【作者】 景秀娟；

【导师】 张兰桐；

【作者基本信息】 河北医科大学 ， 药物分析学， 2008， 硕士

【摘要】 间尼索地平(m-nisoldipine)[化学名称:1, 4-二氢-2, 6-二甲基-4-(3-硝基苯基)-3, 5-吡啶二羧酸甲酯异丁酯]是河北医科大学药学院首次合成的一种新的二氢吡啶类钙拮抗剂。间尼索地平与尼索地平(nisoldipine)为同分异构体,尼索地平用于防治冠心病、高血压和慢性充血性心衰及心源性休克,疗效显著,但是对光不稳定,通过结构改造,间尼索地平不仅保留了尼索地平的药理作用,而且光稳定性也明显增。目前,间尼索地平的含量测定方法主要有铈量法,紫外分光光度法以及高效液相色谱法等。本研究首次采用气相色谱法(GC),气相色谱-质谱联用(GC-MS)法,以尼莫地平(nimodipine)为内标,测定了间尼索地平原料药的含量。药物代谢研究在新药研发中占有很重要的地位,它可以使我们预知候选的新药化合物在体内可能的代谢物及其潜在的毒性,另一方面可以使我们预知在体外有效的药物是否在体内同样有效,因为许多在体外有效的药物在体内疗效很差或无效,这主要是由于这些化合物在体内的吸收和代谢转运等方面的原因所造成的。因此,这一工作可为新药在体内的代谢过程和毒性评价提供重要的依据,同时根据其代谢物的生物活性和毒性,可以合成更为安全有效的候选化合物。本文从体外到体内两个方向对间尼索地平的代谢进行了初步的研究,发现了多个代谢物。通过微生物转化方法得到一转化产物的纯品,并进行了结构确认。对间尼索地平在大鼠体内的代谢途径有了一定的推断。第一部分GC与GC-MS法测定间尼索地平含量目的:建立GC和GC-MS法测定间尼索地平原料药含量的方法,为其质量控制提供依据。方法:1. GC法:(1)确定色谱条件:选择毛细管色谱柱,优化仪器参数,如进样口温度、柱温、检测器温度等,调整流动相流速,确定分析条件。(2)进行方法学考察,测定样品。2. GC-MS法:(1)确定色谱条件,如:色谱柱的型号、进样口温度、柱温、载气流速、进样模式等。(2)确定质谱条件,如气质接口温度、离子源温度、四级杆温度、EI电离能量、检测模式等。(3)进行方法学考察,测定样品含量。结果:1. GC法:(1)色谱条件如下:固定相为100%二甲基硅氧烷的HP-1毛细管柱(50m×320μm×0.52μm),柱温:265℃;进样口温度:295℃;ECD检测器,检测器温度:300℃;载气为高纯氮气,流速:4.0mL·min-1;进样量1μL,分流进样,分流比20:1。(2)该法重复性良好,RSD为0.1%,平均回收率为99.5%,RSD为0.4%(n=9)。3批样品的含量均大于97%。2. GC-MS法:(1)色谱条件:色谱柱DB-1ms毛细管柱(30m×250μm×0.25μm);载气为高纯氦气,流速0.8mL·min-1;升温程序:在200℃的基础上20℃·min-1升温至260℃,恒温5min,然后10℃·min-1升至300℃,恒温3min结束,运行时间15min;进样口温度250℃;不分流进样。(2)质谱条件:气质接口温度250℃;离子源温度230℃;四级杆温度150℃;EI电离方式,电子能量70ev;检测模式为SIM模式;用于定量的特征质谱峰为m/z 266.0,210.0(间尼索地平)和m/z 296.0,254.0(内标尼莫地平),溶剂延迟4min。(3)该法重复性的RSD为0.4%,平均回收率为98.9%,RSD为0.3%(n=9)。结论:本文建立了GC和GC-MS法测定间尼索地平原料药含量的方法,该法准确、灵敏、快速,可用于间尼索地平的含量测定。第二部分间尼索地平体外代谢研究目的:研究间尼索地平在大鼠肝微粒体内的酶促反应动力学;通过肝微粒体温孵、微生物转化、肠道菌厌氧培养等多种方法寻找代谢物,并尽可能的制备转化产物单体。方法:1.酶促反应动力学研究:(1)确定色谱条件及样品处理方法。(2)进行方法学验证。(3)考察时间、蛋白浓度、底物浓度等因素对间尼索地平在大鼠肝微粒体中代谢消除速率的影响。2.肝微粒温孵实验:根据文献报道的方法将间尼索地平加入微粒体温孵液中,温孵一定时间后,处理并采用HPLC-PDA和HPLC-MS分析,确认代谢物。3.微生物转化实验:经过筛选,选出转化能力强的菌株对间尼索地平进行转化,制备转化产物单体,通过MS、NMR等波谱数据对其结构进行确认。4.肠道菌培养:取大鼠粪便,利用肠道菌培养的方法,研究间尼索地平的代谢。5.胃内容物温孵:取新鲜大鼠胃内容物加入间尼索地平进行温孵,采用HPLC-PDA分析。结果:1.酶促反应动力学研究:(1)色谱条件:色谱柱:Welchrom C18柱(4.6mm×250mm×5μm),流动相:乙腈-水(62∶38),流速:1.0mL·min-1,检测波长:237nm,柱温:30℃,进样量:20μL。样品处理方法:取温孵体系1.0mL,加入5mol·L-1氢氧化钠溶液200μL,涡旋20s,再加入乙醚-正己烷(1:1)混合液5mL,涡旋2min,于2850×g离心5min,取上层有机相3mL,37℃N2吹干,残留物用甲醇40μL溶解后经HPLC测定。(2)方法精密度好,日内、日间精密度RSD分别为6.1%和1.6%(n=15),准确度高,回收率为93.0%,RSD为3.8%(n=15)。(3)间尼索地平的消除速率受时间、蛋白浓度、底物浓度等因素的影响,因这些因素的不同而不同。2.肝微粒体温孵实验:在大、小鼠肝微粒体中发现了间尼索地平的多个代谢物,但二者有所不同,说明间尼索地平的代谢存在种属差异。3.微生物转化实验:经筛选,发现雅致小克银汉霉(Cunninghamella elegans,AS 3.1207)对间尼索地平的代谢能力强,重现性好,由此得到一转化产物,确认其结构为1, 4-二氢-2, 6-二甲基-4-(3-硝基苯基)- 3, 5-吡啶二羧酸甲酯-(2-甲基-2-羟基)丙基酯(文中均以字母M表示)。4.从肠道菌培养液中发现4个温孵产物,但空白对照实验也有类似产物,对这些产物是否为肠道菌的转化产物应做进一步的研究。5.胃内容物温孵液中没有发现代谢物。结论:通过微生物转化法得到一转化产物单体,确认了其结构,从肝微粒体温孵实验发现间尼索地平的代谢存在种属差异。第三部分间尼索地平体内代谢研究目的:寻找间尼索地平在大鼠体内代谢物的同时研究间尼索地平及其代谢物在大鼠体内的过程,尽可能解释间尼索地平的代谢途径。方法:收集灌胃给予间尼索地平后大鼠的胃、小肠、肝组织、血液、尿液、胆汁、粪便等生物样品,采用HPLC-PDA分析,以紫外吸收光谱为依据,寻找代谢物,对胆汁、粪便等样品进行HPLC-MS分析。结果:体内生物样品中除肝组织、血液、尿液中没有发现代谢物外,胃、小肠、胆汁、粪便均有所发现。结论:间尼索地平在大鼠体内发生广泛的生物转化,在胆汁中能检测到多个代谢物,粪样中也有3个明显的代谢物色谱峰。经实验发现,间尼索地平的主要代谢场所是肝脏,胆汁和粪是间尼索地平及其代谢物的重要排泄途径。更多还原

【Abstract】 m-Nisoldipine[3-iosbutyl-5methyl1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-pyridine-3, 5dicarboxylate] is a new kind of DHPs which was developed by School of Pharmacy of Hebei Medical University. m-Nisoldipine and nisoldipine which is widely used in clinic to treat coronary artery disease, hypertension, Chronic congestive heart failure and cardiac shock etc. with significant effect are isomerides. After structure modification, m-nisoldipine is not only kept the same pharmacological action of nisoldipine, but also has superior photostability to nisoldipine which is not stable when exposed to light. At present there are several assay methods for m-nisoldipine, such as cerium sulfate method, ultraviolet spectrophotometry, high performance liquid chromatography, and so on. Our present paper described the GC and GC-MS methods for the assay of m-nisoldipine with nimodipine as the internal standard for the first time.The understanding of drug metabolism plays an important role in the development of new drug entities. The studies on drug metabolism can predict the probalble metabolites of a new candidate drug and its potential toxicity, on the other hand, we can foresee whether the studied drug owns the permanent effect, as many candidate drugs have therapeutic effect in vitro but weak pharmacologic action or inefficacy in vivo. The main reasons are the problems about absorption and metabolism of the compounds. Therefore the work pertinent metabolism can provid significant clues for the metabolic process and toxicity evaluation of new drugs, which makes it possible that the much more safer and effective compounds are synthetized.Our present paper described the preliminary research of m-nisodipine’s metabolism from two directions in vitro and in vivo and we found several metabolites. In addition, we got a pure product of m-nisoldipine by microbial transformation and confirmed its structure as 3-(2-methyl-2-hydroxyl)-propyl -5-methyl-1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-pyridine-3, 5-dicarboxylat(ewhich was represented by the letter of M in our paper). At last we got some massage about the metabolic pathway of m-nisoldipine in rat.Part one Studies on the assay of m-nisoldipine by GC and GC-MSObjective: To establish methods for the assay of m-nisoldipine by GC and GC-MS.Methods: 1. GC method: (1) Chromatographic conditions’definition: We selected capillary chromatographic column, optimized equipment parameters, such as injector temperature, column temperature and the detector temperature, and adjusted mobile phase flow rate in order to make sure the analytical conditions. (2) Validated the methodology and determinate the samples. 2. GC-MS method: (1) Chromatographic conditions’definition: such as different chromatographic column, injector temperature, column temperature, flow rate of carrier gas, sample injection mode and so on. (2) Mass spectra conditions’definition: such as interface temperature, ion source temperature, quadr-pole temperature, ionization modus, detection mode, and so on. (3) Validated the methodology and determinate the analytical conditions.Results: 1. GC method: (1) Chromatographic conditions were as follows: capillary column was HP-1 (50m×320μm×0.52μm) and the stationary phase was 100% dimethyl-siloxane. Column temperature and injector temperature were controlled at 265℃and 295℃, respectively. The detector was ECD and the temperature was 300℃. The carrier gas was high purity nitrogen with follow rate of 4.0mL·min-1. Split stream sampling and split ratio 20:1 was used. The injection volume was 1μL. (2) The reproducibility of this method was good with RSD of 0.1% and the average recovery was 99.5% with RSD of 0.4%(n=9). The content of three batches of samples were not less than 97%. 2. GC-MS method. (1) Chromatographic conditions were as follows: a DB-1ms capillary column (30m×250μm×0.25μm) was used. The carrier gas was high purity helium with follow rate of 0.8mL·min-1. The programmed column temperature was set as follows: the intial temperature was kept at 200℃, then was raised to 260℃at 20℃·min-1 followed by holding 5min and continually went up to 300℃at 10℃·min-1, and subsequently sustained for 3min, the whole running time was 15min. Injector temperature was 250℃and the sample injection form was spitless. (2) Mass spectra conditions: Interface temperature, ion source temperature and quadr-pole temperature were 250℃, 230℃and 150℃, respectively. Electro-ionization was EI with electron energy of 70ev. Analysis was carried out in SIM mode at m/z 266.0, 210.0 for m-nisoldipine and m/z 296.0,254.0 for internal standard nimoldipine. (3) The RSD for reproducibility of this method was 0.4% and the average recovery was 98.9% with RSD of 0.3%(n=9).Conclusion: The methods we established are proved to be accurate, sensitive and convenient and can be used for the determination of m-nisoldipine, which provided some guideline for qulity control.Part two Studies on metabolism of m-nisoldipine in vitroObjectives: To study the enzymatic reaction kinetics of m-nisoldipine; to find its metabolites by multiple means, such as incubation of liver microsome, microbial transformation, anaerobic culture of intestinal tract bacteria, and prepare pure metabolite as far as possible.Methods: 1. Studies on the enzymatic reaction kinetics of m-nisoldipine: (1) Chromatographic conditions and sample’s disposal methods’definition. (2) To validate the methodology. (3) To inspect the effect factor of m-nisoldipine’s metabolism rate, such as time, concentration of protein and the original concentration substrate. 2. Liver microsome experiment: m-nisoldipine was added to the incubation liquid which was prepared according to the pertinent literature, after some time, the incubation culture was treated and analyzed by HPLC-PDA and HPLC-MS. 3. Microbial transformation experiment: firstly, we selected a robust strain for the transformation of m-nisoldipine by screening seven kinds of strain, then we got a pure trasformation product and confirmed its structure by its MS and NMR data. 4. Culture solution of intestinal bacteria were produced from rat feces and then incubated with m-nisoldipine to find whether there were metabolites. 5. Fresh content in rat stomach and m-nisoldipine were incubated together, then the mixture was analyzed by HPLC-PDA.Results: 1. Studies on the enzymatic reaction kinetics of m-nisoldipine. (1) Chromatographic conditions: column was Welchrom C18 (4.6mm×250mm×5μm). Mobile phase was acetonitrile-water (62:38) with the flow rate of 1.0mL·min-1. Detect wavelength was set at 237nm and column temperature was controlled at 30℃. The injection volumn was 20μl. Sample treatment method: 200μL natrium hydroxydatum solution was added into 1.0mL incubation system and then the mixture was vortexed for 20s. After that 5mL ethylether-n-hexane was spiked with the mixture and vortexed for another 2min. The new mixture was centrifugated for 5min at 2850×g, from which 3mL organic phase was took out. After the organic phase being dried by N2 at 37℃, the residue was resolved by 40μL methanol and determined by HPLC. (2) The method had good precision with RSD of 6.1% and 1.6%(n=15) for intra-day and inter-day RSD. The accuracy (93.0%) for the method was high and the RSD was 3.8%(n=15). (3) The elimination rate of m-nisoldipine was different with the different time, protein concentration and original concentration of substrate. 2. Liver microsome experiment: we found several metabolites in the liver microsome for both rat and mouse, but there was difference between the two, which demonstrated the species difference of m-nisoldipine’s metabolism. 3. Microbial transformation experiment: after screening, Cunninghamella elegans , AS 3.1207 was selected to transform m-nisoldipine for its potential metabolic capability and good reproducibility. We got a metabolite of m-nisoldipine from AS 3.1207 whose chemical name was 3-(2-methyl-2-hydroxyl)-propyl -5-methyl-1, 4-dihydro-2, 6-dimethyl-4-(3-nitrophenyl)-pyridine -3, 5-dicarboxylate. 4. We fonud 4 new products in both culture solution of intestinal bacteria and blank control experiment, which made it difficult to decide whether they were metabolites of m-nisoldipne or not. 5. We found no metabolites in the content of rat stomach.Conclusions: We got a pure product by microbial transformation and confirmed its structure. From liver microsome experiment we found there was species difference for m-nisoldipine’s metabolism. Part three Studies on metabolism of m-nisoldipine in vivoObjective: To study the process of m-nisoldipine and its metabolites in rat in order to illuminate the metabolic pathway of m-nisoldipine.Methods: After taking orally m-nisoldipine, blood, urine, bile, feces, contents of stomach and contents of intestine were pretreated and analyzed by HPLC-PDA. The peaks of m-nisoldipine and its metabolites were identified by comparing their UV spectra. The bile and feces were also analyzed by HPLC-MS.Result: Except hepatic tissue, blood and urine, metablites were found in gastrointestinal tract, bile and feces.Conclusion: m-Nisoldipine was transformed extensively in rat body, metabolites were multiple. The main metabolism site was liver and the principal excretion pathways for m-nisoldipine and its metabolites were bile and feces.更多还原