WIN 18446 (Fertilysin) is a potent inhibitor of aldehyde dehydrogenase 1A2 (ALDH1A2) enzyme with an IC50 value of 0.3μM[1]. WIN 18446 has been made as a candidate for male contraception studies[2]. ALDH1a2 is an enzyme involved in retinoic acid biosynthesis[3]. Therefore, inhibiting ALDH1A2 disrupts retinoic acid synthesis, leading suppression of spermatogenesis[3].
In vitro, purified ALDH1A2 enzyme was obtained from cloned human ALDH1A2 testis cDNA[4]. 0.1μM, 1μM or 3μM of WIN 18446 was added to ALDH1A2 enzyme alone with 2.5, 20.5, 50, 100, 500, 1000 and 2000ng/ml of retinol for 20, 40, 60, 80mins. After 80mins treatment with WIN 18446 (0.1μM, 1μM or 3μM) ALDH1A2 enzyme retained only 2-3% of its activity[4,5]. As the incubation time increased until 80mins, the rate of retinoic acid production reached saturation with increased concentration of retinal. When WIN 18446 inhibits ALDH1A2 enzyme, retinaldehyde accumulates, which might compensate for the loss of ALDH1A2 enzyme activity and promote retinoic acid synthesis to saturation[4]. By adding dithiothreitol (DTT) to retain ALDH1A2 enzyme activity, ALDH1A2 enzyme inhibition assay still could not be reversed, indicating that WIN 18446 might inhibited ALDH1A2 enzyme directly[4].
In vivo, C57BL/6 male mice were fed WIN 18446 (2mg/g diet) or AIN9M (control diet) for 3, 7, 14, 21, and 28 days[6]. Testes weights with WIN 18446 treatment (2mg/g diet) were significantly reduced[6]. When further treated WIN 18446 groups with AIN9M control diet only for recovery assay after 14 days, testes weights remained smaller compared to control groups[3,6]. After 28 days treatment of WIN 18446 (2mg/g diet), most testicular tubules exhibited severe epithelial disruption, characterized by the loss of germ cells and the presence of frequent multinucleated cells[3,6].
References:
[1] Amory JK, Muller CH, Shimshoni JA, Isoherranen N, Paik J, Moreb JS, Amory Sr DW, Evanoff R, Goldstein AS, Griswold MD. Suppression of spermatogenesis by bisdichloroacetyldiamines is mediated by inhibition of testicular retinoic acid biosynthesis. Journal of andrology. 2011 Jan 2;32(1):111-9.
[2] Gray A. Overcoming the challenges in developing male contraceptives. The Pharmaceutical Journal. 2016;296:7890.
[3] Das BC, Thapa P, Karki R, Das S, Mahapatra S, Liu TC, Torregroza I, Wallace DP, Kambhampati S, Van Veldhuizen P, Verma A. Retinoic acid signaling pathways in development and diseases. Bioorganic & medicinal chemistry. 2014 Jan 15;22(2):673-83.
[4] Paik J, Haenisch M, Muller CH, Goldstein AS, Arnold S, Isoherranen N, Brabb T, Treuting PM, Amory JK. Inhibition of retinoic acid biosynthesis by the bisdichloroacetyldiamine WIN 18,446 markedly suppresses spermatogenesis and alters retinoid metabolism in mice. Journal of Biological Chemistry. 2014 May 23;289(21):15104-17.
[5] Hartmann S, Froescheis O, Ringenbach F, Wyss R, Bucheli F, Bischof S, Bausch J, Wiegand UW. Determination of retinol and retinyl esters in human plasma by high-performance liquid chromatography with automated column switching and ultraviolet detection. Journal of Chromatography B: Biomedical Sciences and Applications. 2001 Feb 25;751(2):265-75.
[6] Paik J, Snyder JM, Kim A, Haenisch M, Fogassy K, Amory JK. Kinetics of the inhibition and recovery of spermatogenesis induced by treatment with WIN 18,446, a male contraceptive, in mice. Andrology. 2024.
WIN 18446 (Fertilysin)是乙醛脱氢酶1A2(ALDH1A2)的有效抑制剂，IC50值为0.3μM[1]。WIN 18446已被列为男性避孕研究的候选药物[2]。ALDH1A2是一种参与视黄酸生物合成的酶[3]。因此，抑制ALDH1A2会破坏视黄酸合成，导致精子生成受到抑制[3]。
在体外，从克隆体人类ALDH1A2睾丸cDNA中获得纯化的ALDH1A2酶[4]。将0.1μM，1μM或3μM的WIN 18446单独添加到ALDH1A2酶中，与2.5，20.5，50，100，500，1000和2000ng/ml视黄醇一起作用20，40，60，80分钟，ALDH1A2酶仅保留2-3%的活性[4,5]。随着孵育时间增加至80分钟，随着视黄醛浓度的增加，视黄酸产生率达到饱和。当WIN 18446抑制ALDH1A2酶时，视黄醛积累，这可能补偿ALDH1A2酶活性的损失并促进视黄酸的合成直至饱和[4]。通过添加二硫苏糖醇（DTT）保留ALDH1A2酶活性，ALDH1A2酶抑制实验仍然无法逆转，表明WIN 18446可能直接抑制ALDH1A2酶[4]。
在体内，C57BL/6雄性小鼠喂食WIN 18446（2mg/g 饮食）或AIN9M（对照饮食）3，7，14，21 和 28 天[6]。WIN 18446治疗（2mg/g 饮食）后的睾丸重量显著降低[6]。当14天后用 AIN9M 对照饮食进一步治疗WIN 18446组进行恢复测定时，与对照组相比，睾丸重量仍然较小[3,6]。WIN 18446（2mg/g 饮食）治疗28天后，大多数睾丸小管表现出严重的上皮破坏，其特征是生殖细胞丧失和频繁出现多核细胞[3,6]。