WIN 18446
(Synonyms: N,N'-八亚甲基双二氯乙酰胺) 目录号 : GC10438
WIN 18446 (Fertilysin)是乙醛脱氢酶1A2(ALDH1A2)的有效抑制剂，IC50值为0.3μM。
Cas No.:1477-57-2
Sample solution is provided at 25 µL, 10mM.
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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]。
| Kinase experiment [1,2]: | |
Preparation Method | Human ALDH1A2 cDNA was cloned from human testis RNA to generate human ALDH1A2 enzyme. ALDH1A2 enzyme was incubated in assay buffer containing 20mM Hepes, 150mM KCI and 1mM EDTA[1]. 0.1μM, 1μM or 3μM of WIN 18446 was dissolved in DMSO (concentration of DMSO was less than 1%)[1]. Dissolved WIN 18446 was added to assay buffer with 2.5, 20.5, 50, 100, 500, 1000 and 2000ng/ml of retinol dissolved in ethanol for 20, 40, 60, 80mins as treatment group[2]. Concentration of retinoic acid were determined by HPLC. This protocol only provides a guideline, and should be modified according to your specific needs. |
Reaction Conditions | WIN 18446: 0.1μM, 1μM or 3μM; Retinol: 2.5, 20.5, 50, 100, 500, 1000 and 2000ng/ml; 20, 40, 60, 80mins |
Applications | After 80mins treatment with WIN 18446 (0.1μM, 1μM or 3μM) ALDH1A2 enzyme retained only 2-3% of its activity. As the incubation time increased until 80mins, the rate of retinoic acid production reached saturation with increased concentration of retinal. |
| Animal experiment [3]: | |
Animal models | C57BL/6 male mice |
Preparation Method | C57BL/6 male mice (breeding age, n=60) were fed WIN 18446 (2mg/g diet) with AIN9M (control diet) for 3, 7, 14, 21, and 28 days[3]. Five of the sixty mice per time point were euthanized and testes were collected. After 28 days, the rest of mice were fed with AIN9M (control diet) only for recovery assay for 42 and 56 days (up to 8 weeks) [3]. Five recovery group mice were euthanized per time point and testes were collected. Control group mice (n=15) were fed with AIN9M (control diet) only[3]. Testes specimens were fixed in Davidson's fixative for 24–48h, then transferred to 70% ethanol for testicular histology analysis[3]. This protocol only provides a guideline, and should be modified according to your specific needs. |
Dosage form | WIN 18446: 2mg/g diet for 3, 7, 14, 21, 28, 42 and 56 days; AIN9M: control diet for 3, 7, 14, 21, 28, 42 and 56 days |
Applications | Testes weights from WIN 18446 (2mg/g diet) treated mice group were significantly reduced. Testes weights from recovery group (AIN9M control diet only) still remained smaller compared to control groups. 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. |
[1] 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. | |
| Cas No. | 1477-57-2 | SDF | |
| 别名 | N,N'-八亚甲基双二氯乙酰胺 | ||
| 化学名 | N,N'-(octane-1,8-diyl)bis(2,2-dichloroacetamide) | ||
| Canonical SMILES | ClC(C(NCCCCCCCCNC(C(Cl)Cl)=O)=O)Cl | ||
| 分子式 | C12H20Cl4N2O2 | 分子量 | 366.11 |
| 溶解度 | ≥ 11.4mg/mL in DMSO | 储存条件 | Store at -20°C |
| General tips | 请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。 储备液的保存方式和期限：-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。 为了提高溶解度，请将管子加热至37℃，然后在超声波浴中震荡一段时间。 |
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| Shipping Condition | 评估样品解决方案：配备蓝冰进行发货。所有其他可用尺寸：配备RT，或根据请求配备蓝冰。 | ||
| 制备储备液 | |||
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1 mg | 5 mg | 10 mg |
| 1 mM | 2.7314 mL | 13.6571 mL | 27.3142 mL |
| 5 mM | 0.5463 mL | 2.7314 mL | 5.4628 mL |
| 10 mM | 0.2731 mL | 1.3657 mL | 2.7314 mL |
| 第一步：请输入基本实验信息（考虑到实验过程中的损耗，建议多配一只动物的药量） | ||||||||||
| 给药剂量 | mg/kg |  |
动物平均体重 | g | |
每只动物给药体积 | ul | |
动物数量 | 只 |
| 第二步：请输入动物体内配方组成（配方适用于不溶于水的药物；不同批次药物配方比例不同，请联系GLPBIO为您提供正确的澄清溶液配方） | ||||||||||
| % DMSO % % Tween 80 % saline | ||||||||||
| 计算重置 | ||||||||||
计算结果:
工作液浓度： mg/ml；
DMSO母液配制方法： mg 药物溶于 μL DMSO溶液（母液浓度 mg/mL，
体内配方配制方法：取 μL DMSO母液，加入 μL PEG300，混匀澄清后加入μL Tween 80，混匀澄清后加入 μL saline，混匀澄清。
1. 首先保证母液是澄清的；
2.
一定要按照顺序依次将溶剂加入，进行下一步操作之前必须保证上一步操作得到的是澄清的溶液，可采用涡旋、超声或水浴加热等物理方法助溶。
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