本文選題：DPP-4抑制劑 + GLP-1��； 參考：《浙江大學(xué)》2014年博士論文

【摘要】：背景： 糖尿病是一種由于胰島素分泌功能受損而導(dǎo)致高血糖的代謝性疾病,臨床主要分為1型糖尿病和2型糖尿病,后者人數(shù)占90%以上,患者常伴有脂質(zhì)代謝異常。長(zhǎng)期代謝紊亂將導(dǎo)致胰島功能進(jìn)一步衰退及喪失,并可能導(dǎo)致視網(wǎng)膜、腎臟和神經(jīng)系統(tǒng)病變以及心血管并發(fā)癥的發(fā)生。因此,有效改善糖脂代謝對(duì)于控制糖尿病進(jìn)展、延緩并發(fā)癥發(fā)生、改善糖尿病患者生活質(zhì)量具有重要意義。針對(duì)2型糖尿病的發(fā)病機(jī)制,其藥物治療的主要策略是促進(jìn)胰島素釋放和改善胰島素抵抗,然而目前的口服降血糖藥物尚不能達(dá)到理想效果。此外,現(xiàn)有胰島素促分泌藥物存在低血糖、體重增加等不良反應(yīng),噻唑烷二酮類胰島素增敏藥可能存在增加心衰發(fā)生率、致癌等風(fēng)險(xiǎn),具有胰島素增敏作用的二甲雙胍劑量過(guò)大可發(fā)生乳酸血癥。因此,尋找具有良好的糖脂代謝調(diào)節(jié)作用且不良反應(yīng)少的抗2型糖尿病藥物成為當(dāng)前的研發(fā)重點(diǎn)。本文探討了新型DPP-4抑制劑和新型吲哚類衍生物GY3的抗糖尿病作用,旨在尋找高效低毒,同時(shí)具有糖脂代謝調(diào)節(jié)作用,且具有自主知識(shí)產(chǎn)權(quán)的抗糖尿病先導(dǎo)化合物并開(kāi)展作用機(jī)制研究。 第一部分新型吲哚類衍生物GY3的糖脂代謝調(diào)節(jié)作用及機(jī)制研究 研究目的：糖脂代謝紊亂是糖尿病的重要臨床表現(xiàn),與非酒精性脂肪性肝炎及心血管疾病并發(fā)癥的發(fā)生密切相關(guān),而現(xiàn)有抗糖尿病藥物糖脂代謝調(diào)節(jié)作用療效不足并常伴有多種毒副作用的發(fā)生。因此,開(kāi)發(fā)同時(shí)具有糖脂代謝調(diào)節(jié)作用的新型抗糖尿病藥物具有重要意義。我們?cè)谇捌谘芯恐邪l(fā)現(xiàn),新型吲哚類衍生物GY3具有較好的抗糖尿病作用,本論文對(duì)GY3的糖脂代謝調(diào)節(jié)作用作進(jìn)一步研究,旨在尋找同時(shí)具有糖脂代謝調(diào)節(jié)作用的抗糖尿病先導(dǎo)化合物并開(kāi)展作用機(jī)制研究。 方法與結(jié)果：采用葡萄糖測(cè)定試劑盒檢測(cè)GY3對(duì)HepG2肝細(xì)胞和3T3-L1成熟脂肪細(xì)胞葡萄糖消耗的影響,采用油紅-O染色和甘油三酯測(cè)定試劑盒檢測(cè)GY3對(duì)FFAs誘導(dǎo)的HepG2脂質(zhì)蓄積模型和3T3-L1成熟脂肪細(xì)胞模型脂質(zhì)含量的影響。結(jié)果表明,GY3可促進(jìn)HepG2和3T3-L1細(xì)胞葡萄糖消耗,降低3T3-L1細(xì)胞和FFAs誘導(dǎo)的HepG2細(xì)胞內(nèi)脂質(zhì)含量。采用Western blotting法和P13K抑制劑考察了PI3K/AKT信號(hào)通路在GY3糖脂代謝調(diào)節(jié)中的作用,結(jié)果表明,GY3增加細(xì)胞葡萄糖消耗和降低細(xì)胞內(nèi)脂質(zhì)含量作用不依賴于PI3K/AKT信號(hào)通路。采用Western blotting法結(jié)合光密度分析對(duì)AMPK信號(hào)通路蛋白表達(dá)進(jìn)行檢測(cè),并采用Real Time RT-PCR對(duì)AMPK下游基因SREBP1-c和PEPCK表達(dá)進(jìn)行檢測(cè),結(jié)果表明,AMPK信號(hào)通道可能在GY3促進(jìn)HepG2和3T3-L1細(xì)胞葡萄糖消耗,減少其細(xì)胞內(nèi)脂質(zhì)含量過(guò)程中發(fā)揮了一定作用。為進(jìn)一步明確其作用機(jī)制,采用AMPK抑制劑compound C考察了AMPK通路在其中的作用地位,結(jié)果表明,GY3促進(jìn)細(xì)胞葡萄糖消耗和降低細(xì)胞內(nèi)脂質(zhì)含量作用依賴于AMPK信號(hào)通路。 結(jié)論：GY3可促進(jìn)細(xì)胞葡萄糖消耗,降低細(xì)胞內(nèi)脂質(zhì)含量；其糖脂代謝調(diào)節(jié)作用的發(fā)揮是AMPK信號(hào)通路所介導(dǎo)的。 第二部分新型DPP-4抑制劑YF-220-169的抗糖尿病作用及機(jī)制研究 研究目的：DPP-4抑制劑是一類具有葡萄糖依賴性降血糖作用的新型抗糖尿病藥物,可通過(guò)抑制DPP-4活性,延緩GLP-1降解,進(jìn)而通過(guò)促進(jìn)胰島素分泌、抑制胰高血糖素分泌及抑制胃排空等發(fā)揮降血糖作用,同時(shí)具有胰島保護(hù)作用。目前已上市藥物對(duì)DPP-4的抑制活性及臨床效果尚有待提高,而我們發(fā)現(xiàn)六氫吡咯[3,4-b]吡咯衍生物具有較好的DPP-4抑制活性。因此,我們對(duì)一系列衍生物進(jìn)行篩選,旨在尋找DPP-4抑制活性和選擇性更高,降糖作用更好,且具有自主知識(shí)產(chǎn)權(quán)的DPP-4抑制劑,并開(kāi)展其抗糖尿病作用及機(jī)制研究。 方法與結(jié)果：首先我們采用Gly-Pro-AMC熒光檢測(cè)法對(duì)衍生物體外DPP-4抑制活性及選擇性進(jìn)行篩選,發(fā)現(xiàn)YF-220-169具有優(yōu)于陽(yáng)性藥西格列汀和維格列汀的DPP-4抑制作用和選擇性。液相色譜-串聯(lián)質(zhì)譜法檢測(cè)給藥各時(shí)間點(diǎn)大鼠體內(nèi)血藥濃度表明,YF-220-169與西格列汀相比具有更大的體內(nèi)暴露量和更長(zhǎng)的體內(nèi)半衰期。接著,我們采用正常大鼠、db/db遺傳性糖尿病小鼠及STZ誘導(dǎo)的糖尿病大鼠對(duì)其抗糖尿病作用及機(jī)制進(jìn)行了研究。采用強(qiáng)生穩(wěn)豪型血糖儀測(cè)定血糖,Gly-Pro-AMC熒光檢測(cè)法檢測(cè)血漿DPP-4活性,Elisa法檢測(cè)血漿GLP-1和胰島素水平,HE染色并鏡下拍照觀察動(dòng)物胰島組織形態(tài)。正常大鼠實(shí)驗(yàn)結(jié)果表明：YF-220-169(0.3、1、3和10mg/kg)單次給藥可明顯改善大鼠葡萄糖耐量,相同劑量下較西格列汀強(qiáng)；YF-220-169單次給藥可明顯抑制大鼠葡萄糖刺激下的血漿DPP-4活性,同時(shí)升高血漿GLP-1和胰島素水平,相同劑量下較西格列汀強(qiáng)；YF-220-169單次給藥3h和6h后均可明顯抑制血漿DPP-4活性；在無(wú)葡萄糖刺激的情況下,YF-220-169對(duì)正常大鼠無(wú)降血糖作用。db/db小鼠實(shí)驗(yàn)表明：YF-220-169通過(guò)抑制DPP-4活性、促進(jìn)GLP-1和胰島素分泌而具有較好的降血糖和降血脂等抗糖尿病作用；長(zhǎng)期給藥可能將改善小鼠的胰島素抵抗?fàn)顟B(tài),改善胰島組織形態(tài)和功能；YF-220-169不增加小鼠基礎(chǔ)狀態(tài)下胰島素含量,可降低低血糖發(fā)生的風(fēng)險(xiǎn)；對(duì)體重?zé)o影響,可降低長(zhǎng)期給藥體重增加的風(fēng)險(xiǎn)。對(duì)STZ誘導(dǎo)的糖尿病大鼠實(shí)驗(yàn)表明：YF-220-169可通過(guò)抑制DPP-4活性、促進(jìn)GLP-1和胰島素分泌而具有較好的抗糖尿病作用；YF-220-169長(zhǎng)期給藥可能改善STZ誘導(dǎo)的糖尿病大鼠胰島組織形態(tài)和功能,從而升高基礎(chǔ)狀態(tài)下胰島素絕對(duì)缺乏的糖尿病大鼠模型血漿胰島素水平；YF-220-169長(zhǎng)期給藥對(duì)大鼠體重?zé)o影響,可降低長(zhǎng)期給藥體重增加的風(fēng)險(xiǎn)。 結(jié)論：(1)具有自主知識(shí)產(chǎn)權(quán)的六氫吡咯[3,4-b]吡咯衍生物YF-220-169表現(xiàn)出優(yōu)秀的體外DPP-4選擇性抑制作用,較好的體內(nèi)藥代動(dòng)力學(xué)特性以及降糖降脂作用。(2)YF-220-169長(zhǎng)期給藥可能改善機(jī)體胰島素抵抗?fàn)顟B(tài),同時(shí)可改善胰島組織形態(tài)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 .
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R965

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