本文選題：藥物代謝 + 間尼索地平��； 參考：《河北醫(yī)科大學(xué)》2014年碩士論文

【摘要】：間尼索地平（m-nisoldipine）作為新型二氫吡啶類鈣離子通道拮抗劑，主要用于高血壓和心臟病的防治。以往研究表明其多以代謝物形式排出體外。肝臟作為藥物代謝的重要器官，是多數(shù)Ⅰ相和Ⅱ相反應(yīng)的發(fā)生場(chǎng)所，細(xì)胞色素P450酶是參與藥物代謝和生物活化的主要酶類，了解藥物與CYP450酶的作用關(guān)系能夠?yàn)樗幬镒饔脵C(jī)制的研究及聯(lián)合用藥提供理論依據(jù)。 本試驗(yàn)利用液質(zhì)聯(lián)用（HPLC-MS/MS）技術(shù)，采用人肝微粒體溫孵、CYP450重組酶溫孵及化學(xué)抑制劑試驗(yàn)，對(duì)參與間尼索地平生物轉(zhuǎn)化的CYP450酶亞型進(jìn)行鑒別。對(duì)灌胃給藥大鼠組織中的間尼索地平及其代謝產(chǎn)物進(jìn)行分析，為進(jìn)一步理解其作用機(jī)制提供依據(jù)和技術(shù)支持。采用大鼠肝微粒體體外溫孵及大鼠體內(nèi)代謝的方法，初步探究間尼索地平Ⅱ相代謝中的葡萄糖醛酸結(jié)合反應(yīng)。對(duì)間尼索地平對(duì)映體在人肝微粒體中的酶促反應(yīng)動(dòng)力學(xué)進(jìn)行研究，優(yōu)化反應(yīng)條件，并比較兩者的酶促反應(yīng)動(dòng)力學(xué)參數(shù)。 第一部分間尼索地平在人源CYP450酶各亞型中的代謝研究 目的：通過人肝微粒體溫孵、CYP450重組酶溫孵和化學(xué)抑制劑試驗(yàn)，對(duì)參與間尼索地平生物轉(zhuǎn)化的CYP450酶亞型進(jìn)行鑒別。 方法：將溫孵樣品分別在EMS-IDA-EPI、 MRM-IDA-EPI和PREC-IDA-EPI模式下進(jìn)行檢測(cè)，分析反應(yīng)生成的間尼索地平代謝產(chǎn)物。 人肝微粒體溫孵試驗(yàn)，將間尼索地平與人肝微粒體進(jìn)行體外溫孵，，應(yīng)用HPLC-MS/MS技術(shù)檢測(cè)代謝產(chǎn)物，獲得二級(jí)質(zhì)譜碎片信息，推測(cè)代謝產(chǎn)物結(jié)構(gòu)。 化學(xué)抑制劑試驗(yàn)，CYP450特異性抑制劑與人肝微粒體溫孵30min后加入間尼索地平，一定時(shí)間后測(cè)定體系中代謝產(chǎn)物生成量，并與未加入抑制劑的陰性對(duì)照溶液比較。 CYP450重組酶溫孵試驗(yàn)，將間尼索地平與CYP450重組酶進(jìn)行體外溫孵，尋找代謝產(chǎn)物并比較各亞型酶中間尼索地平的代謝情況。 結(jié)果：在人肝微粒體溫孵體系中共發(fā)現(xiàn)8種代謝產(chǎn)物，其中間尼索地平氧化脫氫、3位側(cè)鏈羥基化及兩者結(jié)合的3種代謝產(chǎn)物生成量遠(yuǎn)高于其他5種代謝產(chǎn)物。 化學(xué)抑制劑試驗(yàn)中發(fā)現(xiàn)，鹽酸噻氯匹定（CYP2C19抑制劑）能夠明顯抑制3位側(cè)鏈羥基化產(chǎn)物生成，而酮康唑（CYP3A4抑制劑）對(duì)氧化脫氫產(chǎn)物的抑制作用明顯。 CYP450重組酶試驗(yàn)中發(fā)現(xiàn)，CYP2C19和CYP3A4對(duì)間尼索地平的催化活性明顯高于其他4種亞型，CYP2C19主要催化生成羥基化產(chǎn)物，CYP3A4對(duì)氧化反應(yīng)的催化能力強(qiáng)。 結(jié)論：化學(xué)抑制劑試驗(yàn)及CYP450重組酶試驗(yàn)結(jié)果一致表明，CYP2C19和CYP3A4是人肝微粒體中參與間尼索地平生物轉(zhuǎn)化的主要酶亞型。 第二部分HPLC-MS/MS法研究大鼠組織中間尼索地平及其代謝產(chǎn)物 目的：通過對(duì)大鼠6種組織樣品中間尼索地平代謝產(chǎn)物的分析，總結(jié)藥物代謝規(guī)律，為進(jìn)一步闡明其作用機(jī)制提供依據(jù)。 方法：雄性SD大鼠灌胃給予間尼索地平1h后，取心、肝、脾、肺、腎和腦組織，加入生理鹽水制成勻漿，經(jīng)乙醚萃取后應(yīng)用HPLC-MS/MS對(duì)樣品進(jìn)行分析。結(jié)合本實(shí)驗(yàn)室前期研究中已經(jīng)進(jìn)行結(jié)構(gòu)確證的代謝產(chǎn)物，通過比較保留時(shí)間和二級(jí)質(zhì)譜碎片信息推測(cè)所測(cè)得代謝產(chǎn)物的結(jié)構(gòu)。 結(jié)果：在6種組織樣品中均檢測(cè)到間尼索地平的代謝產(chǎn)物，其中在肝組織中發(fā)現(xiàn)9種代謝產(chǎn)物，且含量較高；在脾和腎組織中發(fā)現(xiàn)5種代謝產(chǎn)物；在心和肺組織中發(fā)現(xiàn)4種代謝產(chǎn)物；在腦組織中僅檢測(cè)到2種代謝產(chǎn)物且含量很低。 結(jié)論：間尼索地平在生物體內(nèi)極易發(fā)生生物轉(zhuǎn)化而生成代謝產(chǎn)物，肝臟是其代謝的主要場(chǎng)所，代謝途徑主要為吡啶環(huán)氧化脫氫、吡啶環(huán)3位側(cè)鏈羥基化及酯鍵的水解。 第三部分間尼索地平Ⅱ相代謝葡萄糖醛酸化反應(yīng)的初步探究 目的：通過對(duì)大鼠肝微粒體體外溫孵樣品和膽汁、尿液的分析，初步探究間尼索地平Ⅱ相代謝的葡萄糖醛酸化反應(yīng)。 方法：向體系中加入U(xiǎn)DPGA、丙甲菌素、MgCl2等輔酶，將間尼索地平與大鼠肝微粒體進(jìn)行體外溫孵，大鼠灌胃給予間尼索地平后收集36h內(nèi)膽汁和尿液。應(yīng)用HPLC-MS/MS技術(shù)對(duì)生物樣品進(jìn)行分析，在EMS-IDA-EPI、MRM-IDA-EPI和PREC-IDA-EPI模式下檢測(cè)間尼索地平的葡萄糖醛酸代謝產(chǎn)物。 結(jié)果：通過與空白樣品比較，上述樣品中未能檢測(cè)到葡萄糖醛酸代謝產(chǎn)物，膽汁和尿液中只檢測(cè)到Ⅰ相代謝物。 結(jié)論：間尼索地平僅二氫吡啶環(huán)上仲胺含活潑H可與葡萄糖醛酸結(jié)合，但吡啶環(huán)極易氧化失去活潑H，同時(shí)試驗(yàn)中的反應(yīng)條件、液相色譜-質(zhì)譜條件也可能影響試驗(yàn)結(jié)果。因此，在今后的試驗(yàn)中，應(yīng)盡可能優(yōu)化試驗(yàn)條件，以進(jìn)一步探明間尼索地平的葡萄糖醛酸結(jié)合反應(yīng)。 第四部分間尼索地平對(duì)映體在人肝微粒體中的酶促反應(yīng)動(dòng)力學(xué)研究 目的：研究間尼索地平對(duì)映體在人肝微粒體中的酶促反應(yīng)動(dòng)力學(xué)，計(jì)算其酶促反應(yīng)動(dòng)力學(xué)參數(shù)，并對(duì)R型和S型間尼索地平的酶促反應(yīng)動(dòng)力學(xué)過程進(jìn)行比較。 方法：以尼莫地平為內(nèi)標(biāo)，Sapphire C18色譜柱（4.6mm×150mm，5μm），乙腈-0.1%甲酸水（90:10），等度洗脫，電噴霧離子源（ESI源），負(fù)離子模式下MRM監(jiān)測(cè)，分別測(cè)定人肝微粒體體外溫孵后R型、S型間尼索地平含量。采用Linewrave-Burk作圖法處理數(shù)據(jù)，計(jì)算酶促動(dòng)力學(xué)參數(shù)Km和Vmax。 結(jié)果：間尼索地平對(duì)映體在人肝微粒體中最佳溫孵時(shí)間為30min，蛋白濃度為1mg·mL-1，底物濃度為25μmol·L-1。R-間尼索地平Km=79.20μmol·L-1，Vmax=2.727μmol·(min·mg protein)-1；S-間尼索地平Km=83.8μmol·L-1，Vmax=2.892μmol·(min·mg protein)-1。 結(jié)論：該方法簡(jiǎn)單、可靠，適用于間尼索地平的體外代謝研究，R型和S型間尼索地平的酶促反應(yīng)動(dòng)力學(xué)過程無明顯差異。
[Abstract]:M-nisoldipine is a new two hydropyridine calcium channel antagonist, which is mainly used in the prevention and treatment of hypertension and heart disease. Previous studies showed that it was expelled in the form of metabolites. The liver, as an important organ for drug metabolism, is the place where the majority of phase I and II are opposite. Cytochrome P450 enzyme is involved in the drug. Understanding the relationship between drugs and CYP450 enzymes can provide a theoretical basis for the research of drug action mechanism and the combination of drugs.
In this experiment, we used liquid mass spectrometry (HPLC-MS/MS) technique to identify the CYP450 enzyme subtypes of the biotransformation of nisodipine by using the temperature hatching of human liver particles, incubation of CYP450 recombinant enzyme and chemical inhibitor test, and the analysis of the nisodipine and its metabolites in the rat tissues of gavage to further understand the mechanism of its action. To provide basis and technical support. The methods of incubating rat liver microsomes in vitro and metabolism in rats were used to explore the glucuronic acid binding reaction in the phase II metabolism of nisoxdipine. The enzyme reaction kinetics of the enantiomer in human liver microsomes was studied, the reaction conditions were optimized, and the enzymatic reaction of the two was compared. Dynamic parameters.
Part one metabolism of nisodipine in human CYP450 subtypes
Objective: to identify CYP450 enzyme subtypes involved in the bioconversion of nicardipine through incubation of human liver microparticles, incubation of CYP450 recombinase and chemical inhibitor test.
Methods: the samples were incubated in EMS-IDA-EPI, MRM-IDA-EPI and PREC-IDA-EPI mode respectively, and the metabolites of the reaction were analyzed.
The temperature incubation experiment of human liver microparticles was incubated with the human liver microsomes in vitro, and the metabolites were detected by HPLC-MS/MS technique, and the two level mass spectra of mass spectra were obtained, and the structure of the metabolites was speculated.
The chemical inhibitor test, the CYP450 specific inhibitor and the body temperature of the human liver were incubated with the temperature of the human liver microparticle for 30min. After a certain time, the amount of metabolites in the system was measured and compared with that of the negative control solution, which was not added to the inhibitor.
CYP450 recombinant enzyme incubated in vitro and incubated between nisodipine and CYP450 recombinant enzyme in vitro to find metabolites and compare the metabolism of nisopodipine in the middle of each subtype.
Results: 8 kinds of metabolites were found in the body temperature Incubating System of human liver particles, including the oxidative dehydrogenation of nippine, the 3 side chain hydroxylation and the combination of the 3 kinds of metabolites were much higher than the other 5 kinds of metabolites.
In the chemical inhibitor test, it was found that tilopidine hydrochloride (CYP2C19 inhibitor) could obviously inhibit the formation of 3 side chain hydroxylation products, while ketoconazole (CYP3A4 inhibitor) inhibited the oxidative dehydrogenation products.
In the CYP450 recombinant enzyme test, it was found that the catalytic activity of CYP2C19 and CYP3A4 on the inter nisodipine was significantly higher than that of the other 4 subtypes. CYP2C19 mainly catalyzed the formation of hydroxylation products, and CYP3A4 had a strong catalytic ability to the oxidation reaction.
Conclusion: the results of chemical inhibitor test and CYP450 recombinant enzyme test show that CYP2C19 and CYP3A4 are the main subtypes of biotransformation of nisisodipine in human liver microsomes.
The second part is to study the relationship between nndipine and its metabolites in rat tissues by HPLC-MS/MS.
Objective: through the analysis of the metabolites of the 6 tissue samples in the middle of the rat, the regulation of drug metabolism was summarized and the basis for further clarifying the mechanism of its action was provided.
Methods: after the male SD rats were fed with neldipine 1H, the heart, liver, spleen, lung, kidney and brain tissue were taken into the homogenate, and the homogenate was made by adding physiological saline. After the ether extraction, the samples were analyzed with HPLC-MS/MS. The metabolites confirmed by the structure in the previous study in this laboratory were compared, and the retention time and the two grade mass spectrometry fragment letter were compared. The structure of the metabolites was measured by the interest speculates.
Results: the metabolites of interdipine were detected in 6 tissue samples, of which 9 metabolites were found in the liver tissue with high content; 5 metabolites were found in the spleen and kidney tissue; 4 metabolites were found in the heart and lung tissues; only 2 metabolites were detected in the brain tissue and the content was very low.
Conclusion: biotransformation of nxdipine in organism is very easy to produce biotransformation and produce metabolites. The liver is the main place for its metabolism. The main metabolic pathways are oxidative dehydrogenation of pyridine ring, 3 side chain hydroxylation of pyridine ring and hydrolysis of ester bond.
A preliminary study of third phase naloxidipine II metabolism glucuronization reaction
Objective: To explore the glucuronization reaction of phase II metabolism of nicoslodipine by analyzing the samples and bile from rat liver microsomes incubated in vitro.
Methods: UDPGA, methyloxin, MgCl2 and other coenzyme were added into the system to incubate the inter nosdipine with rat liver microsomes in vitro. The rats were given the bile and urine in 36h after gavage, and the biological samples were analyzed by HPLC-MS/MS, and the nnso was detected in the EMS-IDA-EPI, MRM-IDA-EPI and PREC-IDA-EPI modes. A glucuronic acid metabolite of the horizon.
Results: glucuronide metabolites were not detected in the above samples compared with blank samples. Only phase I metabolites were detected in bile and urine.
Conclusion: the active H in the two hydropyridine ring is only active with glucuronic acid, but the pyridine ring is very easy to oxidize and lose active H. At the same time, the conditions of the reaction in the test and the liquid chromatography-mass spectrum condition may also affect the test results. Therefore, in the future experiment, we should optimize the test conditions as far as possible to further explore the intern Nile. The glucuronic acid binding reaction of the horizon.
Kinetics of enantiomeric reaction of fourth nisodipine enantiomers in human liver microsomes
Objective: To study the enzyme catalyzed kinetics of nisedipine enantiomers in human liver microsomes, to calculate the kinetic parameters of the enzyme reaction, and to compare the kinetics of the enzymatic reaction between R and S type nisodipine.
Methods: with nimodipine as the internal standard, Sapphire C18 column (4.6mm x 150mm, 5 mu m), acetonitrile -0.1% formate water (90:10), ISO elution, electrospray ion source (ESI source) and MRM monitoring under negative ion mode, the content of R and S type nxodipine was measured in vitro after incubating in human liver microsomes, and the data were processed by Linewrave-Burk mapping method and enzymatic method was used to calculate enzyme promotion. Dynamic parameters Km and Vmax.
Results: the best incubation time of the enantiomers in human liver microsomes was 30min, the concentration of the protein was 1mg mL-1, the concentration of the substrate was 25 mu mol / L-1.R-, Km=79.20 Mu mol / L-1, and Vmax=2.727 micron mol.
Conclusion: the method is simple, reliable and suitable for the in vitro metabolism study of nicardipine. There is no significant difference in the enzymatic reaction kinetics between nimodipine and type R S.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R965;O657.63

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