No abstract

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).

Three positional isomers of mono-PEGylated sCTs were purified and characterized. Of these, the resistance to proteolytic degradation was highest for the Lys18-residue modified mono-PEG-sCT. These studies demonstrate that the in vivo stability of PEGylated sCTs is highly dependent on the site of PEG molecule attachment.

No abstract

The effects of the hindered and non-hindered water soluble long-chain disulfide bonds on the stability and cytotoxicity of the ricin A chain (RTA) immunotoxin were examined. The RTA immunotoxins were prepared with the Fab fragments of anti-common acute lymphoblastic leukemia antigen (CALLA) monoclonal antibody (Fab-RTA) using sulfosuccinimidyl-6-[(-methyl-(-(2-pyridyldithio)toluamido]hexanoate (S-LC-SMPT) and sulfosuccinimidyl-6-[3-(2-pyridyldithio)-propionamido]hexanoate (S-LC-SPDP). The prepared Fab-RTA immunotoxins were evaluated for their conjugation yield, immunoreactivity, thermal and disulfide bond stability and cytotoxicity. The conjugation yield of the Fab-RTA immunotoxin from the water soluble long chain crosslinking agents, S-LC-SMPT and S-LC-SPDP, were comparable. Both Fab-RTA immunotoxins exhibited a similar immunoreactivity and thermal stability in aqueous solution. However, S-LC-SMPT -mediated Fab-RTA, sterically hindered, showed an enhanced disulfide bond stability in vitro over S-LC-SPDP mediated one. In the cytotoxicity against antigenic cell Daudi, the S-LC-SMPT -mediated RTA immunotoxin maintained a comparable cytotoxicity, compared with S-LC-SPDP mediated Fab-RTA immunotoxin.