Conjugation of salmon calcitonin to a combed-shaped end functionalized poly(poly(ethylene glycol) methyl ether methacrylate) yields a bioactive stable conjugate.
Salmon calcitonin (sCT) was conjugated via its N-terminal cysteine to a comb-shaped end-functionalized poly(poly(ethylene glycol) methyl ether methacrylate) (PolyPEG, 6.5 kDa), and to linear PEG (5 kDa). Conjugate molecular weight and purity was assessed by SEC-HPLC and MALDI-TOF MS. Bioactivity of conjugates was measured by cyclic AMP assay in T47D cells. Calcium and calcitonin levels were measured in rats following intravenous injections. Stability of conjugates was tested against serine proteases, intestinal and liver homogenates and serum. Cytotoxicity of conjugates was assessed by lactate dehydrogenase (LDH) assay and by haemolytic assay of rat red blood cells. Results showed that the two conjugates were of high purity with molecular weights similar to predictions. Both conjugates retained more than 85% bioactivity in vitro and had nanomolar EC(50) values similar to sCT. While both sCT-PolyPEG(6.5 K) and sCT-PEG(5 K) were resistant to metabolism by serine proteases, homogenates and serum, PolyPEG (6.5 K) was more so. Although both conjugates reduced serum calcium to levels similar to those achieved with sCT, PolyPEG(6.5 K) extended the T(1/2) and AUC of serum sCT over values achieved with sCT-PEG and sCT itself. None of PolyPEG, PEG or methacrylic acid displayed significant cytotoxicity. PolyPEG may therefore have potential to improve pharmacokinetic profiles of injected peptides.
Ryan
Sinéad M
SM
UCD School of Agriculture, Food Science and Veterinary Medicine and UCD Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland.
Wang
Xuexuan
X
Mantovani
Guiseppe
G
Sayers
Claire T
CT
Haddleton
David M
DM
Brayden
David J
DJ
D000818:Animals; D050071:Bone Density Conservation Agents / Q000008:administration & dosage* / Q000009:adverse effects / Q000737:chemistry / Q000493:pharmacokinetics; D018938:Caco-2 Cells; D002116:Calcitonin / Q000008:administration & dosage* / Q000009:adverse effects / Q000737:chemistry / Q000493:pharmacokinetics; D002118:Calcium / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D000242:Cyclic AMP / Q000378:metabolism; D004337:Drug Carriers / Q000737:chemistry*; D004355:Drug Stability; D004912:Erythrocytes / Q000187:drug effects; D006461:Hemolysis / Q000187:drug effects; D006801:Humans; D007413:Intestinal Mucosa / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008099:Liver / Q000187:drug effects / Q000378:metabolism; D008689:Methacrylates / Q000737:chemistry*; D008970:Molecular Weight; D011092:Polyethylene Glycols / Q000737:chemistry*; D011109:Polymethacrylic Acids; D051381:Rats; D017208:Rats, Wistar
D050071:Bone Density Conservation Agents; D004337:Drug Carriers; D008689:Methacrylates; D011109:Polymethacrylic Acids; C528061:poly(poly(ethylene glycol)methacrylate); D011092:Polyethylene Glycols; C028815:salmon calcitonin; D002116:Calcitonin; D000242:Cyclic AMP; D002118:Calcium