Improved intestinal delivery of salmon calcitonin by Lys 18-amine specific PEGylation: Stability, permeability, pharmacokinetic behavior and in vivo hypocalcemic efficacy

Abstract

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 μM in a Caco-2 cell monolayer transport system. In particular, tissue distribution results showed that 125I-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.