The Appraising And Pharmacologic Mechanism Study Of Novel PPAR Agonist P633 And C333

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.