Novel bioadhesive chitosan-EDTA conjugate protects leucine enkephalin from degradation by aminopeptidase N.

PURPOSE

To develop a novel bioadhesive polymer that protects peptide drugs from luminal degradation by aminopeptidase N and to evaluate the system in vitro on porcine mucosa.

METHODS

EDTA was covalently bound to chitosan in order to combine the bioadhesive properties of the polymer with the well known capacity of EDTA to complexed metal ions which are essential for the enzymatic activity of proteases. The inhibitory effect of this polymer conjugate was evaluated by using leucine enkephalin (Leu enkephalin) as a model drug. The degree of Leu enkephalin degradation caused by aminopeptidase N (EC 3.4.11.2), as well as porcine mucosa, in the presence of the polymer conjugate, was quantified by HPLC analysis.

RESULTS

The chitosan-EDTA conjugate is capable of binding 2.01 +/- 0.12 mmole of zinc per gram of polymer at pH 6.5 (n = 3; +/-S.D.). As zinc is an essential co-factor for aminopeptidase N, enzyme activity (48 mU/ml) could be completely inhibited under the use of 1.0% chitosan-EDTA conjugate. The inhibitory effect of 1.0% chitosan-EDTA conjugate on the degradation of Leu enkephalin on porcine mucosa within 3 h at 37 degrees C was even 2.9-fold higher than that of a recently developed zinc complexing bacitracin-poly(acrylic acid) conjugate of the same concentration. The novel polymer conjugate is more bioadhesive than unmodified chitosan and is easily hydratable in water and basic aqueous solutions exhibiting quick swelling properties.

CONCLUSIONS

The bioadhesive polymer conjugate described here seems to be a useful tool in overcoming enzymatic degradation by aminopeptidase N.