口服FSHβ2385-CAN纳米粒子靶向调节Pim1和Wnt4表达作为男性避孕药的机制

It is necessary to develop male target contraceptive agents with high compliance, for the severe lack of male contraceptives on current market. Pim1 and Wnt4 are important proteins in tight junction between the adjacent Sertoli cells, and responsible for the maturation and development of spermatozoa. Our preliminary work found that the synthesized compound H2-AF2385 could inhibit the maturation of sperm cell by interfering with the expression of Pim1 and Wnt4, therefore, the compound may be a novel male contraceptive. Because of the male follicle-stimulating hormone receptor (FSHr) is expressed only in the Sertoli cells of the blood testis barrier, thus the FSHr can be introduced as the new target for male contraceptives delivery. Herein, this project is intended to conjugate H2-AF2385 with synthesized FSHβ peptide (without hormonal activity) by carbodiimide, and multi-coated with chitosan and alginate, to prepare the male oral contraceptive nanoparticles of FSHβ-H2-AF2385@chitosan@alginate nanoparticle (FSHβ2385-CAN) to achieve Sertoli cell active targeting. The internal mechanism between FSHβ2385-CAN and Pim1, Wnt4 genome/protein will be studied by processes of co-culture with Sertoli cell, gene modification, transmembrane resistance, and other biological technologies. In vivo rat model will be used to test the relationships between FSHβ2385-CAN treatment and the changes of Pim1, Wnt4 genome/proteins. The antifertility effects, pharmacokinetics, and toxic effects of FSHβ2385-CAN will also be verified in animal model. Thus, the underline mechanism of FSHβ2385-CAN to antifertility may be disclosed. The discoveries of the new target and novel agent should provide new insights of improving male antifertility in particular in humans, and will lay down solid theoretical basis to develop male oral nano-targeted contraceptives, so that it is a very important proposal in social and economic fields.

目前市场上男性避孕药非常匮乏，开发具有高度依从性的男性靶向避孕药十分必要。Pim1和Wnt4是支持细胞紧密连接处的重要蛋白，负责精子的发育和成熟。我们前期研究发现，化合物H2-AF2385可干扰Pim1和Wnt4的表达，抑制精子的成熟，是新型男性避孕药。男性体内卵泡刺激素受体（FSHr）仅在支持细胞表达，因此FSHr可作为男性避孕药的递送靶点。本项目拟使用非激素活性的FSHβ为靶向载体分子，经碳二亚胺将FSHβ与H2-AF2385偶联和壳聚糖/海藻酸双层包覆，制备具有支持细胞靶向性的FSHβ2385-CAN口服纳米避孕药；经与大鼠支持细胞共培养、Pim1及Wnt4基因修饰、跨膜电阻测试等技术，探索该口服纳米避孕药与Pim1、Wnt4基因及蛋白质变化的内在关系；通过大鼠灌胃，研究其在体内的抗生育效果、药代动力学和毒副作用，揭示其抗生育作用的潜在机制，以期为男性口服避孕药的开发奠定理论基础。

目前已有的化合物，在雄性避孕中均存在一定程度的毒副作用，或避孕效果不可逆，或生物利用度和依从性较差等问题。而且对氯尼达明衍生物的研究多局限于adjudin和gamendazole等，应用效果并不满意。因此，有必要合成新型化合物（H2-AF2385），并对其与大鼠睾丸ES的作用进行更深入的分子机制研究，并同时进行靶向、缓释和口服纳米制剂的研究，以期早日实现男性口服靶向避孕药的市场化。.借助之前的研究成果（“姜黄素水溶性改性及自组装胶束”，文章及专利），项目制备了含活性AF-2364和AF-2385的口服纳米制剂，并对这些纳米制剂在体内对BTB“开关”过程中的作用进行了深入研究，现已基本阐明由AF-2385作用后产生的小分子活性肽（F5和CAMSAP2/2TIP /mTORC1/rpS6）在支持细胞（Sertoli cells）基底及ES区对雄性精子的发生的调控机制。本研究制备的雄性口服靶向避孕制剂，有望在未来推广和应用。.在项目执行过程中，本项目组还进行了人类脂肪组织来源细胞、尿液细胞等，经小分子技术体外定向诱导为Leydig样细胞并成功分泌睾酮，为睾丸间质细胞缺损或缺如的移植治疗提供了一定的实验基础；在研究过程中还揭示了尼古丁和黄曲霉毒B1素对人类睾丸间质细胞损害的分子路径，为男性生殖障碍提供了一定的预防医学基础。