PK/PD modelling of comb-shaped PEGylated salmon calcitonin conjugates of differing molecular weights

Abstract

Salmon calcitonin (sCT) was conjugated via cysteine-1 to novel comb-shaped end-functionalised (poly(PEG) methyl ether methacrylate) (sCT-P) polymers, to yield conjugates of total molecular weights (MW) inclusive of sCT: 6.5, 9.5, 23 and 40kDa. The conjugates were characterised by HPLC and their in vitro and in vivo bioactivity was measured by cAMP assay on human T47D cells and following intravenous (i.v.) injection to rats, respectively. Stability against endopeptidases, rat serum and liver homogenates was assessed. There were linear and exponential relationships between conjugate MW with potency and efficacy respectively, however the largest MW conjugate still retained 70% of E(max) and an EC(50) of 3.7nM. In vivo, while free sCT and the conjugates reduced serum [calcium] to a maximum of 15-30% over 240 min, the half-life (T(1/2)) was increased and the area under the curve (AUC) was extended in proportion to conjugate MW. Likewise, the polymer conferred protection on sCT against attack by trypsin, chymotrypsin, elastase, rat serum and liver homogenates, with the best protection afforded by sCT-P (40kDa). Mathematical modelling accurately predicted the MW relationships to in vitro efficacy, potency, in vivo PK and enzymatic stability. With a significant increase in T(1/2) for sCT, the 40kDa MW comb-shaped PEG conjugate of sCT may have potential as a long-acting injectable formulation.