CN116919951A - 小分子药物indisulam在制备治疗自身免疫病的药物中的应用 - Google Patents
小分子药物indisulam在制备治疗自身免疫病的药物中的应用 Download PDFInfo
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Abstract
本发明公开了小分子药物indisulam在制备治疗自身免疫病的药物中的应用，本发明进一步验证了该分子的安全性，长期连续使用该药物不会导致动物体重下降，且体重变化与正常对照小鼠无差异；多器官的组织切片表明该药物对各器官无明显的毒副作用。此外该药物可促进调节性T细胞的分化，但不影响其他免疫细胞的功能。因此我们将该药物用于治疗自身免疫性疾病，发现效果良好。
Description
技术领域
本发明涉及医药领域，具体涉及小分子药物indisulam在制备治疗自身免疫病的药物中的应用。
背景技术
肿瘤的形成与免疫系统有关，免疫细胞功能被抑制或者耗竭，无法有效杀伤肿瘤，是肿瘤发生、逃逸、转移复发的关键原因。与此相反，自身免疫性疾病是机体免疫细胞功能亢进或无法有效被抑制，导致免疫细胞攻击自身正常组织细胞导致的疾病，患者往往需要终身服用免疫抑制药物，导致正常的免疫细胞功能也被抑制，从而大大增加患者感染和罹患肿瘤的风险。而在人体内，天然存在一群免疫调节性T细胞，它能适时适度的抑制其他免疫细胞的功能，从而维持机体的免疫稳态，既保持对外界病原微生物和肿瘤细胞的清除能力，又不会导致自身免疫性疾病。
小分子药物具有使用广泛、理论成熟等优势。据统计，在常用药物中，小分子药物的数量可占总量的98％。小分子药物结构具有良好的空间分散性，其化学性质决定了其良好的成药性能和药物代谢动力学性质。这些特点就使得小分子药物在药物研发过程及其它药物领域中表现出巨大优势。小分子药物通常是信号传导抑制剂，能够特异性地阻断肿瘤生长、增殖过程中所必需的信号传导通路，从而达到治疗的目的。简单来说，小分子作为传统药物，可以口服，能够进入细胞内作用于胞内靶点，而大分子药物，一般作用于细胞表面的靶点，抑制蛋白相互作用，特异性强，但一般不能口服，难以进入细胞内。小分子药物indisulam在临床前的研究中具有很好的肿瘤抑制效果，并已经通过临床二期研究，但在三期临床试验中却没有抗肿瘤效果。而小分子药物indisulam是否用于其他疾病的治疗目前并无报道。因此，亟需对小分子药物indisulam的其它治疗用途进行开发。
发明内容
有鉴于此，本发明的目的之一在于提供一种小分子药物indisulam在制备治疗自身免疫病的药物中的应用。
为达到上述目的，本发明提供如下技术方案：
1、小分子药物indisulam在制备治疗自身免疫病的药物中的应用。
本发明优选的，所述自身免疫病为自身免疫性脑脊髓炎。
本发明优选的，所述小分子药物indisulam在制备提高自身免疫病的调节性T细胞比例的药物中的应用。
本发明优选的，所述小分子药物indisulam在制备改善自身免疫病患者脊髓组织病变的药物中的应用。
本发明的有益效果在于：本发明公开了小分子药物indisulam在制备治疗自身免疫病的药物中的应用，本发明进一步验证了该分子的安全性，长期连续使用该药物不会导致动物体重下降，且体重变化与正常对照小鼠无差异；多器官的组织切片表明该药物对各器官无明显的毒副作用。此外该药物可促进调节性T细胞的分化。因此我们将该药物用于治疗自身免疫性疾病，发现效果良好。
附图说明
为了使本发明的目的、技术方案和有益效果更加清楚，本发明提供如下附图进行说明：
图1为对照组与实验组在注射第1天和注射后29天时体重变化；
图2为小分子药物indisulam对小鼠器官的影响；
图3为Indisulam治疗组小鼠脾脏(SP)和淋巴结(LN)CD4 T细胞中调节性T细胞(Treg)比例；
图4为HPCD注射组和Indisulam治疗组小鼠自身免疫性脑脊髓炎评分及两组小鼠脊髓组织H&E染色结果。
图5为自身免疫性脑脊髓炎小鼠经HPCD注射和Indisulam治疗后，脾脏调节性T细胞的比例。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明，以使本领域的技术人员可以更好的理解本发明并能予以实施，但所举实施例不作为对本发明的限定。
小分子药物indisulam的获得：购买于上海瀚香生物有限公司，CAS：165668-41-7。
实施例1、自身免疫性脑脊髓炎(EAE)模型建立
按每只小鼠30μl 10mg/ml髓寡树突细胞糖蛋白(MOG)+100μl 1×PBS+100μl 5mg/ml完全弗氏佐剂(CFA)准备各组分。
乳液配置：连接两个螺纹注射器在三通阀中，拔下其中一个的推杆，依次加入PBS、MOG和CFA，安装好注射器，通过开关三通阀推动注射器排除气体，在冰上来回推动至完全乳化，随后转移到一个注射器中，至于冰上暂存(在烧杯中加入一滴乳液，液滴将稳定保持为团块，缓慢消散，说明乳化完成)。
小鼠免疫：用异氟烷将小鼠麻醉，并用2ml注射器于小鼠背部腰段两侧行皮下各注射100μl乳液。随后用胰岛素注射器在小鼠眼球静脉注射100μl百日咳毒素(PTX:2μl100mg/ml PTX+98μl 1×PBS)，并在48h后重复注射一次。
称重并评分：从免疫当天开始，隔天称重小鼠，在有发病迹象后开始每天称重并评分(正常0分，鼠尾无力1分，后肢无力2分，后肢瘫痪3分，前肢瘫痪4分，死亡5分，中间状态可设0.5分)。
成功建模的标准：小鼠在致敏后7-14天开始发病，症状表现为，运动不协调、肢体瘫痪等临床多发性硬化症的表现。疾病等级标准分为5级。1级：动物尾部无力；2级：尾部无力加肢体无力；3级：肢体轻度麻痹；4级：肢体严重麻痹，被动翻身后不能复原；5级：濒死状态。
实施例2、小分子药物indisulam对正常小鼠体重或器官的影响
环糊精配置：将羟丙基β环糊精(药用级)按0.2g/ml溶于无菌生理盐水中。
Indisulam配置：将Indisulam(100mg/ml)与上述HPCD按1：19稀释。
注射：取正常小鼠每只隔天腹腔注射100μl羟丙基β环糊精(HPCD)(对照组)或Indisulam(实验组)，连续四周，注射前(day1)与注射四周(day29)后对每只小鼠称重并统计，结果如图1所示。结果显示，实验组与对照组在注射后29天时体重以及体重与第一天的比值无显著性差异。
然后分别取对照组和实验组处理的心、肝、脾、肺、肾、大肠和小肠进行H&E染色，H&E染色委托生物公司进行，包括脱水、修剪、包埋、切片、染色、封片等，最后镜检，结果如图2所示。结果显示，在实验组和对照组之间没有明显差异，说明药物对各器官没有明显的毒副作用。
实施例3、小分子药物indisulam对自身免疫性疾病的影响
造模成功后按实施例2方式注射药物，29天后取部分脑、腰段脊髓进行H&E染色。剩余脑、脊髓组织分别进行淋巴细胞分离，使用流式细胞术分析细胞情况。
流式细胞术(FACS)具体步骤如下：
1.细胞收获：
①淋巴结细胞获取：将小鼠断颈处死，放置在铺了70％酒精消毒擦手纸的泡沫板上，剪开小鼠外皮，取腋下、腋窝、腹股沟淋巴结各两个，在1×PBS缓冲液中用尼龙细胞过滤网研磨过滤，离心(500g，5min)后用1×PBS缓冲液重悬。
②脾脏细胞获取：取脾脏，步骤同上，研磨过滤完成后离心(500g，5min)，用1mlACK Lysing Buffer重悬，室温静止5min裂解红细胞，离心(500g，5min)后用1×PBS缓冲液重悬。
③脑、脊髓细胞获取：取大脑和脊髓，步骤同上，将脑脊髓充分剪碎后研磨过滤，离心(500g，5min)后用50％ Percoll重悬，缓慢滴加至70％Percoll上层，离心(18℃，500g，30min，缓升缓降)后吸取中间层，离心(500g，5min)后用1×PBS缓冲液重悬。
2.细胞死亡染色：将细胞悬液离心(500g，5min)，弃上清，每个样品用100μl死活染液(zombie yellow:1×PBS缓冲液1:1000)重悬，室温避光孵育15min，离心(500g，5min)后用1×PBS缓冲液重悬。
3.细胞表面染色：按表面抗体:FACS Buffer(0.5％FBS-PBS)1:400配置表面抗体预混液，将悬液离心(500g，5min)，弃上清，每个样品用50μl表面抗体预混液重悬，冰上避光孵育30min，离心(500g，5min)后用FACS Buffer重悬。
4.细胞因子/转录因子染色：将细胞悬液离心(500g，5min)，弃上清，每个样品用50μl对应的细胞因子/转录因子固定液重悬，冰上避光孵育30min；离心(10000g，1min)后每个样品用200μl相应的Wash Buffer重悬，离心(10000g，1min)后每个样品用50μl细胞因子/转录因子染液(细胞因子抗体:细胞因子Wash Buffer 1:200，转录因子抗体:转录因子WashBuffer1:100)重悬，冰上避光孵育30min，离心(10000g，1min)后每个样品用200μl相应的Wash Buffer重悬。(若同时染细胞因子和转录因子，可单独使用转录因子Kit)
5.收样：将染色完成的悬液离心(未固定的500g，5min，固定过的10000g，1min)，弃上清，用200-500μl的FACS重悬，用尼龙网过滤至流式样品管中，用流式细胞仪进行流式荧光检测。
结果如图3所示。结果显示，Indisulam注射组小鼠脾脏(SP)和淋巴结(LN)CD4 T细胞中调节性T细胞(Treg)比例显著提高。
H&E染色委托生物公司进行，取小鼠的脊髓进行组织切片染色：包括脱水、修剪、包埋、切片、染色、封片等，最后镜检，结果如图4所示。结果显示，自身免疫性脑脊髓炎(EAE)小鼠隔天注射环糊精与Indisulam，发现Indisulam可显著降低模型小鼠疾病的发生，并显著改善小鼠体重下降的情况，同时脊髓组织的病变情况也显著好于HPCD组。
通过流式细胞技术分析发现，与HPCD治疗组相比，经Indisulam治疗的自身免疫性脑脊髓炎小鼠，脾脏中调节性T细胞的比例显著上升。
上述结果表明Indisulam可显著增加自身免疫性脑脊髓炎小鼠脾脏的调节性T细胞比例，进而降低模型小鼠疾病的发生，并显著改善小鼠体重下降的情况，同时脊髓组织的病变情况也显著好于HPCD组。
将造模成功的自身免疫性脑脊髓炎小鼠经HPCD注射和治疗Indisulam后，检测脾脏调节性T细胞的比例，结果如图5所示。结果显示，Indisulam治疗组脾脏调节性T细胞的比例更高。
以上所述实施例仅是为充分说明本发明而所举的较佳的实施例，本发明的保护范围不限于此。本技术领域的技术人员在本发明基础上所作的等同替代或变换，均在本发明的保护范围之内。本发明的保护范围以权利要求书为准。
Claims (4)
1.小分子药物indisulam在制备治疗自身免疫病的药物中的应用。
2.根据权利要求1所述的应用，其特征在于：所述自身免疫病为自身免疫性脑脊髓炎。
3.根据权利要求1所述的应用，其特征在于：所述小分子药物indisulam在制备提高自身免疫病的调节性T细胞比例的药物中的应用。
4.根据权利要求1所述的应用，其特征在于：所述小分子药物indisulam在制备改善自身免疫病患者脊髓组织病变的药物中的应用。
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