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系統識別號 U0026-2707201011103500
論文名稱(中文) 比較amiodarone 和 dronedarone對於細胞色素3A4在轉錄層次的抑制探討
論文名稱(英文) Comparing the inhibition of CYP3A4 transcription by amiodarone and dronedarone
校院名稱 成功大學
系所名稱(中) 藥理學研究所
系所名稱(英) Department of Pharmacology
學年度 98
學期 2
出版年 99
研究生(中文) 林佳蓁
研究生(英文) Chia-Chen Lin
學號 s2697109
學位類別 碩士
語文別 中文
論文頁數 66頁
口試委員 指導教授-黃金鼎
口試委員-賴明亮
口試委員-周辰熹
中文關鍵字 細胞色素 
英文關鍵字 CYP3A4  PXR  amiodarone  dronedarone 
學科別分類
中文摘要 細胞色素P450為藥物代謝機制中,負責將藥物進行氧化作用的酵素。其中以CYP3A4含量最多,並且參與代謝約百分之五十的臨床用藥,已知Pregnane X receptor (PXR) 為CYP3A4上游的調控基因。PXR會受到外來藥物或內生性物質的活化,並與共同活化子 steroid receptor coactivator-1(SRC-1)以及hepatocyte nuclear factor 4α (HNF4α) 間產生交互作用,進而去調控參與在藥物或內生性物質代謝機制中的酵素以及運輸器,可能造成臨床上藥物交互作用的原因之一。
已知抗黴菌藥物ketoconazole 不僅抑制CYP3A4的酵素活性,並會藉由破壞 PXR和SRC-1或HNF4α 的交互作用,降低CYP3A4在mRNA 以及蛋白質的表現量。所以我們預測有其他的藥物也會藉由抑制PXR和其他因子的交互作用而降低CYP3A4的表現量。利用HepG2細胞,以acid phosphatase (ACP) assay確保細胞維持80%以上存活率的藥物濃度進行實驗。接著以CYP3A4 reporter assay 篩選廣為人知的CYP3A4 inhibitor,從中發現第三類抗心律不整藥: amiodarone會抑制CYP3A4 reporter activity,並且抑制CYP3A4的mRNA 和蛋白質表現量,但不影響CYP3A4酵素活性。接著用同樣的實驗方法,針對不含碘的amiodarone類似藥物dronedarone 進行研究,發現dronedarone和amiodarone 一樣會抑制CYP3A4 reporter activity和 CYP3A4的mRNA 以及蛋白質表現量。同樣的,dronedarone 對CYP3A4的酵素活性沒有抑制。接著利用mammalian two-hybrid assay了解amiodarone和dronedarone並非透過破壞PXR和SRC-1的交互作用達到對CYP3A4的抑制,但是隨著amiodarone濃度增加破壞PXR和HNF4α的交互作用的程度增加。
研究結果顯示,amiodarone和dronedarone 會藉由影響PXR對CYP3A4的
調控而抑制CYP3A4轉錄轉譯程度,可以解釋不會抑制CYP3A4酵素活性的amiodarone卻有許多藥物交互作用的情形。而dronedarone為新核准的抗心律不整用藥,臨床用藥濃約為amiodarone以及本實驗濃度(5μM)的1/10 (0.1-0.5μM),所以dronedarone可能帶來的藥物交互作用遠小於amiodarone,這個結果可以用來作為臨床給藥的一項參考。另外,我們也認為PXR的調控能力透過藥物的抑制,會影響代謝酵素或是運輸器的表現,進而引起藥物間的交互作用。
英文摘要 Pregnane X receptor (PXR) is one of the important factors in drug-drug interaction. PXR functions a xenobiotic sensor which recruits transcription coactivator such as steroid receptor coactivator-1 (SRC-1) and interacts with hepatocyte nuclear factor 4α (HNF-4α) upon ligand binding and then induces gene expression. PXR regulates drug metabolic enzymes or transporters such as CYP3A4 or P-glycoprotein. In the previous study, ketoconazole, a clinically used antifungal compound, not only inhibits CYP3A4 through enzymatic level, but also through the transcription of gene regulated by disrupting PXR interaction with SRC-1 and HNF-4α. We predicted other CYP3A4 inhibitors may disrupt interaction between PXR and other factors resulting in drug-drug interaction because of lower CYP3A4 expression. We established CYP3A4 reporter assay in HepG2 cell line to screen popular inhibitor of CYP3A4. We found amiodarone, an antiarrhythmic agent, decreased CYP3A4 reporter activity, mRNA and protein expression in a dose dependent manner. In addition, the same approaches were used to characterize dronedarone, a benzofuran derivate of amiodarone. CYP3A4 reporter activity, mRNA and protein expression of CYP3A4 were inhibited by dronedarone. The mammalian two-hybrid assay showed the mechanism of CYP3A4 inhibition by amiodarone or dronedarone is not mediated by the disruption of PXR and SRC-1 interaction. We proved that amiodarone disrupted the interaction between PXR and HNF-4α. The CYP3A4 enzyme activity wasn’t inhibited by amiodarone or dronedarone. To compare the inhibitory effect of amiodarone or dronedarone and understanding the rifampin-induced PXR-mediated mechanism of CYP3A4 was suppressed by amiodarone or dronedarone may improve therapeutic drug used and then prevent adverse effect. Furthermore, realizing the inhibition of PXR regulation by drug may prevent that other metabolic enzyme or transporter involve in drug-drug interaction.
論文目次 中文摘要 I
Abstract III
Acknowledgement V
Contents VI
Table index VIII
Figure index IX
Abbreviations X
Chapter 1 Introduction 1
I . Foreword 1
II . Cytochrome P450 (CYP450 or CYPs) 1
III . Cytochrome 3A4 (CYP3A4) 2
IV . Pregnane X receptor (PXR) 4
V . Steroid receptor coactivator-1 (SRC-1) 6
VI . Hepatocyte nuclear factor 4α (HNF4α) 8
VII . Antiarrhythmic drug: Amiodarone and Dronedarone 9
VIII . Purpose 11
Chapter 2 Materials 12
I . Chemicals and reagents 12
II . Kits 15
III . Antibody of western blotting 16
IV . Enzyme 16
V . Bacterial culture accessories and antibiotics 16
VI . Material of cell culture 17
VII . Instruments 18
VIII . Others 18
Chapter 3 Experimental methods 20
I . Chemicals 20
II . Cell culture 20
III . Cell viability assay 21
IV . CYP3A4 enzyme activity assay (P450 GloTM assay) 21
V . Plasmids construction 21
VI . CYP3A4 reporter assay 22
VII . Mammalian two-hybrid assay 22
VIII . RNA extraction and reverse transcription-PCR 23
IX . Western blotting 24
Chapter 4 Results 26
I . Drug screen to search the inhibitor of CYP3A4 26
II . Inhibition of amiodarone and dronedarone on rifampin-mediated activation of CYP3A4 gene expression. 26
III . Effect of amiodarone or dronedarone on rifampin-mediated binding of PXR to SRC-1 in HepG2 cells. 27
IV . PXR and HNF4α interaction on amiodarone or dronedarone treatment in HepG2 cells. 28
V . Cell-base CYP3A4 enzyme activity assessment on amiodarone or dronedarone treatment 29
Chapter 5 Discussion 30
References 35

參考文獻 Argyrokastritis A, Kamakari S, Kapsetaki M, Kritis A, Talianidis I, Moschonas NK. Human hepatocyte nuclear factor-4 (hHNF-4) gene maps to 20q12-q13.1between PLCG1 and D20S17. Hum Genet. 1997; 99(2):233-6.
Bertilsson G, Heidrich J, Svensson K, Asman M, Jendeberg L, Sydow-Bäckman M, Ohlsson R, Postlind H, Blomquist P, Berkenstam A. Identification of a human nuclear receptor defines a new signaling pathway for CYP3A induction. Proc. Natl. Acad. Sci. USA.1998; 95, 12208–12213.
Chen D, Ma H, Hong H, Koh SS, Huang SM, Schurter BT, Aswad DW, Stallcup MR. Regulation of transcription by a protein methyltransferase. Science. 1999; 284(5423):2174-7.
Chen H, Lin RJ, Schiltz RL, Chakravarti D, Nash A, Nagy L, Privalsky ML, Nakatani Y, Evans RM. Nuclear receptor coactivator ACTR is a novel histone acetyltransferase and forms a multimeric activation complex with P/CAF and CBP/p300.Cell. 1997; 90(3):569-80.
Duniec-Dmuchowski Z, Ellis E, Strom SC, Kocarek TA. Regulation of CYP3A4 and CYP2B6 expression by liver X receptor agonists. Biochem Pharmacol. 2007; 74(10):1535-40.
Fernandez-Rachubinski FA, Weiner JH, Blajchman MA. Regions flanking exon 1 regulate constitutive expression of the human antithrombin gene. J Biol Chem. 1996; 271(46):29502-12.
Goodwin B, Hodgson E, D'Costa DJ, Robertson GR, Liddle C. Transcriptional regulation of the human CYP3A4 gene by the constitutive androstane receptor. Mol Pharmacol. 2002; 62(2):359-65.
Goodwin B, Hodgson E, Liddle C. The orphan human pregnane X receptor mediates the transcriptional activation of CYP3A4 by rifampicin through a distal enhancer module. Mol Pharmacol. 1999; 56(6):1329-39.
Guengerich FP. Cytochrome P-450 3A4: regulation and role in drug metabolism. Annu Rev Pharmacol Toxicol. 1999; 39:1-17.
Guengerich FP. Cytochromes P450, drugs, and diseases. Mol interv. 2003; 3(4): 94-204.
Hadzopoulou-Cladaras M, Kistanova E, Evagelopoulou C, Zeng S, Cladaras C, Ladias JA.Functional domains of the nuclear receptor hepatocyte nuclear factor 4. J Biol Chem. 1997; 272(1):539-50.
Hariparsad N, Chu X, Yabut J, Labhart P, Hartley DP, Dai X, Evers R. Identification of pregnane-X receptor target genes and coactivator and corepressor binding to promoter elements in human hepatocytes. Nucleic Acids Res. 2009; 37(4):1160-73.
Hashimoto H, Toide K, Kitamura R, Fujita M, Tagawa S, Itoh S, Kamataki T. Gene structure of CYP3A4, an adult-specific form of cytochrome P450 in human livers, and its transcriptional control. Eur J Biochem. 1993; 218(2):585-95.
He K, Iyer KR, Hayes RN, Sinz MW, Woolf TF, Hollenberg PF. Inactivation of cytochrome P450 3A4 by bergamottin, a component of grapefruit juice. Chem Res Toxicol. 1998; 11(4):252-9.
Huang H, Wang H, Sinz M, Zoeckler M, Staudinger J, Redinbo MR, Teotico DG, Locker J, Kalpana GV, Mani S. Inhibition of drug metabolism by blocking the activation of nuclear receptors by ketoconazole. Oncogene. 2007; 26(2):258-68.
Istrate MA, Nussler AK, Eichelbaum M, Burk O. Regulation of CYP3A4 by pregnane X receptor: The role of nuclear receptors competing for response element binding. Biochem Biophys Res Commun. 2010; 393(4):688-93.
Jover R, Bort R, Gómez-Lechón MJ, Castell JV. Cytochrome P450 regulation by hepatocyte nuclear factor 4 in human hepatocytes: a study using adenovirus-mediated antisense targeting. Hepatology. 2001; 33(3):668-75.
Kalitsky-Szirtes J, Shayeganpour A, Brocks DR, Piquette-Miller M. Suppression of drug-metabolizing enzymes and efflux transporters in the intestine of endotoxin-treated rats. Drug Metab Dispos. 2004; 32(1):20-7.
Kamiya A, Inoue Y, Gonzalez FJ. Role of the hepatocyte nuclear factor 4alpha in control of the pregnane X receptor during fetal liver development. Hepatology. 2003; 37(6):1375-84.
Kamiyama Y, Matsubara T, Yoshinari K, Nagata K, Kamimura H, Yamazoe Y. Role of human hepatocyte nuclear factor 4alpha in the expression of drug-metabolizing enzymes and transporters in human hepatocytes assessed by use of small interfering RNA. Drug Metab Pharmacokinet. 2007; 22(4):287-98.
Katoh M, Nakajima M, Yamazaki H, Yokoi T. Inhibitory effects of CYP3A4 substrates and their metabolites on P-glycoprotein-mediated transport. Eur J Pharm Sci. 2001; 12(4):505-13.
Khojasteh-Bakht SC, Koenigs LL, Peter RM, Trager WF, Nelson SD. (R)-(+)-Menthofuran is a potent, mechanism-based inactivator of human liver cytochrome P450 2A6. Drug Metab Dispos. 1998; 26(7):701-4.
Kliewer SA, Goodwin B, Willson TM. The nuclear pregnane X receptor: a key regulator of xenobiotic metabolism. Endocr Rev. 2002; 23(5):687-702.
Kliewer SA, Moore JT, Wade L, Staudinger JL, Watson MA, Jones SA, McKee DD, Oliver BB, Willson TM, Zetterström RH, Perlmann T, Lehmann JM. An orphan nuclear receptor activated by pregnanes defines a novel steroid signaling pathway. Cell 1998; 92(1):73-82.
Koh SS, Chen D, Lee YH, Stallcup MR. Synergistic enhancement of nuclear receptor function by p160 coactivators and two coactivators with protein methyltransferase activities. J Biol Chem. 2001; 276(2):1089-98.
Lehmann JM, McKee DD, Watson MA, Willson TM, Moore JT, Kliewer SA. The human orphan nuclear receptor PXR is activated by compounds that regulate CYP3A4 gene expression and cause drug interactions. J Clin Invest. 1998; 102(5):1016-23.
Li AP, Kaminski DL, Rasmussen A. Substrates of human hepatic cytochrome P450 3A4. Toxicology. 1995; 104(1-3):1-8.
Li J, O'Malley BW, Wong J. p300 requires its histone acetyltransferase activity and SRC-1 interaction domain to facilitate thyroid hormone receptor activation in chromatin. Mol Cell Biol. 2000; 20(6):2031-42.
Li T, Chiang JY. Rifampicin induction of CYP3A4 requires pregnane X receptor crosstalk with hepatocyte nuclear factor 4alpha and coactivators, and suppression of small heterodimer partner gene expression. Drug Metab Dispos. 2006; 34(5):756-64.
Liddle C, Goodwin BJ, George J, Tapner M, Farrell GC. Separate and interactive regulation of cytochrome P450 3A4 by triiodothyronine, dexamethasone, and growth hormone in cultured hepatocytes. J Clin Endocrinol Metab. 1998 ; 83(7):2411-6.
Lim YP, Huang JD. Interplay of pregnane X receptor with other nuclear receptors on gene regulation. Drug Metab Pharmacokinet. 2008; 23(1):14-21.
Lim YP, Kuo SC, Lai ML, Huang JD. Inhibition of CYP3A4 expression by ketoconazole is mediated by the disruption of pregnane X receptor, steroid receptor coactivator-1, and hepatocyte nuclear factor 4alpha interaction. Pharmacogenet Genomics. 2009; 19(1):11-24.
Martínez-Jiménez CP, Jover R, Donato MT, Castell JV, Gómez-Lechón MJ. Transcriptional regulation and expression of CYP3A4 in hepatocytes. Curr Drug Metab. 2007; 8(2):185-94.
Matheny CJ, Lamb MW, Brouwer KR, Pollack GM. Pharmacokinetic and pharmacodynamic implications of P-glycoprotein modulation. Pharmacotherapy. 2001; 21(7):778-96.
Matic M, Mahns A, Tsoli M, Corradin A, Polly P, Robertson GR. Pregnane X Receptor: Promiscuous regulator of detoxification pathways. Int J Biochem Cell Biol. 2007; 39(3), 478-83.
Nelson DR, Kamataki T, Waxman DJ, Guengerich FP, Estabrook RW, Feyereisen R, Gonzalez FJ, Coon MJ, Gunsalus IC, Gotoh O, et al. The P450 superfamily: update on new sequences, gene mapping, accession numbers, early trivial names of enzymes, and nomenclature. DNA Cell Biol. 1993; 12(1):1-51.
Nelson DR, Koymans L, Kamataki T, Stegeman JJ, Feyereisen R, Waxman DJ, Waterman MR, Gotoh O, Coon MJ, Estabrook RW, Gunsalus IC, Nebert DW. P450 superfamily: update on new sequences, gene mapping, accession numbers and nomenclature. Pharmacogenetics. 1996; 6(1):1-42.
Nelson DR. Cytochrome P450 and the individuality of species. Arch Biochem Biophys. 1999; 369(1):1-10.
Ohmori S, Ishii I, Kuriya S, Taniguchi T, Rikihisa T, Hirose S, Kanakubo Y, Kitada M. Effects of clarithromycin and its metabolites on the mixed function oxidase system in hepatic microsomes of rats. Drug Metab Dispos. 1993; 21(2):358-63.
Ohyama K, Nakajima M, Suzuki M, Shimada N, Yamazaki H, Yokoi T. Inhibitory effects of amiodarone and its N-deethylated metabolite on human cytochrome P450 activities: prediction of in vivo drug interactions. Br J Clin Pharmacol. 2000; 49(3):244-53.
Oñate SA, Tsai SY, Tsai MJ, O'Malley BW. Sequence and characterization of a coactivator for the steroid hormone receptor superfamily. Science. 1995; 270(5240):1354-7.
Pascussi JM, Drocourt L, Fabre JM, Maurel P, Vilarem MJ. Dexamethasone induces pregnane X receptor and retinoid X receptor-alpha expression in human hepatocytes: synergistic increase of CYP3A4 induction by pregnane X receptor activators. Mol Pharmacol. 2000; 58(2):361-72.
Plant N. The human cytochrome P450 sub-family: transcriptional regulation, inter-individual variation and interaction networks. Biochim Biophys Acta. 2007; 1770(3):478-88.
Roten L, Schoenenberger RA, Krähenbühl S, Schlienger RG. Rhabdomyolysis in association with simvastatin and amiodarone. Ann Pharmacother. 2004; 38(6):978-81.
Schmidt GA, Hoehns JD, Purcell JL, Friedman RL, Elhawi Y. Severe rhabdomyolysis and acute renal failure secondary to concomitant use of simvastatin, amiodarone, and atazanavir. J Am Board Fam Med. 2007; 20(4):411-6.
Shiau AK, Barstad D, Loria PM, Cheng L, Kushner PJ, Agard DA, Greene GL. The structural basis of estrogen receptor/coactivator recognition and the antagonism of this interaction by tamoxifen. Cell. 1998; 95(7):927-37.
Sladek FM, Ruse MD Jr, Nepomuceno L, Huang SM, Stallcup MR. Modulation of transcriptional activation and coactivator interaction by a splicing variation in the F domain of nuclear receptor hepatocyte nuclear factor 4alpha1. Mol Cell Biol. 1999; 19(10):6509-22.
Späth GF, Weiss MC. Hepatocyte nuclear factor 4 expression overcomes repression of the hepatic phenotype in dedifferentiated hepatoma cells. Mol Cell Biol. 1997; 17(4):1913-22
Spencer TE, Jenster G, Burcin MM, Allis CD, Zhou J, Mizzen CA, McKenna NJ, Onate SA, Tsai SY, Tsai MJ, O'Malley BW.Steroid receptor coactivator-1 is a histone acetyltransferase. Nature. 1997; 389(6647):194-8.
Stanley LA, Horsburgh BC, Ross J, Scheer N, Wolf CR. PXR and CAR: nuclear receptors which play a pivotal role in drug disposition and chemical toxicity. Drug Metab Rev. 2006; 38(3):515-97.
Stoykov I, van Beeren HC, Moorman AF, Christoffels VM, Wiersinga WM, Bakker O. Effect of amiodarone and dronedarone administration in rats on thyroid hormone-dependent gene expression in different cardiac components. Eur J Endocrinol. 2007; 156(6):695-702.
Sueyoshi T, Negishi M. Phenobarbital response elements of cytochrome P450 genes and nuclear receptors. Annu Rev Pharmacol Toxicol. 2001; 41:123-43.
Takeshita A, Taguchi M, Koibuchi N, Ozawa Y. Putative role of the orphan nuclear receptor SXR (steroid and xenobiotic receptor) in the mechanism of CYP3A4 inhibition by xenobiotics. J Biol Chem. 2002; 277(36):32453-8.
Tegude H, Schnabel A, Zanger UM, Klein K, Eichelbaum M, Burk O. Molecular mechanism of basal CYP3A4 regulation by hepatocyte nuclear factor 4alpha: evidence for direct regulation in the intestine. Drug Metab Dispos. 2007; 35(6):946-54.
Thummel KE, Wilkinson GR. In vitro and in vivo drug interactions involving human CYP3A. Annu Rev Pharmacol Toxicol. 1998; 38:389-430. Review.
Tirona RG, Kim RB. Nuclear receptors and drug disposition gene regulation. J Pharm Sci. 2005; 94(6):1169-86.
Tirona RG, Lee W, Leake BF, Lan LB, Cline CB, Lamba V, Parviz F, Duncan SA, Inoue Y, Gonzalez FJ, Schuetz EG, Kim RB. The orphan nuclear receptor HNF4alpha determines PXR- and CAR-mediated xenobiotic induction of CYP3A4. Nat Med. 2003; 9(2):220-4.
Voegel JJ, Heine MJ, Tini M, Vivat V, Chambon P, Gronemeyer H. The coactivator TIF2 contains three nuclear receptor-binding motifs and mediates transactivation through CBP binding-dependent and -independent pathways. EMBO J. 1998; 17(2):507-19.
Wacher VJ, Wu CY, Benet LZ. Overlapping substrate specificities and tissue distribution of cytochrome P450 3A and P-glycoprotein: implications for drug delivery and activity in cancer chemotherapy. Mol Carcinog. 1995; 13(3):129-34.
Watkins RE, Davis-Searles PR, Lambert MH, Redinbo MR. Coactivator binding promotes the specific interaction between ligand and the pregnane X receptor. J Mol Biol. 2003; 331(4):815-28.
Wu RC, Smith CL, O'Malley BW. Transcriptional regulation by steroid receptor coactivator phosphorylation. Endocr Rev. 2005; 26(3):393-9.
Xie W, Uppal H, Saini SP, Mu Y, Little JM, Radominska-Pandya A, Zemaitis MA. Orphan nuclear receptor-mediated xenobiotic regulation in drug metabolism. Drug Discov Today. 2004; 9(10), 442-449.
Xu J, Li Q. Review of the in vivo functions of the p160 steroid receptor coactivator family. Mol Endocrinol. 2003; 17(9):1681-92.
Yamreudeewong W, DeBisschop M, Martin LG, Lower DL. Potentially significant drug interactions of class III antiarrhythmic drugs. Drug Saf. 2003; 26(6):421-38.
Yao TP, Ku G, Zhou N, Scully R, Livingston DM. The nuclear hormone receptor coactivator SRC-1 is a specific target of p300. Proc Natl Acad Sci U S A. 1996; 93(20):10626-31.
Zhang J, Kuehl P, Green ED, Touchman JW, Watkins PB, Daly A, Hall SD, Maurel P, Relling M, Brimer C, Yasuda K, Wrighton SA, Hancock M, Kim RB, Strom S, Thummel K, Russell CG, Hudson JR Jr, Schuetz EG, Boguski MS. The human pregnane X receptor: genomic structure and identification and functional characterization of natural allelic variants. Pharmacogenetics. 2001; 11(7):555-72.
Zhou SF. Drugs behave as substrates, inhibitors and inducers of human cytochrome P450 3A4. Curr Drug Metab. 2008; 9(4):310-22.
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