran-4-yl)oxy)pyrimidine-2-carboxamide (PF-04937319) is biotransformed in humans to N-methyl-5-((2-methyl-6-((5-methylpyrazin-2-yl)carbamoyl)benzofuran-4-yl)oxy)pyrimidine-2-carboxamide (M1), accounting for ∼65% of total exposure at steady state. 2. As the disproportionately abundant nature of M1 could not be reliably predicted from in vitro metabolism studies, we evaluated a chimeric mouse model with humanized liver on TK-NOG background for its ability to retrospectively predict human disposition of PF-04937319. Since livers of chimeric mice were enlarged by hyperplasia and contained remnant mouse hepatocytes, hepatic intrinsic clearances normalized for liver weight, metabolite formation and liver to plasma concentration ratios were plotted against the replacement index by human hepatocytes and extrapolated to those in the virtual chimeric mouse with 100% humanized liver. 3. Semi-physiological pharmacokinetic analyses using the above parameters revealed that simulated concentration curves of PF-04937319 and M1 were approximately superimposed with the observed clinical data in humans. 4. Finally, qualitative profiling of circulating metabolites in humanized chimeric mice dosed with PF-04937319 or M1 also revealed the presence of a carbinolamide metabolite, identified in the clinical study as a human-specific metabolite. The case study demonstrates that humanized chimeric mice may be potentially useful in preclinical discovery towards studying disproportionate or human-specific metabolism of drug candidates.

The pharmacokinetics of cytochrome P450 probes in humans can be extrapolated from corresponding data in cynomolgus monkeys using simplified physiologically based pharmacokinetic (PBPK) modeling. In the current study, despite some species difference in drug clearances, this modeling methodology was adapted to estimate human plasma concentrations of P450 probes based on data from commonly used medium-sized experimental animals, namely dogs and minipigs. Using known species allometric scaling factors and in vitro metabolic clearance data, the observed plasma concentrations of slowly eliminated caffeine and warfarin and rapidly eliminated omeprazole, metoprolol and midazolam in two young dogs were scaled to human oral monitoring equivalents. Using the same approach, the previously reported pharmacokinetics of the five P450 probes in minipigs was also scaled to human monitoring equivalents. The human plasma concentration profiles of the five P450 probes estimated by the simplified human PBPK models based on observed/reported pharmacokinetics in dogs/minipigs were consistent with previously published pharmacokinetic data in humans. These results suggest that dogs and minipigs, in addition to monkeys, could be suitable models for humans during research into new drugs, especially when used in combination with simple PBPK models.

1. 3-N-Demethylation of caffeine (1,3,7-trimethylxanthine) is mediated by human cytochrome P450 1A2, whereas 7-N-demethylation and C-8-hydroxylation are reportedly catalyzed by monkey P450 2C9 and rat P450 1A2, respectively. 2. Roles of marmoset P450 enzymes in caffeine oxidation were investigated using nine marmoset liver microsomes and 14 recombinantly expressed marmoset P450 enzymes. 3. Predominant caffeine 7-N-demethylation and C-8-hydroxylation activities in marmoset liver microsomes were moderately (r = 0.78, p

1. Pharmacokinetics of human cytochrome P450 probes (caffeine, racemic warfarin, omeprazole, metoprolol and midazolam) were investigated after single intravenous and oral administrations at doses of 0.20 and 1.0 mg kg(-1), respectively, in combination to three young (3-year-old) and three aged (16-year-old) cynomolgus monkeys. 2. The plasma concentrations of caffeine and R-/S-warfarin decreased slowly in a monophasic manner, but those of omeprazole, metoprolol and midazolam decreased rapidly, in a similar manner to those as reported for pharmacokinetics in humans. 3. The mean maximum concentrations of R- and S-warfarin (4.6 and 3.7 µg/mL, respectively) in aged monkeys after oral administration were significantly higher than those in young monkeys (3.3 and 2.7 µg/mL). The mean clearance (CL) values of midazolam in aged monkeys (9.5 mL/min/kg) were significantly lower than those in young monkeys (13 mL/min/kg). 4. Individual intrinsic CL values for omeprazole (r = 0.29) and metoprolol (r = 0.30) of individual monkey livers were inversely correlated with their ages significantly (p 0.05) in liver microsomes prepared from 55 cynomolgus monkeys. 5. These results suggest that cynomolgus monkeys could be a good model for humans, especially with particular characteristics in reduced CLs of some human P450 substrates by aging.

1. An increasing number of studies have indicated the roles of CYP4 proteins in drug metabolism; however, CYP4 expression has not been measured in cynomolgus monkeys, an important animal species for drug metabolism studies. 2. In this study, cynomolgus CYP4A11, CYP4F2/3, CYP4F11 and CYP4F12, along with CYP2J2, were immunoquantified using selective antibodies in 28 livers and 35 small intestines, and their content was compared with CYP1A, CYP2A, CYP2B6, CYP2C9/19, CYP2D, CYP2E1, CYP3A4 and CYP3A5, previously quantified. 3. In livers, CYP2J2, CYP4A11, CYP4F2/3, CYP4F11 and CYP4F12, varied 1.3- to 4.3-fold, represented 11.2, 14.4, 8.0, 2.7 and 0.3% of total immunoquantified CYP1-4 proteins, respectively. 4. In small intestines, CYP2J2, CYP4F2/3, CYP4F11 and CYP4F12, varied 2.4- to 9.7-fold, represented 6.9, 36.4, 2.4 and 9.3% of total immunoquantified CYP1-4 proteins, respectively, making CYP4F the most abundant P450 subfamily in small intestines. CYP4A11 was under the detection limit in all of the samples analyzed. 5. Significant correlations were found in liver for CYP4A11 with lauric acid 11-/12-hydroxylation and for CYP4F2/3 and CYP4F11 with astemizole hydroxylation. 6. This study revealed the relatively abundant contents of cynomolgus CYP2J2, CYP4A11 and CYP4Fs in liver and/or small intestine, suggesting their potential roles for the metabolism of xenobitotics and endogenous substrates.

1. The pharmacokinetics of acetaminophen (marker of gastric emptying), antipyrine (marker of hepatic metabolic activity and total body water), diazepam (lipophilic and highly distributed), diphenhydramine (hepatic blood flow-limited and alpha-1 acid glycoprotein bound) and ofloxacin (renally eliminated) were evaluated in cynomolgus monkeys (3-18 years old) and beagle dogs (2-11 years old) as models in elderly persons. 2. Gastric pH fluctuated with aging in monkeys and dogs. The concentration of alpha-1 acid glycoprotein appeared to be increased by aging. There were no age-related differences in the absorption rates of the drugs under the conditions used in the study. Total body fat increased and water decreased in monkeys, but these parameters did not change in dogs. 3. Hepatic blood flow decreased in both species, but a significant decrease of hepatic clearance was only seen in monkeys. Renal clearance decreased significantly with age in monkeys and showed a tendency to decrease in dogs. 4. Age-related alterations of physiological parameters in monkeys are in agreement with clinical observations in humans, except for the lack of a change in the plasma albumin concentration. Therefore, this study suggests that monkey might be a suitable animal model for prediction of age-related changes in pharmacokinetics in humans.

1. The purpose of this study was to investigate the inhibitory effects of diclofenac on human cytochrome P450 1A2-, 2C19- and 3A4-mediated drug oxidations and to evaluate the drug interaction potential of diclofenac and 4'-hydroxydiclofenac. 2. Diclofenac was converted to 4'-hydroxydiclofenac by recombinantly expressed human P450 1A2 with Km and Vmax values of 33 µM and 0.20 min(-1), respectively. Diclofenac and 4'-hydroxydiclofenac suppressed flurbiprofen 4'-hydroxylation by P450 2C9 strongly and moderately, respectively; however, they did not affect P450 2C19-dependent S-mephenytoin hydroxylation or P450 3A4-dependent midazolam hydroxylation. 3. Although the caffeine 3-N-demethylation activity of liver microsomal P450 1A2 was inhibited by simultaneous incubation with diclofenac, the riluzole N-hydroxylation activities of recombinant P450 1A2 and human liver microsomes were inhibited after preincubation with diclofenac or 4'-hydroxydiclofenac for 20 min in the presence of NADPH. Using the inhibition constant (37 µM) of diclofenac on caffeine 3-N-demethylation and the reported 95th percentiles of maximum plasma concentration (10.5 µM) after an oral dose of diclofenac, the in vivo estimated increase in area under the plasma concentration-time curve was 29%. 4. These results suggest that diclofenac could inhibit drug clearance to a clinically important degree that depends on P450 1A2. Clinically relevant drug interactions in vivo with diclofenac are likely to be invoked via human P450 1A2 function in addition to those caused by the effect of diclofenac on P450 2C9.

Caffeine (1,3,7-trimethylxanthine) is a phenotyping substrate for human cytochrome P450 1A2. 3-N-Demethylation of caffeine is the main human metabolic pathway, whereas monkeys extensively mediate the 7-N-demethylation of caffeine to form pharmacological active theophylline. Roles of monkey P450 enzymes in theophylline formation from caffeine were investigated using individual monkey liver microsomes and 14 recombinantly expressed monkey P450 enzymes, and the results were compared with those for human P450 enzymes. Caffeine 7-N-demethylation activity in microsomes from 20 monkey livers was not strongly inhibited by α-naphthoflavone, quinidine or ketoconazole, and was roughly correlated with diclofenac 4'-hydroxylation activities. Monkey P450 2C9 had the highest activity for caffeine 7-N-demethylation. Kinetic analysis revealed that monkey P450 2C9 had a high Vmax/Km value for caffeine 7-N-demethylation, comparable to low Km value for monkey liver microsomes. Caffeine could dock favorably with monkey P450 2C9 modeled for 7-N-demethylation and with human P450 1A2 for 3-N-demethylation. The primary metabolite theophylline was oxidized to 8-hydroxytheophylline in similar ways by liver microsomes and by recombinant P450s in both humans and monkeys. These results collectively suggest a high activity for monkey liver P450 2C9 toward caffeine 7-N-demethylation, whereas, in humans, P450 1A2-mediated caffeine 3-N-demethylation is dominant.

1. The distribution of an anti-cancer agent carboplatin to brains was investigated in combination with hyperbaric oxygenation treatment in rats. 2. After intravenous administration of carboplatin (30 mg kg(-1)) to male Wistar rats, elimination curves of plasma drug concentrations plotted against a time of 45 min were not different with or without hyperbaric oxygenation (at 0.20-0.25 MPa for last 20 min) treatments. 3. Carboplatin concentrations of livers, lungs and kidneys in each group were similar at the endpoint of hyperbaric oxygenation treatment. 4. Under these atmosphere conditions (at 0.10 MPa), carboplatin concentration was at an undetectable level in rat brains (

The aim of this study was to investigate the inter-individual variations in cytochrome P4502J2 (CYP2J2) and its typical drug oxidation activities in human liver microsomes in both Japanese and Caucasian populations. CYP2J2 contents were determined immunochemically in liver microsomes from 20 Japanese and 29 Caucasian samples using recombinant CYP2J2 commercially available as a standard. Ebastine hydroxylation and astemizole O-demethylation activities were compared. The CYP2J2 genotype was determined by direct sequencing of liver genomic DNA. The mean expression levels of CYP2J2 determined immunochemically in liver microsomes from Japanese and Caucasian samples were 2.0 +/- 1.5 and 1.2 +/- 2.1 pmol CYP2J2 mg-1 protein (mean +/- standard deviation), respectively, accounting for 1.8 +/- 1.1% and 0.52 +/- 0.65% of the total hepatic P450 content (0.15 +/- 0.19 and 0.27 +/- 0.14 nmol P450 mg-1 protein, respectively). The individual variation of the two marker drug oxidation activities could not be fully accounted for by the CYP2J2 contents or currently known CYP2J2 genotypes. The amounts of CYP2J2 in liver microsomes with the CYP2J2*7 allele (-76GT) were decreased to 39% compared with those of liver microsomes from other individuals. The results indicate that CYP2J2 accounts for approximately 1-2% of total P450 in human liver microsomes. The information about large inter-individual variation of the CYP2J2 suggests that this enzyme plays a significant role in the metabolism of xenobiotics and may be useful in in-silico simulations of drug disposition.

Potential differences in drug clearance between Japanese and Caucasians were investigated by integrating data on demography, liver size, the abundance of the major cytochromes P450 and in vitro metabolic parameters. Eleven drugs (alprazolam, caffeine, chlorzoxazone, cyclosporine, midazolam, omeprazole, sildenafil, tolbutamide, triazolam, S-warfarin and zolpidem) fulfilled the entry criteria of the study (i.e. the necessary in vitro metabolism data were available and clearance values had been reported both in Caucasians and Japanese). Values of relevant biological variables were obtained from the literature, and clearance predictions were made using the Simcyp Population-Based ADME Simulator. The ratios of observed oral clearance (CLp.o.) values in Caucasians compared with Japanese ranged from 0.6 to 2.8 (integrating data from 82 sources). The CLp.o. values for alprazolam, caffeine and zolpidem were not statistically different between Caucasian and Japanese (p>0.05), whereas those for chorzoxazone, cyclosporine, omeprazole, tolbutamide and triazolam were higher in Caucasians (p

Using a newly developed real-time reverse transcriptase-polymerase chain reaction method, mRNAs were quantitated for CYP3A4, CYP3A5 and CYP3A7 in adult livers from 24 Japanese and 24 Caucasian subjects to elucidate the potential ethnic differences in the expression levels of human cytochrome P450 (CYP) 3As. The expression level of CYP3A4 mRNA in Japanese livers (n = 24) was approximately three times higher than that in Caucasian livers (n = 24, p < 0.001). The mean level of CYP3A5 mRNA was approximately twice higher in Japanese (n = 9) than in Caucasians (n = 5) heterozygous for the CYP3A5 *1 allele (p = 0.057). The CYP3A7 mRNA level was twice higher in Japanese (n = 24) than in Caucasians (n = 22) carrying the CYP3A7 *1A/ *1A genotype (p = 0.042). The level of CYP3A4 mRNA did not correlate with those of CYP3A5 (r = 0.044, n = 24) or CYP3A7 (r = 0.21, n = 24) mRNAs in Japanese livers in contrast to co-regulatory expression of CYP3A4, CYP3A5 and CYP3A7 in Caucasian livers. The results indicate that there are ethnic differences in the expression levels of adult liver CYP3A mRNAs between Japanese and Caucasians, and that the mechanism(s) regulating the hepatic CYP3A expression may be different between these ethnic groups.

1. Cytochrome P450 (P450, CYP) enzymes involved in drug oxidations in mouse intestines were characterized for their role in the first-pass metabolism of xenobiotics. 2. Preparation of mouse intestinal microsomes using a buffer containing glycerol and protease inhibitors including (p-amidinophenyl) methanesulphonyl fluoride, EDTA, soybean trypsin inhibitor, aprotinin, bestatin and leupeptine gave the highest testosterone 6beta-hydroxylase activity among several preparation buffers tested in this study. Testosterone 6beta-hydroxylase activity catalysed by mouse intestinal microsomes subjected to freezing and thawing was lower than that catalysed by unfrozen intestinal microsomes. 3. Low but significant catalytic activities of nifedipine oxidation, midazolam 1'- and 4-hydroxylation, chlorzoxazone 6-hydroxylation, bufuralol 1'- and 6-hydroxylations and tolbutamide methylhydroxylation were observed in mouse intestinal microsomes. Testosterone 6beta-hydroxylation, chlorzoxazone 6-hydroxylation, and bufuralol 1'- and 6-hydroxylations were inhibited by ketoconazole, diethyldithiocarbamate and quinine respectively. 4. Immunoblot analysis using anti-rat CYP3A antibodies demonstrated two immunoreactive bands showing similar migration in mouse intestinal and hepatic microsomes, although studies using anti-CYP1A, anti-CYP2C, anti-CYP2D and anti-CYP2E1 antibodies did not detect any band in mouse intestinal microsomes. 5. The results suggest that mouse intestinal microsomes should be prepared with glycerol and several protease inhibitors and that Cyp3a enzymes probably play an important role in drug oxidations catalysed by mouse intestine.

1. Two different types of genetic polymorphisms in each of CYP2C18 and CYP2C19 genes were examined and compared with respect to their frequencies in distribution in liver DNA of 39 Japanese and 45 Caucasians. 2. Individuals who were classified into CYP2C19m1 (as detected with SmaI digestion) in exon 5 of CYP2C19 gene were found to display a CYP2C18m1 polymorphism (as detected with DdeI digestion) in the 5'-flanking region of CYP2C18 gene in Japanese and Caucasian populations. The Japanese subjects who were classified into CYP2C19m2 (as detected with BamHI digestion) in exon 4 of CYP2C19 gene were found to have a CYP2C18m2 genetic polymorphism (as detected with Tsp509I digestion) in exon 2 of CYP2C18 gene. None of the Caucasians had the CYP2C18m2 nor CYP2C19m2 alleles. 3. Frequencies in two types (C416T in exon 3, A1061C in exon 7) of CYP2C9 genetic polymorphism were found to be independent to those of CYP2C18 and CYP2C19 genetic polymorphisms in these samples. 4. Thus, the results suggest that the CYP2C18 gene is localized very closely to the CYP2C19 gene on the same human chromosome.

1. The effects of treatment of rat with roxithromycin, erythromycin and troleandomycin as well as other chemicals including typical cytochrome P450 inducers were examined in rat and human liver microsomes. 2. Erythromycin and troleandomycin but not roxithromycin caused slight increases in CYP3A1 levels and the N-demethylation of roxithromycin, erythromycin and troleandomycin and oxidation of nifedipine in rat, but none of these chemicals induced significantly CYP2B1 levels or benzphetamine N-demethylation activities. 3. Dexamethasone and pregnenolone 16 alpha-carbonitrile induced CYP3A1 levels and N-demethylation of roxithromycin, erythromycin and troleandomycin but not of benzphetamine, in rat liver microsomes. Treatment of rat with phenobarbital caused increases in both CYP2B1 and 3A1 levels and all of the N-demethylation activities examined. Phenytoin and metyrapone produced increases in contents of 2B1 and activities of benzphetamine N-demethylation as well as of roxithromycin, erythromycin and troleandomycin, although these two inducers did not induce 3A1 protein significantly. 4. In man, a liver sample that was high in CYP3A4 and nifedipine oxidation activity was found to be the most active in N-demethylation activities towards these substrates examined. In addition, recombinant 3A4 catalysed very efficiently the N-demethylation of roxithromycin, erythromycin and troleandomycin in reconstituted monooxygenase systems. 5. These data suggest that erythromycin and troleandomycin, but not roxithromycin, were able to induce CYP3A1 in rat liver microsomes, and that N-demethylation of roxithromycin, erythromycin and troleandomycin were catalysed mainly by 3A1 (and partly by 2B1) in rat and by 3A4 in man.