CN115919860B - 氟班色林在制备治疗雄激素脱发药物中的用途 - Google Patents
氟班色林在制备治疗雄激素脱发药物中的用途 Download PDFInfo
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Abstract
本发明公开了氟班色林在制备治疗雄激素脱发药物中的用途。细胞模型结果显示,低浓度的氟班色林即可抑制Lncap细胞的增殖,显示出细胞水平的药效活性。动物实验结果显示,氟班色林可以逆转雄激素介导的雄激素脱发损伤,可治疗雄激素脱发,恢复毛发的生长。因此,氟班色林可以在制备治疗雄激素脱发药物中应用。
Description
技术领域
本发明属于小分子药物领域,涉及氟班色林在制备治疗雄激素脱发药物中的用途。
背景技术
雄激素性脱发(androgenic alopecia,AGA)是一种以头发密度进行性减少为特点的疾病,通常始于额发线的双颞叶衰退,然后在顶点弥漫性头发稀疏,最终在顶点中心完全脱发,是一种常染色体显性遗传病。与位于枕叶区域的毛囊相比,男性和女性在头皮额叶区域的雄激素受体和5α-还原酶I型和II型活性水平较高。额毛囊中的α-还原酶I型和II型活性在男性中是女性的三倍。因此,男性雄激素性脱发被认为是一种雄激素依赖性疾病,在中国,男性总体患病率为21.3%,但随着年龄的增长而增加。AGA严重影响患者的社交和心理,患者求治心切。AGA的病理特点包括毛囊小型化、毛囊生长期缩短和休止期延长。在临床上表现为绒毛代替末端毛发(绒毛定义为直径<30μm,长度<30μm;过渡性头发宽度在30至40μm之间;末端毛发>40μm)和总毛发密度(头发/平方厘米)降低作为主要的临床特征。在组织学上也可以看到终末毛囊被绒毛囊取代,同时巨噬细胞的毛囊周围浸润,皮脂腺大小增加和真皮变薄。毛囊的周期性生长分为四个时期:生长期(anagen),位于真皮乳头细胞(dermalpapilla cells.DPCs)周国的毛基质角质形成细胞增殖,毛干生长;退行期(catagen),毛囊发生受严格控制的细胞调亡和细胞外基质重塑:休止期(telogen),毛干附着在毛囊上皮球状基部,可因梳洗而脱落;活跃脱毛期(exogen),这是休止期毛发的脱落阶段。睾酮(testosterone,T)是人体内主要的性激素之一,DPCs中表达的5α-还原酶(5a-reductase,5GR)催化T转化为更具活性的5α-二氢睾酮(5α-dihvdrotestosterone,DHT)。DHT和毛发基质细胞上的雄激素受体(androgen receptor,AR)结合,形成DHT-AR复合物进入细胞核,作为转录因子诱导毛囊毛乳头细胞分泌多种细胞因子,如转化生长因子-B(transforminggrowth factor B.TGF-B)、白细胞介素-1a(interleukin 1a.1L-1a)和肿瘤坏死因子-a(tumor necrosis facior a.TNF-a),可诱导毛囊生长期提前终止,导致AGA。雄激素性脱发是头皮毛囊对循环雄激素异常敏感性的结果,在雄激素的诱导下,雄激素受体表达增加,从而改变毛囊内的间充质-上皮细胞相互作用,影响毛发生长、真皮大小、真皮细胞以及角质形成细胞和黑色素细胞的活性,Wnt信号通路调节真皮中的细胞,可能在雄激素对头发生长的作用中起关键作用。然而,雄激素相关作用的潜在分子机制在很大程度上仍然未知。
截止目前,对于雄激素脱发治疗经过FDA批准的药物只有两种,为口服药物非那雄胺和外用涂抹剂米诺地尔。非那雄胺为5α还原酶抑制剂,通过减少体内DHT含量治疗脱发。米诺地尔为血管扩张剂,通过改善毛囊微循环,达到治疗脱发的效果。其他治疗手段包括激光治疗,毛发移植,干细胞治疗等非药物治疗手段。但是这些治疗方法都存在很大的副作用,如性功能障碍,多毛症,费用高等问题。因此寻找新的且副作用更小的治疗雄激素脱发药物具有重要意义。
发明内容
本发明的目的针对现有技术的上述不足,提供氟班色林在制备治疗雄激素脱发药物中的用途。
本发明的目的可通过以下技术方案实现:
氟班色林在制备治疗雄激素脱发的药物中的应用。
作为本发明的一种优选,氟班色林能够增加毛发长度、生长期与休止期比例、表皮与真皮厚度比例、单位面积内的毛囊数以及毛囊的直径。
作为本发明的一种优选,氟班色林在制备逆转雄激素介导的雄激素脱发损伤,治疗雄激素脱发,恢复毛发生长的的药物中的应用。
氟班色林在制备抑制雄激素受体二聚体,阻止其进入细胞核的药物中的应用。
雄激素受体二聚体作为治疗靶点在筛选治疗雄激素脱发药物中的用途。氟班色林即为结合雄激素受体二聚体的结构利用计算机模拟技术筛选得到的治疗雄激素脱发的化合物。
有益效果:
本发明结合计算机模拟筛选技术与体内外药物筛选实验筛选得到一种雄激素受体二聚体的抑制剂,一方面在结构上该化合物对雄激素受体二聚体的结构具有结合活性,相对于米诺地尔作用靶点明确,特异性强,另一方面,该化合物为雄激素受体二聚体的抑制剂,与雄激素受体二聚体的结合可抑制雄激素受体二聚体的入核,抑制该复合体下游激活因子的招募及与雄激素受体反应原件的结合,从而阻断下游的基因响应,治疗雄激素脱发。该药物与非那雄胺相比,作用条件温和,不会引起其他部位的雄激素受体介导的下游反应,因此可大大降低雄激素脱发治疗药物的副作用。
细胞模型结果显示,低浓度的氟班色林即可抑制Lncap细胞的增殖,显示出细胞水平的药效活性。动物实验结果显示,与模型组相比,氟班色林及非那雄胺给药组小鼠的毛发均有不同程度的恢复且与对照组及完全空白组毛生长情况相近。氟班色林组动物毛发恢复有效率达85.7%,非那雄胺组达71.4%。与模型组相比,HE组织切片染色显示氟班色林组的毛发长度增加,生长期与休止期比例增加,小鼠表皮与真皮厚度比例增加,单位面积内的毛囊数增加,毛囊的直径增加。因此本次结果显示,氟班色林可以逆转雄激素介导的雄激素脱发损伤,可治疗雄激素脱发,恢复毛发的生长。
附图说明
图1:抑制二氢睾酮刺激下的前列腺癌细胞Lncap细胞的增殖
图2:氟班色林在AGA模型小鼠中的毛长统计图
图3:氟班色林在AGA模型小鼠上的毛发再生率柱状图
图4:氟班色林对AGA模型小鼠毛囊生长期与休止期比例柱状图
图5:氟班色林对AGA模型小鼠真皮层与表皮层厚度比例柱状图
图6:氟班色林在AGA模型小鼠中单位面积毛囊数目统计柱状图
图7:氟班色林在AGA模型小鼠中毛囊直径统计柱状图
图8:氟班色林对AGA模型小鼠的毛发恢复图
图9:氟班色林抑制雄激素受体二聚体入核的核浆分离蛋白图
具体实施方式
实施例1
细胞相关实验涉及的试剂与细胞:二氢睾酮,批号:CHB16A802100G。DMSO,批号:#RNBK5094,氟班色林,批号:W16O8Z45834,将5mg氟班色林溶于1.28mL的DMSO中配成浓度为10000uM的母液。Lncap细胞系为人前列腺癌细胞系,购自厦门逸漠生物科技有限公司,样本编号为:20210106-06,生物安全等级:BSL-1,上皮样,贴壁生长;单细胞及松散贴壁的细胞簇。培养条件为:RPMI Medium 1640(Invitrogen,11875093)88ml+FBS(Gibco)10ml+Glutamax(Invitrogen,35050)1ml+Sodium Pyruvate 100mM Solution(Invitrogen,11360070)1ml,该细胞对二氢睾酮(生长调节并生成酸性磷酸酶ACP)有响应。
将二氢睾酮溶解于DMSO配置成0.01mol/L的母液,用培养基依次稀释为10-5M,10- 6M,10-7M,10-8M,10-9M,10-10M的浓度梯度,加入到贴壁的Lncap细胞中,培养48h,72h,96h,用MTT测定Lncap细胞的增殖,结合RT-PCR探索不同浓度下,下游基因KLK3,KLK2的响应,将促增殖效果最明显且下游基因响应最显著对应的二氢睾酮的浓度10-9M作为Lncap细胞最佳的雄激素造模浓度。在雄激素造模后的Lncap细胞中加入不同浓度梯度的药物10-3uM,10-1uM,1uM,10uM,50uM,100uM分别共培养24h,48h,96h,MTT法测定氟班色林对Lncap细胞的增殖抑制率。结果显示,低浓度的氟班色林即可抑制Lncap细胞的增殖,显示出细胞水平的药效活性。
本实验结果以Excel软件处理处理,实验结果见图1及下表:
表1:不同浓度DHT对Lncap细胞的增殖促进48h
实验结果显示,不同浓度的DHT对Lncap细胞的促进增殖情况不同,10-9uM的DHT对Lncap细胞的增值促进最明显。因此选择此浓度作为药物造模的最佳浓度。
表2:不同浓度Fibanserin对Lncap细胞的增殖抑制48h
实验结果显示,以10-9M DHT造模后加入不同浓度的氟班色林,对Lncap细胞的增殖抑制随氟班色林浓度的升高而升高,因此在细胞水平,氟班色林具有药效活性。
实施例2
动物实验相关试剂:丙酸睾酮,批号:T818615。大豆油,批号:A23GS146219。将80mg丙酸睾酮溶解于40mL大豆油配置成2mg/mL的溶液。非那雄胺,批号:A16GS145548。将0.28mg溶解于20mL生理盐水配成1.4X10-2mg/mL的溶液,氟班色林,批号:W16O8Z45834,将0.32mg溶解于20mL生理盐水配成1.6X10-2mg/mL的溶液
试验动物为雄性C58BL/6小鼠,非近交系封闭群,体重18-22g,5周龄,合格证号:No.202207119,由扬州大学比较医学中心提供。实验室室温20-22℃,相对湿度40%-60%,换气扇通气,自然光源12h/日,笼养,每笼7只,每三日清扫笼舍一次。
在注射丙酸睾酮造模前,先将小鼠固定,用电推推掉小鼠背部毛发,再用脱毛膏脱掉绒毛的预处理。按照7只/笼进行分组,分为完全空白组(除脱毛外不作任何处理),对照组(注射大豆油0.2mL+灌胃生理盐水0.1mL),模型组(注射丙酸睾酮溶液0.2mL+灌胃生理盐水0.1mL),模型组+非那雄胺(注射丙酸睾酮溶液0.2mL+灌胃非那雄胺溶液0.1mL),模型组+氟班色林(注射丙酸睾酮溶液0.2mL+灌胃氟班色林溶液0.1mL),其中在灌胃之前,先注射丙酸睾酮溶液造模21天,通过观察小鼠背部毛发的生长情况判断造模的成功率。在造模成功后,在继续注射丙酸睾酮溶液的基础上灌胃给药处理,给药4周。给药结束后,统计药物的有效率(毛发恢复小鼠与小鼠总数比例的百分比),统计小鼠长毛部位的面积与脱毛部位面积的比例,在小鼠背部毛发恢复的不同部位多点取小鼠毛发进行长度测量与统计,脱颈处死小鼠,取其背部皮肤,一部分冻存于-80℃用于提取蛋白质进行核浆分离实验探索药物对雄激素受体二聚体的抑制入核情况,一部分用4%多聚甲醛,货号:AF030,固定48h后,进行组织切片,做HE染色,显微镜下统计小鼠背部皮肤单位面积内的毛囊数,毛乳头直径,毛囊生长期与休止期的比例,与模型组,完全空白组,对照组及阳性药非那雄胺比较统计氟班色林的药效。判断标准:对小鼠脱毛后背部脱毛面积进行测量记为A,给药结束后小鼠背部长毛面积记为B,对每组中A/B的数值取平均值作为动物毛发恢复的有效率。
结果显示,与模型组相比,氟班色林及非那雄胺给药组小鼠的毛发均有不同程度的恢复且与对照组及完全空白组毛生长情况相近(图2)。氟班色林组动物毛发恢复有效率达85.7%,非那雄胺组达71.4%(图3)。与模型组相比,HE组织切片染色显示氟班色林组的毛发长度增加,生长期与休止期比例增加,小鼠表皮与真皮厚度比例增加,单位面积内的毛囊数增加,毛囊的直径增加(图4-图7)。因此本次结果显示,氟班色林可以逆转雄激素介导的雄激素脱发损伤,可治疗雄激素脱发,恢复毛发的生长(图8)。
表3:对照组与非那雄胺与氟班色林的动物有效率
组别 | 对照组 | 非那雄胺 | 氟班色林 |
药物有效率 | 100% | 71.4% | 85.7% |
实施例3
按照酶消化法提取小鼠毛乳头原代细胞,培养条件为L-DMEM(凯基生物)88ml+FBS(森贝伽生物)10ml+Glutamax(Invitrogen,35050)1ml 1ml,培养10天,原代细胞长满T25培养瓶,进行传代,一部分用于流式细胞数实验进行细胞表面标志物的鉴定,鉴定结果为CD56,CD133,CD140a阳性,CD73,CD90阴性,为毛乳头细胞。将细胞接种到12孔板中,分别按照空白组,DHT组(10-8mol/L),DHT(10-8mol/L)+非那雄胺组(5umol/L),DHT(10-8mol/L)+高剂量氟班色林组(5umol/L),DHT(10-8mol/L)+低剂量氟班色林组(1umol/L)进行加药处理,48h后利用碧云天核蛋白与浆蛋白分离试剂盒,按照说明书进行核浆分离实验,将分离的核蛋白与浆蛋白分别进行SDS-PAGE凝胶电泳。
实验结果显示:H3为核蛋白的内参,β-Tublin为浆蛋白的内参,从蛋白图可以看出,核蛋白组只有H3蛋白,浆蛋白组只有β-Tublin蛋白,说明核浆分离实验成功。DHT组相对于空白组,雄激素受体表达上调。非那雄胺组与两种剂量的氟班色林组相对于DHT组雄激素受体含量无显著性差异,说明非那雄胺与氟班色林不改变雄激素受体的总量,但是两种剂量的氟班色林组相对于非那雄胺组与DHT组的雄激素受体在细胞核中的含量均减少,在细胞浆中的含量均相对增加,说明氟班色林能抑制雄激素受体的入核(图9)。
Claims (3)
1.氟班色林在制备治疗雄激素脱发的药物中的应用。
2.根据权利要求1所述的应用,其特征在于氟班色林能够增加毛发长度、生长期与休止期比例、表皮与真皮厚度比例、单位面积内的毛囊数以及毛囊的直径。
3.根据权利要求1所述的应用,其特征在于氟班色林在制备逆转雄激素介导的雄激素脱发损伤的药物中的应用。
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