Improved intestinal delivery of salmon calcitonin by Lys18-amine specific PEGylation: stability, permeability, pharmacokinetic behavior and in vivo hypocalcemic efficacy.

Peptides like salmon calcitonin (sCT) are subjected to aggressive proteolytic attack by various intestinal enzymes, and fractions that enter the systemic circulation via the intestinal route are rapidly inactivated by tissue accumulation and glomerular filtration. Here, we describe the beneficial effects of the Lys(18)-amine specific PEGylation of sCT on the intestinal delivery of sCT. Two key properties were enhanced by the PEGylation process: (i) the resistance of sCT to intestinal enzymes and (ii) the systemic clearance of sCT that had entered the circulation. Initially, we evaluated the cAMP-secreting activities of PEG(2K)-sCT isomers substituted at Cys(1)-, Lys(11)- or Lys(18)-amine position in T47D cells, and found that sCT PEGylated at Lys(18)-amine (Lys(18)-PEG(2K)-sCT) had the highest bioactivity. We then investigated the stability of Lys(18)-PEG(2K)-sCT in the presence of intestinal enzymes, its abilities to traverse the intestinal membrane, its pharmacokinetic behavior and in vivo hypocalcemic efficacy. Results show that Lys(18)-PEG(2K)-sCT has significantly increased resistance to pancreatic peptidases and brush-border peptidases. Despite the molecular size increase caused by PEGylation, Lys(18)-PEG(2K)-sCT was found to have an intestinal permeability similar to that of unmodified sCT (p>0.59) over an apical concentration range 12.5-100 microM in a Caco-2 cell monolayer transport system. In particular, tissue distribution results showed that (125)I-labeled Lys(18)-PEG(2K)-sCT markedly resists liver accumulation and glomerular filtration; levels were reduced by 75% and 50% vs. sCT. Finally, the hypocalcemic efficacy of intestinally administered Lys(18)-PEG(2K)-sCT, measured as total serum calcium in a rat model, was 5.8 and 3.0 times that of sCT at 100 and 200 IU/kg (p<0.025). Our findings suggest that this site-specific conjugation of peptides with PEG of proper size enhances pharmacokinetic properties by increasing their abilities to resist both proteolysis and systemic clearance without significantly reducing their membrane permeabilities or bioactivities. We believe that this concept, namely, dual effects by PEGylation, has great potential value because it presents a practical means of enhancing the efficacies of the peroral/intestinal pharmacologic route.