The hypoglycemic effect of the PEGylated FGF-21

Abstract

Fibroblast growth factor 21 (FGF-21) is a member of FGF family. Recently, it is discovered as a non-insulin-dependent cytokine to regulate blood glucose and a potential drug candidate for treatment of type 2 diabetes mellitus. However, the stability of FGF-21 is poor and its half-life in vivo is short, which severely affect its application in clinical practice. In order to solve this problem, the N-terminus of mFGF-21 was PEGylated in a site-specific manner by methoxy poly-ethylene glycol (mPEG) propionaldehydes with an average molecular mass of 20 ku for improving its biological properties including increasing half-life in vivo, and decreasing immunogenicity. The effects of pH, reaction time, protein concentrations and mass ratio between the reactants on the PEGylation of mFGF-21 were analyzed. PEG-mFGF-21 was isolated by Capto Q anion exchange chromatography or Superdex 75 gel filtration chromatography. As a result, the optimal reaction method for mFGF-21 PEGylation and purification processes of PEG-mFGF-21 were established. Then we study the physical and chemical properties, immunogenicity, in vivo half-life, in vitro biological activity and in vivo hypoglycemic effects of PEG-mFGF-21. We found that the temperature stability and anti-protease ability of mFGF-21 were significantly improved after PEGylation. The indirect ELISA results for detection of serum antibody levels against mFGF-21 and the target protein concentration showed that PEGlyation of mFGF-21 significantly reduced its immunogenicity and increased its half-life in vivo. The glucose uptake assay results in HepG2 cells demonstrated that the in vitro activity of PEG-mFGF-21 did not decline. However, with the increment of stimulating time, the glucose uptake in cells treated with PEG-mFGF-21 significantly increased than mFGF-21. The experimental results of short-term blood glucose regulation in type 2 diabetic animals showed that the hypoglycemic speed of mFGF-21 was faster than PEG-mFGF-21, but it lasted shorter. The long-term blood glucose regulation experimental results showed that PEG-mFGF-21 had better hypoglycemic effect than mFGF-21. Surprisingly, blood glucose of PEG-mFGF-21-treated mice remained at low level for several days after the drug was withdrawn. In conclusion, under the premise of remaining its biological activity in vitro, PEGylation of mFGF-21 can improve its physical stability and anti-protease ability, decrease its immunogenicity and increase its in vivo half-life. It can also prolong the hypoglycemic effect of mFGF-21 in diabetic animals. This study provides an important technology platform for drug development of FGF-21.