【作者】 陈伟；

【导师】 王莉莉； 宫泽辉；

【作者基本信息】 中国人民解放军军事医学科学院 ， 药理学， 2009， 博士

【摘要】 糖尿病是一种严重威胁人类健康的内分泌代谢紊乱性疾病,其中90%以上为2型糖尿病(T2DM)。T2DM常与肥胖、脂代谢紊乱等并存,并与代谢综合症(MS)一起成为了一种流行性、日益严重的世界公共卫生难题。现有口服降糖药物不能有效改善糖尿病伴发的脂代谢紊乱,且存在长期使用的副作用问题。因此,研发新型、安全性更好的创新药物具有重要意义。过氧化合物酶体增殖因子活化受体(PPAR),是核受体超家族成员之一,有三种亚型:α、β/δ和γ。其中PPARα和PPARγ分别是降脂药物贝特类和糖尿病治疗药物噻唑烷二酮类的作用靶标。鉴于PPARα和γ激动剂各自具有的药理学作用特点,具有PPARα和PPARγ双重激动活性的化合物理论上可能具有各亚型激动剂的优势,并能够因此避免多种药物合用的相互作用问题。因此,以PPARα、PPARγ为靶标,开发兼具PPARα/γ双重激动活性的药物成为继罗格列酮之后,又一个“重磅炸弹”式T2DM预防和治疗药物的研究重点和热点。P633和C333是基于PPARα和PPARγ受体结构,设计合成的全新结构化合物。前期实验结果表明,两者均具有明确的PPARα和PPARγ的激动活性(P633的EC50分别是0.012μM和0.032μM,C333的EC50分别是0.399μM和0.155μM)和降低实验动物血糖水平的作用,同时具有良好的成药性。本课题的研究目标是通过对上述两种初步确定的结构全新的候选药物进行系统的药效评价和深入的作用机制研究,以期充分了解P633和C333的有效性和安全性,确定具有开发价值的候选新药。本研究工作由四部分内容组成:第一部分,化合物体外生物活性及作用机制研究,从核受体水平、细胞水平对P633和C333与受体的结合作用、调节作用、及对核转录调控因子的影响,在靶细胞上的生物学活性进行了研究;第二、三部分,在两种遗传性2型糖尿病和两种胰岛素抵抗性实验动物模型上,分别以PPARγ激动剂罗格列酮和/或PPARα/γ双重激动剂GW409544作为对照药物,评价了P633和C333的药效;深入探讨了其靶标相关和非相关作用机制。第四部分,针对罗格列酮临床使用出现的常见副反应,在C57 BL /6J小鼠上,初步观察了C333的相关作用。第一部分,分别采用表面等离子体共振分析技术和哺乳动物细胞双杂交实验方法,观察化合物与PPARγ蛋白的结合作用,以及对PPARγ2与核共激活因子(CBP)、共抑制因子(NCOR1)相互作用的影响;在前脂肪细胞分化和肝细胞脂肪酸摄取实验上,分别观察了化合物对PPARγ和PPARα靶标激活相关的细胞功能的影响及其对靶基因的调控作用。结果表明,P633和C333对PPARγ具有较高的亲和特性,其KD值分别为6.74×10-7 M和4.50×10-6 M(与PPARγ激动剂曲格列酮相近,KD值为2.74×10-7M);两化合物均能够促进核共激活因子CBP与PPARγ2结合、促进共抑制因子NCOR1从PPARγ2解离,但其作用强度均弱于PPARγ激动剂罗格列酮和PPARα/γ双重激动剂GW409544;在小鼠3T3-L1前脂肪细胞和HepG2肝细胞上,0.1-10μM的P633和C333均剂量相关的促进前脂肪细胞分化、促进肝细胞摄取游离脂肪酸、促进PPARγ下游直接靶基因ap2、LPL、GLUT4和PPARα下游直接靶基因ACO表达。因此,P633和C333可以与PPARγ结合,有效调节PPARγ2与核辅助调控因子的相互作用,并通过调节PPARα、PPARγ的靶基因实现其靶标作用相关的生物学功能。也进一步证实,与转译激活水平筛选结果相一致,新结构类型的PPARα/γ激动剂P633和C333是通过作用于PPARα、PPARγ实现其药理学作用。第二部分,在具有代谢综合征特征的MSG诱导肥胖小鼠上,10mg/kg的P633给药2周,能够改善模型小鼠的葡萄糖不耐受、餐后高血糖、以及高瘦素和高胰岛素血症,升高血中HDLc水平。在遗传性糖尿病db/db小鼠上,2.5 mg/kg和10mg/kg P633给药2周能够改善db/db小鼠胰岛素敏感性、高血糖、高瘦素和高胰岛素血症,降低血中TG、TC和FFA水平,但不产生罗格列酮和GW409544样的增加体重和脂肪重的作用;在遗传性糖尿病KKAy小鼠上,给药5天时出现降糖趋势,至第10天时不再降糖,至2周后出现升糖趋势,并剂量依赖性的升高血清胰岛素,但低剂量(1 mg/kg和2.5 mg/kg)可部分改善血脂紊乱。对P633用药后在KKAy出现的异常现象,采用组织病理学、电镜超微结构和免疫组化及定量PCR的方法,从胰岛素分泌组织到胰岛素作用的主要靶组织进行了分析。结果表明:10 mg/kg的P633剂量依赖性的显著上调肝脏糖异生关键酶PEPCK表达;显著促进胰岛增生、胰岛细胞核产生畸形样改变,线粒体肿胀,嵴消失;但P633降低小鼠肝脂含量,改善肝脏脂肪变性和肝细胞肥大,也未表现出脂肪组织胰岛素抵抗加重。可见,新结构化合物P633除具有PPARγ和PPARα激活作用外,还具有激活肝糖异生途径的靶标外作用的活性。提示,在基于靶标的新药发现过程中,全面评价候选分子的药理学作用十分必要。第三部分,在前期KKAy小鼠、饮食诱导的胰岛素抵抗大鼠模型药效评价的基础上,在db/db小鼠和MSG诱导肥胖大鼠模型上,评价C333对糖尿病及其并发症的治疗作用。结果表明:在MSG大鼠模型上,3 mg/kg和10mg/kg C333给药40天能够增强MSG肥胖大鼠的胰岛素敏感性,促进葡萄糖钳夹时的葡萄糖输注量,改善肝脏和胰腺病理改变,同时在该模型上无罗格列酮样的增加体重和棕色脂肪重,及非诺贝特样的增加肝重的不良作用。在db/db小鼠上,10 mg/kg和20mg/kg C333显著改善胰岛素敏感性、降低其高血糖及血TG、LDLc水平和TC/HDLc比值,在20mg/kg时其降糖作用略强于或相当于罗格列酮,无罗格列酮和GW409544样的增加体重和促进脂肪增加的副作用。采用组织病理学、电镜超微结构、定量PCR、western blot等实验方法进行深入分析,结果表明C333在体内能够激活PPARα/γ,调解(促进或抑制)肝脏(如PEPCK和CPT1等)和脂肪(如adiponectin和LPL等)内糖、脂代谢关键靶基因表达、促进骨骼肌和脂肪组织胰岛素受体蛋白表达和脂肪组织葡萄糖转运体蛋白表达,显著抑制肝脏、肌肉和脂肪组织内的11βHSD1基因表达,对肝脏11HSD1的下调作用最强,这提示抑制局部糖皮质激素的再活化也是其改善胰岛素的敏感性作用机制之一。第四部分,在C57BL/6J小鼠上观察了C333对正常小鼠体重及水钠潴留的影响,结果表明给药4周10-20 mg/kg的C333对正常小鼠的体重、脏器重量、血糖及血中乳酸含量均无明显影响,不上调介导水钠潴留作用的肾脏钠通道ENaCγ的蛋白表达;以上动物实验结果提示:C333促体重增加、产生水钠潴留和水肿等副作用的可能性较低,具有较好的安全性特征。综上所述,本课题取得的主要进展如下:1)证实新型PPARα/γ激动剂P633和C333在分子水平与PPARγ蛋白分子结合并具有较高的亲和特性。2)成功建立了两种转录辅助调控因子与PPARγ2相互作用、细胞双杂交分析的新模型。发现P633和C333剂量依赖的促进核共激活因子CBP与PPARγ2结合、促进共抑制因子NCOR1从PPARγ2解离,但其作用强度弱于PPARγ激动剂罗格列酮和PPARα/γ双重激动剂GW409544。3)证实P633和C333具有促进3T3-L1前脂肪细胞分化、促进肝细胞摄取游离脂肪酸、调节PPARγ和PPARα直接靶基因表达的靶标特异性作用特征。4)完成了候选化合物C333有效增加MSG诱导肥胖大鼠、db/db和KKAy小鼠的胰岛素敏感性,降低血糖水平、改善脂代谢紊乱的药效学评价,确证其作用机制主要是激活肝脏、脂肪组织PPARγ和PPARα,调节糖、脂代谢靶基因表达。C333在实验动物上无增重、促进水钠潴留的副作用,具有良好的安全性特征。确定C333是一个具有开发价值的候选新药。5)完成P633的药效学评价及作用机制研究,发现P633改善MSG诱导肥胖小鼠和db/db小鼠的胰岛素敏感性、高血糖、高瘦素和高胰岛素血症,并部分改善脂代谢紊乱症状,而无增重副作用。然而,P633却时间依赖性的升高KKAy小鼠的血糖,原因是非靶向的促进肝脏糖异生所致。6)在基于特定机制和靶标的新药发现过程中,在开发早期即进行多模型、多指标的综合性评价十分必要,有利于及早发现潜在问题提高研发效率。更多还原

【Abstract】 Diabetes, an endocrine metabolic disease, threatens human’s health and life seriously. More than ninety percent diabetics are type 2 diabetes (T2DM), which characterized by insulin resistance, hyperglycemia, and often accompanied with dyslipidemia and obesity. Together with metabolic syndrome (MS), they have become an epidemic and worldwide public health conundrum. However, the marketed anti-diabetic drugs still can’t meet the clinical demands, which include they can’t effectively ameliorate the accompanied dyslipidemia, and have serious side effects related to its long term dosing. Therefore, novel therapeutic drugs with better safety and tolerability are emergently needed, and which may possess a great prospect.Peroxisome proliferators activated receptors (PPARs) are a kind of nuclear receptor, which include three subtypes, i.e.α、β/δandγ, and modulate the expression of a series of target genes that play critical roles in regulating glucose, lipid and cholesterol metabolism. These have made PPARs as attractive therapeutic targets for T2DM and MS. Current PPARαagonists (fibrates) on the market are effective hypolipidemic agents, which decrease the progression of atherosclerosis while improving glucose intolerance in type 2 diabetic patients. Activation of PPARγby thiazolidinediones (TZDs) improves insulin sensitivity and decreases hyperglycemia in diabetic patients. Therefore, simultaneously activation of both PPARαand PPARγmight combine the advantages of the PPARαand PPARγagonists, and avoid some disadvantages of pure PPARγagonist. Therefore, researches in past few years have been focusing on the study of novel structural PPARα/γdual agonists, which might provide a broad spectrum of beneficial metabolic effects.Novel compounds P633 and C333, were designed and synthesized based on the spatial structures of PPARα/PPARγproteins in our lab. Preliminary studies indicated that they activate both PPARαand PPARγ, with EC50 values of 0.012μM and 0.032μM for P633 respectively, and 0.399μM and 0.155μM for C333 respectively, decrease blood glucose of diabetic animal models, and have good drug-like characteristics. In order to confirm the real candidate drug, systematic pharmacologic evaluations and mechanism analysis on P633 and C333 were carried out in the present study.The present work is composed of four parts. In the first part, using surface plasmon resonance (SPR) and mammalian two-hybrid assays, the binding activity and characteristics between the two compounds and PPARγprotein, the impacts to the interaction between PPARγ2 and co-activator CBP or co-repressor NCOR1 were observed. 3T3-L1 preadipocyte differentiation and free fatty acid uptake in HepG2 hepatic cell were used to study their bioactivity related to the activation of PPARαand PPARγ, likewise regulation in the target genes expression were used to probe the mechanisms related to PPARαand PPARγby RT-PCR. On molecular level, the binding constant, in terms of KD, between P633, C333 and PPARγwere 6.74×10-7 M and 4.50×10-6 M, respectively, closed to that of troglitazone(2.74×10-7 M), a classic PPARγagonist. P633 and C333 both induced the recruitment of co-activator CBP with PPARγ2, and dissociated co-repressor NCOR1 from PPARγ2, but their effects were lowered than that of PPARγagonist rosiglitazone and PPARα/γdual agonist GW409544 at the same concentrations. On celluar level, P633 and C333 from 0.1 to 10μM dose related promoted the differentiation of 3T3-L1 preadipocyte and free fatty acid uptake in HepG2 cells, and concomitantly up-regulated the expression of target genes of PPARα(ACO) and PPARγ(ap2、LPL and GLUT4), respectively. Therefore, P633 and C333 could bind to PPARγ, effectively regulated the interaction between PPARγ2 and nuclear transcriptional assistant regulators, and activate the target and produced related bioactivity through regulation the target genes expression. These confirmed the results in cellular transactivation assay, together demonstrated that P633 and C333, the structurally novel PPARα/γagonists, executed their pharmacologic effects through activation of PPARαand PPARγ.In the second and third parts, with rosiglitazone and/or GW409544 as control drugs, the pharmacodynamics and on-target or off-target mechanisms of P633 and C333 were performed on two kinds of T2DM diabetic and two kinds of insulin resistant rodent models.The second part, In MSG induced metabolic syndrome mice model, after treatment for 2 weeks, 10 mg kg-1 P633 improved the glucose intolerance, increased blood glucose, hyperleptinemia and hyperinsulinemia. 2.5 mg kg-1and 10 mg kg-1 P633 ameliorated the insulin sensitivity, blood glucose, hyperleptinemia and hyperinsulinemia, decreased serum TG, TC and FFA levels, without significant increasement of body weight and adipose weight as that of rosiglizone and GW409544 in genetic diabetic db/db mice. However, in the diabetic KKAy mice, sequential blood glucose monitoring indicated that blood glucose level showed initial descending tendency on the fifth day, but relapsed on the tenth day, and rose above that of control animals from the fifteenth day on. The anabatic increased blood glucose level showed no sign of descending relative to that of controls till the 25th day, and P633 also dose dependently increased serum insulin levels. At the same time, 1 mg/kg and 2.5 mg/kg P633 showed partial improvement to the lipid dysfunctions.To probe the reasons behind KKAy mice, the pancreatic and insulin sensitive target organs were analysed by histopathological, ultra structure observation, immunohistological and quantative PCR assays. The results indicated that 10 mg kg-1 P633 dose dependently up-regulated the expression of hepatic gluconeogenesis enzyme of PEPCK, significantly promoted pancreatic islet hyperplasticity, disrupted the structure of the pancreaticβcells, increased anomalous nuclei and swollen mitochondria and the losing of ridges. Simultaneously, P633 decreased the hepatic lipid contents, ameliorated hepatic steatosis and hypertrophy of hepatocytes, and insulin sensitivity in adipose tissue was not affected. Therefore, besides activate PPARγand PPARα, the structurally novel compound P633 still possesses off-target effect of up-regulating hepatic gluconeogenesis.Which hints us that comprehensive pharmacologic evaluations of candidate molecules is extremely essential in the process of drug development that based on special targets.In the third part, we focused on appraising the pharmacologic effects of C333 in db/db mice and MSG induced rat models. The results demonstrated that, during the 40days experiment, 3 mg/kg and 10mg/kg C333 enhanced the insulin sensitivity of MSG rat, increased the glucose infusion rate in the euglycemic hyperinsulinemic clamp experiment, and improved hepatic and pancreatic pathologic changes. In diabetic db/db mice, 10 mg/kg and 20mg/kg C333 effectively improved insulin sensitivity, decreased blood glucose and serum TG and LDLc and the ratio of TC/HDLc. 20mg/kg C333 was comparative to that of rosiglitazone. As well there are no obvious increasement of body weight and adipose weight in the two strains as that of rosiglitazone. Further studies with histopathological analysis, transmission electron microscope analysis, quantative PCR method and western blot, discovered that C333 could activate PPARα/γin vivo, regulate key target genes of glucose and lipid metabolism in liver (such as PEPCK and CPT1) and adipose (such as adiponectin and LPL), and increase the protein expression of insulin receptor in skeletal muscle and adipose and glucose transporter 4 in adipose tissue. Additionally, it had also been found that C333 significantly down-regulated the mRNA expression of 11βHSD1 in liver, skeletal muscle and adipose tissue, especially in the liver, which indicated that inhibition the regeneration of local glucocoticoid is also one of the mechanisms in improving insulin sensitivity of C333.In the last part, more attentions were paid on the question if C333 exists the latest reported body weight gain and fluid retention side effect as that of pure PPARγagonist rosiglitazone by in vivo experiment on C57 BL /6J mice. After 4 weeks administration with 10 and 20 mg/kg C333, the body weight, major organ weights, blood glucose and lactate content were not impacted. As well, the expression of ENaCγin kidney that mediated the fluid retention of rosiglitazone was not up-regulated by C333. The above results together signified that C333 is a candidate drug with good safety characters and few chances of increase body weight and fluid retention.In summarize, the major advancements of present work are as follows:1) Novel PPARα/γagonists P633 and C333 showed higher affinity to PPARγprotein on molecular level.2) Established two mammarian hybrid assay models for studying the interaction of PPARγ2 with transcriptional assistant regulators, and found that P633 and C333 both dose related induced the recruitment of co-activator CBP to PPARγ2, and dissociated co-repressor NCOR1 from PPARγ2, but the intensity under same concentrations was lowered than that of rosiglitazone and GW409544.3) P633 and C333 promoted 3T3-L1 preadipocyte differentiation, free fatty acid uptake of HepG2 hepatic cell and concomitantly up-regulated the target genes expression of PPARαin liver and PPARγin adipose cells, respectively.4) Novel PPARα/γagonist C333 could effectively ameliorate insulin sensitivity, decrease blood glucose, and partially improve dyslipidemia in MSG or DIO rats and db/db or KKAy mice, but doesn’t affect the body weight, and increase fluid retention in rodent models. The pharmacological mechanism is mainly related to activation of the PPARαand PPARγin the target tissues, ie, liver and adipose. In addition, down-regulation to 11βHSD1 may also contribute to the benefical effects of C333. Therefore, C333 is a candidate drug worth further developed.5) Novel PPARα/γagonist P633 could ameliorate insulin sensitivity, decrease blood glucose, hyperleptinemia and hyperinsulinemia, and partially improve dyslipidemia in MSG induced mice and db/db mice, doesn’t increase the body weight. Unexpected, P633 showed increase of blood glucose in genetic KKAy mice in time-dependent manner. The reason behind is the off-target effect of P633 in up-regulating hepatic gluconeogenesis.6) Comprehensive pharmacologic and bioactivity evaluations are extremely essential in drug discovery and development, especially the candidates come from definite mechanisms or target-based modes.Which would be contribute to facilitate the efficiency of new drug development.更多还原