Influence of Five Herbal Medicines on Cytochrome P450 3A4 Drug-Metabolizing Enzyme Activity

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1 29 4 ( ) J Korean Oriental Med 2008;29(4): Original Article 고재언 1 황진우 2 고호연 3 최유경 1 박종형 1 고성규 4 전찬용 1 1 경원대학교한의과대학내과학교실 2 국립의료원한방진료부한방내과 3 세명대학교한의과대학내과학교실 4 경희대학교한의과대학예방의학교실 Influence of Five Herbal Medicines on Cytochrome P450 3A4 Drug-Metabolizing Enzyme Activity Jae-Eon Go 1 Jin-Woo Hwang 2 Ho-Yeon Go 3 You-Kyung Choi 1 Jong-Hyung Park 1 Seong-Gyu Ko 4 Chan-Yong Jun 1 1 Department of internal Medicine College of Oriental Medicine Kyungwon University 2 Department of Oriental internal Medicine National Medical Center 3 Department of internal Medicine College of Oriental Medicine Se-Myung University 4 Department of Preventive Medicine College of Oriental Medicine Kyunghee University Objectives: The aim of this study was to investigate the influence of five herbal medicines on cytochrome P450 (CYP) 3A4 drug-metabolizing enzymes in human liver microsomes Methods: By using of human liver microsomes we extracted Cnidium officinale Makino Rhus verniciflua Stokes Prunus persica Batsch Corydalis remota Fisch Carthamus tinctorius Linne which are called Hwalhyulgeoouhyak( ) Then they were incubated and measured for relative enzyme activity under incubation conditions compared to ketoconazole which is known as a representative inhibitor of Results: We showed that all of five traditional herbal medicines had no inhibition effect of at and 50 / doses in human liver microsomes although Rhus verniciflua Stokes (RVS) showed a little inhibition as about 95% enzyme activity of control However this result was not enough to prove that RVS has a inhibition effect Moreover we can t confirm that those rates have significant induction effect on Conclusions: The result of this study could support that those herbal medicines are more reliable than chemical drugs even if this is a basic step to prove that result Key Words : Cytochrome P450 3A4 Hwalhyulgeoouhyak ketoconazole Herb-drug interaction 서론 1) ) 2) ( alcohol 접수 :2008년 6월 25일 수정 :2008년 8월 11일 채택 :2008년 8월 22일 교신저자 : 전찬용 (Chan-Yong Jun) 서울시송파구송파동 20-8 경원대학교서울한방병원 Tel : Fax : joncy@kyungwonackr 104

2 6 : Cytochrome P450 3A4 (687) Cytochrome P450 (CYP450) steroid hormones fatty acids prostaglandins ( ) (oxidative metabolism) (heme) CYP 450 CYP 4) 50% St John s wort inducer St John s wort 3) 5) 6) 11) (Corydalis remota Fisch) < > 10) < > 10) 11) (Carthamus tinctorius Linne) < > 10) Ketoconazole 3A4 inhibitor Macrolide amlodipine Ketoconazole 11) CYP CYP Liquid- Chromatography / Mass Spectrometry 9) 8) 실험 (Cnidium officinale Makino) < > 10) 11) (Rhus verniciflua Stokes) < > 10) (Prunus persica Batsch) 11) 1 재료 1) (Cnidium officinale Makino) (Rhus verniciflua Stokes) (Prunus persica Batsch) (Corydalis remota Fisch) (Carthamus tinctorius Linne) 5 (Kyungbuk Korea) 100 g 1 L 80% sonication Filter 2 (Whatman Maidstone England) 105

3 (688) 29 4 ( ) Table 1 No Scientific name Korean name Part of using yield 1 Cnidium officinale Makino 천궁 川芎 Roots 1838% 2 Rhus verniciflua Stokes 건칠 乾漆 Bark of tree 630% 3 Prunus persica Batsch 도인 桃仁 Seed and Branch 785% 4 Corydalis remota Fisch 현호색 玄胡索 Roots 472% 5 Carthamus tinctorius Linne 홍화 紅花 Flowers 2407% (Eyela Tokyo Japan) (Freezedryer Matsushita Japan) 1838g 63g 785g 472g 2407g ( : 1838% 63% 785% 472% 2407%) 40 / in DMSO stock stock DMSO ( : / ) (Table 1) 2) Human Liver Microsome Microsome ( : BD Biosciences) Vial / 250 mm sucrose -80 Ultra low freezer (Sanyo Japan) 3) chemical (Dimethyl sulfoxide (DMSO) 1 M Ketoconazole in DMSO : index inhibitor 10 mm midazolam in DMSO : CYP 3A4 substrate 16 um Terfenadine : Internal standard 1 M Kpi buffer (ph 74) 01 M Glucose-6-phosphate (G-6-P) 10 mm NADP + 1 unit/ Glucose-6-phosphate dehydrogenase (G-6-P dehydrogenase) Sigma 01% Acetic Acid 100% Methanol (HPLC grade) 100% Acetonitrile (HPLC grade) 01% Formic Acid JC Baker 3 (KIST) Solid Phase Extraction Oasis SPE Kit (Waters Korea ) CYP protocol glass tubes 15 microtubes LC/MS-MS Auto-sampling inserts vials KIST 2 방법 1) Cytochrome P450 cassette assay [75 ( ) + 6 ( Control + Ketoconazole ) + 19( ) ] Kpi-buffer mM Ketoconazole 10mM midazolam voltexing Glass tube Labeling premixture ( M Kpi buffer mM midazolam 100 ) pre-mixture Glass tube 159 Control 3 glass tube 1 DMSO Negative Control 3 glass tube 1 1mM Ketoconazole tube 1 106

4 6 : Cytochrome P450 3A4 (689) Ultra low freezer Human Liver Microsome glass tube 10 voltexing 5 37 water bath pre-incubation ice bucket ice on ice NADPH Generating System (NGS) 01 M G-6-P 10mM NADP + G-6-P dehydrogenase 2 : 1 : voltexing G-6-P dehydrogenase ice bucket mixture G-6-P dehydrogenase 01M G-6-P 10mM NADP + enzyme activity 5 pre-incubation 30 NGS voltexing 30 waterbath incubation enzyme activity stop solution 01% Acetic acid LC/MS-MS peak Internal Standard 016uM Methanol Terfenadine Terfenadine final concentration 016uM 400 Acetic Acid 100 [75 ( = + 6 (Control + Ketoconazole ) + 19 (extra)] (016uM Final concentration / 16uM stock) = 400 IS (terfenadine in MeOH) voltexing 30 incubation NGS stop solution Solid Phase Extraction (SPE) N Evaporator MeOH 01% Formic Acid 100% Acetonitrile 85 : 15 solution 15 microtube 50 5 voltexing LC/MS-MS insert Autosampling vial LC/MS-MS API 2000 (Agilent USA) sample metabolite 2) Solid Phase Extraction Stop solution enzyme activity glass tube SPE 96 well plate filtration MeOH activation SPE 96 well plate SPE cassette waste tray (total 81 ) well 1 MeOH sealing tape pad MeOH filter activation 01% Acetic Acid 1 2 Activation well sample sample filter filter 01% Acetic Acid 1 washing 1 HPLC grade (purity 99%) MeOH well sealing elution waste tray 96 well sample collection plate sample pipetting 15 microtube N Evaporator Evaporation 3) Human Liver Microsome Kpi buffer 3 glass tube DMSO 1 Control inhibitor 1mM Ketoconazole 1 glass tube Negative Control 40 / stock DMSO / DMSO glass tube 1 107

5 (690) 29 4 ( ) NADPH generating system 1 M G-6-P 10mM NADP G-6-P dehydrogenase LC/MS-MS DMSO control 100% 1mM Ketoconazole / control 100% Fig 1 Midazolam I'-hydroxymidazolam 4) Cytochrome P450 enzyme inhibition inhibition 50% 50% Cytochrome P450 Cytochrome P450 cassette enzyme assay 3A4 inhibitor Ketoconazole inhibition 90% Ketoconazole Assay Cytochorme P450 Mean ± Standard Deviation 3 Descriptive Analysis window SPSS/PC version 110 결과 1 효소반응으로인한 Midazolam 의 변환 Midazolam LC/MS Cytochrome P450 3A4 substrate enzyme activity midazolam 1-OH-midazolam metabolite 13) (Fig1) 2 다섯가지활혈거어약의 활성도측 정결과 1) (Cnidium officinale Makino) Control DMSO enzyme activity 100 % negative control Ketoconazole 622% enzyme activity (Cnidium officinale Makino) / enzyme activity ± 069% ± 047% ± 148% ± 049 Enzyme Activity (%) Fig Cnidium officinale Makino Con Ket Concentration(ug/ml) 108

6 6 : Cytochrome P450 3A4 (691) ± 126% Fig / negative control Ketoconazole enzyme activity 622% Ketoconazole inhibition control DMSO enzyme activity (Fig2) 2) (Rhus verniciflua Stokes) Enzyme Activity (%) Prunus persica Batsch Con Ket Concentration(ug/ml) Control DMSO enzyme activity 100% negative control Ketoconazole 622% enzyme activity (Rhus verniciflua Stokes) / enzyme activity ± 488% 9161 ± 920% 9685 ± 951% 9510 ± ± 183% Fig / control DMSO inhibition negative control Ketoconazole inhibition / control DMSO (Fig3) 3) (Prunus persica Batsch) Control DMSO enzyme activity 100% Fig 4 negative control Ketoconazole 622% enzyme activity (Prunus persica Batsch) / enzyme activity ± 103% ± 148% ± 071% ± 064% ± 116% Fig / negative control Ketoconazole enzyme activity 622% Ketoconazole inhibition control DMSO enzyme activity (Fig4) Enzyme Activity (%) Fig Rhus verniciflua Stokes Con Ket Concentration(ug/ml) 4) (Corydalis remota Fisch) Control DMSO enzyme activity 100 % negative control Ketoconazole 622% enzyme activity (Corydalis remota Fisch) / enzyme activity ± 177% ± 247% ± 132% ± 004% ± 145% Fig / negative control Ketoconazole enzyme activity 622% Ketoconazole inhibition control DMSO enzyme 109

7 (692) 29 4 ( ) Enzyme Activity (%) Corydalis remota Fisch Con Ket Concentration(ug/ml) / negative control Ketoconazole enzyme activity 622% Ketoconazole inhibition control DMSO enzyme activity (Fig6) 고찰 Fig 5 activity (Fig5) 5) (Carthamus tinctorius Linne) Control DMSO enzyme activity 100 % negative control Ketoconazole 622% enzyme activity (Carthamus tinctorius Linne) / enzyme activity ± 063% ± 064% ± 224% ± 067% ± 044% Fig6 Enzyme Activity (%) Fig Carthamus tinctorius Linne Con Ket Concentration(ug/ml) CYP Klingenberg Garfinke microsome NADH dithionide (hydrosulfide) 450nm 1962 Omura Sato cytochrome heme CYP450 13) (endoplasmic reticulum) CYPs MDR(Multi Drug Resistance) isoform CYP450 15) Liver Microsome 14) isoform Human 110

8 6 : Cytochrome P450 3A4 (693) (Cnidium officinale Makino) ( ; Umbelliferae) 11) 16) 17) (Rhus verniciflua Stokes) ( ; Anacardiaceae) 11) 18) 19) 25) 26) 27) Human Liver Microsome / CYP450 activity LC/MS-MS 11) (Prunus persica Batsch) ( ; Rosaceae) / / 20) 11) 21) 22) (Corydalis remota Fisch) ( ; Papaveraceae) 11) 23) 24) CYP450 (Carthamus tinctorius Linne) ( ; Compositae) 50% (induction) Kinetic Study RNA RNA 111

9 (694) 29 4 ( ) CYP450 CYP450 CYP450 in vitro in vivo 결론 / Ketoconazole 50% Ketoconazole DMSO (n=3) 참고문헌 1 Guengerich FP Cytochrome P450s and other enzymes in drug metabolism and toxicity AAPS J 2006;8(1): : ; 2006 p Guengerich FP Cytochromes P450 drugs and diseases Mol Interv 2003;3(4): Guengerich FP Characterization of human cytochrome P450 enzymes FASEB J 1992;6(2): Iwata H Tezuka Y Usia T Kadota S Hiratsuka A Watabe T Inhibition of human liver microsomal and CYP 2D6 by extracts from 78 herbal medicines Journal of Traditional Medicines 2004;21(1): Pal D Mitra AK MDR- and -mediated drug-herbal interactions Life Sci 2006;78(18): Zhou S Gao Y Jiang W Huang M Xu A Paxton JW Interactions of herbs with cytochrome P450 Drug Metab Rev 2003;35(1): Jiang X Blair EY McLachlan AJ Investigation of the effects of herbal medicines on warfarin response in healthy subjects: a population pharmacokinetic-pharmacodynamic modeling approach J Clin Pharmacol 2006 ;46(11): Lee SK Jun IH Yoo HH Kim JH Seo YM Kang MJ Lee SH Jeong TC Kim DH Characterization of in vitro metabolites of deoxypodophyllotoxin in human and rat liver microsomes using liquid chromatography/tandem mass spectrometry Rapid Commun Mass Spectrom 2008;22(1): : ; 2005 p : ; 2000 p Emoto C Iwasaki K Relative roles of CYP2C19 and /5 in midazolam 1 -hydroxylation Xenobiotica 2007;37(6): Pharmacy Newsletter of Drug Information 2004;24(9) 14 Flockhart DA Desta Z Mahal SK Selection of drugs to treat gastro-oesophageal reflux disease: the role of drug interactions Clin Pharmacokinet 2000;39(4): Martignoni M Groothuis GM de Kanter R Species differences between mouse rat dog monkey and human CYP-mediated drug metabolism inhibition and induction Expert Opin 112

10 6 : Cytochrome P450 3A4 (695) Drug Metab Toxicol 2006;2(6): ;18(4): ( ) ( ) ( ) MCAO ( ) ( ) ( ) ( ) 2005;22(3): ( ) 2006;19(3): ( ) 2006;20(3): ( ) 2007;21(1): ( ) 2004;7(3): ;13(2): ;15(3): ;19(1): ; 17(1): ;28(1): ( ) ( ) ( ) ( ) ( ) ( ) 2006;9 (3):