本文關(guān)鍵詞：優(yōu)克那非的體內(nèi)外代謝研究　出處：《吉林大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 優(yōu)克那非 細(xì)胞色素P450酶 代謝穩(wěn)定性 “Cocktail”探針底物法 代謝產(chǎn)物鑒定

【摘要】：優(yōu)克那非(Yonkenafil)是一種新型磷酸二酯酶(Phosphodiesterase,PDE)V抑制劑,其結(jié)構(gòu)為環(huán)磷酸鳥苷(Cyclic Guanosine monophosphate,cGMP)的五元并六元雜環(huán)酮類,由我國科研工作者獨(dú)立研發(fā),并具有自主知識產(chǎn)權(quán)。前期對優(yōu)克那非藥效學(xué)試驗(yàn)結(jié)果表明,優(yōu)克那非對PDE V具有很好的選擇性抑制作用,相比于現(xiàn)有的磷酸二酯酶V抑制劑類藥物伐地那非和西地那非,其具有更明顯的優(yōu)勢。目前,本實(shí)驗(yàn)室已完成對優(yōu)克那非在大鼠和比格犬中的藥代動力學(xué)研究,而存在于肝微粒體中的CYP450酶是負(fù)責(zé)機(jī)體內(nèi)藥物代謝的主要酶系,在新藥的臨床前藥代動力學(xué)研究中,對藥物與CYP450酶之間相互作用的研究有助于實(shí)現(xiàn)對新藥的篩選、對臨床用藥效果的預(yù)測及為臨床合理用藥提供可靠的依據(jù)。因此本論文在之前的研究基礎(chǔ)上,對優(yōu)克那非進(jìn)行體外代謝研究,并對體內(nèi)外的代謝產(chǎn)物進(jìn)行鑒定。本文建立了測定人肝微粒體(HLMs)孵育體系中優(yōu)克那非及其代謝物M1、M2、M3濃度的LC-MS/MS定量分析方法。通過對孵育時間、肝微粒體蛋白濃度、底物優(yōu)克那非濃度的優(yōu)化,使CYP450酶催化優(yōu)克那非代謝的過程能夠在最佳的孵育條件下進(jìn)行。最終確定的最佳孵育條件如下:孵育時間為15min,體系中肝微粒體蛋白濃度為0.5mg/mL,底物優(yōu)克那非濃度為8μg/mL。為了進(jìn)行優(yōu)克那非在人肝微粒體中的酶促反應(yīng)動力學(xué)研究,對底物優(yōu)克那非設(shè)置一系列濃度梯度,測得其代謝速率,運(yùn)用Lineweaver-Burk雙倒數(shù)作圖法計(jì)算出酶促反應(yīng)動力學(xué)參數(shù)Km和Vmax,計(jì)算得到的人肝微粒體催化優(yōu)克那非代謝的Km值為15.4μmol/L,Vmax值為5.56×10-3μmol/(min mg protein)-1,代謝清除率CLint為3.61×10-4 L(min mg protein)-1。通過分別向孵育體系中加入不同CYP酶亞型的特異性抑制劑,判斷優(yōu)克那非在肝微粒體中代謝的代謝途徑,結(jié)果顯示:抑制劑酮康唑和氟康唑均對優(yōu)克那非的代謝產(chǎn)生明顯的抑制作用,說明優(yōu)克那非主要通過CYP3A4和CYP2C9代謝,酮康唑?qū)Υx物M1、M2、M3的抑制率接近100%,氟康唑?qū)Υx物M1、M2、M3的抑制率分別為54.7%、33.1%、65.7%。向孵育體系中分別加入一系列濃度梯度的酮康唑和氟康唑溶液,測定代謝產(chǎn)物的生成量,以抑制劑濃度為橫坐標(biāo),抑制率為縱坐標(biāo),繪制抑制曲線,通過GraphPad Prism 5.0軟件計(jì)算IC50值,測得的酮康唑IC50值為3.24μM,氟康唑IC50值為39.5μM。在人、猴、大鼠、小鼠、比格犬肝微粒體中進(jìn)行優(yōu)克那非的代謝穩(wěn)定性研究,結(jié)果顯示,優(yōu)克那非在5個種屬的肝微粒體中均發(fā)生明顯的代謝,其消除半衰期(t1/2)大小為猴SD大鼠CD小鼠人Beagle犬,其中優(yōu)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為優(yōu)克那非臨床前藥代動力學(xué)研究模型。通過混合探針底物(Cocktail)法研究優(yōu)克那非對人肝微粒體中各CYP酶亞型的抑制作用,計(jì)算優(yōu)克那非對各亞型的IC50值,結(jié)果顯示,優(yōu)克那非對CYP3A4、CYP2C9、CYP2C19、CYP2D6、CYP2E1亞型均有不同程度的抑制作用,但在正常給藥劑量時,優(yōu)克那非不會引起嚴(yán)重的藥物-藥物相互作用。通過LC-TOF MS/MS高分辨質(zhì)譜尋找優(yōu)克那非的未知代謝產(chǎn)物,結(jié)果顯示,在五個種屬肝微粒體孵育樣品及收集的大鼠給藥后膽汁和糞便中均檢測到了除代謝物M1、M2、M3以外的代謝產(chǎn)物,其分子式為C24H33N5O5S,通過對比其與母藥優(yōu)克那非的二級碎片離子差異,推測其可能是哌嗪環(huán)上發(fā)生羥基化的代謝產(chǎn)物,而其結(jié)構(gòu)需要NMR手段的驗(yàn)證。
[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.

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R96

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