Development Of Zwitterionic Polypeptide Conjugated GLP-1 Analogue For Sustained Release

GLP-1(glucagon-like peptide-1)is a safe and effective incretin for lowering blood glucose in type 2 diabetes,and it is an important object of current drug research for type 2 diabetes.However,due to its sensitivity to DPP-Ⅳ enzyme resection and rapid renal clearance,the half-life of the natural GLP-1 is very short.Therefore,GLP-1 analogues replaced by enzyme-resection-sensitive residues are widely used at present,and the improvement of circulating half-life and slow-release mechanism through the conjugation of GLP-1 with macromolecular carriers is the main research direction.However,the former has different degrees of side effects due to high blood drug concentration peaks after injection.The latter,such as Exenatide Microspheres,achieves slow release of the drug through the degradation of polylactic acid to maintain a stable blood concentration,but there are still problems of adverse immune reactions and unstable degradation speed.Based on the excellent anti-protein non-specific adsorption ability,histocompatibility,enzyme degradation and biological metabolism ability of zwitterionic polypeptide material pEK,we design a GLP-1 analogue(pEK-16C-GLP1)modified with hexadecyl stearic acid-terminated polyglutamic acid-lysine zwitterionic polypeptide(pEK-16C).It is expected that pEK-16C-GLP1 can reduce the side effects and adverse immunogenic reactions caused by the peak blood concentration,through the way of subcutaneous sustained release combinate with improving the circulatory half-life of the drug,and improve the overall safety and long-term efficacy of the drug.In this paper,we synthesis and analysis the pEK-16C-GLP1,and study its in vitro release,in vitro activity,pharmacokinetic,therapeutic efficacy,and immunogenicity.The main research contents and results are as follows:(1)pEK-16C-GLP1 with different molecular weights was synthesized,and the successful synthesis of pEK-16C-GLP1 was confirmed through a series of tests.Through the critical micelle concentration(CMC)test,the CMC of pEK-16C-GLP1 is 0.001 mg/mL.Combining transmission electron microscopy(TEM)and dynamic laser light scattering(DLS)tests,shown that pEK-16C-GLP1 forms a micelle with a hydrophobic core of GLP1-16C and a hydrophilic shell of pEK in the water phase,and its hydrodynamic diameter is 500nm.The in vitro drug release results show that when the molecular weight of pEK-16C is 15400 Da,the drug has a good sustained release and reduction Kidney clearance ability.Studies on the biological activity of drugs on INS-1 cells show that pEK-16C-GLP1 retains the physiological activity of GLP-1 to promote insulin secretion and cell proliferation in INS-1 cells.(2)Continuously monitor the plasma GLP-1 concentration after subcutaneous administration in SD rats and the subcutaneous release process of the fluorescently labeled drug pEK-16C-GLP1-Cy5.5.It shows that the drug is released into the blood circulation in a zero-order kinetic mode of the subcutaneous storage,and stably maintains the effective GLP-1 concentration in the body.Through intravenous injection of pEK-16C-GLP1-Cy3,the elimination half-life of the drug was prolonged from 1.5 min of natural GLP-1 to 39.44h of pEK-16C-GLP1,which proved that pEK-16C-GLP1 significantly improved the half-life of GLP-1.Pharmacodynamic studies have shown that pEK-16C-GLP1 achieves 10 days and 24 days blood glucose control in SD rat type 2 diabetes model and spontaneous type 2 diabetes db/db mouse model,Comparing with Polyethylene glycol losena,it has more significant long-term blood sugar control ability.(3)Observation of HE,CD68 immunohistochemistry and Masson stainning sections of the skin at the injection site showed that pEK-16C-GLP1 did not cause obvious inflammation and tissue proliferation.However,a large number of inflammatory cells and more tissue proliferation appeared at the injection site of PEG loxenatide,which proved that pEK-16C-GLP1 has a lower immune response and excellent histocompatibility.