SYNTHESIS AND IN-VITRO EVALUATION OF CHITOSAN-EDTA-PROTEASE-INHIBITORCONJUGATES 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 protease s in the intestine, and to evaluate this polymer in vitro. Methods. Th e serine protease inhibitors antipain, chymostatin and elastatinal wer e covalently linked to chitosan (poly-[1 --> 4]-beta-D-glucosamine). T hereafter, the complexing agent ethylenediaminetetraacetic acid (EDTA) was bound to the remaining primary amino groups of the polymer. The i nhibitory effect of the resulting polymer-conjugate towards trypsin (E C 3.4.21.4), chymotrypsin (EC 3.4.21.1), elastase (3.4.21.36), carboxy peptidase A (EC 3.4.17.1), carboxypeptidase B (EC 3.4.17.2) and aminop eptidase N (EC 3.4.11.2) as well as its mucoadhesive properties were e valuated in vitro. Results. Whereas the novel polymer-conjugate exhibi ted excellent swelling properties, its adhesive force was under our as say conditions 42% lower than that of unmodified chitosan. However, th e polymer-conjugate showed a strong inhibitory activity towards all te sted serine proteases. Due to its additional high binding affinity tow ards bivalent metal ions, it also inhibited the Zn2+-dependent exopept idases carboxypeptidase A, B and aminopeptidase N. Conclusions. The no vel mucoadhesive polymer-conjugate described in this study seems to be a useful tool in overcoming the enzymatic barrier to perorally admini stered therapeutic peptides and proteins.