Preparation and characterization of polyethylene-glycol-modified salmon calcitonins.

The conjugation of salmon calcitonin (sCT) by covalent linkage of polyethylene glycol (PEG) was attempted to overcome several disadvantages of sCT as a therapeutic drug, namely its rapid clearance from blood circulation and enzymatic degradation. The polymer employed was succinimidyl carbonate monomethoxypolyethylene glycol (12 kDa). Superose HR size-exclusion chromatography was applied to separate the PEGylated sCTs (mono-PEG-sCT and di-PEG-sCT) from the unmodified sCT. The PEGylation of sCT was verified by an electrophoresis gel stained with iodine and by MALDI-TOF mass spectrometry. The molecular weights of mono-PEG-sCT and di-PEG-sCT were determined to be 16,094 and 29,077 Da, respectively. PEGylated sCTs showed a substantially improved stability in rat liver homogenates as compared to the intact sCT, indicating that PEG molecules protected sCT from various degrading enzymes. These PEGylated sCTs exhibited similar biological activity to the intact sCT by adenosine cyclic 3',5'-phosphate (cAMP) assay. In clearance studies in the rat, PEGylated sCTs had significantly longer circulating half-lives than the intact sCT (11.2 min for mono-PEG-sCT and 54.0 min for di-PEG-sCT versus 4.7 min for intact sCT).