本文選題：阿那曲唑 + 氨魯米特　； 參考：《北京中醫(yī)藥大學(xué)》2016年博士論文

【摘要】：本論文是國家體育總局反興奮劑中心青年課題(2014QK002)——《非甾體類芳香化酶抑制劑的代謝及其影響內(nèi)源性甾體激素排放規(guī)律的研究》的部分內(nèi)容。目的和意義：芳香化酶抑制劑(Aromatase inhibitors, AIs)作為芳香化酶選擇性抑制劑,可以特異性地抑制芳香化酶的活性,降低雌激素的水平,增加血睪酮的濃度。因此,2001年國際奧林匹克委員會(IOC)將其列入禁用清單,禁止男性運(yùn)動員使用。隨后世界反興奮劑機(jī)構(gòu)(WADA)于2005年規(guī)定該類藥物在女性運(yùn)動員中也禁止使用。氨魯米特、來曲唑和阿那曲唑為市場上最常見的三種非甾體類芳香化酶抑制劑。目前興奮劑檢測的方法主要包括直接法和間接法。直接法是通過檢測運(yùn)動員尿液中禁用藥物的原型或代謝物來判斷運(yùn)動員是否服用該種藥物,而間接法則是利用監(jiān)控運(yùn)動員自身生理生化指標(biāo)的變化幅度來判定運(yùn)動員是否有興奮劑違規(guī)行為。本研究旨在用高靈敏度儀器(UPLC-Q-Exactive Plus Orbitrap Quotation)尋找新代謝物,并在完善直接法檢測的同時研究探尋非甾體類芳香化酶抑制劑影響人體內(nèi)源性甾體激素的變化規(guī)律,將高特異性、高靈敏度的生物指標(biāo)用于運(yùn)動員生物護(hù)照(ABP)分析。研究方法：本研究在獲得了國家體育總局反興奮劑中心倫理委員會批準(zhǔn)后,組織人體受試。在北京中醫(yī)藥大學(xué)和北京體育大學(xué)共組織招募志愿者41名(其中：男性21名,女性：20名),隨機(jī)分為A、B、C三組,其中A組12人(6名男性,6名女性),B組14人(8名男性,6名女性),C組15人(7名男性,8名女性),分別口服氨魯米特、來曲唑和阿那曲唑三種藥物。A組口服氨魯米特(250 mg×2/天),連續(xù)服用3天,B、C兩組分別口服來曲唑(2.5 mg×1/天)和阿那曲唑(1.0 mg×1/天),連續(xù)服用5天。服藥前,收取7天全部尿樣作為空白對照,服藥期間及停藥后的3天留取全部尿樣,停藥后第四天開始至第28天收取晨尿。代謝物分析的尿樣前處理方法采用醋酸鉛沉淀后直接進(jìn)樣液質(zhì)聯(lián)用儀進(jìn)行分析,使用代謝物篩查軟件Mass Frontier 7.0 SR尋找代謝產(chǎn)物,根據(jù)軟件分析結(jié)果和二級譜圖的分析推導(dǎo)代謝物結(jié)構(gòu)。內(nèi)源性甾體激素定量的尿樣前處理采用酶水解后,乙醚提取、吹干、衍生化進(jìn)樣氣質(zhì)聯(lián)用儀進(jìn)行分析。對于氣質(zhì)聯(lián)用儀定量后得到的數(shù)據(jù),首先采用探索性數(shù)據(jù)分析對人體的8個內(nèi)源性甾體激素濃度指標(biāo)逐一進(jìn)行直觀的圖像分析,從圖像中查看數(shù)據(jù)的規(guī)律,再運(yùn)用假設(shè)檢驗中的wilcoxon秩和檢驗找出與服藥前無顯著差別的數(shù)據(jù)點(diǎn),由此得到該指標(biāo)判定服藥的有效窗口期。統(tǒng)計分析所使用的統(tǒng)計分析軟件為Rstudio Version 0.99.467。統(tǒng)計指標(biāo)包括：睪酮(T)、表睪酮(ET)、雄酮(AN)、本膽烷醇酮(ETIO)、5α-雄烷二醇(5α-diol)、5β-雄烷二醇(5β-diol)、去氫表雄酮(DHEA)和雌酮(Estrone)以及它們的比值。研究成果：(a)本研究中發(fā)現(xiàn)主要代謝物共計33個。包括氨魯米特代謝物24個,來曲唑代謝物4個和阿那曲唑代謝物5個。其中24個代謝物未在文獻(xiàn)中報道,為首次在人尿中發(fā)現(xiàn)的代謝產(chǎn)物。(b)服藥后雄激素(T, ET, AN, ETIO,5α-diol,5β-diol和DHEA)濃度升高,雌激素(Estrone)濃度下降,停藥后逐漸恢復(fù)正常。該趨勢在女性志愿者中變化不如在男性志愿者中顯著。(c)比值指標(biāo)分析中,先使用空白尿樣的數(shù)值計算出95%和99%的分位點(diǎn),再分別使用該分位點(diǎn)對服藥后的陽性尿樣進(jìn)行判定并比較判出率,結(jié)果如下：在95%分位點(diǎn)時,新指標(biāo)AN/Estrone判出率為57.86%,ETIO/Estrone判出率為58.25%,T/Estrone判出率為33.86%；在99%分位點(diǎn)時AN/Estrone判出率為42.14%,ETIO/Estrone判出率為45.30%,T/Estrone判出率為24.79%。WADA技術(shù)文件中所包含指標(biāo)的判出率：在95%分位點(diǎn)時,T/ET判出率為5.21%,5α-diol/5β-diol判出率為5.49%,AN/T判出率為10.34%,AN/ETIO判出率為4.90%,5α-diol/ET判出率為3.78%。在99%分位點(diǎn)時,T/ET判出率為3.05%,5α-diol/5β-diol判出率為0.30%,AN/T判出率為5.86%, AN/ETIO判出率為1.37%,5α-diol/ET判出率為1.10%。新指標(biāo)的判出率明顯高于WADA旨標(biāo)的判出率。(d)使用運(yùn)動員生物護(hù)照(ABP)的分析思路,建立三個新指標(biāo)的加權(quán)移動平均控制圖(EWMA)模型,并將該模型用于單個志愿者指標(biāo)波動分析。結(jié)果顯示,服藥后這三個指標(biāo)波動明顯,可以用于判定可疑樣品。(e)對這三個新指標(biāo)進(jìn)行實(shí)驗驗證：1名男性志愿者單次口服甾體類芳香化酶抑制劑—依西美坦(25mg),服藥后留取3天全部尿樣,第4天至第15天留取晨尿。驗證實(shí)驗表明使用依西美坦后指標(biāo)T/Estrone出現(xiàn)大幅度波動,直到第10天以后才恢復(fù)正常,而使用現(xiàn)有的檢測依西美坦代謝物的方法進(jìn)行檢測,窗口期僅為25.5小時。由此可見,該指標(biāo)同樣適用于監(jiān)控甾體類芳香化酶抑制劑的濫用。結(jié)論及意義：通過對非甾體類芳香化酶抑制劑的代謝及其對人體內(nèi)源性甾體激素排放規(guī)律的研究,發(fā)現(xiàn)了33個代謝產(chǎn)物(其中24個為本研究中首次發(fā)現(xiàn))。整理、歸納、計算出適用于判斷芳香化酶抑制劑濫用的新指標(biāo)(AN/Estrone、ETIO/Estrone和T/Estrone);實(shí)驗結(jié)果表明,該指標(biāo)特異性強(qiáng)、靈敏度高,有效延長了對該類藥物的檢測窗口期,同時也對運(yùn)動員生物護(hù)照項目的推動提供了可靠依據(jù)和有益補(bǔ)充。
[Abstract]:This paper is part of the youth project of the Anti Doping center of the State General Administration of sports (2014QK002) - the metabolism of non steroid aromatase inhibitors and the study on the regulation of endogenous steroid hormone emissions. Purpose and significance: the aromatase inhibitor (Aromatase inhibitors, AIs) as an aromatase selective inhibitor, In order to specifically inhibit the activity of aromatase, reduce the level of estrogen and increase the concentration of blood testosterone, the 2001 International Olympic Committee (IOC) put it in a forbidden list to prohibit the use of male athletes. The World Anti Doping Agency (WADA) subsequently banned the use of the drugs in female athletes in 2005. Mitter, letrozole and analtriazol are the three most common non steroid aromatase inhibitors in the market. The methods of doping at present include direct and indirect methods. The direct method is to judge whether the athletes take the drug by detecting the prototype of the banned drugs in the urine of the athletes or by the metabolites. The indirect law is the benefit. To determine whether an athlete has a doping violation by monitoring the changes in the physiological and biochemical indexes of the athletes. This study aims to find new metabolites with a highly sensitive instrument (UPLC-Q-Exactive Plus Orbitrap Quotation) and to study the influence of non steroidal aromatase inhibitors on the human body at the same time as the direct method is perfected. The variation of source steroid hormone is used to analyze the biological passport (ABP) of athletes with high specificity and sensitivity. Research methods: This study organized human subjects after obtaining the approval of the national sports administration Anti Doping center ethics committee. The recruitment of volunteers was organized at the Beijing University of Chinese Medicine and the Beijing Sport University. 41 men (21 men, 20 women) were randomly divided into groups A, B, and C three, of which 12 were in group A (6 men, 6 women), 14 in group B (8 men, 6 women), 15 (7 men, 8 women) in group B, respectively, oral ammoniac and alacrozole.A group,.A group, orally taken orally, Mg * 2/ days, and B, C. The groups were taken orally with letrozole (2.5 mg x 1/ days) and analtriazole (1 mg x 1/ days) for 5 days. Before taking the medicine, all urine samples were collected for 7 days as blank control, the whole urine samples were collected during and 3 days after the drug withdrawal, and the morning urine was collected from fourth days to twenty-eighth days after the drug stopped. The urine sample for metabolite analysis was precipitated after lead acetate precipitation. The metabolites were found using the metabolite screening software Mass Frontier 7 SR, and the metabolite structure was derived from the analysis results of the software and the analysis of the two level spectrum. The endogenous steroid quantitative urine sample pretreatment was separated by enzymatic hydrolysis, ether extraction, blow drying, and derivatization into sample GC-MS. For the data obtained by GC-MS, first of all, we use exploratory data analysis to analyze the 8 endogenous steroid hormone concentrations of human body by visual image analysis, view the law of the data from the image, and then use the Wilcoxon rank sum test in the hypothesis test to find out the data points that have no significant difference before the medicine. The statistical analysis software used by the statistical analysis is Rstudio Version 0.99.467. statistical indexes including testosterone (T), eptestosterone (ET), androsterone (AN), benzalkanone (ETIO), 5 alpha androtanediol (5 alpha), 5 beta androdiol (5 beta -diol), dehydroepiandrosterone (DHEA) and estrone (Estrone), and they Results: (a) 33 major metabolites were found in this study, including 24 amino metabolites, 4 metabolites of letrozole and 5 analtriazole metabolites. 24 metabolites were not reported in the literature for the first time in human urine. (b) androgens (T, ET, AN, ETIO, 5 alpha -diol, 5 beta -diol) The concentration of estrogen (DHEA) and the concentration of estrogen (Estrone) decreased and gradually returned to normal after withdrawal. The trend was less significant in the female volunteers than in the male volunteers. (c) in the ratio index analysis, the number of 95% and 99% loci was calculated using the blank urine sample, and the positive urine sample after the drug was used respectively. The results are as follows: at the 95% point, the rate of AN/Estrone is 57.86%, the rate of ETIO/Estrone is 58.25%, the rate of T/Estrone is 33.86%, the rate of AN/Estrone is 42.14% at the 99% point site, the rate of ETIO/Estrone is 45.30%, and the rate of T/Estrone is the index contained in the 24.79%.WADA Technology file. Rate of judgment: at the 95% points, the rate of T/ET was 5.21%, the 5 alpha -diol/5 beta -diol rate was 5.49%, the AN/T judgment rate was 10.34%, the AN/ETIO judgment rate was 4.90%, the 5 alpha -diol/ET judgment rate was 3.05%, 5 a -diol/5 beta -diol determination rate was 0.30%, AN/T judgment rate was 5.86%, AN/ETIO judgment rate was 1.37%, AN/ETIO judgment rate was 1.37%, 5 alpha Marxism The rate of T judgment was significantly higher than that of the new index of 1.10%.. (d) the weighted moving average control chart (EWMA) model of three new indexes was established by using the analysis idea of athlete's biological passport (ABP), and the model was used to analyze the fluctuation of individual volunteers. The results showed that the three indexes fluctuated obviously after taking the medicine. To determine suspicious samples. (E) experimental verification of these three new indicators: 1 male volunteers took a single oral steroid aromatase inhibitor, eticietam (25mg), to take 3 days after taking the medicine for 3 days and leave the morning urine for fourth days to fifteenth days. Tenth days later, it was not restored to normal, and was detected with the current test of the metabolite of etanettan, with a window period of only 25.5 hours. Thus, this indicator also applies to monitoring the abuse of steroid aromatase inhibitors. Conclusions and significance: metabolism of non steroidal aromatase inhibitors and its internal source to human body. The study of sexual steroid hormone emission laws found that 33 metabolites (24 of them were first discovered in this study). Collation, induction, and calculation are applicable to the new indicators of aromatase inhibitor abuse (AN/Estrone, ETIO/Estrone and T/Estrone). The experimental results show that the index is specific and sensitive and effectively prolongs the class The drug detection window period also provides reliable basis and beneficial supplement for the promotion of athlete's biological passport project.

【學(xué)位授予單位】：北京中醫(yī)藥大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R87

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