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Objectives: With the growing popularity of herbal and natural medicinal products, attention has turned to possible interactions between these products and pharmaceutical drugs. In this study, we examined whether astragaloside IV (AGS-IV) could inhibit the activity of CYP1A2 in rat liver microsomes in vitro and in vivo., Methods: The effect of AGS-IV on CYP1A2 activity was investigated using probe substrates: phenacetin in vitro and theophylline in vivo. Phenacetin was incubated in rat liver microsomes with or without AGS-IV, and the mechanism, kinetics and type of inhibition were determined. The inhibitory effect of AGS-IV on CYP1A2 activity in rats was also determined using theophylline in vivo. The pharmacokinetics of theophylline were observed after a single or week-long treatment with AGS-IV., Key Findings: AGS-IV was found to be a competitive inhibitor with a K(i) value of 6.29 μM in vitro. In the multiple-pretreatment rat group, it was found to have a significantly higher area under the concentration-time curve (AUC) for theophylline, as well as a lower apparent oral total body clearance value (CL/F). In contrast, no significant difference in metabolism of theophylline was found for the single pretreatment group., Conclusions: These findings suggest that AGS-IV is a potent inhibitor of CYP1A2. This work offers a useful reference for the reasonable and safe use of clinically prescribed herbal or natural products to avoid unnecessary herb-drug interactions., (© 2012 The Authors. JPP © 2012. Royal Pharmaceutical Society.)

Cynomolgus macaques, frequently used in drug metabolism studies, are bred mainly in the countries of Asia; however, comparative studies of drug metabolism between cynomolgus macaques bred in these countries have not been conducted. In this study, hepatic gene expression profiles of cynomolgus macaques bred in Cambodia (mfCAM), China (mfCHN), and Indonesia (mfIDN) were analyzed. Microarray analysis revealed that expression of most hepatic genes, including drug-metabolizing enzyme genes, was not substantially different between mfCAM, mfCHN, and mfIDN; only 1.1% and 3.0% of all the gene probes detected differential expression (>2.5-fold) in mfCAM compared with mfCHN and mfIDN, respectively. Quantitative polymerase chain reaction showed that the expression levels of 14 cytochromes P450 (P450s) important for drug metabolism did not differ (>2.5-fold) in mfCAM, mfCHN, and mfIDN, validating the microarray data. In contrast, expression of CYP2B6 and CYP3A4 differed (>2.5-fold, p < 0.05) between cynomolgus (mfCAM, mfCHN, or mfIDN) and rhesus macaques, indicating greater differences in expression of P450 genes between the two lineages. Moreover, metabolic activities measured using 14 P450 substrates did not differ substantially (<1.5-fold) between mfCAM and mfCHN. These results suggest that gene expression profiles, including drug-metabolizing enzyme genes such as P450 genes, are similar in mfCAM, mfCHN, and mfIDN.

Objective: ACAT2 is a major cholesterol esterification enzyme specifically expressed in hepatocytes and may control the amount of hepatic free (unesterified) cholesterol available for secretion into bile or into HDL. This study aims to further elucidate physiologic roles of ACAT2 in human hepatic cholesterol metabolism., Methods and Results: Liver biopsies from 40 normolipidemic, non-obese gallstone patients including some gallstone-free patients (female/male, 18/22) were collected and analyzed for microsomal ACAT2 activity, protein and mRNA expression. Plasma HDL-cholesterol (HDL-C) was significantly higher in females than in males, while triglycerides were significantly lower. ACAT2 activity in females was significantly lower than observed in males, regardless of the presence of gallstone disease. Moreover, the activity of ACAT2 correlated negatively with plasma levels of HDL-C (r=-0.57, P<0.05) and with Apo AI (r=-0.49, P<0.05)., Conclusion: This is the first description of a gender-related difference in hepatic ACAT2 activity in normolipidemic non-obese Chinese patients suggesting a possible role for ACAT2 in the regulation of cholesterol metabolism in humans. The negative correlation between ACAT2 activity and HDL-C or Apo AI may reflect this regulation. Since ACAT2 activity generally has been found to be pro-atherogenic in animal models, the observed sex-related difference may contribute to female protection from complications of coronary heart disease (CHD).

Aim: To characterize the metabolism of cardamonin by the P450 enzymes in human and animal liver microsomes., Methods: Cardamonin was incubated with both human and animal liver microsomal incubation systems containing P450 reaction factors. High performance liquid chromatography coupled with ion trap mass spectrometry was used to identify the metabolites. Serial cardamonin dilutions were used to perform a kinetic study in human liver microsomes. Selective inhibitors of 7 of the major P450 isozymes were used to inhibit cardamonin hydroxylation to identify the isozymes involved in cardamonin metabolism. The cardamonin hydroxylation metabolic capacities of human and various other animals were investigated using the liver microsomal incubation system., Results: Two metabolites generated by the liver microsome system were detected and identified as hydroxylated cardamonin. The Km and Vmax values for cardamonin hydroxylation were calculated as 32 micromol/L and 35 pmol x min(-1) x mg(-1), respectively. Furafylline and clomethiazole significantly inhibited cardamonin hydroxylation. Guinea pigs showed the highest similarity to humans with respect to the metabolism of cardamonin., Conclusion: CYP 1A2 and 2E1 were identified as the P450 isozymes involved in the metabolism of cardamonin in human liver microsomes. Furthermore, our research suggests that guinea pigs could be used in the advanced pharmacokinetic studies of cardamonin in vivo.

The aim of this study was to determine the effects of the aqueous extract of Salvia miltiorrhiza Bge (danshen in Chinese) on the pharmacokinetics of diazepam and on liver microsomal cytochrome P450 enzyme activity in rats. Rats (n = 5) were pretreated with danshen extract (100 mg kg(-1) per day, p.o.) for 15 consecutive days. Control rats (n = 5) received saline at the same time. Each rat was then administered a single oral dose of 15 mg kg(-1) diazepam. The pharmacokinetic parameters of diazepam were significantly different between the two groups. In the danshen pretreated group, the maximum concentration of diazepam and the area under the plasma concentration-time curve were reduced to about 72.7% and 44.4%, respectively, while the total body clearance was markedly increased by 2-fold. To help explain the results, liver microsomal suspensions were obtained from rats that were randomly divided into the control group (n = 10), and the low- (20 mg kg(-1) for 15 days, p.o., n = 10) and high-dose groups (100 mg kg(-1) for 15 days, p.o., n = 10) pretreated with danshen extract. Compared with the control rats, the microsomal protein content, cytochrome P450 enzyme level and erythromycin N-demethylase activity of pretreated rats were significantly increased. These results indicate that danshen extract can stimulate the activity of cytochrome P450 isoforms, and changes in the pharmacokinetics of diazepam resulting from danshen extract are related to an increase in metabolic activity of cytochrome P450.

Aim: To compare the level and/or activity of several important cytochrome P-450 (CYP) enzymes in liver microsomes prepared from different Chinese subjects., Methods: Individual CYP contents, including CYP1A2, CYP2C9, and CYP3A4, in liver microsomes of 17 Han, 17 Zhuang, and 8 Miao subjects were determined by using Western blot analysis and densitometric scanning. The substrates for measuring the activity of individual CYP in vitro included phenacetin, tolbutamide, debrisoquine, and omeprazole., Results: There was a large interindividual variability in the content and activity of CYP1A2, 2C9 and 3A4. And the activity of CYP2D6 also varied greatly between individual samples. CYP3A4 (32 %) is the most abundant CYP in Chinese liver microsomes, and the levels of CYP2C9 (19 %) and CYP1A2 (16 %) were also considerable. No clear ethnic, sex- and age-related differences in individual CYP content and catalytic activity were detected in 42 Chinese liver samples, except that there were somewhat ethnic and sex-related differences in the content and activity of CYP1A2. Good correlation between enzyme protein content and activity was found for CYP1A2, 2C9 and 3A4., Conclusion: Our results may provide useful information for the study of drug metabolism by liver microsomes in Chinese.

It has been previously reported that omeprazole (OP) oxidation is mediated by CYP2C19 and CYP3A4 in human livers. In this study, we assessed their relative contributions with human liver microsomal fractions from Chinese populations that were genotyped by CYP2C19 and recruited from two ethnic groups, Han and Zhuang. The kinetics of 5-hydroxyomeprazole (5-OH-OP) formation was best described by the two-enzyme and single-enzyme Michaelis-Menten equations for liver microsomes from CYP2C19 extensive (EMs) and poor metabolizers, respectively. At a low substrate concentration that may be encountered in vivo, the monoclonal antibody to CYP2C8/9/19 strongly inhibited 5-OH-OP formation in EM microsomes, whereas troleandomycin (TAO) eliminated most of the formation at a high substrate concentration. In poor metabolizer microsomes, either TAO or anti-CYP3A4 could alone abolish 5-OH-OP formation. Furthermore, there were differences between homozygous and heterozygous EMs in the percentage of inhibition by TAO and the antibodies. At the low substrate concentration, OP 5-hydroxyaltion was correlated well with S-mephenytoin 4'-hydroxylation and CYP2C19 contents in liver microsomes of 34 Chinese individuals. Moreover, in these individuals, obviously genetic and somewhat ethnic differences in OP 5-hydroxylation were observed between different CYP2C19 genotypes (wt/wt > wt/m1 > m1/m1) and between Han and Zhuang (Han > Zhuang), respectively. The results indicate that CYP2C19 is a high-affinity enzyme for OP 5-hydroxylation by liver microsomes from Chinese individuals and that its contribution is CYP2C19 gene dependent and ethnically related. Similar studies indicate that OP sulfoxidation is mediated mainly by CYP3A4 and independent of CYP2C19 genotype status.

We conducted a nested case-control study to examine the association of microsomal epoxide hydrolase polymorphism with spontaneous abortion. The analysis included 127 cases and 107 controls from a rural community in China. The prevalence of the homozygous wild-type (AA), the heterozygous variant (Aa), and the homozygous variant (aa) in exon 3 of epoxide hydrolase was 13.4%, 34.6%, and 52.0% in cases and 27.1%, 30.8%, and 42.1% in controls, respectively. In contrast, the variant genotypes in exon 4 of epoxide hydrolase were less frequent in cases than controls. Using women with genotype AA as the referent, the adjusted odds ratio of spontaneous abortion was 2.69 [95% confidence interval (CI) = 1.33-5.42] for those with genotype Aa or aa in exon 3; but it was 0.45 (95% CI = 0.22-0.94) for those with genotype Aa or aa in exon 4, indicating that the two variants have opposite associations with spontaneous abortion. The findings persisted after adjustment for age, education, parity, smoking, alcohol use, occupation, and pesticide exposure, as well as in subgroup analysis. Moreover, for the variant genotypes Aa or aa in exon 3, the odds ratio was twice as great in those cases with three or more spontaneous abortions than in those with fewer spontaneous abortions.

The objectives of this study were to determine the effects of feeding of four vegetables commonly consumed in Thailand, namely, flowers of the neem tree (Azadirachta indica var. siamensis), fruits of Thai and the Chinese bitter gourd (Momordica charantia Linn.) and leaves of sweet basil (Ocimum basilicum Linn) on the levels of phase I enzymes, which include cytochrome P450 (P450), aniline hydroxylase (ANH) and aminopyrine-N-demethylase (AMD) as well as the capacity to activate the mutagenicities of aflatoxin B1 (AFB1) and benzo[a]pyrene (BaP), and to induce the phase II enzymes [i.e. glutathione S-transferase (GST)] in rat liver. It was found that feeding of the diets containing 12.5% neem flowers and Thai bitter gourd fruits for 2 weeks strongly enhanced GST activity, 2.7- and 1.6- fold of the pair-fed control values, respectively, while resulting in a marked reduction of the levels of most phase I reactions. Fruits of the Chinese bitter gourd, which is in the same species as Thai bitter gourd, had no effect on GST activity but decreased AMD activity and the in vitro metabolic activation of AFB1 and BaP. On the other hand, however, dietary sweet basil leaves caused a significant increase in the levels of both GST and all phase I enzymes. Results in the present study clearly demonstrate that neem flowers and Thai bitter gourd fruits contain monofunctional phase II enzyme inducers and compounds capable of repressing some monooxygenases, especially those involved in the metabolic activation of chemical carcinogens, while sweet basil leaves contain compounds, probably bifunctional inducers, capable of inducing both phase I and phase II enzymes and Chinese bitter gourd fruits contain only compounds capable of repressing some monooxygenases. These results therefore suggest that neem flowers and Thai bitter gourd fruits may possess chemopreventive potential, while those of Chinese bitter gourd fruits and sweet basil leaves are uncertain.

Aims: In vitro data indicate that imipramine (IMI), a widely used tricyclic antidepressant drug, is N-demethylated by several isoforms of cytochrome P450, which include CYP3A4. The aim of this study was to investigate the role of CYP3A in the in vivo N-demethylation of IMI., Methods: Healthy subjects were given troleandomycin (TAO), a selective inhibitor of CYP3A, 250 mg daily for 2 days before a single oral dose of 100 mg IMI was administered., Results: Pretreatment with TAO significantly increased the AUC of IMI by 59% (1971 +/- 938 vs 3134 +/- 2000 microg l(-1) h, 95% confidence interval for difference between means: 218 to 2108 microg l(-1) h, P < 0.05) and decreased its oral clearance by 30% (60.9 +/- 27.4 vs 42.5 +/- 22.7 l h(-1), 95% confidence internal for difference between means: 7.2 to 31.7 l h(-1), P < 0.05)., Conclusions: We conclude that CYP3A may play an important role in the in vivo N-demethylation of IMI.

1. Cytochrome P450 isozymes from Asian (31 Chinese subjects) and Caucasian (14 Finnish subjects) livers were examined for their roles in the metabolism of toluene (rates of benzyl alcohol, o- and p-cresol formation) and styrene (rates of styrene glycol formation). 2. For toluene, the overall rate of metabolism was higher in samples from Finnish than from Chinese subjects. At 0-20 mM toluene, the rate of o-cresol formation was significantly higher in Finnish microsomes than in Chinese ones. The formation rates of benzyl alcohol and p-cresol in Finnish samples were also higher than those of Chinese samples, but only at a high substrate concentration (5.0 mM). For styrene metabolism, the Chinese liver microsomes showed higher metabolic rates than the Finnish ones at 0.085 mM styrene, but not at the higher substrate concentration. 3. Mean expression levels of immunochemically detected CYP1A2/1 and CYP2B6 were almost 3-fold higher in Finnish microsomes, whereas CYP2E1 was 1.7-fold higher in Chinese samples. 4. Correlation analysis showed that CYP2E1 (benzyl alcohol formation) and CYP1A2/1 (o-cresol formation) contributed to the metabolism of toluene at the low substrate concentration, whereas CYP2C8 was the form more actively involved at the higher toluene concentrations. At the higher concentration (1.8 mM) of styrene, CYP2B6 was most active isozyme to catalyse the formation of styrene oxide from styrene. 5. These results suggest that CYP2E1 and CYP1A2/1 are the main isoforms responsible for the metabolism of toluene at low substrate concentrations in human liver microsomes, CYP2E1 at low styrene concentration, and CYP2C8 and CYP2B6 at high concentrations of toluene and styrene respectively.

In order to clarify the expression of cytochrome P450 and glutathione S-transferase in human esophagus, 41 samples of human esophagus with squamous-cell carcinoma were investigated by immunoblot analysis and enzyme assays. Cytochrome P450 1A2/1 was clearly expressed in microsomes, and the amount in samples with tumorous tissue was significantly greater than that in samples without tumourous tissues or in liver; cytochrome P450 2B6 and 3A4/3 were expressed polymorphically. Aryl hydrocarbon hydroxylase activity was detected in microsomes and was greater in samples from smokers than non-smokers. Patients who both smoked and drank alcohol, however, had activity similar to that of patients without these habits. Glutathione S-transferase M1 and A1/2 protein existed polymorphically in cytosol, and glutathione S-transferase P1-1 was detected in all samples. The frequency of expression of the glutathione S-transferase A1/2 protein was greater in patients with M1 protein than in those without; no difference in the expression was seen for glutathione S-transferase P1-1. Neither smoking nor drinking influenced the expression or activity of glutathione S-transferase. Our data support the idea that some carcinogens can be directly activated or inactivated in human esophageal epithelium.

Seven human liver samples were collected in this study. Five of them were obtained from donors died from non-disease accident, one was taken from part of the normal tissue of a liver specimen obtained by partial hepatectomy in a case of hepatic cavernous hemangioma and the last one from postmortem died by road accident. All the liver pieces were stored at -75 degrees C immediately after removed from the body. Liver microsomes was prepared by differential ultracentrifugation. The contents of cytochrome P450 and cytochrome b5 in Chinese adult liver microsomes were 0.36 +/- 0.08 and 0.23 +/- 0.05 nmol.mg-1 protein (n = 7). The activities of aminopyrine and ethylmorphine N-demethylase, 7-ethoxycoumarin-O-deethylase, nifedipine oxidase and (-)-praziquantel hydroxylase were 1.07 +/- 0.23, 1.82 +/- 0.31, 0.30 +/- 0.10, 0.43 +/- 0.18 and 0.69 +/- 0.43 nmol.mg-1.min-1 (n = 7), respectively.