非甾体类芳香化酶抑制剂的代谢研究及其专属性指标模型的建立

发布时间：2018-04-24 00:00

本文选题：阿那曲唑 + 氨鲁米特　；参考：《北京中医药大学》2016年博士论文

【摘要】：本论文是国家体育总局反兴奋剂中心青年课题(2014QK002)——《非甾体类芳香化酶抑制剂的代谢及其影响内源性甾体激素排放规律的研究》的部分内容。目的和意义：芳香化酶抑制剂(Aromatase inhibitors, AIs)作为芳香化酶选择性抑制剂,可以特异性地抑制芳香化酶的活性,降低雌激素的水平,增加血睾酮的浓度。因此,2001年国际奥林匹克委员会(IOC)将其列入禁用清单,禁止男性运动员使用。随后世界反兴奋剂机构(WADA)于2005年规定该类药物在女性运动员中也禁止使用。氨鲁米特、来曲唑和阿那曲唑为市场上最常见的三种非甾体类芳香化酶抑制剂。目前兴奋剂检测的方法主要包括直接法和间接法。直接法是通过检测运动员尿液中禁用药物的原型或代谢物来判断运动员是否服用该种药物,而间接法则是利用监控运动员自身生理生化指标的变化幅度来判定运动员是否有兴奋剂违规行为。本研究旨在用高灵敏度仪器(UPLC-Q-Exactive Plus Orbitrap Quotation)寻找新代谢物,并在完善直接法检测的同时研究探寻非甾体类芳香化酶抑制剂影响人体内源性甾体激素的变化规律,将高特异性、高灵敏度的生物指标用于运动员生物护照(ABP)分析。研究方法：本研究在获得了国家体育总局反兴奋剂中心伦理委员会批准后,组织人体受试。在北京中医药大学和北京体育大学共组织招募志愿者41名(其中：男性21名,女性：20名),随机分为A、B、C三组,其中A组12人(6名男性,6名女性),B组14人(8名男性,6名女性),C组15人(7名男性,8名女性),分别口服氨鲁米特、来曲唑和阿那曲唑三种药物。A组口服氨鲁米特(250 mg×2/天),连续服用3天,B、C两组分别口服来曲唑(2.5 mg×1/天)和阿那曲唑(1.0 mg×1/天),连续服用5天。服药前,收取7天全部尿样作为空白对照,服药期间及停药后的3天留取全部尿样,停药后第四天开始至第28天收取晨尿。代谢物分析的尿样前处理方法采用醋酸铅沉淀后直接进样液质联用仪进行分析,使用代谢物筛查软件Mass Frontier 7.0 SR寻找代谢产物,根据软件分析结果和二级谱图的分析推导代谢物结构。内源性甾体激素定量的尿样前处理采用酶水解后,乙醚提取、吹干、衍生化进样气质联用仪进行分析。对于气质联用仪定量后得到的数据,首先采用探索性数据分析对人体的8个内源性甾体激素浓度指标逐一进行直观的图像分析,从图像中查看数据的规律,再运用假设检验中的wilcoxon秩和检验找出与服药前无显著差别的数据点,由此得到该指标判定服药的有效窗口期。统计分析所使用的统计分析软件为Rstudio Version 0.99.467。统计指标包括：睾酮(T)、表睾酮(ET)、雄酮(AN)、本胆烷醇酮(ETIO)、5α-雄烷二醇(5α-diol)、5β-雄烷二醇(5β-diol)、去氢表雄酮(DHEA)和雌酮(Estrone)以及它们的比值。研究成果：(a)本研究中发现主要代谢物共计33个。包括氨鲁米特代谢物24个,来曲唑代谢物4个和阿那曲唑代谢物5个。其中24个代谢物未在文献中报道,为首次在人尿中发现的代谢产物。(b)服药后雄激素(T, ET, AN, ETIO,5α-diol,5β-diol和DHEA)浓度升高,雌激素(Estrone)浓度下降,停药后逐渐恢复正常。该趋势在女性志愿者中变化不如在男性志愿者中显著。(c)比值指标分析中,先使用空白尿样的数值计算出95%和99%的分位点,再分别使用该分位点对服药后的阳性尿样进行判定并比较判出率,结果如下：在95%分位点时,新指标AN/Estrone判出率为57.86%,ETIO/Estrone判出率为58.25%,T/Estrone判出率为33.86%；在99%分位点时AN/Estrone判出率为42.14%,ETIO/Estrone判出率为45.30%,T/Estrone判出率为24.79%。WADA技术文件中所包含指标的判出率：在95%分位点时,T/ET判出率为5.21%,5α-diol/5β-diol判出率为5.49%,AN/T判出率为10.34%,AN/ETIO判出率为4.90%,5α-diol/ET判出率为3.78%。在99%分位点时,T/ET判出率为3.05%,5α-diol/5β-diol判出率为0.30%,AN/T判出率为5.86%, AN/ETIO判出率为1.37%,5α-diol/ET判出率为1.10%。新指标的判出率明显高于WADA旨标的判出率。(d)使用运动员生物护照(ABP)的分析思路,建立三个新指标的加权移动平均控制图(EWMA)模型,并将该模型用于单个志愿者指标波动分析。结果显示,服药后这三个指标波动明显,可以用于判定可疑样品。(e)对这三个新指标进行实验验证：1名男性志愿者单次口服甾体类芳香化酶抑制剂—依西美坦(25mg),服药后留取3天全部尿样,第4天至第15天留取晨尿。验证实验表明使用依西美坦后指标T/Estrone出现大幅度波动,直到第10天以后才恢复正常,而使用现有的检测依西美坦代谢物的方法进行检测,窗口期仅为25.5小时。由此可见,该指标同样适用于监控甾体类芳香化酶抑制剂的滥用。结论及意义：通过对非甾体类芳香化酶抑制剂的代谢及其对人体内源性甾体激素排放规律的研究,发现了33个代谢产物(其中24个为本研究中首次发现)。整理、归纳、计算出适用于判断芳香化酶抑制剂滥用的新指标(AN/Estrone、ETIO/Estrone和T/Estrone);实验结果表明,该指标特异性强、灵敏度高,有效延长了对该类药物的检测窗口期,同时也对运动员生物护照项目的推动提供了可靠依据和有益补充。
[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.

【学位授予单位】：北京中医药大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R87

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