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Abstract: Matrine and oxymatrine are the major bioactive compounds extracted from the root of Sophora flavescens Ait, which have been widely used in ...
Basic & Clinical Pharmacology & Toxicology, 107, 906–913

Doi: 10.1111/j.1742-7843.2010.00596.x

Effects of Matrine and Oxymatrine on Catalytic Activity of Cytochrome P450s in Rats Fang Yuan1*, Jie Chen2*, Wen-jin Wu3, Su-zhen Chen1, Xue-ding Wang1, Zeng Su4 and Min Huang1 1

Institute of Clinical Pharmacology, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, China, 2Department of Pharmacy, First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China, 3Institution of Chinese Medical Science, University of Macau, Macao, China, and 4College of Pharmaceutical Sciences, Zhejiang University (Received 4 December 2009; Accepted 18 March 2010)

Abstract: Matrine and oxymatrine are the major bioactive compounds extracted from the root of Sophora flavescens Ait, which have been widely used in traditional Chinese medicines. The objective of the study was to investigate the effects of matrine or oxymatrine on hepatic cytochrome P450 (CYP450) and the underlying molecular mechanisms. Matrine (15, 75 and 150 mg ⁄ kg) or oxymatrine (36, 180 and 360 mg ⁄ kg) was administered to rats for 14 days and the activities of CYP450 were measured by the quantification of the metabolites from multiple CYP450 probe substrates, using validated liquid chromatography coupled with liquid chromatography-tandem mass spectrometry detection (LC-MS ⁄ MS) and high-performance liquid chromatography methods. The mRNA and protein expression levels of CYPs were determined by quantitative real-time reverse-transcription polymerase chain reaction and Western blotting analysis respectively. Interactions between matrine or oxymatrine and human constitutive androstane (CAR), pregnane X receptor were evaluated by means of the reporter gene assay in CV-1 cells. Our study showed that matrine and oxymatrine significantly induced the activity and gene expression of CYP2B1 in a dose-dependent manner; matrine (150 mg ⁄ kg) slightly induced the mRNA and protein expression of CYP2E1 and mildly inhibited the mRNA and protein expression of CYP3A1 in rats. Matrine or oxymatrine could activate human CAR and induce the CYP2B reporter construct in CV-1 cells. These results reveal that matrine and oxymatrine can induce the activity and expression of CYP2B1 ⁄ 2 in rats, and the underlying mechanism may be related to the activation of CAR.

In recent years, there has been a globally increasing application of herbal medicines in the treatment of various diseases and the promotion of health. Many herb–drug interactions lead to pharmacokinetic interactions mediated by drugmetabolizing enzymes or transporters [1]; therefore, in order to avoid unexpected drug-herb interactions in a clinical situation, it is essential to investigate the potential effects of herbs on cytochrome P450 (CYP450) and to obtain more valid evidence. The root of Sophora flavescens Ait (Leguminosae) has been used as an antipyretic, diuretic and anthelmintic in traditional Chinese medicines [2,3]. Matrine and oxymatrine (structure shown in fig. 1) are the major pharmacologically active quinolizidine alkaloids in the root of S. flavescens [4]. Oxymatrine was transformed into matrine by human intestinal bacteria metabolism in vitro [5]. After Intraperitoneal injection (i.p.) of 40 mg ⁄ kg oxymatrine, the 0–24 hr urine samples of the rats were analysed by liquid chromatographytandem mass spectrometry (LC-MS ⁄ MS), the structures of six phase I metabolites were identified and the main metabolite was confirmed to be matrine in the rat urine [6]. Matrine was effective in anti-tumour [7] and antipyretic activity [3]. Oxymatrine exhibited anti-inflammatory effect against

Author for correspondence: Min Huang, Institute of Clinical Pharmacology, School of Pharmaceutical Sciences, Sun Yat-sen University, 74 Zhongshan Road II, Guangzhou 510080, China (fax +86 20 87331782, e-mail [email protected]). *These authors contributed equally to this work.

ischaemic and reperfusion injury in brain and liver [8]. Several independent studies demonstrated that both matrine and oxymatrine exhibited antivirus activity against hepatitis B and hepatitis C viruses (HCV) [9–11]. Oxymatrine was effective in inhibiting proliferation of HCV and antagonizing liver fibrosis in patients [12]. A clinical study indicated that matrine could effectively reduce serum levels of aspartate aminotransferase (ALT), glutamic-oxal(o)acetic transaminase (AST) and c-glutamyl transpeptidase in patients with chronic hepatitis B [13]. At present, matrine and oxymatrine are the most commonly prescribed drugs for anti-hepatitis virus in China. CYP450 is an important superfamily of hemoproteins responsible for the monooxygenation of various xenobiotics, including therapeutic drugs, environmental pollutants, carcinogens, as well as many endogenous substrates such as steroids, prostaglandins, arachidonic acids and leukotrienes [14]. Rat CYP1A2, 2C7, 2C11, 2D2, 2E1, 2B1 ⁄ 2 and 3A1 ⁄ 2 are the major CYPs for drug metabolism [15]. Almost all human and rat CYPs are subject to induction and inhibition by a variety of xenobiotics and endogenous compounds. Many herb–drug interactions result from the inhibition or induction of CYP450 [16]. Despite their widespread application, there are few reports on the drug interactions between matrine or oxymatrine and other medications. The purpose of the current study was to investigate the effects of matrine and oxymatrine on major CYPs in rats and the underlying molecular mechanisms.

 2010 The Authors Basic & Clinical Pharmacology & Toxicology  2010 Nordic Pharmacological Society

MATRINE AND OXYMATRINE AND RAT CYPS

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Materials and Methods Materials. Matrine, oxymatrine, dexamethasone, phenobarbital and rifampin were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Bovine serum albumin, 6-(4-chlorophenyl)-imidazo[2,1-b] [1,3]thiazole5-carbaldehyde-O-(3,4-dichlorobenzyl)-oxime (CITCO) and b-naphthoflavone (b-NF) were purchased from Sigma-Aldrich (St Louis, MO, USA). Bicinchoninic acid assay kits were purchased from Beyotime Biotechnology (Shanghai, China). NADPH was obtained from Roche Diagnostics Ltd. (Beijing, China). Trizol reagent was from Invitrogen (Carlsbad, CA, USA). M-MLV and Taq enzymes were obtained from Promega (Madison, WI, USA) and Takara Medical Co., Ltd. (Kyoto, Japan) respectively. Oligonucleotide primers were synthesized by Sangon Co. (Shanghai, China). Antibodies of rabbit anti-rat CYP3A1, CYP2B1 ⁄ 2 and CYP2E1, horseradish peroxidaselabelled anti-goat IgG and anti-rabbit IgG were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Antibodies of mouse anti-GADPH Mouse mAb were obtained from Calbiochem (San Diego, CA, USA). Methanol and formic acid of HPLC grade were purchased from Tedia Inc. (Beijing, China). All other reagents were of analytical grade or HPLC grade. Plasmids. Promoter reporter gene plasmid of CYP2B-1.6k ⁄ PB ⁄ XREM has been described previously [17]. CYP2B-1.6k ⁄ PB ⁄ XREM was kindly provided by Dr Hongbing Wang (Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, USA), pcDhCAR2 [18] by Dr Oliver Burk (Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart and University of Tuebingen, Germany), ptk-(3A4)3-Lu [19] and [Gal-hPXR ligand-binding domain (LBD)] [20] by Dr Xie Wen (Center for Pharmacogenetics and Department of Pharmaceutical Sciences, University of Pittsburgh, USA) and Prof. Zhu Xinqiang (Institute of Medical Nutrition and Food Hygiene, Zhe Jiang University, China). Animals. Healthy male Sprague–Dawley rats (weighing 240–290 g) were obtained from the Experimental Animal Center of Sun Yat-sen University, Guangzhou, China. Rats housed in cages were kept in a room under controlled temperature (23–24C) and 12-hr light : dark cycle. The rats had free access to tap water and regular diet. All procedures were approved by the Animal Ethical Committees of the Sun Yat-sen University, in accordance with the National Institute of Health and Nutrition Guidelines for the Care and Use of Laboratory Animals. Both matrine and oxymatrine were dissolved in water. The control group received the same volume of water. Preparation of rat hepatic microsomes. In a separate experiment, rats were divided into control vehicle, matrine (15, 75 and 150 mg ⁄ kg), oxymatrine (36, 180 and 360 mg ⁄ kg) and positive control group respectively. Dexamethasone was used as the positive control for CYP3A1 and CYP2D2, phenobarbital for CYP2B1 and 2C7, b-naphthoflavone for CYP1A2 and isoniazid for CYP2E1. (Rats were given intragastric matrine or oxymatrine for 14 days. Dexamethasone at 100 mg ⁄ kg ⁄ day and b-naphthoflavone at

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80 mg ⁄ kg ⁄ day for 3 days, i.p., phenobarbital at 80 mg ⁄ g ⁄ day and isoniazid at 5 mg ⁄ kg ⁄ day for 4 days, i.p.) On day 15, the rats were killed and livers were collected, weighed and transferred to ice immediately after the surgical excision, cut into pieces, and the microsomes were prepared as described previously [21] and stored at )80C. The microsomal protein concentration in the supernatant was determined with the bicinchoninic acid assay according to Liu et al. (Beyotime Biotechnology) [22]. Measurements of CYP activity for probe substrates. Phenacetin (1A2), tolbutamide (2C7), dextromethorphan (2D2), chlorzoxazone (2E1), resorufin (2B1 ⁄ 2) and nifedipine (3A1) were selected as the CYP isoform probe substrates for the study [23]. The metabolites of CYP1A2, CYP2C7, CYP2D2, CYP2E1 and CYP3A1 probe substrates from all incubations were determined by our previously reported LC ⁄ MS ⁄ MS method [24]. A typical incubation system (500 ll) including 0.1 M potassium phosphate buffer (pH 7.4), 10 mM MgCl2, 0.25 mg ⁄ ml microsomal protein, and the five probe substrates was applied. The mixture was pre-incubated in a 37C water bath with gentle shaking for 5 min. The reaction was initiated by the addition of 1 mM NADPH and terminated with the addition of 2 ml ice-chilled ethyl acetate after 20-min. incubation. The CYP2B1 ⁄ 2 activities were determined by the method described by Lubet et al. [25] with slight modification. Briefly, 450 ll of the substrate 0.1mg ⁄ ml pentoxyresorufin in 0.1 M potassium phosphate buffer (pH 7.4, 10 mM MgCl2 and 0.25 mg ⁄ ml microsomal protein) was pre-incubated for 5 min. at 37C. The reaction was started by the addition of 50 ll of 1 mM NADPH, and incubated for 20 min. at 37C, then terminated by adding 2 ml of cold methanol. The mixture was vigorously mixed, and centrifuged at 10,000 · g for 10 min. Resorufin formed by the reaction reflects the CYP2B activity. The supernatant was injected into HPLC for detecting resorufin (Waters, Milford, MA, USA). The HPLC conditions were as follows: hypersil BDS C18 column [5 lm, 4.6mm·150 mm (Dalian Elite Scientific Instruments, Dalian, China)]; mobile phase, 20 mM phosphate buffer (pH 4.66)-methanol-acetonitrile (52:45:3, v ⁄ v); flow rate, 1.0 ml ⁄ min; column temperature, 4C; fluorescence detection, excitation at 560 nm and emission at 585 nm. Real-time quantitative PCR. The rats were killed under anaesthesia and the livers immediately excised. A small portion of the liver sample was snap-frozen in liquid nitrogen and stored at –80C before extraction of total RNA. Approximately 100–200 mg of liver sample was homogenized in 8 ml TRIzol reagent to extract total RNA. The total RNA was quantified by the absorbance at 260 nm. The purity of RNA was assessed by the absorbance ratio (260 ⁄ 280 nm). For reverse transcription, the typical reaction mixture (final volume 20 ll) was prepared with final concentrations as follows: 4 units M-MLV reverse transcriptase, 1· RT buffer, 10 U RNase inhibitor, 2.5 lM random hexamer primers, 0.5 mM each of dATP, dGTP, dCTP and dTTP and 2 lg of total RNA extract. The RT reaction was performed for 60 min. at 42C and then 5 min. at 93C to stop the reaction. Primers for CYP2B1, CYP3A1, CYP2E1 and 18s RNA were 5¢-GGG ATG GGA AAG AGG AGT-3¢ (S), 5¢-ATG GAG CAG ATG ATG TTG G-3¢ (A); 5¢-CTG CAT TGG CAT GAG GTT TGC TCT-3¢ (S), 5¢-TCA TCC CGT GGC ACA ACC TTT AGA-3¢ (A); 5¢-AAC TGA GAC CAC CAG CAC AAC TCT3¢ (S), 5¢-ACC ACA GCA TCC ATG TAG GGC ATA-3¢ (A); and 5¢-TGC TGC TCA GTC AGT TCA TC-3¢ (S), 5¢-TCA CGC ACT CCT CCA TTT-3¢ (A) respectively. The primers were designed using Primer Express Version 2.0 and entered into the NCBI Blast to ensure specificity as the Rattus norvegicus CYP450, family 2, locus. Quantitative real-time PCR assays for rat CYP mRNA levels were performed using an iQTM5Multicolor Real-time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA). The mRNA expression of CYP2B1, CYP3A1 and CYP2E1 was normalized against that of rat 18S rRNA, which was detected using a pre-developed primer mixture following the instruction manual of SYBR Premix Ex TaqTM (TaKaRa). The folds of induction of CYP2B1 ⁄ 2, CYP2E1 and CYP3A1 were calculated using the following equation:

 2010 The Authors Basic & Clinical Pharmacology & Toxicology  2010 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 107, 906–913

FANG YUAN ET AL.

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Fold of induction ¼ 2DD where

DDCt ¼ DCt (treated)  DCt (control)

Table 1. Effects of matrine and oxymatrine on CYP2B1 ⁄ 2 activity in rats ( x ± S.D., n = 6).

Group (n = 6)

Liver weight ⁄ body weight (%)

Control Phenobarbital Matrine 15 mg ⁄ kg Matrine 75 mg ⁄ kg Matrine 150 mg ⁄ kg Oxymatrine 36 mg ⁄ kg Oxymatrine 180 mg ⁄ kg Oxymatrine 360 mg ⁄ kg

3.2 5.8 3.4 4.3 3.3 3.6 3.5 4.4

DCt (treated) ¼ DCt (CYP)  DCt (18S) for the compound treatment group. DCt (control) ¼ DCt (CYP)  DCt (18S) for the vehicle control group.

Western blotting analysis. The levels of CYP3A1, 2E1, 2B1 ⁄ 2 and GADPH in rat liver microsomes were determined by Western blotting. Thereafter, the microsomal protein (10 lg) for each sample was separated by SDS-polyacrylamide gel electrophoresis, and SDS-polyacrylamide gel was electrophoretically transferred to nitrocellulose membranes using a Mini-Protean II system (Bio-Rad). The membrane was blocked with 10% milk and incubated overnight at 4C with a 1:2000 or 1:3000 dilution of a primary antibody of either rabbit anti-rat CYP3A1, CYP2B1 ⁄ 2 and CYP2E1 or anti-GADPH mouse mAb coupled to secondary antibodies, goat anti-mouse IgG and goat anti-rabbit IgG. The membrane was immersed in the enhanced chemiluminescence solution for 60 sec. The gel images were visualized using Chem-Doc (Bio-Rad), and densitometric analysis was performed with Quantity One 1-D Analysis software (Bio-Rad). Cell culture, transient transfection and reporter gene assay. CV-1 cell lines were obtained from the Shanghai Institutes for Biological Sciences (Shanghai, China). The mechanism of matrine or oxymatrine on the activity of CYP450 was analyzed through transient transfection of CV-1 for the selective activation of pregnane X receptor (PXR), constitutive androstane receptor (CAR). The cells were cultured in Dulbecco’s modified Eagle’s medium supplemented by 10% foetal bovine serum and 100 U ⁄ ml penicillin and 100 lg ⁄ ml streptomycin at 37C, 5% CO2. For the reporter assay, cells were seeded into 24-well plates at 1.5 · 105 cells per well in 500 ll of DMEM without antibiotics and incubated overnight. The cells in each well were transiently transfected with 200 ng of respective luciferase reporter construct CYP2B1.6 kb ⁄ PB ⁄ XREM or ptk-(3A4)3-Luc, 5 ng internal control plasmid (PhRL-CMV) and 10 ng of the respective nuclear receptor expression plasmid hCAR or (Gal-hPXR LBD) or empty expression vector pcDNA3 (Invitrogen) using lipofectamineTM 2000 reagent according to the manufacturer’s procedures. Six hours after transfection, the cells were washed with phosphate-buffered saline to remove LiptofectamineTM 2000 complexes and then supplied with fresh medium (without phenol red and supplemented with foetal calf serum) and treated with solvent (0.1% DMSO) or test compounds at the concentration of rifampicin (20 lM) or CITCO (50 lM), matrine and oxymatrine (100, 300 lM) for 24 hr. The MTT assay showed that the drugs used above did not affect the cell viability at the concentrations of co-culture with transfected CV-1 cells. Subsequently, luciferase activities were measured with cell lysates using the Dual Luciferase Reporter reagents according to the manufacturer’s instruction (Promega). Statistics. Data are expressed as mean € S.D. One-way analysis of variance (ANOVA) was used for statistical comparisons. Differences between the two groups were analysed using unpaired Student’s t-test. Statistical significance was set as p < 0.05.

Results Effects of matrine and oxymatrine pre-treatment on rat CYP450 activities. During treatment of rats with matrine and oxymatrine for 14 days, compared to the control group, CYP2B1 ⁄ 2 activi-

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0.5 1.2** 0.1 0.3 0.1 0.3 0.1 0.3

CYP2B1 activity (pmol ⁄ mg ⁄ min) 4.3 43.9 15.0 17.5 28.4 4.9 8.1 13.9

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1.0 17.1** 1.8** 3.7** 8.2** 0.9 3.4* 3.6**

*p < 0.05, **p < 0.01 compared with the control group.

ties were significantly increased, i.e. 9.3-fold by phenobarbital, 2.5-, 3.1- and 5.7-fold by 15, 75 and 150 mg ⁄ kg matrine and 89%, 2.3-fold by 180 and 360 mg ⁄ kg oxymatrine respectively (table 1, fig. 2). Rat CYP2E1 activities were increased 4.5-fold by isoniazid and 21% by 150 mg ⁄ kg matrine, compared to the control group. Matrine at a higher dose (150 mg ⁄ kg), but not 17 or 75 mg ⁄ kg, caused a slight increase in rat CYP2E1 activity and a decreased CYP3A1 activity, i.e. only 15%. Rat CYP1A2, CYP2D2 and CYP2C7 activities were not significantly altered by matrine or oxymatrine (fig. 2). Treatment with 15, 75 and 150 mg ⁄ kg matrine or 36, 180 and 360 mg ⁄ kg oxymatrine for 14 days had no significant effect on rat body weight and liver weight (table 1). Effects of matrine and oxymatrine on CYP mRNA expression in SD rats. To investigate whether CYP3A1, CYP2B1 ⁄ 2 and CYP2E1 mRNA levels were regulated by matrine or oxymatrine, realtime PCR was performed. The liver microsomes of rats pretreated with different dosages of matrine or oxymatrine were used to detect the expression of mRNA. Matrine pre-treatment at 15, 75 and 150 mg ⁄ kg increased the mRNA expression of CYP2B1 ⁄ 2 by 74%, 4.3- and 5.5-fold respectively. The mRNA expression of CYP2B1 ⁄ 2 increased 59% and 4.2-fold by oxymatrine at 180 and 360 mg ⁄ kg. The mRNA expression of CYP2E1 was increased by 39% and CYP3A1 decreased by 20% when rats were treated with matrine 150 mg ⁄ kg respectively. The effects of matrine and oxymatrine on CYP2B1 ⁄ 2, 3A1 and 2E1 were consistent with the effect of matrine and oxymatrine on rat CYP450 activities respectively (fig. 3). Effects of matrine and oxymatrine on CYP protein expression in SD rats. As the expression of CYP2B1 ⁄ 2 mRNA was induced by matrine or oxymatrine, the effects of matrine and oxymatrine on the expression of protein were further investigated in rat liver microsomes. Western blotting analysis showed significantly increased expression of CYP2B1 ⁄ 2 in rats treated with matrine or oxymatrine. The expression of the CYP2B1 ⁄ 2 protein in rats treated with matrine 15, 75 and 150 mg ⁄ kg

 2010 The Authors Basic & Clinical Pharmacology & Toxicology  2010 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 107, 906–913

MATRINE AND OXYMATRINE AND RAT CYPS

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Fig. 2. Effects of matrine and oxymatrine on liver enzyme activity of CYP3A1, 2B1, 2E1, 2C7, 2D2 and 1A2 in rats (x € S, n = 6). I: CYP2B and CYP2C7; II: CYP3A1 and CYP2D2; III: CYP2E1 and CYP1A2; Positive controls: Dexamethasone for CYP3A1 and CYP2D2; Isoniazid for CYP2E1; b-naphthoflavone for CYP1A2; Phenobarbital for CYP2B1 ⁄ 2 and CYP2C7. *p < 0.05, **p < 0.01 compared with the control group.

increased 2.1-, 5.3- and 6.2-fold, respectively, compared with control rats. Oxymatrine at 180 and 360 mg ⁄ kg increased the expression of the CYP2B1 protein of rats 1.9- and 4.4- fold respectively. The protein expression of CYP2E1 increased by 49% in the matrine 150 mg ⁄ kg group, the protein expression of CYP3A1 decreased at 20% in the matrine 150 mg/kg group respectively. These results were generally consistent with those observed in the real-time PCR assay (fig. 4).

Fig. 3. Effects of matrine and oxymatrine on the mRNA expression of liver CYP2B1 ⁄ 2, 3A1 and 2E1 in rats. CYP2B1 ⁄ 2, 3A1 and 2E2 mRNA levels were measured with reverse transcription real-time PCR and normalized to 18sRNA. Results are presented as the mean from three independent experiments and expressed as fold over the control group. I: CYP2B1 ⁄ 2; II: CYP3A1; III: CYP2E1. Positive controls: Dexamethasone for CYP3A1; Isoniazid for CYP2E1; Phenobarbital for CYP2B1 ⁄ 2. *p < 0.05, **p < 0.01compared with the control group.

Roles of matrine or oxymatrine in PXR- or CAR-mediated transcriptional regulation of CYP3A4 or CYP2B6 expression. We then examined whether matrine or oxymatrine was involved in the transcription regulation of CYP3A4 or CYP2B6 mediated by CAR or PXR. Reporter gene assay was performed. Our results indicated that the CYP2B6 reporter activity was elevated roughly 1.7 fold for 100 lM matrine, 3.1 fold for 300 lM matrine and increased 91% for

 2010 The Authors Basic & Clinical Pharmacology & Toxicology  2010 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 107, 906–913

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Fig. 4. Effects of matrine and oxymatrine on the protein expression of liver CYP2B1 ⁄ 2, 3A1 and 2E1 in rats. CYP2B1 ⁄ 2, 3A1 and 2E1 protein levels were measured with Western blotting and normalized to GADPH. Results are presented as the mean from three independent experiments and expressed as fold over the control group. I: Lanes 1-5 of matrine are the protein expression of control, positive control, matrine (15, 75 and 150 mg ⁄ kg); Lanes 1–5 of oxymatrine are control, positive control, oxymatrine (36, 180 and 360 mg ⁄ kg) respectively. II: relative protein expression of CYP2B1 ⁄ 2 (A), CYP3A1 (B) and CYP2E1 (C) by matrine and oxymatrine. Positive controls: Dexamethasone for CYP3A1; Isoniazid for CYP2E1; Phenobarbital for CYP2B1 ⁄ 2. *p < 0.05, **p < 0.01 compared with the control group.  2010 The Authors Basic & Clinical Pharmacology & Toxicology  2010 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 107, 906–913

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MATRINE AND OXYMATRINE AND RAT CYPS

Fig. 5. Effects of matrine and oxymatrine on CAR-mediated transcriptional regulation of CYP2B6. CV-1 cells were transfected with hCARII, CYP2B-1.6 kb ⁄ PB ⁄ XREM and PhRL-CMV. Six hours later, the cells were treated with 0.1% DMSO (control), 50 lM CITCO (positive control), matrine (100, 300 lM) or oxymatrine (100, 300 lM). Following 24 hr treatment, firefly luciferase activity was determined and normalized against Renilla luciferase activity. Values are expressed as the mean of the fold increase in activity in DMSOtreated cells from three independent experiments. *p < 0.01, compared with the control group. **p < 0.05, compared with the control group.

300 lM oxymatrine, respectively, compared with 0.1% DMSO-treated cells (fig. 5). No significant change in the CYP3A4 reporter activity was observed in the presence of matrine or oxymatrine (fig. 6). These observations demonstrate that matrine and oxymatrine were responsible for the trans-activation of the CYP2B6 reporter construct via CAR. Discussion

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The possibility of herb–drug interactions has received more attention than ever before because of the increasingly widespread application of herbal products in medical treatment [26]. Understanding xenobiotic induction of CYP450 at the transcriptional level plays a significant part in predicting herb–drug interactions and preventing herb-induced hepatotoxicity [1]. Although matrine and oxymatrine are widely

Fig. 6. Effects of MT and OMT on PXR-mediated transcriptional regulation of CYP3A4. CV-1 cells were transfected with Gal-hPXR ligand-binding domain, ptk-(3A4)3-Luc and PhRL-CMV. Six hours later, the cells were treated with 0.1%DMSO (control), 20 lM Rifampicin (positive control) (100, 300 lM) matrine or (100, 300 lM) oxymatrine. Following 24 hr treatment, firefly luciferase activity was determined and normalized against Renilla luciferase activity. Values are expressed as the mean of the fold increase in activity that in DMSO-treated cells from three independent experiments. **p < 0.01, compared with the control group.

911

used in the treatment of viral hepatitis in China [9,10], there are few reports about the effects of matrine and oxymatrine on CYP450s. Our results demonstrated that matrine and oxymatrine could induce CYP2B activity in a dose-dependent manner in rats. In contrast, CYP1A2, CYP2D2 and CYP2C7 activities of rats were not significantly altered by matrine or oxymatrine. At the dose of 150 mg ⁄ kg, matrine induced CYP2E1 and inhibited CYP3A1 slightly. Furthermore, the results of Western blotting and real-time PCR assay suggested that matrine and oxymatrine can induce the expression of mRNA and protein of CYP2B1 ⁄ 2. The mRNA and protein expression of CYP2E1 and CYP3A1 can be induced and inhibited by matrine at 150 mg ⁄ kg respectively. Reporter assays showed that matrine and oxymatrine could activate CAR and induce the CYP2B reporter construct. To our knowledge, this is the first time that the effects of matrine and oxymatrine on CYPs in rats have been investigated systematically. The clinical daily doses of oxymatrine and matrine range from 0.4 to 1.8 g and from 0.2 to 0.8 g respectively. According to the body surface area ratio of rats to humans [27], the dosage conversion coefficient for rats to humans is 6.25. Thus, the dose ranges of oxymatrine and matrine in a 60-kg person are equivalent to 40–180 mg ⁄ kg oxymatrine and 20– 80 mg ⁄ kg matrine, respectively, in rats. Treatment of rats with matrine at 15 mg ⁄ kg [28] or oxymatrine at 40 mg ⁄ kg [29] for pharmacokinetic studies was also reported. Taking these factors into account, the doses of 15, 75, 150 and 180 mg ⁄ kg matrine or 36, 180, 360 and 430 mg ⁄ kg oxymatrine for 14 days were employed in the preliminary tests. Furthermore, treatment of rats with 180 mg ⁄ kg matrine or 430 mg ⁄ kg oxymatrine resulted in an increase in serum AST and ALT activities, which indicated the impairment of liver function (data not shown). Therefore, doses of 15, 75 and 150 mg ⁄ kg matrine or 36, 180 and 360 mg ⁄ kg oxymatrine were employed in the present research. As matrine or oxymatrine is normally used in the treatment of hepatitis for more than 1 month [30] and the duration of CYP450 induction experiments is conventionally 4–14 days [31], the duration of 5, 10 and 14 days was used to investigate the CYP450inducing effects of matrine and oxymatrine in rats in our preliminary tests. The peak induction in the tests happened in 14 days (data not shown); therefore, the duration of 14 days was used as the administration time. Ueng et al. reported that oxymatrine contributed partly to the CYP2B, CYP3A and CYP2A induction by S. flavescens in mice [32]. Our results demonstrated that CYP2B could also be induced by oxymatrine; however, oxymatrine failed to induce CYP3A, which is in contrast to the findings of Ueng et al. The different results between the above reports and our studies can be due to the different drug concentrations and the various sources and purity of oxymatrine and matrine. Furthermore, species difference between mice and rats in response to matrine and oxymatrine may also be a reasonable explanation. Human CYP2B6 is involved in the metabolism of 4% of drugs among the top 200 drugs such as bupropin, efavirenz

 2010 The Authors Basic & Clinical Pharmacology & Toxicology  2010 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 107, 906–913

FANG YUAN ET AL.

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and cyclophosphamide (CPA) and ketamine [33]. CPA exerts its chemotherapeutic effect after biotransformation to 4-hydroxy-CPA by CYP2B6 [34]. The synergism of the antitumour effect was observed in Ehrlich carcinoma-bearing mice with 100 mg ⁄ kg oxymatrine plus CPA because the level of 4-hydroxy-CPA was increased via oxymatrine [35]. Therefore, the induction of CYP 2B1 ⁄ 2 via oxymatrine is attributed to the increase in 4-hydroxy-CPA and the improvement in the chemotherapeutic effect of CPA was expected. The CYP2B1 ⁄ 2 induction by matrine and oxymatrine suggested that possible drug interactions between matrine or oxymatrine and cytochrome substrates should be noticed. Further studies should be undertaken to determine whether matrine and oxymatrine can affect human CYP2B6 isozymes due to a clear species difference between humans and rodents in relation to some drugs. Our results revealed that the activity and expression of CYP2E1 and CYP3A1 were affected slightly after matrine administration at a high dose. Based on the conventional dosage used in the clinic, it is safe to combine matrine or oxymatrine with the drug metabolized by CYP2E1 and CYP3A. Although matrine can slightly induce CYP2E1 and inhibit CYP3A1, the structurally similar compound oxymatrine did not alter the activity of CYP2E1 and CYP3A1. The reasons underlying this phenomenon are unclear. Due to the partial transformation of oxymatrine into matrine, a possible explanation is that matrine is more effective than oxymatrine in altering the activity of CYP450 [6]. PXR and CAR are closely related nuclear receptors that play important roles in regulating the expression of CYP3A or CYP2B [36]. Our results showed that both matrine and oxymatrine were identified as potent activators of human CAR. The cross-regulation in the regulation of CYP2B and CYP3A reveals that xenobiotic-mediated induction of the CYP2B gene is regulated by PXR and CAR [37]. Further research is needed to elucidate the role of PXR in the activation of CYP2B by matrine and oxymatrine. To sum up, our data suggest that matrine and oxymatrine induced the activity and expression of CYP2B1 ⁄ 2, and that the underlying mechanism is related to the activation of CAR. The results displayed that there is a herb–drug interaction potential of matrine and oxymatrine, and that the induction of CYP2B by both matrine and oxymatrine may pose a potential problem in patients. However, further clarification is to be made by detailed pharmacokinetic studies in vivo. Acknowledgements The authors appreciate the financial support provided by the National Major Projects for science and technology development from Science and Technology Ministry of China (grant no. 2009ZX09304-003). References 1 Tomlinson B, Hu M, Lee VW. In vivo assessment of herb–drug interactions: possible utility of a pharmacogenetic approach? Mol Nutr Food Res 2008;52:799–809.

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MATRINE AND OXYMATRINE AND RAT CYPS 23 Etheridge AS, Black SR, Patel PR, So J, Mathews JM. An in vitro evaluation of cytochrome P450 inhibition and P-glycoprotein interaction with goldenseal, Ginkgo biloba, grape seed, milk thistle, and ginseng extracts and their constituents. Planta Med 2007;73:731–41. 24 He F, Bi HC, Xie ZY, Zuo Z, Li JK, Li X et al. Rapid determination of six metabolites from multiple cytochrome P450 probe substrates in human liver microsome by liquid chromatography ⁄ mass spectrometry: application to high-throughput inhibition screening of terpenoids. Rapid Commun Mass Spectrom 2007;21:635–43. 25 Lubet RA, Mayer RT, Cameron JW, Nims RW, Burke MD, Wolff T et al. Dealkylation of pentoxyresorufin: a rapid and sensitive assay for measuring induction of cytochrome(s) P-450 by phenobarbital and other xenobiotics in the rat. Arch Biochem Biophys 1985;238:43–8. 26 Qiu F, Zhang R, Sun J, Jiye A, Hao H, Peng Y et al. Inhibitory effects of seven components of danshen extract on catalytic activity of cytochrome P450 enzyme in human liver microsomes. Drug Metab Dispos 2008;36:1308–14. 27 Spiers DE, Candas V. Relationship of skin surface area to body mass in the immature rat: a reexamination. J Appl Physiol 1984;56:240–3. 28 Wu W, Huang J, Liu S, Li X, Cai Y. Study on pharmacokinetics and tissue distribution of stealth matrine liposomes in rats. Zhongguo Zhong Yao Za Zhi 2009;34:751–5. 29 Wang X, Zhang W, Fan LY, Hao B, Ma AN, Cao CX et al. Sensitive quantitative determination of oxymatrine and matrine in rat

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 2010 The Authors Basic & Clinical Pharmacology & Toxicology  2010 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 107, 906–913