布格呋喃与CYP450/P-糖蛋白的相互作用及生物学效应

【作者】 李恩；

【导师】 李燕； 程桂芳； 陈晓光； 申竹芳；

【作者基本信息】 中国协和医科大学 ， 药理学， 2010， 博士

【摘要】 药物代谢是新药研究过程中必不可少的重要环节,口服药物的生物利用度和药物相互作用是评价药物代谢特征的重要指标。已知在药物代谢过程中,当两种或多种药物经同一代谢酶代谢时,药物间则可能由于对药酶的竞争而发生相互作用,致使血药浓度显著增加,导致严重的不良反应。此外,药物进入体内在受到药酶代谢转化的同时,也可诱导或抑制某些CYP450同工酶的表达水平和代谢活性,从而调节自身和其它化合物的代谢转化,以致改变新药的安全性和疗效。因此,在新药研发的临床前阶段明确药物的主要代谢途径以及对药酶的诱导和抑制,对于认识个体间代谢差异和预测潜在的药物-药物相互作用具有重要意义,此外,还有助于明确某些代谢产物的药理学特隆和进一步开发的价值。P-糖蛋白是一类ATP依赖性膜蛋白,在小肠粘膜、血脑屏障、肝细胞、肾及睾丸等器官均有分布,是上述生理屏障的重要组成部分并发挥泵出作用,阻碍某些有害物质进入这些特定部位,同时也可加快药物从这些组织部位的消除,从而影响药物的吸收、分布、代谢和排泄。药物对上述器官P-糖蛋白的调控可引发非代谢性的药物相互作用。因此,有必要在新药研发阶段关注药物与P-糖蛋白之间的相互影响。虽然肝脏是人体最主要的代谢器官,参与大多数药物和毒物的生物转化,但除肝脏之外,肠道和肾脏作为吸收和排泄器官也表达部分CYP同工酶和转运蛋白,在药物代谢中的作用同样不可忽视。布格呋喃是由中国医学科学院药物研究所研发的抗焦虑新药。前期体内药代动力学结果显示,大鼠口服布格呋喃后具有半衰期短,生物利用度较低,靶器官分布少的特点。布格呋喃可经CYP450代谢,同时也是肠道P-糖蛋白的底物,但参与布格呋喃代谢的同工酶亚型、布格呋喃与CYP450以及P-糖蛋白相互作用的分子机制和由此产生的生物学效应还有待进一步明确。为此,本研究可分为以下几方面内容：1.采用大鼠、人肝微粒体和重组人源CYP450同工酶温孵法,应用GC-MS分析技术,研究参与布格呋喃Ⅰ相代谢的同工酶类型和产物生成的相关性。2.从酶活性水平、mRNA转录水平和蛋白表达水平评价多次口服不同剂量布格呋喃对大鼠肝脏CYP的调控作用。3.应用探针底物法、RT-PCR、western-blotting方法从细胞、亚细胞组分、mRNA和蛋白水平考察布格呋喃对小肠CYP3A、P-糖蛋白和肾CYP2C11、2E1以及P-糖蛋白的影响。4.应用整体动物、原位脑灌流模型及构建P-糖蛋白高表达MDCK细胞株研究P-糖蛋白对布格呋喃脑分布以及多次口服布格呋喃对脑P-糖蛋白的影响。5.根据布格呋喃对药物代谢酶和外排蛋白的研究结果,选取可能与布格呋喃合用的四种临床药物,考察布格呋喃与其他药物合用对药代动力学的影响。研究结果表明：一、布格呋喃在大鼠／人肝脏微粒体主要代谢产物鉴定及比较1.1本研究应用的GC-MS分析方法可有效分离布格呋喃在微粒体温孵体系中的代谢产物并初步鉴定其结构类型。1.2布格呋喃在大鼠肝微粒体中可代谢为一羟基、一羰基和二羟基代谢产物。布格呋喃在人肝微粒体中的代谢特征与大鼠相似,同样生成一羟基、一羰基和二羟基类型代谢产物,但代谢速率较大鼠慢,生成的代谢产物种类较大鼠中少。1.3应用CYP同工酶选择性抑制剂研究结果显示,在肝微粒体加入CYP3A4和CYP2E1的抑制剂酮康唑和戒酒硫后,布格呋喃消除速率减慢,各代谢产物生成量明显减少。体外重组人源CYP3A4和CYP2E1温孵结果表明,布格呋喃在重组人源CYP3A4中可迅速被代谢,代谢产物主要为一羰基和二羟基产物。综合以上结果,可初步判断CYP3A4是参与布格呋喃代谢的主要CYP同工酶,一羰基和二羟基化是其催化的主要反应。二、布格呋喃对大鼠肝脏CYP450s的调控2.1布格呋喃(4、16、64 mg/kg,1次／日×7)多次给药可提高大鼠肝脏CYP1A2和2E1活性、基因和蛋白表达水平也明显增加,并呈一定剂量-效应关系,但诱导强度弱于经典诱导剂β-萘黄铜和乙醇。2.2多次口服布格呋喃可抑制大鼠肝脏CYP2D活性,对CYP2C6和CYP2C11活性均有一定程度的诱导作用,但对CYP3A活性无明显影响。2.3布格呋喃在自身处理的肝微粒体中代谢速率有所提高,提示布格呋喃诱导自身代谢与其对肝脏药物代谢酶的诱导有关。2.4布格呋喃在浓度为1-10μM时对CYP450同工酶存在不同程度抑制作用,但浓度降为0.5gM时对CYP450同工酶则无显著性影响。三、布格呋喃对大鼠肠道和肾脏主要CYP450s和P-糖蛋白的调控3.1布格呋喃可上调大鼠小肠CYP3A的催化活性和蛋白表达,诱导人源肠道LS-174T细胞CYP3A4蛋白含量升高,提示布格呋喃可通过调控小肠CYP3A蛋白含量提高其的催化活性。同时,布格呋喃亦可上调肠道P-糖蛋白含量。应用Caco-2细胞Transwell模型研究发现,布格呋喃100μM处理72h可明显降低地高辛在Caco-2单层细胞的渗透系数,同时增加其外排系数,此改变可被P-糖蛋白抑制剂环孢菌素所逆转；另一方面,布格呋喃100μM可抑制P-糖蛋白对地高辛的外排。以上结果提示,布格呋喃可诱导／抑制肠道P-糖蛋白功能。3.2布格呋喃多次口服可上调肾脏CYP2C11、2E1和P-糖蛋白的水平,并且对肾CYP2E1的上调倍数大于肝脏。四、布格呋喃与大鼠脑P-糖蛋白的相互作用4.1应用大鼠原位脑灌流模型研究发现,布格呋喃在脑中的分布在0-8min内呈线性增加；P-糖蛋白抑制剂环孢菌素可使布格呋喃自血液向脑组织的单向转运明显增加,提示布格呋喃是脑P-糖蛋白的底物,血脑屏障中的P-糖蛋白可对布格呋喃脑分布产生阻碍作用。4.2应用RT-PCR、western blot和罗丹明脑-血分布方法检测大鼠脑MDR1基因的mRNA和P-糖蛋白表达及功能发现,多次口服布格呋喃可上调P-糖蛋白表达和功能。五、布格呋喃对联合用药的药代动力学影响研究5.1非那西丁为常用解热镇痛药,体内主要经CYP1A2代谢生成代谢产物扑热息痛。当布格呋喃与非那西丁联合用药时,单次给予布格呋喃组非那西丁AUC、峰浓度升高,清除率下降,而其代谢产物扑热息痛的AUC和达峰浓度均有所下降；多次给药组非那西丁的AUC下降,MRT减小,而清除率提高,同时扑热息痛的AUC和峰浓度均较对照组升高。以上结果与布格呋喃对肝脏CYP1A2的诱导／抑制相关。5.2氯唑沙宗是中枢性肌松剂,在体内主要经CYP2E1代谢。体内药代动力学结果表明,多次口服布格呋喃组氯唑沙宗的AUC下降,MRT减小,清除率提高,而6-羟基氯唑沙宗的AUC和峰浓度均较对照组升高,提示布格呋喃多次给药可在一定程度上加快氯唑沙宗的消除,可能与对肝脏CYP2E1的诱导密切相关。但单次给予布格呋喃组氯唑沙宗各药代动力学参数无显著性变化,说明布格呋喃单次口服对氯唑沙宗体内过程影响不大。5.3咪达唑仑属于苯二氮卓类镇静催眠药,主要由CYP3A同工酶代谢,其清除率主要取决于CYP3A活性。大鼠体内药代动力学研究结果表明,口服布格呋喃后咪达唑仑达峰浓度提高,推测与前述布格呋喃对CYP3A的体外抑制作用有关。而多次口服布格呋喃的大鼠表现为咪达唑仑体内AUC、达峰浓度显著降低,清除大大加快,但相应体内代谢产物也减少,提示其体内代谢还有其它因素参与。据报道,咪达唑仑是CYP3A和P-糖蛋白共同的底物,布格呋喃多次给药对小肠P-糖蛋白也有诱导作用,当两药合用时,布格呋喃可通过诱导P-糖蛋白减少咪达唑仑的吸收,导致其血药浓度降低,同时代谢产物减少。5.4地高辛为一种中效强心苷,为P-糖蛋白的底物,治疗安全范围较小,毒性反应严重时会危及生命。前期研究表明,布格呋喃对P-糖蛋白有诱导／抑制作用,因此本文对二者合用时地高辛的血药浓度进行了监测。实验结果表明,布格呋喃单次口服可明显升高地高辛血药浓度(峰浓度升高约25%,AUC升高85%),因此两药合用时,注意避免出现毒副作用。布格呋喃多次给药可使地高辛达峰浓度和AUC分别降至对照组的70%和80%,提示在长期服用布格呋喃后使用其他P-糖蛋白的底物药应注意药效学的监控。综上所述,本文应用体外亚细胞组分、细胞、在体器官和整体动物模型,较系统地研究了布格呋喃对肝脏、小肠、肾、脑CYP同工酶和P-糖蛋白的诱导和抑制以及参与其代谢的主要CYP450同工酶,并研究了布格呋喃对合用药体内药代动力学的影响。上述研究不仅可阐明布格呋喃与CYP450和P-糖蛋白相互作用的分子机制,同时为预测临床药物相互作用和合理用药、针对药代特性进行新药的结构改造提供科学依据。更多还原

【Abstract】 Drug metabolism is an important part in new drug development and the bioavailability of oral drugs and the drug-drug interaction are the pivotal guideline to evaluate the pharmacokinetics. The drug-drug interaction may occur when they combine the same CYP isozyme competitively which leads to enhancing plasma concentration and serious adverse reaction subsequently. In the other hand, drugs can induce and/or inhibit certain CYP450 in expression or activity levels when they enter the body, thereby modify the biotransformation of themselves and other drugs and change the security and curative effect. Therefore, the illumination of the metabolic pathway and the induction/inhibition on metabolic enzymes of new drugs plays an important role to investigate the difference of individual metabolism and the pharmacodynamics of some metabolites for exploration ulteriorly in the preclinical phase of drug development.P-glycoprotein (P-gp) was identified as an ATP-dependent transporter. It has been found in intestinal mucosa, blood-brain-barrier, hepatic cell, kidney and testicle etc. P-gp can pump the toxic compound and numerous drugs out of these tissues and impact the absorption, disposition, metabolism and elimination. The modification of drugs to P-gp may lead to the nonmetabolism drug-drug interaction. So the connection between new drugs and P-gp should be noticed during the developing stage.Moreover, previous studies have shown that, second to liver, intestine and kidney express several metabolic enzymes and transporters which are involved in the drug metabolism.Buagafuran is a synthetic derivative of agarofuran, which showed significant antianxiety activity in several animal models, with higher potency and lower toxicity compared with diazepam and buspirone. The possible antianxiety mechanism of buagafuran was related to the modulation of central monoamine neurotransmitters. The preclinical pharmacokinetics studies indicated that the absorption of buagafuran was extremely poor with an absolute bioavailability below 9.5%. The highest radioactivity of 3H-buagafuran was found in gastrointestinal tract, followed by liver and kidney (unpublished results), but less in brain (target organ). Several rats liver CYP enzymes and intestine P-glycoprotein involved the biotransformation of buagafuran, but it has not been demonstrated that the type of metabolites, the CYP isoforms involved in the biotransformation of buagafuran, the interaction of buagafuran between CYP and P-gp. So, in this thesis the following study results were reported:1. Investigate the CYP isoforms and metabolites of buagafuran with rat and human liver microsomes, recombination human supersomes and CG-MS analysis.2. Evaluate the effect of buagafuran on the liver CYPs in mRNA, protein and activity levels.3. Inspect the impact of buagafuran on the intestinal CYP3A/P-gp and renal CYP2C11,2E1 and P-gp in mRNA, protein and activity levels.4. Using the intact animal and in situ brain perfusion to study the interaction of buagafuran and P-gp in brain.5. According to the results of regulation of buagafuran to CYPs and P-gp, we chose the drugs which may be prescribed combined with buagafuran to study the possible impacts of buagafuran on other drugs from the view of pharmacokinetics in vivo.The results were shown as follows:1. The identification and comparison of the metabolites of buagafuran in rat and human liver microsomes. 1.1 The GC-MS analysis method for the buagafuran and its metabolites in biological samples showed good sensitivity, and high specialization and appropriate for study in microsomes incubation system.1.2 Buagafuran could be transformed to one hydroxyl-, one carbonyl-and two hydroxyl-metabolism in rat liver microsomes. The main metabolites in human liver microsomes were similar as in rats, but the number was less and the rate of production was slower.1.3 Ketoconazole and disulfiram are the selective inhibitors to CYP3A4 and CYP2E1, they could reduce the elimination rate of buagafuran and the generation of the metabolites, and buagafuran could be metabolized in the CYP3A4 supersome. Summarization above results, the CYP3A4 was the main CYP isoform involved in the catalysis of translation of buagafuran into one hydroxyl-and two hydroxyl-metabolites.2. The regulation of buagafuran to rat liver microsomes CYP450s2.1 Buagafuran (4,16,64mg/kg) administered by gavage for 7 continuous days significant increased the activity of CYP1A2 and CYP2E1 in a dose dependent manner (1.56-,1.89-,3.07-fold and 1.4-,1.48-,1.79-fold), while the mRNA and protein levels of CYP 1A2 and 2E1 were elevated in certain extents.2.2 CYP2C6, CYP2C11 were also slightly induced by buagafuran, while buagafuran had no effect on liver CYP3 A2 in rats.2.3 The metabolic rate of buagafuran in self-treated rat liver microsomes was faster than control group, which indicated that the induction of self-metabolism might be related to the up-regulation of liver CYPs.2.4 Buagafuran in 1-10μM showed inhibition on rat liver CYPs in different extents, but the 0.5μM of buagafuran has no inhibit effect in vitro.3. The regulation of buagafuran on CYP450s and P-gp in rat intestine and kidney.3.1 Buagafuran could increase activity and expression of CYP3A in rat intestine and induce expression of CYP3A4 protein in LS-174T. The induction of P-gp expression was found in rat intestine by buagafuran. According the result of digoxin transfer experiments performed in Caco-2 transwell model, buagafuran showed induction/inhibition on function of P-gp.3.2 Multiple oral administration of buagafuran could up-regulate the CYP2C11、2E1 and P-gp in rat kidney, and the extent was more than in liver.4. The interaction between buagafuran and P-gp in rat brain4.1 The efflux of buagafuran in brain was studied in rat in situ brain perfusion model and the result demonstrated that the P-gp inhibitor could enhance the penetration of buagafuran from blood to brain.4.2 The effect of buagafuran on brain P-gp was measured by RT-PCT, western-blotting and the blood-brain ratio of rodanmin123 in rat. The result improved the induction of buagafuran on brain P-gp in mRNA, protein and function levels.5. PK effect of buagafuran on clinical drugs5.1 Phenacetin is a common used antipyretic analgesic and can be transformed to acetaminophen mediated by CYP1A2. After single administration of buagafuran, the PK profiles exhibited the enhenced AUC, Cmax and reduced CL/F. After multiple administration buagafuran the AUC and MRT decreased. The concentration of acetaminophen in plasma was reduced by multiple and increased by single administration of buagafuran. The result above was correlation with the induction and inhibition of buagafuran on liver CYP1A2.5.2 Chlorzoxazone, a centrally acting muscle relaxant, was mainly metabolized by CYP2E1 in vivo. Because of the significant induction on CYP2E1 after multiple doses of buagafuran, the PK interaction of chlorzoxazone and buagafuran coadministration was studied in vivo. The result demonstrated that multiple dosages could induce the metabolism of chlorzoxazone by changing AUC, MRT and CL/F and enhance the 6-hydroxylchlorzoxazone, while single dose had no obvious effect on chlorzoxazone PK profile. The result suggested that multiple doses of buagafuran could accelerate chlorzoxazone metabolism through induction of CYP2E1 activity and protein expression.5.3 It is reported that CYP3A is mainly involved in the hepatic metabolism of a short acting benzodiazepine derivative agent midazolam. The PK interaction between midazolam and buagafuran was investigated just because of the inhibition on CYP3A in vitro. The results showed the inhibition on the midazolam metabolism by single dosage appearing as the increasing plasma concentration, which was consisitant with the inhibition study. Multiple dosages of buagafuran could decrease the AUC, Cmax of midazolam as well as 1’-hydroxyl midazolam. The results might be due to the involvement of P-gp in the biotransformation of midazolam.5.4 Digoxin, an intermediate-acting cardiac glycoside, was a substrate of P-gp. Because of its narrow therapeutic window and unexpected toxic effect, blood drug concentration should be monitored in clinical application. Previous study showed that buagafuran could induce/inhibit the intestinal P-gp, under the above condition, the P-gp mediated interaction between them may happen in all probability. Our result showed that the blood concentration of digoxin was elevated significantly (Cmax increased by 25%, AUC increased by 85%) by single dosing of buagafuran and multiple dosages could reduce the Cmax and AUC of digoxin to 70% and 80% compare with control group which magnified the possibility of adverse effect. Thus, coadministration interaction should be concerned between those drugs, especially the drugs with narrow therapeutic window, and pharmaceutics from buagafuran. Comprehensive analysis should be given to personalize drug therapy.In conclusion, this report included the studies of the induction/inhibition of buagafuran on main CYP450 and P-gp in liver, intestine, kidney and brain systematically by various subcellular fractions, cell lines, intact animal and in situ animal models. The pharmacokinetics was performed on buagafuran and coadministration drugs in order to provide reliable references for prediction of clinical drug-drug interaction. The present study may demonstrate the molecular mechanisms of the interaction between buagafuran and CYPs/P-gp, likewise provide the valuable data for clinical application and structure reformation of buagafuran.更多还原