Preparation And Biological Activity Of Long-acting GLP-1 Analogue Modified By Zwitterionic Polypeptides

Glucagon like peptide-1(GLP-1)was widely studied as an important target for the treatment of type ? diabetes.Due to its short in-vivo half-life,only 2?3 minutes,some synthetic polymers like PEG and PLGA have been investigated by researchers to improve the pharmacokinetics(PK)and pharmacodynamics(PD)of GLP-1.But these polymers have been discovered to trigger an adverse immune response.Zwitterionic polypeptides have the advantages of nonfouling,advanced enzyme degradation and normal metabolism.In order to achieve a more stable in vivo release and an excellent circulation,improve the therapeutice fficacy and bioavailability,and avoid adverse immune reactions,the GLP-1 derivative(poly(EK)-lira)modified by zwitterionic polypeptide(poly(EK))witch made up of lysine(K)and glutamic acid(E)was designed,and its physicochemical properties,in vitro bioactivity,therapeutic efficacy,pharmacokinetics and immunogenicity were studied.The main contents and conclusions of this thesis were described as below:1.N?-(8-benzyl-L-glumatyl)-N?-benzyloxycarbonyl-L-lysine(poly(EK))was condensed by EDC·HC1/HOBt and N?-boc-S-trityl-L-cysteine serves as the end-group,and then deprotected by a mixed solution of 33 wt%HBr/HOAc and TFA.Lira-MA was prepared by the reaction of Lira-mainchain with Fmoc-protected and maleic anhydride.After the Click reaction of Lira-MA and Poly(EK)and the deprotection of the Fmoc group,poly(EK)-Lira has been synthesized.The structure of poly(EK),Lira-MA,poly(EK)-Lira were confirmed by 1 H NMR,and the MW and MW distribution of the poly(EK)were determined by GPC.Through a series of tests and characterization,including dynamic light scattering(DLS),transmission electron microscopy(TEM),circular dichroism(CD)and critical micelle concentration(CMC),it is confirmed that poly(EK)-Lira can disperse in water to form the micelles.2.Poly(EK)-Lira,lira-mainchain and PBS were added to INS1 cells to compare the different effect on the relative growth rate,as well as insulin secretion,and the relationship between the relative growth rate,as well as the insulin secretion,and the concentration of these peptides have been studyed.Therefore,the advanced bioactivity of poly(EK)-Lira has been proved.3.Via measuring the blood glucose of type ? diabetic rats after s.c.injection of poly(EK)-Lira,Liraglutide and PBS,the excellent in-vivo therapeutic efficacy of poly(EK)-Lira has been proved.By measuring the content of GLP-1 in blood at different time points after s.c.injection,the elimination half-life of poly(EK)-Lira was calculated.The retention of poly(EK)-Lira with Cy5.5 labeled in subcutaneous was studied by in vivo imaging technology,which indicated that poly(EK)-Lira had long-acting effect.4.After subcutaneously s.c.injection of poly(EK)-Lira for three weeks,HE-stained slices of surrounding tissues were observed.The mitigation of inflammatory immune responses with poly(EK)-Lira has been confirmed.And compared to PEG-Lira and PBS,it was confirmed that the modification of poly(EK)disolved the original problem of inflammatory immune responses.By testing the anti-poly(EK)antibody in serums of SD rats which were given subcutaneously s.c.injection of poly(EK)-Lira for three weeks,it has been proved that poly(EK)-Lira has the better biocompatibility.