常用抗肿瘤药物体内监测方法及环磷酰胺与酮康唑相互作用研究

发布时间：2018-03-03 15:35

本文选题：抗肿瘤药物　切入点：UHPLC-MS/MS　出处：《第二军医大学》2014年硕士论文　论文类型：学位论文

【摘要】：肿瘤是临床上致死率最高的疾病之一，临床上常用的治疗方案有化疗，手术以及放射治疗，其中化疗治疗方案较为成熟。传统上，根据常用抗肿瘤药物来源以及其作用机制可以分为如下几类：烷化剂、抗代谢物类、抗生素、植物激素等。近来，随着制药技术的发展，抗肿瘤药物的靶向性得以提高，但是其治疗引起的毒副作用以及不同病人个体临床药效的差异却不可避免。根据药物的药物代谢动力学研究，可以得知药物的临床差异性表现很大一部分由原型药物在体内的毒性代谢产物或者活性代谢产物决定的，因此对这些原型药物以及其具有代表性的代谢产物进行定量分析，了解其及时的血药浓度，有助于我们了解特定药物在体内的代谢情况，从而对药物的合理使用进行更加权威科学的分析，促进个体化用药的发展。 TDM （therapeutic drug monitoring），即临床血药浓度监测，是以药代动力学原理为指导，借助气相色谱、液相色谱或者气质（液质）联用等常用分析手段，定量分析体内原型药物及其代谢产物的血药浓度，从而评价药物疗效或调整给药方案，达到临床上合理化用药。抗肿瘤化疗药物具有治疗窗窄，在不同患者体内代谢差异性较大，易发生药物相互作用等特点，因此对其原型及代谢产物进行血药浓度监测有很大的临床意义。本课题中研究主要分为两大部分，第一部分为借助于UHPLC-MS/MS（ultrahigh performance liquid chromatography tandem mass spectrometry）对几种常用抗肿瘤药及其主要代谢产物进行TDM研究，他们如下，吉西他滨和二氟脱氧脲苷，异长春花碱和脱乙酰基异长春花碱，依托泊苷和脱甲基依托泊苷，UHPLC-MS/MS（ultra highperformance liquid chromatography tandem mass spectrometry）是近来兴起的一项热门技术，它的优势体现在快速，分离度高，样本用量少上面。方法学的验证内容包括特异性，线性，提取回收率和基质效应，稳定性等，前处理方法则采用了经典的蛋白沉淀法（三倍体积的含0.1%甲酸的乙腈或者甲醇溶液作为沉淀剂）。方便快捷的样品前处理方法，较短的洗脱时间以及较好的色谱分离保证了该方法适用于临床上的高通量分析。第二部分从环磷酰胺的毒副产物代谢途径出发，为避免环磷酰胺治疗引起的神经毒副反应，而进行的环磷酰胺和酮康唑体内外相互作用的研究。环磷酰胺是一种常见的酰胺氮芥类细胞毒作用药物。临床上其神经毒性主要是由脱氯乙基代谢途径引起的，该代谢途径为部分体内游离的环磷酰胺经过CYP3A4作用，产生脱氯乙基环磷酰胺和氯乙醛。其中氯乙醛被认为是环磷酰胺临床上神经毒性作用的生物标记物。临床上，通过有效地影响CYP酶家族的活性而实现药物间的相互作用是常用的联合用药策略之一，，针对环磷酰胺的药物相互作用研究，尝试了潜在CYP3A4抑制剂如氟康唑、磺胺甲VA唑和酮康唑等常用抗真菌药物，最终选取了酮康唑作为试药。酮康唑是经典的CYP3A4抑制剂，已知的药物相互作用模型中，酮康唑能够减少咪达唑仑经CYP3A4介导的代谢产物2-羟基咪达唑仑的生成。类似的，我们进行了酮康唑与环磷酰胺的体内外相互作用研究，希望通过酮康唑抑制CYP3A4的活性而阻断环磷酰胺对应的毒副产物生成途径。体外酶孵育环境中，借助于人肝微粒体模拟人体内药物代谢环境，成功地验证了环磷酰胺的脱氯乙基过程，同时在酮康唑作为试药时，脱氯乙基环磷酰胺的生成量也相应减少，说明酮康唑在体外有效地抑制了环磷酰胺的脱氯乙基过程。接着在大鼠体内进一步验证了这一相互作用，UHPLC-MS/MS对大鼠血药浓度的监测结果显示，与空白实验组对比（静脉注射环磷酰胺10mg/kg，酮康唑用量为0mg/kg），联合用药组（静脉注射环磷酰胺10mg/kg，酮康唑用量分别为10，20和40mg/kg）脱氯乙基环磷酰胺对应的AUC0-∞（药时曲线面积）值降低51.19%以上。换言之，当酮康唑和环磷酰胺两药合用时，环磷酰胺在体内经CYP3A4介导的脱氯乙基代谢途径被酮康唑很大程度的阻断。这一结果提示我们环磷酰胺联合CYP3A4抑制剂例如酮康唑时，会大大减少毒性产物脱氯乙基环磷酰胺的生成，这提示我们可以从代谢角度而对临床上环磷酰胺的毒副作用进行治疗干预，提出更加合理化的治疗方案。当然临床用药相当复杂多变，很多临床病症并非只是两药合用的情形；并且参与药物的代谢酶并非只有一种，这里我们只是对占主要地位的CYP3A4进行讨论，也不能反应出药物代谢性相互作用的全貌。但是，本课题提出的基于细胞模型的体外和基于动物模型的体内相互作用研究，不失为临床用药提供了合理用药研究思路。
[Abstract]:The tumor is one of the highest mortality rate of clinical treatment, commonly used in clinical chemotherapy, surgery and radiotherapy, and chemotherapy in the treatment program is more mature. Traditionally, according to the commonly used antitumor drug sources and its mechanism is divided into the following categories: alkylating agent, anti metabolites, antibiotics, hormones. Recently, with the development of pharmaceutical technology, anti-tumor drug targeting can be improved, but the treatment induced toxicity and clinical efficacy of different individual differences is inevitable. According to the study of pharmacokinetics of drugs, the clinical difference of drug performance in large part by the prototype of drugs in vivo the toxic metabolites or active metabolite determined, so the prototype drug and its representative metabolites quantitative analysis, understand its timely Blood concentration helps us understand the metabolism of certain drugs in the body, so that we can make a more authoritative and scientific analysis of the rational use of drugs, and promote the development of personalized medicine.
TDM (therapeutic drug monitoring), the clinical blood concentration monitoring in pharmacokinetics principle, by means of gas chromatography, liquid chromatography (LC) or temperament combined with other commonly used analysis methods, quantitative analysis of blood concentration of the drug and its metabolites in vivo prototype, so as to evaluate drug efficacy or adjust to the treatment plan, to clinical rational medication. Anticancer drugs with narrow therapeutic window in different patients with metabolic characteristics of large, prone to drug interactions, so the prototype and metabolites have great clinical significance of blood concentration monitoring. The study mainly consists of two parts. The first part is based on the UHPLC-MS/MS (ultrahigh performance liquid chromatography tandem mass spectrometry) TDM research of several kinds of antineoplastic drugs and its metabolites, they are as follows, Gemcitabine and two fluorodeoxyglucose urea glycosides, ISO Changchun flower base and deacetylation of Changchun flower base, Tuo Jiaji and etoposide etoposide, UHPLC-MS/MS (ultra highperformance liquid chromatography tandem mass spectrometry) is a popular technology recently, it embodies the advantages of fast, high degree of separation, less sample quantity. The content and method of verification the specificity, linearity, recovery rate and matrix effect, stability, pretreatment methods using classical protein precipitation method (containing 0.1% formic acid acetonitrile or three times the volume of methanol solution as the precipitating agent). Pretreatment method of convenient sample, eluting shorter time and better the chromatographic separation ensures high throughput analysis this method is applicable to clinical practice. The second part embarks from the side product of metabolism caused by cyclophosphamide, to avoid cyclophosphamide treatment The neurotoxic side effects and the study of the interaction of cyclophosphamide and ketoconazole in vitro and in vivo.
Cyclophosphamide is a common amide nitrogen mustard cytotoxic drugs. The clinical neurotoxicity is mainly caused by the dechlorination of ethyl metabolic pathway, the metabolic pathway for body free cyclophosphamide after CYP3A4, produced the dechlorination of cyclophosphamide and ethyl Trichloroacetaldehyde. Trichloroacetaldehyde is considered to be the biomarker of neurotoxic effects of cyclophosphamide clinical. Clinically, and drug interactions is one of the commonly used combination strategy through the effective effect of CYP enzyme family activity for cyclophosphamide drug interaction research, try to potential CYP3A4 inhibitors such as fluconazole, ketoconazole, miconazole and sulfamethoxazole VA commonly used antifungal agents, chose ketoconazole as a reagent. The CYP3A4 inhibitor ketoconazole is the classic models of known drug interactions, ketoconazole can reduce midazolam by CYP3A The 4 generation mediated by metabolites of 2- hydroxymidazolam. Similarly, we conducted a study the interaction of ketoconazole and cyclophosphamide in vitro and in vivo, to inhibit CYP3A4 activity of ketoconazole by blocking toxic byproducts. The corresponding ways of generating cyclophosphamide enzyme incubated in vitro in the environment, with the help of human liver microsomes simulation of the human body drug metabolism, successfully tested ethyl chloride removal process of cyclophosphamide, at the same time as the production of reagents ketoconazole, dechlorination ethyl cyclophosphamide also reduced, indicating ketoconazole in vitro effectively inhibited the ethyl chloride removal process of cyclophosphamide.
Then in rats in vivo was further verified by the interaction, monitoring on rat blood concentration results UHPLC-MS/MS showed that compared with the blank experiment group (intravenous cyclophosphamide 10mg/kg, ketoconazole dosage of 0mg/kg), combination group (intravenous cyclophosphamide 10mg/ kg, ketoconazole dosage were 10,20 and 40mg/kg) for AUC0- dechlorination cyclophosphamide (ethyl corresponding concentration time curve area) values lower than 51.19%. In other words, when ketoconazole and cyclophosphamide combination of two drugs, the dechlorination of ethyl metabolic pathway in vivo by cyclophosphamide CYP3A4 mediated by blocking ketoconazole greatly. This result suggests that cyclophosphamide combined with CYP3A4 inhibitors such as ketoconazole, will generate greatly to reduce the toxic byproducts of dechlorination of ethyl cy, suggesting that we can from the perspective of clinical and metabolic side effects of cyclophosphamide treatment intervention And put forward the treatment more reasonable clinical medication. Of course very complex, a lot of clinical symptoms is not just a combination of two drugs; drug metabolizing enzymes and participate in not only one, here we just on the dominant CYP3A4 are discussed, also can not reflect the metabolic drug interactions in the picture. However, this paper is based on the cell model in vitro and study based on the interaction of animal model in vivo, provides rational drug research ideas is the clinical medication.

【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R96

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