de Gooyer Marcel E, Oppers-Tiemissen Hendrika M, Leysen Dirk, Verheul Herman A M, Kloosterboer Helenius J
N.V. Organon, Research & Development Laboratories, Molenstraat 110, PO Box 20, 5340 BH, Oss, The Netherlands.
Steroids. 2003 Mar;68(3):235-43. doi: 10.1016/s0039-128x(02)00184-8.
To exclude that aromatization plays a role in the estrogenic activity of tibolone, we studied the effect tibolone and metabolites on the aromatization of androstenedione and the aromatization of tibolone and its metabolites to 7alpha-methyl-17alpha-ethynylestradiol (7alpha-MEE) by human recombinant aromatase. Testosterone (T), 17alpha-methyltestosterone (MT), 19-nortestosterone (Nan), 7alpha-methyl-19-nortestosterone (MENT) and norethisterone (NET) were used as reference compounds. Sensitive in vitro bioassays with steroid receptors were used to monitor the generation of product and the reduction of substrate. LC-MSMS without derivatization was used for structural confirmation. A 10 times excess of tibolone and its metabolites did not inhibit the conversion of androstenedione to estrone by human recombinant aromatase as determined by estradiol receptor assay whereas T, MT, Nan, and MENT inhibited the conversion for 75, 53, 85 and 67%, respectively. Tibolone, 3alpha- and 3beta-hydroxytibolone were not converted by human aromatase whereas the estrogenic activity formed with the Delta4-isomer suggests a conversion rate of 0.2% after 120 min incubation. In contrast T, MT, Nan, and MENT were completely converted to their A-ring aromates within 15 min while NET could not be aromatized. Aromatization of T, MT, Nan and MENT was confirmed with LC-MSMS. Structure/function analysis indicated that the 17alpha-ethynyl-group prevents aromatization of (19-nor)steroids while 7alpha-methyl substitution had no effect. Our results with the sensitive estradiol receptor assays show that in contrast to reference compounds tibolone and its metabolites are not aromatized.
为排除芳香化作用在替勃龙雌激素活性中所起的作用，我们研究了替勃龙及其代谢产物对雄烯二酮芳香化作用的影响，以及人重组芳香化酶将替勃龙及其代谢产物芳香化为7α-甲基-17α-乙炔基雌二醇（7α-MEE）的作用。睾酮（T）、17α-甲基睾酮（MT）、19-去甲睾酮（诺龙）、7α-甲基-19-去甲睾酮（MENT）和炔诺酮（NET）用作参考化合物。采用对类固醇受体敏感的体外生物测定法监测产物的生成和底物的减少。未衍生化的液相色谱-串联质谱法用于结构确证。通过雌二醇受体测定法确定，替勃龙及其代谢产物过量10倍时，不会抑制人重组芳香化酶将雄烯二酮转化为雌酮，而T、MT、诺龙和MENT分别抑制该转化的比例为75%、53%、85%和67%。人芳香化酶不会将替勃龙、3α-和3β-羟基替勃龙转化，而与Δ4-异构体形成的雌激素活性表明，孵育120分钟后的转化率为0.2%。相比之下，T、MT、诺龙和MENT在15分钟内完全转化为其A环芳香化产物，而NET无法进行芳香化。通过液相色谱-串联质谱法确证了T、MT、诺龙和MENT的芳香化。结构/功能分析表明，17α-乙炔基可阻止（19-去甲）类固醇的芳香化，而7α-甲基取代则无此作用。我们采用敏感雌二醇受体测定法的结果表明，与参考化合物不同，替勃龙及其代谢产物不会发生芳香化。
