Synthesis and in vitro evaluation of chitosan-EDTA-protease-inhibitor conjugates which might be useful in oral delivery of peptides and proteins.

PURPOSE

To develop a novel mucoadhesive polymer that protects peptide drugs from degradation by secreted as well as membrane-bound proteases in the intestine, and to evaluate this polymer in vitro.

METHODS

The serine protease inhibitors antipain, chymostatin and elastatinal were covalently linked to chitosan (poly-[1-->4]-beta-D-glucosamine). Thereafter, the complexing agent ethylenediaminetetraacetic acid (EDTA) was bound to the remaining primary amino groups of the polymer. The inhibitory effect of the resulting polymer-conjugate towards trypsin (EC 3.4.21.4), chymotrypsin (EC 3.4.21.1), elastase (3.4.21.36), carboxypeptidase A (EC 3.4.17.1), carboxypeptidase B (EC 3.4.17.2) and aminopeptidase N (EC 3.4.11.2) as well as its mucoadhesive properties were evaluated in vitro.

RESULTS

Whereas the novel polymer-conjugate exhibited excellent swelling properties, its adhesive force was under our assay conditions 42% lower than that of unmodified chitosan. However, the polymer-conjugate showed a strong inhibitory activity towards all tested serine proteases. Due to its additional high binding affinity towards bivalent metal ions, it also inhibited the Zn(2+)-dependent exopeptidases carboxypeptidase A, B and aminopeptidase N.

CONCLUSIONS

The novel mucoadhesive polymer-conjugate described in this study seems to be a useful tool in overcoming the enzymatic barrier to perorally administered therapeutic peptides and proteins.