本文選題：抗腫瘤藥物　切入點(diǎn)：UHPLC-MS/MS　出處：《第二軍醫(yī)大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：腫瘤是臨床上致死率最高的疾病之一，臨床上常用的治療方案有化療，手術(shù)以及放射治療，其中化療治療方案較為成熟。傳統(tǒng)上，根據(jù)常用抗腫瘤藥物來源以及其作用機(jī)制可以分為如下幾類：烷化劑、抗代謝物類、抗生素、植物激素等。近來，隨著制藥技術(shù)的發(fā)展，抗腫瘤藥物的靶向性得以提高，但是其治療引起的毒副作用以及不同病人個(gè)體臨床藥效的差異卻不可避免。根據(jù)藥物的藥物代謝動力學(xué)研究，可以得知藥物的臨床差異性表現(xiàn)很大一部分由原型藥物在體內(nèi)的毒性代謝產(chǎn)物或者活性代謝產(chǎn)物決定的，因此對這些原型藥物以及其具有代表性的代謝產(chǎn)物進(jìn)行定量分析，了解其及時(shí)的血藥濃度，有助于我們了解特定藥物在體內(nèi)的代謝情況，從而對藥物的合理使用進(jìn)行更加權(quán)威科學(xué)的分析，促進(jìn)個(gè)體化用藥的發(fā)展。 TDM （therapeutic drug monitoring），即臨床血藥濃度監(jiān)測，是以藥代動力學(xué)原理為指導(dǎo)，借助氣相色譜、液相色譜或者氣質(zhì)（液質(zhì)）聯(lián)用等常用分析手段，定量分析體內(nèi)原型藥物及其代謝產(chǎn)物的血藥濃度，從而評價(jià)藥物療效或調(diào)整給藥方案，達(dá)到臨床上合理化用藥。抗腫瘤化療藥物具有治療窗窄，在不同患者體內(nèi)代謝差異性較大，易發(fā)生藥物相互作用等特點(diǎn)，因此對其原型及代謝產(chǎn)物進(jìn)行血藥濃度監(jiān)測有很大的臨床意義。本課題中研究主要分為兩大部分，第一部分為借助于UHPLC-MS/MS（ultrahigh performance liquid chromatography tandem mass spectrometry）對幾種常用抗腫瘤藥及其主要代謝產(chǎn)物進(jìn)行TDM研究，他們?nèi)缦�，吉西他濱和二氟脫氧脲苷，異長春花堿和脫乙�；愰L春花堿，依托泊苷和脫甲基依托泊苷，UHPLC-MS/MS（ultra highperformance liquid chromatography tandem mass spectrometry）是近來興起的一項(xiàng)熱門技術(shù)，它的優(yōu)勢體現(xiàn)在快速，分離度高，樣本用量少上面。方法學(xué)的驗(yàn)證內(nèi)容包括特異性，線性，提取回收率和基質(zhì)效應(yīng)，穩(wěn)定性等，前處理方法則采用了經(jīng)典的蛋白沉淀法（三倍體積的含0.1%甲酸的乙腈或者甲醇溶液作為沉淀劑）。方便快捷的樣品前處理方法，較短的洗脫時(shí)間以及較好的色譜分離保證了該方法適用于臨床上的高通量分析。第二部分從環(huán)磷酰胺的毒副產(chǎn)物代謝途徑出發(fā)，為避免環(huán)磷酰胺治療引起的神經(jīng)毒副反應(yīng)，而進(jìn)行的環(huán)磷酰胺和酮康唑體內(nèi)外相互作用的研究。 環(huán)磷酰胺是一種常見的酰胺氮芥類細(xì)胞毒作用藥物。臨床上其神經(jīng)毒性主要是由脫氯乙基代謝途徑引起的，該代謝途徑為部分體內(nèi)游離的環(huán)磷酰胺經(jīng)過CYP3A4作用，產(chǎn)生脫氯乙基環(huán)磷酰胺和氯乙醛。其中氯乙醛被認(rèn)為是環(huán)磷酰胺臨床上神經(jīng)毒性作用的生物標(biāo)記物。臨床上，通過有效地影響CYP酶家族的活性而實(shí)現(xiàn)藥物間的相互作用是常用的聯(lián)合用藥策略之一，，針對環(huán)磷酰胺的藥物相互作用研究，嘗試了潛在CYP3A4抑制劑如氟康唑、磺胺甲VA唑和酮康唑等常用抗真菌藥物，最終選取了酮康唑作為試藥。酮康唑是經(jīng)典的CYP3A4抑制劑，已知的藥物相互作用模型中，酮康唑能夠減少咪達(dá)唑侖經(jīng)CYP3A4介導(dǎo)的代謝產(chǎn)物2-羥基咪達(dá)唑侖的生成。類似的，我們進(jìn)行了酮康唑與環(huán)磷酰胺的體內(nèi)外相互作用研究，希望通過酮康唑抑制CYP3A4的活性而阻斷環(huán)磷酰胺對應(yīng)的毒副產(chǎn)物生成途徑。體外酶孵育環(huán)境中，借助于人肝微粒體模擬人體內(nèi)藥物代謝環(huán)境，成功地驗(yàn)證了環(huán)磷酰胺的脫氯乙基過程，同時(shí)在酮康唑作為試藥時(shí)，脫氯乙基環(huán)磷酰胺的生成量也相應(yīng)減少，說明酮康唑在體外有效地抑制了環(huán)磷酰胺的脫氯乙基過程。 接著在大鼠體內(nèi)進(jìn)一步驗(yàn)證了這一相互作用，UHPLC-MS/MS對大鼠血藥濃度的監(jiān)測結(jié)果顯示，與空白實(shí)驗(yàn)組對比（靜脈注射環(huán)磷酰胺10mg/kg，酮康唑用量為0mg/kg），聯(lián)合用藥組（靜脈注射環(huán)磷酰胺10mg/kg，酮康唑用量分別為10，20和40mg/kg）脫氯乙基環(huán)磷酰胺對應(yīng)的AUC0-∞（藥時(shí)曲線面積）值降低51.19%以上。換言之，當(dāng)酮康唑和環(huán)磷酰胺兩藥合用時(shí)，環(huán)磷酰胺在體內(nèi)經(jīng)CYP3A4介導(dǎo)的脫氯乙基代謝途徑被酮康唑很大程度的阻斷。這一結(jié)果提示我們環(huán)磷酰胺聯(lián)合CYP3A4抑制劑例如酮康唑時(shí)，會大大減少毒性產(chǎn)物脫氯乙基環(huán)磷酰胺的生成，這提示我們可以從代謝角度而對臨床上環(huán)磷酰胺的毒副作用進(jìn)行治療干預(yù)，提出更加合理化的治療方案。當(dāng)然臨床用藥相當(dāng)復(fù)雜多變，很多臨床病癥并非只是兩藥合用的情形；并且參與藥物的代謝酶并非只有一種，這里我們只是對占主要地位的CYP3A4進(jìn)行討論，也不能反應(yīng)出藥物代謝性相互作用的全貌。但是，本課題提出的基于細(xì)胞模型的體外和基于動物模型的體內(nèi)相互作用研究，不失為臨床用藥提供了合理用藥研究思路。
[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.

【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R96

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相關(guān)期刊論文 前10條

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2 朱業(yè)晉,張e

本文編號：1561628