Ⅰ Preparation Of Exenatideand Liraglutide Using Solid Phase Peptide Synthesis Methods Ⅱ Evaluation Of Exenatide Pharmacodynamics

Objective:This paper was designed to establish feasible routes of synthesizing exenatide andliraglutide and to purify these products using high performance liquid chromatography.The therapeutic effects of exenatide on stimulating islet beta cell’s proliferation and onhypoglycemic effects were investigated as well.Methods:Fmoc strategy and solid phase peptide synthesis (SPSS) methods were employed tosynthesize exenatide and liraglutide with6-chloro-1-hydroxy-1H-benzotriazole(Cl-HOBt)as coupling reagent; the vector were Rink amide MBHA resin (loading:0.23mmol/L) andFmoc-Gly-Wang resin (loading:0.27mmol/L) respectively and with trifluoroaceticacid(TFA) as cleaving solution. Whether the processes accomplished were determined byninhydrin test. The products were purified by high performance liquid chromatographyusing Agilent ZORBAX SB-C18(9.4*250mm) columns and the mobile phase consisted ofA(water containing0.1%TFA) and B(acetonitrile containing0.1%TFA) with a flow rateof4.0mL/min. Mass spectrometry was applied to determine the contents and molecularweight.To investigate the effect of exenatide synthesized experimentally on islet beta cellswith exenatide injection (Lilly Co. products) as control, INS-1islet beta cell line werecultured in the presence of exenatide pretreatment, and the cells survival rate weredetermination by MTT method in elisa plates. To investigate bioequivalence of these twoexenatide products, diabetes models established by streptozotocin administrated i.p in mice,which were divided into4groups (A: blank group; B: model group; C: control group; D:medication group) randomly, and the effect of both produts on blood glucose were evaluted.During exenatide administrated for15days once a day, the levels of blood glucose weredetermined using blood glucose meter. The glucose tolerance test was performed in a week after exenatide drawn while the model mice obtained drinking water freely withoutchowing for16hours.Results:The elution gradient of exeantide was as follows: A accounted for90%for initialgradient and90%A to67%in15min and to58%A in50min and to0%A in5min, thenkeep run0%A for another10min and the product peak was appeared in25min. The elutiongradient of liraglutide was as follows: A accounted for85%for initial gradient and85%Ato70%in5min and to50%A in25min and to0%A in10min, then keep run0%A foranother10min and the product peak was appeared in33min. The amount of exeantide as aproduct was51.5mg with purity of99.73%and yield of12.3%, and39.4mg liraglutide wasobtained finally with purity of93.5%and yield of10.5%. The molecular weights of bothproducts (exeantide and liraglutide) determined by mass spectrometry was4186and3751,respectively. This result had no difference from actual molecular weights of exeantide andliraglutide.According to the dose-effect relationship of exentide stimulating beta cell’sproliferation, exenatide stimulated beta cell’s proliferation with an efficacy concentrationof120nmol/L. The activity of this experimental product was equal to the exenatideinjection of Lilly Co.. Compared with diabetes model mice there were significanthypoglycemic effects in diabets mice administred by exenatide for15days (P＜0.05), andthere were no significant different therapeutic effects between two exenatide products. Theglucose tolerance test showed that exenatide improved the diabetic mice’s glucosetolerance significantly.Conclusion:The two processes of synthesizing exenatide and liraglutide proved practically andeffectively. Pharmacodynamics experiment showed that exenatide synthesized presentlystimulated beta cell’s proliferation and had hypoglycemic effects in diabetic mice.