新型吲哚类衍生物和DPP-Ⅳ抑制剂的抗糖尿病作用及其机制研究

发布时间：2018-07-02 20:58

本文选题：DPP-4抑制剂 + GLP-1　；参考：《浙江大学》2014年博士论文

【摘要】：背景：糖尿病是一种由于胰岛素分泌功能受损而导致高血糖的代谢性疾病,临床主要分为1型糖尿病和2型糖尿病,后者人数占90%以上,患者常伴有脂质代谢异常。长期代谢紊乱将导致胰岛功能进一步衰退及丧失,并可能导致视网膜、肾脏和神经系统病变以及心血管并发症的发生。因此,有效改善糖脂代谢对于控制糖尿病进展、延缓并发症发生、改善糖尿病患者生活质量具有重要意义。针对2型糖尿病的发病机制,其药物治疗的主要策略是促进胰岛素释放和改善胰岛素抵抗,然而目前的口服降血糖药物尚不能达到理想效果。此外,现有胰岛素促分泌药物存在低血糖、体重增加等不良反应,噻唑烷二酮类胰岛素增敏药可能存在增加心衰发生率、致癌等风险,具有胰岛素增敏作用的二甲双胍剂量过大可发生乳酸血症。因此,寻找具有良好的糖脂代谢调节作用且不良反应少的抗2型糖尿病药物成为当前的研发重点。本文探讨了新型DPP-4抑制剂和新型吲哚类衍生物GY3的抗糖尿病作用,旨在寻找高效低毒,同时具有糖脂代谢调节作用,且具有自主知识产权的抗糖尿病先导化合物并开展作用机制研究。第一部分新型吲哚类衍生物GY3的糖脂代谢调节作用及机制研究研究目的：糖脂代谢紊乱是糖尿病的重要临床表现,与非酒精性脂肪性肝炎及心血管疾病并发症的发生密切相关,而现有抗糖尿病药物糖脂代谢调节作用疗效不足并常伴有多种毒副作用的发生。因此,开发同时具有糖脂代谢调节作用的新型抗糖尿病药物具有重要意义。我们在前期研究中发现,新型吲哚类衍生物GY3具有较好的抗糖尿病作用,本论文对GY3的糖脂代谢调节作用作进一步研究,旨在寻找同时具有糖脂代谢调节作用的抗糖尿病先导化合物并开展作用机制研究。方法与结果：采用葡萄糖测定试剂盒检测GY3对HepG2肝细胞和3T3-L1成熟脂肪细胞葡萄糖消耗的影响,采用油红-O染色和甘油三酯测定试剂盒检测GY3对FFAs诱导的HepG2脂质蓄积模型和3T3-L1成熟脂肪细胞模型脂质含量的影响。结果表明,GY3可促进HepG2和3T3-L1细胞葡萄糖消耗,降低3T3-L1细胞和FFAs诱导的HepG2细胞内脂质含量。采用Western blotting法和P13K抑制剂考察了PI3K/AKT信号通路在GY3糖脂代谢调节中的作用,结果表明,GY3增加细胞葡萄糖消耗和降低细胞内脂质含量作用不依赖于PI3K/AKT信号通路。采用Western blotting法结合光密度分析对AMPK信号通路蛋白表达进行检测,并采用Real Time RT-PCR对AMPK下游基因SREBP1-c和PEPCK表达进行检测,结果表明,AMPK信号通道可能在GY3促进HepG2和3T3-L1细胞葡萄糖消耗,减少其细胞内脂质含量过程中发挥了一定作用。为进一步明确其作用机制,采用AMPK抑制剂compound C考察了AMPK通路在其中的作用地位,结果表明,GY3促进细胞葡萄糖消耗和降低细胞内脂质含量作用依赖于AMPK信号通路。结论：GY3可促进细胞葡萄糖消耗,降低细胞内脂质含量；其糖脂代谢调节作用的发挥是AMPK信号通路所介导的。第二部分新型DPP-4抑制剂YF-220-169的抗糖尿病作用及机制研究研究目的：DPP-4抑制剂是一类具有葡萄糖依赖性降血糖作用的新型抗糖尿病药物,可通过抑制DPP-4活性,延缓GLP-1降解,进而通过促进胰岛素分泌、抑制胰高血糖素分泌及抑制胃排空等发挥降血糖作用,同时具有胰岛保护作用。目前已上市药物对DPP-4的抑制活性及临床效果尚有待提高,而我们发现六氢吡咯[3,4-b]吡咯衍生物具有较好的DPP-4抑制活性。因此,我们对一系列衍生物进行筛选,旨在寻找DPP-4抑制活性和选择性更高,降糖作用更好,且具有自主知识产权的DPP-4抑制剂,并开展其抗糖尿病作用及机制研究。方法与结果：首先我们采用Gly-Pro-AMC荧光检测法对衍生物体外DPP-4抑制活性及选择性进行筛选,发现YF-220-169具有优于阳性药西格列汀和维格列汀的DPP-4抑制作用和选择性。液相色谱-串联质谱法检测给药各时间点大鼠体内血药浓度表明,YF-220-169与西格列汀相比具有更大的体内暴露量和更长的体内半衰期。接着,我们采用正常大鼠、db/db遗传性糖尿病小鼠及STZ诱导的糖尿病大鼠对其抗糖尿病作用及机制进行了研究。采用强生稳豪型血糖仪测定血糖,Gly-Pro-AMC荧光检测法检测血浆DPP-4活性,Elisa法检测血浆GLP-1和胰岛素水平,HE染色并镜下拍照观察动物胰岛组织形态。正常大鼠实验结果表明：YF-220-169(0.3、1、3和10mg/kg)单次给药可明显改善大鼠葡萄糖耐量,相同剂量下较西格列汀强；YF-220-169单次给药可明显抑制大鼠葡萄糖刺激下的血浆DPP-4活性,同时升高血浆GLP-1和胰岛素水平,相同剂量下较西格列汀强；YF-220-169单次给药3h和6h后均可明显抑制血浆DPP-4活性；在无葡萄糖刺激的情况下,YF-220-169对正常大鼠无降血糖作用。db/db小鼠实验表明：YF-220-169通过抑制DPP-4活性、促进GLP-1和胰岛素分泌而具有较好的降血糖和降血脂等抗糖尿病作用；长期给药可能将改善小鼠的胰岛素抵抗状态,改善胰岛组织形态和功能；YF-220-169不增加小鼠基础状态下胰岛素含量,可降低低血糖发生的风险；对体重无影响,可降低长期给药体重增加的风险。对STZ诱导的糖尿病大鼠实验表明：YF-220-169可通过抑制DPP-4活性、促进GLP-1和胰岛素分泌而具有较好的抗糖尿病作用；YF-220-169长期给药可能改善STZ诱导的糖尿病大鼠胰岛组织形态和功能,从而升高基础状态下胰岛素绝对缺乏的糖尿病大鼠模型血浆胰岛素水平；YF-220-169长期给药对大鼠体重无影响,可降低长期给药体重增加的风险。结论：(1)具有自主知识产权的六氢吡咯[3,4-b]吡咯衍生物YF-220-169表现出优秀的体外DPP-4选择性抑制作用,较好的体内药代动力学特性以及降糖降脂作用。(2)YF-220-169长期给药可能改善机体胰岛素抵抗状态,同时可改善胰岛组织形态和功能。
[Abstract]:Background :

Diabetes mellitus is a metabolic disease that causes hyperglycemia due to impaired insulin secretion . It is mainly divided into type 1 diabetes mellitus and type 2 diabetes mellitus , the latter accounts for more than 90 % . The main strategy of drug therapy is to promote insulin release and improve insulin resistance .

Study on the Regulation and Mechanism of Glycolipid Metabolism in the First Part of New indole Derivatives GY3

The aim of this study is to investigate the important clinical manifestation of glycolipid metabolism disorder , which is closely related to the occurrence of non - alcoholic steatohepatitis and cardiovascular disease complications . It is of great significance to develop a new type of anti - diabetic drug with glycolipid metabolism regulating effect . In this paper , we find that the novel indole derivative GY3 has better anti - diabetic effect .

The effects of GY3 on glucose consumption in HepG2 and 3T3 - L1 adipocytes were detected by using glucose measurement kit . The results showed that GY3 could promote the glucose consumption of HepG2 and 3T3 - L1 cells and decrease the lipid content in 3T3 - L1 adipocytes . The results showed that GY3 could promote the glucose consumption of HepG2 and 3T3 - L1 cells and decrease the lipid content in 3T3 - L1 adipocytes .

Conclusion : GY3 can promote cell glucose consumption and decrease intracellular lipid content .
The function of glycolipid metabolism regulation is mediated by AMPK signal pathway .

Study on the Anti - diabetic Effects and Mechanism of the Second Part of the New Type of DPP - 4 Inhibitors

Research purposes : DPP - 4 inhibitor is a new type of anti - diabetic drug with glucose - dependent blood glucose lowering effect . It can inhibit the degradation of GLP - 1 by inhibiting the activity of DPP - 4 . The inhibition activity and clinical effect of DPP - 4 have yet to be improved by promoting insulin secretion , inhibiting glucagon secretion and inhibiting gastric emptying .

METHODS AND RESULTS : First , we used Gly - Pro - AMC fluorescence detection method to screen the inhibitory activity and selectivity of DPP - 4 in vitro . It was found that the plasma levels of DPP - 4 and insulin in rats were higher than that of sitagliptin .
The single dose of the F - 220 - 169 significantly inhibited plasma DPP - 4 activity in rat glucose , while increasing plasma GLP - 1 and insulin levels , which were higher at the same dose than in sitagliptin ;
The plasma DPP - 4 activity was significantly inhibited after 3 h and 6 h after single administration .
The experimental results of db / db mice showed that the inhibition of DPP - 4 activity , promoting GLP - 1 and insulin secretion , and lowering blood lipid and other anti - diabetes effects were observed in db / db mice .
Long - term administration may improve the insulin resistance of mice and improve the morphology and function of pancreatic islets ;
The risk of hypoglycemia can be reduced without increasing the insulin content in the basal state of mice .
No effect on body weight can reduce the risk of long - term administration of body weight gain . The experimental results of the diabetic rats with the induced diabetes show that the inhibition of DPP - 4 activity and promoting the secretion of GLP - 1 and insulin has better anti - diabetic effect .
The long - term administration of F - 220 - 169 could improve the morphology and function of islet tissue of diabetic rats , which induced diabetic rats , thereby increasing the plasma insulin level in diabetic rats with absolutely no insulin deficiency in basal state ;
The long - term administration of the F - 220 - 169 has no effect on the weight of the rats and can reduce the risk of prolonged administration of the drug .

Conclusion : ( 1 ) The selective inhibition of DPP - 4 in vitro , the pharmacokinetics of 4 - b pyrrolopyrrole derivatives in vitro and the effects of reducing sugar and lipid - lowering are shown in ( 1 ) The selective inhibition of DPP - 4 in vitro , and ( 2 ) the long - term administration of F - 220 - 169 may improve the insulin resistance status and improve the morphology and function of islet tissue .
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R965

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