肝臟細胞色素P450 2B1/2對靜脈麻醉藥物PROPOFOL代謝所扮演之角色探討

ROLE OF HEPATIC CYTOCHROME P450 2B1/2 IN PROPOFOL METABOLISM

林怡伶

指導教授 ： 陳瑞明 ； 陳大樑

臺北醫學大學/醫學院/醫學科學研究所/碩士(2004年)

摘要

Propofol是臨床常用之靜脈麻醉藥物，主要用於手術時之麻醉誘導與持續，亦可用於加護病房病人，做為鎮靜藥物。先前研究證實，肝臟細胞色素P450s (cytochrome P450s; CYPs)對此藥物之代謝扮演重要角色。但對於藥理及毒理研究常用之實驗動物模式，大鼠肝臟中，是何種CYP異構酶(isoforms)參與propofol之代謝，則尚無相關研究被提出。所以本研究主要將探討，於大鼠肝臟微粒體中，主要是何種CYP異構酶參與propofol之代謝。此外，肝細胞內CYPs對藥物代謝之效率，與藥物於細胞內之運輸情形有關，而細胞支架(cytoskeleton)能影響細胞的藥物運輸，因此，本研究另一研究目標，將探討細胞支架是否會影響肝臟CYPs之酵素活性。 已知在不同物種中，CYPs對propofol代謝均扮演著重要之角色，先前研究亦指出在人類及犬科動物，主要代謝propofol之CYP異構酶分別為CYP2B6及CYP2B11，皆屬於CYP2B次家族，因此本研究首要目標將探討大鼠肝臟中，CYP2B1/2是否參與於propofol代謝。利用CYP2B誘導劑phenobarbital (PB)，以腹腔注射方式，先對雄性Wistar大鼠(rat)連續施打7天，然後進行動物犧牲，取出肝臟並製備成微粒體。經PB處理後，大鼠肝臟微粒體之pentoxyresorufin O-dealkylase (PROD)活性明顯增加，以免疫蛋白轉印分析顯示，經PB處理之大鼠肝臟微粒體，其CYP2B1/2蛋白量亦顯著增加。若以PB處理之大鼠肝臟微粒體，進行propofol之體外代謝，並以HPLC分析，可偵測到propofol代謝產物之形成，將代謝產物進一步以LC/MS分析後，證實此代謝產物為propofol經氧化代謝所形成之4-hydroxypropofol (4-OH-PPF)。相對於未經藥物處理之控制組，經PB處理之大鼠肝臟微粒體，有較強之propofol氧化代謝能力，4-OH-PPF產量較高。更進一步以CYP2B1/2化學抑制劑orphenadrine處理大鼠肝臟微粒體，發現4-OH-PPF產量顯著下降，顯示其propofol氧化代謝能力降低。接著以CYP2B1/2專一性抗體處理大鼠肝臟微粒體，經抗體處理後，同樣造成4-OH-PPF產量顯著下降，顯示其propofol氧化代謝能力亦降低。 本研究之另一探討方向，以人類肝癌細胞株HepG2細胞為研究模式，探討當細胞支架的F-actin受到調控時，CYP2B活性是否會受影響。以抑制F-actin聚合藥物cytochalasin D處理細胞，以螢光標定物進行F-actin染色，利用共軛焦顯微鏡觀察並記錄細胞內的變化。經cytochalasin D處理後，HepG2細胞中F-actin結構受到干擾而呈點狀分佈。而利用共軛焦顯微鏡進行活細胞之代謝分析，記錄連續時間下，細胞內螢光產物形成之動態變化可知，經cytochalasin D處理後，同時會造成細胞的PROD活性降低，顯示HepG2細胞內由CYP2B參與之pentoxyresorufin代謝能力受到了抑制。 本研究結果顯示，經PB處理後，大鼠肝臟之CYP2B1/2活性及蛋白量都會增加。以PB處理之大鼠肝臟微粒體，能有效率的將propofol氧化代謝成4-OH-PPF。而此氧化代謝反應會受到CYP2B1/2化學抑制劑及專一性抗體所抑制。因此得知，CYP2B1/2對大鼠肝臟代謝propofol扮演著重要的角色，為主要參與propofol氧化代謝之CYP異構酶。另外，經本研究結果亦得知，當HepG2細胞中細胞支架的F-actin聚合被干擾，細胞中CYP2B代謝活性亦會受到抑制，細胞支架F-actin可能與CYP2B代謝受質的能力有關。

關鍵字

2 ； 6-雙異丙烷酚 ； 細胞色素P450s ； 代謝 ； 細胞支架

並列摘要

Propofol, an intravenous anesthetic agent, is widely used for induction and maintenance of anesthesia during surgical procedures. Propofol can be used as a sedative agent for intensive care unit patients. Previous studies had reported that human cytochrome P450s (CYPs) play critical roles in the metabolism of propofol. Rat is a common animal model for pharmacological and toxicological studies of drugs. However, it is not determined which CYP isoforms contribute to the metabolism of propofol in rat liver. Thus, one of specific aims of this study is to determine which CYP isoforms in rat liver has a major role in propofol metabolism. The drug transport from extracellular environment to intracellular endoplasmic reticulums will determine if CYPs can effectively metabolize these drugs. Previous study has reported that cytoskeleton could modulate drug transport. Another specific aim of this study is to evaluate the role of cytoskeleton in CYP activities. Previous studies revealed that CYP2B6 and CYP2B11 are the major CYP isoforms participating in propofol metabolism in human and in canine, respectively. Male Wistar rats were intraperitoneally injected with 80 mg/kg body weight/day phenobarbital (PB) for 7 days. Liver microsomes from control- and PB-treated rats were prepared. Our data revealed that PB significantly increased pentoxyresorufin O-dealkylase (PROD) activity and CYP2B1/2 protein levels in rat liver microsomes. Data from HPLC analysis revealed that PB-treated liver microsomes had much efficacy on propofol hydroxylation than control ones. LC/MS analysis demonstrated that the major metabolite of propofol biotransformation by PB-treated liver microsomes was 4-hydroxypropofol (4-OH-PPF). Hydroxylation of propofol by PB-treated rat liver microsomes was significantly suppressed by orphenadrine, a CYP2B1/2-specific inhibitor. Pretreatment with a polyclonal antibody against rat CYP2B1/2 protein significantly reduced propofol hydroxylation by PB-treated liver microsomes. In this study, we further evaluated that that the modulation of F-actin cytoskeleton could affect CYP2B activity in HepG2 cells. Confocal microscopic analysis showed that cytochalasin D, an inhibitor of F-actin polymerization, could disrupt F-actin cytoskeleton of HepG2 cells. In parallel with the modulation of F-actin polymerization, cellular PROD activity was significantly decreased. This study shows that PB can increase CYP2B1/2 activity and protein levels. Propofol is metabolized by PB-treated rat liver microsomes to 4-OH-PPF. And, a CYP2B1/2-specific inhibitor and antibody significantly inhibit the hydroxylation. Therefore, in rat liver microsomes, CYP2B1/2 plays a major role in propofol hydroxylation. This study has also shown that the modulation of F-actin cytoskeleton can regulate CYP2B activity in HepG2 cells. F-actin cytoskeleton may involve in the metabolism of its substrates by CYP2B.

並列關鍵字

propofol ； cytochrome P450s ； metabolism ； cytoskeleton

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