MPEGylation Of Recombinat Insulin Analogues And Characterization Of Conjugates’ Biology Activity

Global upward trend in the number of patients with diabetes, diabetes prevention andTreatment situation is still grim, insulin is the only in vivo hormones to low blood sugar,which is especially Critical for the treatment of diabetes,but the use of insulin facedfrequent risk of hypoglycemia, weight gain and other problems in patients. To reduce theInconvenience and pain brought by frequent injections of insulin to diabetic patients, whilereducing hypoglycemic events, especially nocturnal hypoglycemia events, more effectivestable long-acting insulin analogues are needed in clinical.In this study, base on human insulin with deletion of ThrB30of Bchain(named:DET),a single20kDa methoxy-PEG-succinimidyl propionate(mPEG-succinimidyl propionate, mPEG-SPA) was attached to the1st annimo Gly of Achain by acylation reaction, form a Conjugates insulin analogues single modified at Glyof A chain(code: PEG-DET-A1). Finally, Establish a successed Preparation process andquality control, structural Confirmation, characterize its biological activity in vivo and invetro, verification for the preparation of a safe, controllable quality insulin conjugates，then laid a preliminary foundation of a new drug.MethodStudy on the Preparation of PEG-DET-A1Direct modification: without protection on amino, use20kDa mPEG-SPA specificmodify alpha amino group of Gly of DET, vary some factors,such as the ratio of insulinand mPEG-SPA, modified buffer systems, protein concentration, time of modification,temperature to optimized the perencents of PEG-DET-A1.BOC: find a suitable reagent conditions to protect two amino acids of insulin: B1amino acid (phenylalanine), and (or) B29amino acid (lysine) by BOC (t-butyldicarbonate), Insulin A1amino acid (glycine) is not protected, purified to give (B1, B29)-two BOC insulin under different conditions.A1the desired site for amino acid (glycine)modification reaction, then subjected to further modification of A1, and then deprotectedBOC off the B1and B29groups,so we got PEG-DET-A1.The structure of PEG-DET-A1was analysised by mass spectrometry, peptidemapping,other products after BOC were analysised too.Evaluation biological activity of PEG-DET-A1in vivo and In vitroBiological activity in vivo: insulin potency is defined according to method (rabbit blood method)In vitro biological activity: the use of rat adipocytes insulin on glucose consumptionwas measured in vitro biological activity, namely POD (glucose oxidase) method for thedetermination of glucose consumption rate to calculate the effect of insulinResultDirect modification of the method, it is detected SAX-HPLC purity of90%, polymeralike ornaments exist in electrophoresis.BOC process’conjugate have a band consistent with the PEG-DET-B29, no polymerobserved,and with a SAX-HPLC purity of93.4%.MALDI-TOF Mass mass spectrometry molecular weight determination is25245.07DaAfter the CD was measured at192nm having positive absorption peak and at210nmhaving characteristic negative absorption peak; non-reducing peptide mapping show the1st peptide disappeared,another new peptide appeared in56min.Extracellular glucose consumption was measured with POD by adipocytes,calculated in vitro biological potency of the active PEG-DET-A1was6.75U/mg.Units of insulin in rabbits was defined experimentally,determined potency in vivoactivity is PEG-DET-A1is37/90×27=11U/mg. Inconsistency in vivo activity may bedue to the presence of the complex of drug absorption, transport in the body, metabolicprocesses. More sensitive to the body obvious.ConclusionBOC process products get a higher purity, less relevant material than directlymodifying process modifications of BOC has proved the correct desired structure, thecorrect modification sites, a PEG molecules are coupled in position A1, and it showedgood biological activity in vivo and in vitro, in vivo the time of control blood sugar islonger than the standard insulin.