Studdy Of Pegylation And Biological Activity Of Insulinotropic Analogues

Objective: TO obtain a long-acting insulinotropic peptide conjugate,a novel GLP-1 conjugate(PEG-Ex4C) was constructed by PEG modification of Byetta analogue. Ex4C was obtained by chemical synthsis with cysteine substitution in the C terminals of Byetta, and then specific modified with three different size of MAL-mPEG and purified by anion-exchange and reverse phase chromatography. The biological activity of conjugation (MAL-mPEG-Ex4C) was investigated by measuring cAMP production in RINm5f cells and evaluating its glucose-lowing efficacy in db/db diabete rat.Methods:(1) Ex4C was obtained by chemical synthsis with cysteine substitution in the C terminals of Byetta.①The purity of Ex4C was analysied by RP-HPLC,SEC-HPLC.②The quality research of Ex4C was investigated by mass spectrum, validate whether the free sulfhydryl of it’s cysteine was exposured.⑶The quality of MAL-mPEG was analysied by RP-HPLC-ELSD,SDS-PAGE.(2) The study of Ex4C modified with three different size of MAL-mPEG.(3) conjugation (MAL-mPEG-Ex4C) was purified by anion-exchange、SEC-HPLC and reverse phase chromatography(4) The pharmacokinetics of MAL-mPEG-Ex4C was research on rhesus monkey and The biological activity of conjugation(MAL-mPEG-Ex4C) was investigated by measuring cAMP production in RINm5f cells and evaluating its glucose-lowing efficacy in db/bd diabete rat.Results:(1) The molecular weight of Ex4C was identical with theoretical which purity is more than 98% and the free thiol of cysteine is all exposed. The purity of MAL-mPEG was more than 92% analysied by SDS-PAGE and SEC-HPLC.(2) Ex4c was modified with MAL-mPEG which have high conjugate ratio.(3) MAL-mPEG-Ex4C purifition method was established. Ex4C,DI-Ex4C,and free MAL-mPEG was removal by anion-exchange、SEC-HPLC and reverse phase chromatography,the purity of MAL-mPEG-Ex4C was more than 98%. (4) The pharmacology of MAL-mPEG-Ex4C indicate that he ED50 of cAMP production in RINm5f cells stimulated by 5kDa、20kDa and 35kDa PEG modified Ex4C was 1.61ng/ml,15.68ng/ml,32.95ng/ml, respectively.Monkey was Subscutanous administration with 20k-MAL-mPEG-Ex4C show the half-life was 2.63 day, and can significantly lower the blood glucose in db/db diabetes mouse models.