title: Synthesis, characterization and in vivo activity of salmon calcitonin coconjugated with lipid and polyethylene glycol.
abstract: An irreversible lipidized salmon calcitonin (sCT) analog, Mal-sCT, was previously shown to have comparable hypocalcemic activity to sCT in vivo. This study reports on the synthesis, characterization and pharmacological activity of novel PEGylated Mal-sCT analogs. Mal-sCT, prepared by conjugating sCT via thio-ether bonds with aqueous-soluble palmitic acid derivative at Cys 1 and Cys 7, was reacted with mPEG-succinimide (mPEG-Suc, 5 kDa). The products were purified and then identified by MALDI-TOF MS and HPLC. Mal-sCT was conjugated with 1 (1PEG-Mal-sCT) or 2 (2PEG-Mal-sCT) PEG chains at Lys 11 and Lys 18, the former being the preferred site of conjugation at higher mPEG-Suc/Mal-sCT ratio. Circular dichroism analysis showed the PEGylated Mal-sCT analogs to possess a robust helical conformation, while size measurement by dynamic light scattering indicated a propensity of the peptides to self-aggregate in aqueous solutions. Both 1PEG-Mal-sCT and 2PEG-Mal-sCT were more stable in rodent intestinal fluids than sCT or Mal-sCT. However, 1PEG-Mal-sCT had comparable hypocalcemic activity to Mal-sCT when injected subcutaneously in the rat, while 2PEG-Mal-sCT was inactive. 1PEG-Mal-sCT was inactive when administered orally in the rat. This study suggested PEGylation of Mal-sCT increased the stability of the lipidized peptide to enzyme degradation, but did not enhance its hypocalcemic activity.
authors.affiliation: Department of Pharmacy, National University of Singapore, 18 Science Drive 4, 117543 Singapore, Singapore.
authors.forename: Weiqiang
authors.initials: W
authors.lastname: Cheng
authors.forename: Lee-Yong
authors.initials: LY
authors.lastname: Lim
mesh_terms: D000284:Administration, Oral; D000818:Animals; D002116:Calcitonin / Q000008:administration & dosage / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology*; D002118:Calcium / Q000097:blood / Q000378:metabolism*; D002942:Circular Dichroism; D004355:Drug Stability; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D007279:Injections, Subcutaneous; D007413:Intestinal Mucosa / Q000378:metabolism; D019308:Palmitic Acid / Q000737:chemistry*; D010316:Particle Size; D011092:Polyethylene Glycols / Q000737:chemistry*; D011487:Protein Conformation; D051381:Rats; D017208:Rats, Wistar; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D019308:Palmitic Acid; D011092:Polyethylene Glycols; C028815:salmon calcitonin; D002116:Calcitonin; D002118:Calcium