CN111068041A - 奥曲肽在制备治疗溃疡性结肠炎药物中的应用 - Google Patents
奥曲肽在制备治疗溃疡性结肠炎药物中的应用 Download PDFInfo
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Abstract
本发明涉及生长抑素类似物奥曲肽治疗溃疡性结肠炎的口服给药途径,属于药学领域。本发明提供了口服给予奥曲肽对溃疡性结肠炎小鼠的治疗作用,溃疡性结肠炎小鼠在口服给予奥曲肽后炎症水平显著下降。本发明提供了口服给药剂量不同会引起的副作用(剂量过高会引起毒性)。本发明还提供了在有预给药情况下,口服途径给予奥曲肽会出现与未预给药不同的药效情况,并通过药物组织分布实验证实了预给药情况下的口服奥曲肽的药物累积。本实验还提供了溃疡性结肠炎小鼠口服奥曲肽吸收程度要高于正常小鼠的口服药动学行为。本发明增加了生长抑素类似物奥曲肽治疗溃疡性结肠炎的口服给药途径,提高了患者的顺应性。
Description
技术领域
本发明属于医药技术,特别是涉及生长抑素类似物奥曲肽治疗溃疡性结肠炎的给药途径的改变,具体地说是发现了奥曲肽的口服给药途径,提高药品使用的安全性及顺应性。
背景技术
随着生命科学的发展,活性多肽类药物已经成为制药行业重点研究领域,多肽类药物的研发和上市出现了逐步加速的趋势。目前,全世界获批上市的多肽类药物已经超过了50个,有约140个多肽类药物在临床研究中,而在临床前研发阶段中的多肽类药物达到500至600个。但由于多肽药物口服时受其本身的物理化学性质的限制,比如在胃酸中不稳定,易被胃肠道中的酶降解而失去生物活性,不易透过胃肠道粘膜等,限制了多肽药物的口服给药,降低了患者的顺应性,而严重限制了它的市场潜力。
生长抑素是在中枢及外周神经系统、内分泌腺体、消化系统,甚至一些肿瘤中广泛分布的多肽类激素,通过与生长抑素受体不同亚型(sstr1-5)结合发挥广泛的生理作用,包括抑制腺体和激素分泌、调节神经传导和细胞分化等。此外对抑制肿瘤,特别是神经内分泌肿瘤也有重要作用。天然的生长抑素在体内不稳定,半衰期短(2-3min)。一系列人工合成的生长抑素类似物如奥曲肽、兰瑞肽、伐普肽等保留了天然生长抑素的活性基团氨基酸序列Phe-Trp-Lys-Thr,并运用醇化、酰胺化、D型氨基酸对多肽进行修饰,以克服天然生长抑素的不足,并且成为已上市的较为成功的肽类药物。
同绝大多数的肽类药物一样,国内外目前生长抑素类似物多肽的给药途径为注射给药。由于患者对注射给药的顺应性大大低于口服给药,因此开发生长抑素类似物多肽的口服给药途径对患者来说具有重要意义。
发明内容
发明目的:针对上述现有技术存在的技术问题,本发明提供了一种奥曲肽在制备治疗溃疡性结肠炎药物中的应用。
技术方案:本发明公开了奥曲肽在制备治疗溃疡性结肠炎药物中的应用。
所述生长抑素类似物奥曲肽为通过一个二硫键连接的环型多肽,并且对肽链骨架进行了一定的修饰,奥曲肽的结构为:D-Phe—Cys—Phe—D-Trp—Lys—Thr—Cys—Thr(ol)。其中第一位的苯丙氨酸和第四位的色氨酸为D型氨基酸,第八位的苏氨酸改造为苏氨醇。肽链骨架环状结构的特征,以及D型氨基酸,非天然氨基酸等的引入,使得奥曲肽在胃肠道内具有一定的抗酶解能力,因此可以通过口服给药。
本发明还公开了奥曲肽在制备治疗溃疡性结肠炎口服药物中的应用。所述药物中奥曲肽的含量为低剂量,预给药情况下为1mg/kg;正常给药情况下为10mg/kg。
本发明是通过对生长抑素类似物奥曲肽的口服给药途径在溃疡性结肠炎C57BL/6小鼠体内以及人正常结肠上皮细胞NCM-460的炎症模型的药效研究实现的。生长抑素类似物奥曲肽的口服给药途径可以显著降低LPS引起的人正常结肠上皮细胞NCM-460的炎症水平,可以显著降低由DSS引起的C57BL/6小鼠的溃疡性结肠炎的炎症水平。
有益效果:本发明开发了生长抑素类似物奥曲肽治疗溃疡性结肠炎的新给药途径,证明了的口服给药途径是可行的,将生长抑素类似物奥曲肽治疗溃疡性结肠炎的单一注射给药途径的应用扩大到了口服给药途径,增加了生长抑素类似物奥曲肽的应用范围。生长抑素类似物奥曲肽治疗溃疡性结肠炎的口服给药途径具有两面性,低剂量可以改善炎症水平,高剂量会加重炎症水平。通过体内口服药动学证明,在预给药的情况下,药物会出现累积,导致是否预给药与其药效发挥紧密相关。生长抑素类似物奥曲肽的口服给药途径中,其在溃疡性结肠炎小鼠结肠吸收显著优于正常小鼠。
附图说明
图1A为奥曲肽对体外肠炎细胞的炎症因子TNF-α的调节作用;
图1B为奥曲肽对体外肠炎细胞的炎症因子IL-6的调节作用;
图1C为奥曲肽对体外肠炎细胞的炎症因子IL-1β的调节作用;
图2A为奥曲肽的口服给药途径对溃疡性结肠炎小鼠的结肠炎症因子TNF-α的调节作用;
图2B为奥曲肽的口服给药途径对溃疡性结肠炎小鼠的结肠炎症因子IL-6的调节作用;
图2C为奥曲肽的口服给药途径对溃疡性结肠炎小鼠的结肠炎症因子IL-1β的调节作用;
图3为奥曲肽的口服给药途径对溃疡性结肠炎小鼠的体重调节作用;
图4为奥曲肽的口服给药途径对溃疡性结肠炎小鼠的结肠长度调节作用;
图5为奥曲肽的口服给药途径对溃疡性结肠炎小鼠的结肠切片H&E染色;
图6为奥曲肽的口服给药途径在溃疡性结肠炎小鼠肠道内组织分布。
具体实施方式
下面结合附图和实施例,对本发明进行进一步的说明。
实施例1生长抑素类似物奥曲肽的口服给药途径对体外肠炎细胞的炎症因子TNF-α,IL-6,IL-1β的调节作用
第一步:用DMSO配置2mg/ml LPS(购自阿拉丁试剂有限公司)母液,配置1mM奥曲肽(购自无锡迈默拓普生物科技有限公司)母液,配置10mM地塞米松(购自阿拉丁试剂有限公司)母液。
第二步:将状态良好的NCM-460细胞(购自湖南丰晖生物科技有限公司)点至6孔板中,分为空白组,肠炎模型组,10μM奥曲肽给药组,100μM奥曲肽给药组,5μM地塞米松给药组,每组6个孔的细胞。
第三步:待细胞长至70-80%密度时,向除空白组的其余四组中加入LPS母液至中浓度1μg/ml,连同空白组一起于37度孵箱培养4h。
第四步:从孵箱中取出细胞,弃去培养基,并用无菌PBS溶液轻轻清洗细胞3次,更换新的培养基,并分别向10μM奥曲肽给药组,100μM奥曲肽给药组,5μM地塞米松给药组中加入药物至终浓度10μM奥曲肽,100μM奥曲肽,5μM地塞米松。将所有细胞置于37度孵箱中培养24h。
第五步:取出细胞,提取RNA,并通过RT-PCR手段,测定细胞中炎症因子TNF-α,IL-6,IL-1β基因表达水平。
结果分别如图1A、图1B、图1C所示,根据图示可见,NCM-460细胞用LPS处置之后,细胞中炎症因子TNF-α,IL-6,IL-1β基因表达水平显著升高,而给予100μM和10μM奥曲肽治疗后,炎症因子TNF-α,IL-6,IL-1β基因表达水平下降,具有显著性差异。
实施例2生长抑素类似物奥曲肽的口服给药途径对溃疡性结肠炎小鼠炎症水平的调节作用
第一步:将42只雄性6周龄C57BL/6小鼠,18-20g,随机分为7组,每组6只,分别为空白组,DSS造模组,1mg/kg奥曲肽预给药组,10mg/kg奥曲肽预给药组,1mg/kg奥曲肽正常给药组,10mg/kg奥曲肽正常给药组,100mg/kg五氨基水杨酸给药组(阳性药组)。
第二步:分好组后,于1-10天,每天向1mg/kg奥曲肽预给药组,10mg/kg奥曲肽预给药组灌胃给予奥曲肽,其余各组灌胃给予同等体积生理盐水。于11-17天,将除空白组的其余六组小鼠的饮用水更换为2.5%DSS水溶液,并对1mg/kg奥曲肽预给药组,10mg/kg奥曲肽预给药组,1mg/kg奥曲肽正常给药组,10mg/kg奥曲肽正常给药组,100mg/kg五氨基水杨酸给药组进行灌胃给药,其余两组灌胃给予同等体积生理盐水。于1-17天每天统计小鼠体重。结果如图3所示,根据图示可见,模型组小鼠随着造模时间增加,体重显著下降,与对照组小鼠具有显著性差异;而口服给予奥曲肽之后,小鼠体重与造模组相比显著回升。
第三步:于第18天处死小鼠,取小鼠结肠,测量其结肠长度。结果如图4所示,根据图示可见,模型组小鼠结肠长度与对照组相比显著降低;10mg/kg奥曲肽正常给药组结肠长度与模型组相比显著提高。
剪取小鼠结肠末端0.5cm置于多聚甲醛中进行固定48h,固定完成后进行石蜡包埋切片并进行H&E染色。结果如图5所示,根据图示可见,与对照组相比,模型组小鼠出现不同程度的表层上皮细胞脱落、炎性细胞浸润、隐窝损伤等病变;与模型组相比,10mg/kg给药组和1mg/kg预给药组组织病变损伤显著改善。
取部分小鼠结肠用匀浆器匀碎溶于生理盐水中,12000g*10分钟离心,取上清液。用ELISA试剂盒测定结肠匀浆上清液中炎症因子TNF-α,IL-6,IL-1β蛋白表达水平。结果分别如图2A、图2B、图2C所示,根据图示可见,模型组小鼠结肠中炎症因子TNF-α,IL-6,IL-1β表达水平与对照组相比显著升高;1mg/kg奥曲肽预给药组和10mg/kg奥曲肽正常给药组炎症因子TNF-α,IL-6,IL-1β表达水平与模型组小鼠相比显著下降;10mg/kg奥曲肽预给药组炎症因子TNF-α,IL-6,IL-1β表达水平与模型组小鼠相比反而显著升高。
实施例3奥曲肽的口服给药途径在溃疡性结肠炎小鼠肠道内组织分布
第一步:将36只雄性6周龄C57BL/6小鼠,18-20g,随机分为36组,每组12只,分别为空白组多剂量给药组,造模组多剂量给药组,造模组单剂量给药组。
第二步:分好组后,空白组多剂量给药组使用正常饮用水,造模组多剂量给药组和造模组单剂量给药组使用2.5%DSS水溶液作为饮用水。同时于1-6天,每天向空白组多剂量给药组,造模组多剂量给药组灌胃给予25mg/kg奥曲肽,向造模组单剂量给药组灌胃给予同等体积生理盐水。于第七天向三组同时灌胃给予25mg/kg奥曲肽。
第三步:在第七天灌胃给予25mg/kg奥曲肽后0.5h,将三组的各12只小鼠随机每组抽取6只进行处死,眼眶采取血清,并取十二指肠,空肠,回肠,结肠组织置于冰中储存。在第七天灌胃给予25mg/kg奥曲肽后2h,将三组各剩下的6只小鼠处死,眼眶采取血清,并取十二指肠,空肠,回肠,结肠组织置于冰中储存。
第四步:取各肠段组织按1g:10ml加入超纯水进行匀浆,12000g*10分钟离心后取上清。取50μl肠匀浆上清液加入10μl内标物氧化奥曲肽(0.25μg/ml),加入400μl乙腈,震荡30s后18000rpm*10分钟离心,取上清400μl加入140μl水及400μl二氯甲烷,震荡30s后8000rpm*10分钟离心,取上清120μl18000rpm*10分钟离心,取100μl置于内插管于LC-MS进行进样,测定奥曲肽浓度。
测定结果如图6所示,根据图示可见,(1)与对照组相比,模型组结肠与血浆中奥曲肽含量显著升高;(2)与单次给药组相比,多次给药后血浆及各肠段奥曲肽含量显著升高,说明多次给药后奥曲肽会发生蓄积现象,并解释了预给药情况下高剂量奥曲肽组会出现药物毒性而正常给药情况下不会出现的现象。
Claims (5)
1.奥曲肽在制备治疗溃疡性结肠炎药物中的应用。
2.奥曲肽在制备治疗溃疡性结肠炎口服药物中的应用。
3.根据权利要求1或2所述应用,其特征在于,所述奥曲肽结构为:D-Phe—Cys—Phe—D-Trp—Lys—Thr—Cys—Thr(ol)。
4.根据权利要求2所述应用,其特征在于,所述药物中奥曲肽在预给药的情况下口服给药1mg/kg有药效;在正常给药情况下口服给药10mg/kg有药效。
5.根据权利要求2所述应用,其特征在于,所述口服药物降低LPS引起的人正常结肠上皮细胞NCM-460的炎症水平,降低由DSS引起的小鼠的溃疡性结肠炎的炎症水平。
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