Inhibitory Effects of Phytochemicals on Metabolic Capabilities of CYP2D6*1 and CYP2D6*10 Using Cell-Based Models in Vitro
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Acta Pharmacologica Sinica (2014) 35: 685–696 npg © 2014 CPS and SIMM All rights reserved 1671-4083/14 $32.00 www.nature.com/aps Original Article Inhibitory effects of phytochemicals on metabolic capabilities of CYP2D6*1 and CYP2D6*10 using cell-based models in vitro Qiang QU1, 2, Jian QU1, Lu HAN2, Min ZHAN1, Lan-xiang WU3, Yi-wen ZHANG1, Wei ZHANG1, Hong-hao ZHOU1, * 1Institute of Clinical Pharmacology, Hunan Key Laboratory of Pharmacogenetics, Xiangya Hospital, Central South University, Changsha 410078, China; 2Xiangya Hospital, Central South University, Changsha 410008, China; 3Institute of Life Sciences, Chongqing Medical University, Chongqing 400016, China Aim: Herbal products have been widely used, and the safety of herb-drug interactions has aroused intensive concerns. This study aimed to investigate the effects of phytochemicals on the catalytic activities of human CYP2D6*1 and CYP2D6*10 in vitro. Methods: HepG2 cells were stably transfected with CYP2D6*1 and CYP2D6*10 expression vectors. The metabolic kinetics of the enzymes was studied using HPLC and fluorimetry. Results: HepG2-CYP2D6*1 and HepG2-CYP2D6*10 cell lines were successfully constructed. Among the 63 phytochemicals screened, 6 compounds, including coptisine sulfate, bilobalide, schizandrin B, luteolin, schizandrin A and puerarin, at 100 μmol/L inhibited CYP2D6*1- and CYP2D6*10-mediated O-demethylation of a coumarin compound AMMC by more than 50%. Furthermore, the inhibition by these compounds was dose-dependent. Eadie-Hofstee plots demonstrated that these compounds competitively inhibited CYP2D6*1 and CYP2D6*10. However, their Ki values for CYP2D6*1 and CYP2D6*10 were very close, suggesting that genotype- dependent herb-drug inhibition was similar between the two variants. Conclusion: Six phytochemicals inhibit CYP2D6*1 and CYP2D6*10-mediated catalytic activities in a dose-dependent manner in vitro. Thus herbal products containing these phytochemicals may inhibit the in vivo metabolism of co-administered drugs whose primary route of elimination is CYP2D6. Keywords: CYP2D6; drug interaction; herbal medicine; coptisine; bilobalide; schizandrin; luteolin; puerarin Acta Pharmacologica Sinica (2014) 35: 685–696; doi: 10.1038/aps.2013.202 [5] Introduction and also increased Km . Human cytochrome P450 2D6 (CYP2D6) is an extensively As combination therapy using herbal products with pre- characterized polymorphic drug-metabolizing enzyme that scription medications becomes more widely used in the world, catalyzes the bio-conversion of many xenobiotics. CYP2D6 the safety of herb-drug interactions, especially the inhibition metabolizes more than 25% of drugs, including antidepres- of P450-mediated drug metabolism by herb products, has sants, antipsychotics, beta-receptor antagonists, analgesics, aroused intense concerns[6]. The CYP2D6 catalytic activ- antiarrhythmics, etc[1, 2]. CYP2D6 possesses more than 70 sig- ity is possibly decreased as a result of enzyme inhibition by nificant alleles, leading to various catalytic activities[3]. Com- herbal supplements, including piper nigrum fruit, melaleuca pared to the two alleles of 188C and 4268G in CYP2D6*1 (wild leucadendron leaf[7], goldenseal, valerian[8]，black cohosh[8], type), CYP2D6*10 has two single nucleotide polymorphisms and naturally occurring compounds, such as eupatorin[9], (SNP) 188T (rs1065852, Pro34Ser) and 4268C (rs1135840, Ser- epiberberine[10], berberine, and hydrastine[11]. In recent years, 486Thr) (http://www.cypalleles.ki.se/cyp2d6.htm), and the substantial progress has been made in predicting herb-drug frequencies are 38%–70% in Asians[4]. The decreased clearance interactions, but the presence of polymorphic variants of P450s of drugs in vivo by CYP2D6*10 is caused by low expression complicates the prediction. CYP2D6 genotype-dependent herb-drug interactions have been rarely investigated. HepG2 cells are derived from human hepatoma and show * To whom correspondence should be addressed. E-mail [email protected] many liver-specific functions. Because of the presence of Received 2013-10-03 Accepted 2013-11-11 membrane barriers, transport processes and the NADPH sys- npg www.nature.com/aps Qu Q et al 686 tem, HepG2 cells closely reflect the in vivo situation and are and then inserted into pcDNA3.1(–) (Invitrogen) in the pres- more predictive than microsomes in some aspects[12]. How- ence of T4 DNA ligase. Substitutions (188T and 4268C) were ever, HepG2 cells lack almost all CYP450s. HepG2 cells tran- introduced into the pcDNA3.1-CYP2D6*1 vector using the siently or stably transfected with CYP2D6 expression vectors QuikChange Site-directed Mutagenesis Kit (Stratagene, Shang- have been used to investigate the metabolism and toxicity of hai, China). The mutation primers are listed in Supplementary chemicals, as well as to screen drug candidates[13–15]. The fluo- Table 1. The constructs were sequenced to confirm the intro- rogenic probe 3-[2-(N,N-diethyl-N-methyl-ammonium)ethyl]- duction of these mutations. 7-methoxy-4-methylcoumarin (AMMC) is a useful substrate to examine the CYP2D6 activity in transfected-HepG2 cells and Generation of stable cell lines expressing CYP2D6*1 and rat and human CYP2D6 microsomes[14, 16]. CYP2D6 O-demeth- CYP2D6*10 isoforms ylates AMMC to form AMHC (3-[2-(N,N-diethyl-N-methyl- The HepG2 cells (HB-8065, ATCC) were cultured in modified ammonium)ethyl]-7-hydroxy-4-methylcoumarin), which can Eagle’s medium (MEM, Invitrogen) supplemented with 10% be detected by fluorimetry. Thus, our screening model used fetal calf serum (Abcam Labs, Cambridge, MA, USA) at 37 °C stably transfected HepG2 cells expressing CYP2D6*1 or *10 in a humidified atmosphere (5% CO 2) and transfected with and quantified CYP2D6 activity by analyzing the conversion pcDNA3.1-CYP2D6*1, pcDNA3.1-*10 or pcDNA3.1(–) plas- of AMMC to AMHC by flourometry. mids using 10 μL LipofectamineTM 2000 (Invitrogen). Clones The aims of the present study were to screen phytochemicals were selected for G418 resistance for 12 d and subcultured. to determine whether they inhibit CYP2D6 and to understand the effects of these phytochemicals on the catalytic activities of Quantitative real time PCR and Western blotting the CYP2D6 allelic variants, CYP2D6*1 and *10. The investi- Total RNA was extracted from the stable CYP2D6 express- gation of genotype-enzyme kinetics and genotype-dependent ing cells, and the first-strand cDNA was synthesized. The herb-drug interactions may be important for drug discovery cDNA was amplified by performing quantitative RT-PCR for and development, as well as the optimization of drug dosage 40 cycles using SYBR Green (Bio-rad, Hercules, CA, USA) (for among individuals, thereby guiding individual therapy. primer sequences see Supplementary Table 1). The relative expression of the CYP2D6 mRNA was normalized to β-actin. Materials and methods The analysis was carried out using the 2-ΔΔCt method[17]. Chemicals and reagents The whole cell proteins were extracted using the RIPA pro- AMMC was purchased from BD Gentest (Woburn, MA, USA). tein extraction reagent and separated on an 8% SDS-polyacryl- DMSO, quinidine, bufuralol, ketoconazole, amiodarone, chlor- amide gel by electrophoresis. The proteins were transferred promazine, verapamil, cimetidine, quercetin, omeprazole to a polyvinylidene difluoride membrane (Millipore, Bedford, and lovastatin were purchased from Sigma-Aldrich (St Louis, MA, USA), which was incubated for 1 h at room temperature MO, USA). Sixty-three phytochemicals were obtained from with rabbit polyclonal antibodies (1:400 dilution, Abcam Labs) Sigma-Aldrich or the National Institutes for Food and Drug against the CYP2D6 protein. The membranes were washed Control (Beijing, China). Aminoglycoside antibiotic G418 was twice for 10 min and incubated with Dylight 800-labeled anti- purchased from Invitrogen (Carlsbad, CA, USA). All reagents body (1:7500, KPL lnc, Gaithersburg, MD, USA). The mem- were of high-performance liquid chromatography (HPLC) branes were washed twice and analyzed using the Odyssey grade. Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA). The relative expression of CYP2D6 was normalized to Construction of CYP2D6*1 and CYP2D6*10 expression plasmids β-actin. Total RNA was extracted from a surgical specimen of the human liver at the Xiangya Hospital using TRIzol reagent Evaluation of the metabolic kinetics of CYP2D6*1 and *10 in (Invitrogen), and the first-strand cDNA was synthesized using expression cell models using HPLC and fluorimetry a cDNA Synthesis Kit (Takara, Dalian, China). The CYP2D6 Cells that stably expressed either CYP2D6*1 or *10 were cDNA was amplified using primers according to the NCBI diluted to 1×106 cells/mL and cultured in 96-well plates for Reference Sequence (NM_000106.4) (Supplementary Table 1) 24 h. After being washed twice with PBS, cells were incu- Table 1. Kinetic constants for the CYP2D6*1 and *10-mediated metabolic activity. a c CYP2D6 Detection Km Vmax CLint Substrate CL b isoforms Method (μmol/L) (pmol·min-1·mg-1) int ratio CYP2D6*1 AMMC Fluorimetry 12.45±0.65 42.60±0.74 3.42 d d CYP2D6*10 AMMC Fluorimetry 15.58±1.89 13.67±0.59 0.88 25.7% CYP2D6*1 Bufuralol HPLC 12.58±0.79 123.0±2.57 9.77 d d CYP2D6*10 Bufuralol HPLC 16.56±1.39 22.09±0.68 1.33 13.6% a b c d Km and Vmax results are expressed as mean±SD; CLint was calculated by Vmax/Km; CLint ratio was calculated by CLint CYP2D6*10/*1×100%; P<0.05. Acta Pharmacologica Sinica www.chinaphar.com npg Qu Q et al 687 bated with 15 μmol/L AMMC in 100 μL incubation buffer (1 *10-mediated AMMC-O-demethylation, the stably transfected mmol/L Na2HPO4, 137 mmol/L NaCl, 5 mmol/L KCl, 0.5 cells were incubated with 15 μmol/L AMMC and multiple mmol/L MgCl2, 2 mmol/L CaCl2, 10 mmol/L glucose, and 10 concentrations of the inhibitors, ranging from 0 to 80 μmol/L. mmol/L Hepes, pH 7.4) for 30 to 150 min at 37 °C. Seventy- To determine the mode of inhibition, the stably transfected five microliters of the supernatant was withdrawn per well cells were incubated with various concentrations of AMMC and transferred to a new 96-well plate.