【作者】 王伟；

【导师】 颜炜群；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2008， 博士

【摘要】 注射用蟾皮总碱为蟾皮提取液的冻干粉针剂型,是中华大蟾蜍阴干全皮提取精制而成的无菌粉针,具有清热解毒、消肿、止痛、提高机体免疫力等功效。本课题创新性的提出了一种新的吲哚类生物碱提取工艺,使提取物中吲哚类生物碱的含量达到90%以上,并按国家一类新药的标准进行了成药性研究--药效学、一般药理学、毒理学研究。并制定了注射用蟾皮总碱的质量标准,为注射用蟾皮总碱的临床实验建立了理论基础。实验结果表明:注射用蟾皮总碱的生产和制备工艺稳定,适合大规模工业化生产。不同浓度注射用蟾皮总碱对人肿瘤细胞增殖呈现出不同程度的抑制作用,浓度为0.4352 ug/ml的注射用蟾皮总碱对人胃癌细胞SGC-7901、人肝癌细胞SMMC-7721和人宫颈癌细胞Hela的抑制率分别为41.41%、62.41%、80.53%,均高于同剂量的华蟾素注射液组的肿瘤抑制率。注射用蟾皮总碱急性毒性实验检测不到明显的急性毒性反应。长期毒性实验结果显示,高剂量、长时间(170 ug/kg、连续三个月)给药后大鼠血液学检查肾脏尿素氮含量为9.94mmol/L,较正常值明显升高,恢复期一个月时尿素氮含量恢复正常。肾脏病理学检查在三个月时表现为肾小管细胞肿胀、变形、境界模糊,胞核消失,细胞坏死。恢复期一个月时肾脏病理学检查已恢复正常。Beagle犬连续高剂量、长时间(52ug/kg、连续三个月)给药后,血液学检查尿素氮含量为5.54mmol/L ,尿常规检查尿液pH值为5.13、尿维生素C含量为0.75mmol/L,尿沉渣镜检(x400)可见7-9个红细胞,与对照组比较,差异明显;病理学检查显示给药三个月时犬肾小管细胞形状不规则,单位面积细胞数量减少,部分胞体皱缩,胞浆透亮度下降,细胞变圆。恢复期1个月后病理检查恢复正常。实验表明注射用蟾皮总碱较大剂量连续用药3个月对大鼠及Beagle犬肾脏功能有影响,恢复期1个月肾脏各项指标恢复正常。各项实验证明,采用新工艺提取的注射用蟾皮总碱质量工艺稳定,药效显著,无明显及长期毒副作用,可进行临床试用。更多还原

【Abstract】 Tumor is one kind of familiar disease, and with the speed up of aging and pollution, it becomes the most serious disease that endangers the people’s health. The death rate of tumor sufferers is a little less than that of cardiopathy. This makes it the second killer of human health.The alkaloids extracted from toad skin are broadly used as one of anti-tumor drugs at present. INj-alkaloids in toad skin is a safe and effective medicine to treat the PLC followed the metastatic tumer of lung .The research of pharmaceutical included pharmaceutical technology and quality standard. The main purpose of this article mainly works over the new Medicine prescription, manufacture technology, quality standard and pharmacology according to the request of the National New Drug Examination and Approval Method.Studied the extract process of preparation, developed an optimal process of extraction with traditional medicated process and modern technique, the method is water extraction and ethanol precipitation ,water washing-out supernumerary substance in polyamide column, collect ethanol elution. It is obey the rule of Chinese Medical, extract the effective component and simplify the process at the same time. Above all, the best abstract technology of Vapour distillation was determined, INj-alkaloids in toad skin quality standard was established, INj-alkaloids in toad skin is steady.On the other hand, according to the guide rules of Chinese medical injection in new medicine evaluation, to make sure this injection is safety, effective and stability, assurance the quality of the injection, and found the base of injection’s fingerprint. The content of one was determined by HPLC. The method is simple, accurate reproducible and can be applied for quality control. Research of the quantity standard has been completed, standard of drug and preparation has been set up, the result show this method is simple and feasible and can guarantee the stability of this preparation.And anti-tumor efficacy and safety of INj-alkaloids in toad skin by acute-term toxicity test has been researched , in vitro pharmacodynamics test, safety test, pharmacology test and long-term toxicity test and so on.Through to acute-term toxicity test and LD50 determination, give the result of clinical medication, determiones the dose of medicine. About 2min’s the drug poisoning mouse starts to appear the active reduction, peak wool, shuts item, walk haltingly, waggle, jitter. The mouse which are poisoned of large dose have a deep and slow breath, weak and limp. Beyond die the body-righting reflex of dead animals doesn’t disappear, the breath from slow to tenuity. Dead mouse have respiration ceases first and cardiac arrest last, which are dissected have no abnormality acquire of organs. The undead ones who are killed 2 weeks later also have no abnormality acquire of organs.The in vitro experiment of pharmacodynamics shows alkaloids of toad skin’s inhibition on tumor cells cultured in vitro using MTT analysis, and tumor control rate via cell appearance observed by inverted microscope. The results indicate that INj-alkaloids in toad skin inhibits evidently the human tumor cells proliferation in vitro and the inhibition depends a certain dose.Estimate the safety of the medicine, results of hepersensibility test, heamolyticus test and BV stimulus test on INj-alkaloids in toad skin show that INj-alkaloids in toad skin has no allergize on guinea pig, no obviously effect of in vitro heamolyticus and agglomeration in rabbit’s red cell, it also has no obviously stimulation on rabbit’s BV and can not cause pathological change of tissues of BV surrounding.The Pharmacology research uses to appraise the INj-alkaloids in toad skin regarding the animal cardiovascular, the respiratory and the nervous system influence. Through pressure transducer, tension transducer, needle-electrode connectting BL-420E biological function experiment system, determine INj-alkaloids in toad skin to take the different time compartment dogs femoral artery blood pressure, breath and the electrocardiogram around separately: observes the medicine with multi-purpose mouse activity recording instrument to the mouse spontaneous activity influence; observes the medicine with Fall-tiltboard method to the mouse coordination exercise influence; recording medicine to mouse pentobarbital sodium hypnosis function influence in subthreshold dose by Righting reflex deprivation. The results show that the anaesthesial dogs’ systolic pressure reduce slightly, but there has not seen the remarkable difference about heart rate, diastic and the respiratory system.Moreover, INj-alkaloids in toad skin which in each dose has no obviously affects to the mouse coordnation exercise, automatic activities and pentobarbital sodium hypnosis function in subthreshold dose. The results indicate that INj-alkaloids in toad skin has certainly reduces the systolic pressure function which depends dose to the dog and it has no obviously affects regarding dogs’ respiratory system and mouse’s nervous system.Results of long-term toxicity test indicate that INj-alkaloids in toad skin has no effect on animal general activity, weight increased, organs’ coefficient, hemogram and function of liver and kidney; every organ has no change of pathohistology. Stop to using this medicine, organs’ coefficient, hemogram and function of liver and kidney of rats also have not been affected, so it is safe on the clinical application.更多还原