优克那非的体内外代谢研究

发布时间：2018-01-16 14:26

本文关键词：优克那非的体内外代谢研究　出处：《吉林大学》2017年硕士论文　论文类型：学位论文

【摘要】：优克那非(Yonkenafil)是一种新型磷酸二酯酶(Phosphodiesterase,PDE)V抑制剂,其结构为环磷酸鸟苷(Cyclic Guanosine monophosphate,cGMP)的五元并六元杂环酮类,由我国科研工作者独立研发,并具有自主知识产权。前期对优克那非药效学试验结果表明,优克那非对PDE V具有很好的选择性抑制作用,相比于现有的磷酸二酯酶V抑制剂类药物伐地那非和西地那非,其具有更明显的优势。目前,本实验室已完成对优克那非在大鼠和比格犬中的药代动力学研究,而存在于肝微粒体中的CYP450酶是负责机体内药物代谢的主要酶系,在新药的临床前药代动力学研究中,对药物与CYP450酶之间相互作用的研究有助于实现对新药的筛选、对临床用药效果的预测及为临床合理用药提供可靠的依据。因此本论文在之前的研究基础上,对优克那非进行体外代谢研究,并对体内外的代谢产物进行鉴定。本文建立了测定人肝微粒体(HLMs)孵育体系中优克那非及其代谢物M1、M2、M3浓度的LC-MS/MS定量分析方法。通过对孵育时间、肝微粒体蛋白浓度、底物优克那非浓度的优化,使CYP450酶催化优克那非代谢的过程能够在最佳的孵育条件下进行。最终确定的最佳孵育条件如下:孵育时间为15min,体系中肝微粒体蛋白浓度为0.5mg/mL,底物优克那非浓度为8μg/mL。为了进行优克那非在人肝微粒体中的酶促反应动力学研究,对底物优克那非设置一系列浓度梯度,测得其代谢速率,运用Lineweaver-Burk双倒数作图法计算出酶促反应动力学参数Km和Vmax,计算得到的人肝微粒体催化优克那非代谢的Km值为15.4μmol/L,Vmax值为5.56×10-3μmol/(min mg protein)-1,代谢清除率CLint为3.61×10-4 L(min mg protein)-1。通过分别向孵育体系中加入不同CYP酶亚型的特异性抑制剂,判断优克那非在肝微粒体中代谢的代谢途径,结果显示:抑制剂酮康唑和氟康唑均对优克那非的代谢产生明显的抑制作用,说明优克那非主要通过CYP3A4和CYP2C9代谢,酮康唑对代谢物M1、M2、M3的抑制率接近100%,氟康唑对代谢物M1、M2、M3的抑制率分别为54.7%、33.1%、65.7%。向孵育体系中分别加入一系列浓度梯度的酮康唑和氟康唑溶液,测定代谢产物的生成量,以抑制剂浓度为横坐标,抑制率为纵坐标,绘制抑制曲线,通过GraphPad Prism 5.0软件计算IC50值,测得的酮康唑IC50值为3.24μM,氟康唑IC50值为39.5μM。在人、猴、大鼠、小鼠、比格犬肝微粒体中进行优克那非的代谢稳定性研究,结果显示,优克那非在5个种属的肝微粒体中均发生明显的代谢,其消除半衰期(t1/2)大小为猴SD大鼠CD小鼠人Beagle犬,其中优克那非在人、大鼠和小鼠中的消除半衰期相近,提示大鼠和小鼠可以作为优克那非临床前药代动力学研究模型。通过混合探针底物(Cocktail)法研究优克那非对人肝微粒体中各CYP酶亚型的抑制作用,计算优克那非对各亚型的IC50值,结果显示,优克那非对CYP3A4、CYP2C9、CYP2C19、CYP2D6、CYP2E1亚型均有不同程度的抑制作用,但在正常给药剂量时,优克那非不会引起严重的药物-药物相互作用。通过LC-TOF MS/MS高分辨质谱寻找优克那非的未知代谢产物,结果显示,在五个种属肝微粒体孵育样品及收集的大鼠给药后胆汁和粪便中均检测到了除代谢物M1、M2、M3以外的代谢产物,其分子式为C24H33N5O5S,通过对比其与母药优克那非的二级碎片离子差异,推测其可能是哌嗪环上发生羟基化的代谢产物,而其结构需要NMR手段的验证。
[Abstract]:Youkenafei (Yonkenafil) is a new type of phosphodiesterase (Phosphodiesterase, PDE) V inhibitors, the structure of cyclic guanosine monophosphate (Cyclic Guanosine, monophosphate, cGMP) of five yuan and six yuan of heterocyclic ketones, by researchers in China independent research and development, and has independent intellectual property rights. The Youkenafei pharmacodynamic test results show that Youkenafei inhibited the good selectivity to PDE V, compared with the existing V phosphodiesterase inhibitors vardenafil and sildenafil, which has more obvious advantages. At present, the laboratory has completed the study on pharmacokinetics of Youkenafei in rats and beagle dogs, CYP450 in liver microsomal enzyme is the main enzyme responsible for in vivo drug metabolism, in preclinical pharmacokinetic studies of new drugs, research on the interaction between the drug and the CYP450 enzyme helps to achieve Screening of new drugs, prediction of clinical medication effect and provide a reliable basis for clinical rational use of drugs. In this thesis, on the basis of previous studies, in vitro metabolism of Youkenafei, and metabolites in vivo and were identified. The determination of human liver microsomes (HLMs) and its metabolites were Youkenafei M1 the education system in M2, LC-MS/MS quantitative analysis method of M3 concentration. The incubation time and microsomal protein concentration, substrate concentration Youkenafei optimization, the process of CYP450 catalyzed Youkenafei metabolism can in the optimal incubation conditions were as follows. The optimal incubation conditions: final sterile incubation time is 15min. Liver microsomal protein concentration was 0.5mg/mL, concentration of substrate Youkenafei is 8 g/mL. for Youkenafei in human liver microsome enzymatic kinetics of substrate, excellent It set up a series of concentration gradient, measured the metabolic rate of Lineweaver-Burk, using double reciprocal plot method to calculate the enzymatic kinetic parameters of Km and Vmax in human liver microsomes catalyzed Youkenafei metabolism calculated the value of Km is 15.4 mol/L, Vmax = 5.56 * 10-3 mol/ (min mg protein) -1 metabolism the clearance rate of CLint is 3.61 * 10-4 L (min mg protein) -1. by breeding specific inhibitors with different CYP isoforms in the system to hatch, metabolic pathway, metabolism in liver microsomes to determine Youkenafei showed obviously inhibit inhibitor ketoconazole and fluconazole on Youkenafei metabolism, illustrate the main Youkenafei through CYP3A4 and CYP2C9 metabolism, ketoconazole on metabolites of M1, M2, M3, the inhibition rate is close to 100%, fluconazole on the metabolites of M1, M2, M3 inhibition rates were 54.7%, 33.1%, 65.7%. respectively to the incubation system In a series of concentrations of ketoconazole and fluconazole solution, content determination of metabolites, with the inhibitor concentration as abscissa, the inhibition rate as ordinate, draw the inhibition curve by GraphPad Prism 5 software to calculate the IC50 value, the measured value is 3.24 M IC50 ketoconazole, fluconazole IC50 value is 39.5 M. in human, monkey, rat, mouse, study, metabolic stability of Youkenafei beagle dog liver microsomes showed obvious Youkenafei metabolism occurred in 5 species of liver microsomes, the elimination half-life (t1/2) size of monkey SD rats CD mice Beagle dogs, which Youkenafei in the people, rats and mice in the elimination half-life is similar, suggesting that rats and mice can be used as models for kinetic study Youkenafei preclinical pharmacokinetics. By mixing the probe substrate (Cocktail) method to study the Youkenafei of human liver microsome in various CYP isoforms Inhibition of Youkenafei calculation of the subtypes of IC50, results show that Youkenafei on CYP3A4, CYP2C9, CYP2C19, CYP2D6, inhibition of CYP2E1 subtypes in different degrees, but the dosage in normal, Youkenafei will not cause serious drug drug interactions. Unknown metabolites by LC-TOF, high resolution MS/MS for Youkenafei mass spectrometry results showed that five species in liver microsomes and collected samples of rats after administration of bile and feces were detected in the metabolites of M1, M2, metabolites other than M3, its molecular formula is C24H33N5O5S, with the comparison of the two Youkenafei TK fragment ion difference. Probably is a metabolite of hydroxylation occurred on the piperazine ring, and verify the structure needs to be NMR.

【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R96

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