EP-A-O 650 999 discloses chitosan adducts with acidic chelate forming agents. The viscosity of the resulting gels can be adjusted by the addition of salts of polyvalent metals and acids, in which chitosan is only moderately soluble or insolu- ble.

JP-A-01239077 (JAPIO-abstract No. 89-239077) discloses the addition of chitosan and ethylene diamine tetraacetic acid (EDTA) into fertilizers.

In general the present invention relates to chitosan[= poly(D- glucosamine) conjugates comprising the following substructure within their overall structures: L characterized in that R represents a substituent selected from the group of -CH,COO-, -CH2CH2-N(-CH2COO ) 2 and -CH2CH2-N (-CH2C0O ) -CH2CH2-N (-CH2COO ) 2- More particularly, the present invention relates to chitosan- ethylenediaminetetraacetate[= EDTA] conjugates comprising the following substructure within their overall structures: characterized in that chitosan is reacted with EDTA in the presence of carbodiimides, i.e., by a carbodiimide mediated reaction.

In the structural formulae above the negative ionic charge at the carboxylate group may be compensated by the respective positive charge provided by positively charged mono- or polyva- lent counterions, such as protons, Li+, Na+, K+, Mug2+ Ca2+, Zn2+ and other positively charged inorganic or organic cations which may be represented by the cations to be complexed.

The chitosan-conjugates according to the present invention are obtainable by reacting chitosan with an acidic chelate-complex forming agent (chelating agent) selected from the group of nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA) in the presence of carbodiimides. The molar ratio of chitosan to chelating agents preferably is about 1:20 in case that no

crosslinks should be present in the final product. In case that inter- and/or intramolecular crosslinking should be achieved the molar ratio of chitosan to chelating agent should be smaller than about 1:20, as described below. In a preferred embodiment the reaction is carried out in aqueous medium using l-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride as the mediator. The chelating agent is covalently linked to the chitosan-structure by the formation of an amide bond.

More particularly the invention relates to the production of chitosan-ethylenediaminetetraacetate conjugates comprising the following substructure within their overall structures: characterized in that chitosan is reacted with EDTA in the presence of carbodiimides.

Further embodiments of the present invention relate to pharma- ceutical compositions, cosmetical compositions and bioadhesive matrices comprising the chitosan-conjugates described above.

In a further preferred embodiment of this invention the chito- san-conjugates are comprised in a bioadhesive matrix which can be used for the manufacture of medicaments for the administra- tion or delivery of drugs, preferably of peptide- or protein- containing active agents (drugs).

The chitosan-conjugates of the present invention and in par- ticular the chitosan-EDTA-conjugate can be used as a bioadhe- sive matrix system for the administration of peptide and pro- tein drugs, as gelling agent for semi-solid drug dosage forms and cosmetics and as a complexing polymer for mono- or polyva- lent cations, preferably for pharmaceutic and/or cosmetic applications.

Prior to using the conjugates according to the present inven- tion for the final purpose as gelling agent, matrix, complexing agent, etc. it may be necessary to neutralize the carboxylic acid groups in the conjugate using a suitable alkaline agent.

In detail, the present invention relates to chitosan- ethylenediamine tetraacetate conjugates. At least one of the carboxylic acid moieties of EDTA is bound to the amine moiety of chitosan to form an amide. The reaction can be mediated by carbodiimides, such as 1-ethyl-3-(3-dimethylaminopropyl) car- bodiimide hydrochloride [= EDAC]. The amount of EDTA covalently bound to chitosan can be controlled by the ratio of chitosan to EDTA employed. A preferred molar ratio of chitosan to EDTA is about 1:20 inter- and intramolecular crosslinking can be avoided. When the excess of EDTA is smaller than about 1:20 (molar ratio of D-glucosamine units to EDTA) during the cou- pling reaction, additional cross-links are generated since EDTA forms amide linkages with more than one amine moiety of chito- san by the formation of intra- and/or intermolecular links.

Adjusting the ratio of chitosan to chelating agent allows to adjust the viscosity of the resulting gel. This, in turn allows to predetermine the retention/release rate of the active phar- maceutical or cosmetic ingredient which may be incorporated into said gel.

It is noteworthy that the chitosan-conjugates according to the present invention are not susceptible against alkaline media, i.e., pH-values >7.

Thus, as described above through the ratio of chitosan to EDTA employed, the amount of coupled EDTA and the extent of addi- tional cross-linking can be controlled. Consequently, the present invention also relates to such crosslinked chitosan- conjugates containing intra- and/or intermolecular crosslinks.

Said crosslinks are also formed by covalent amide bonds. Of course, the intra- and/or intermolecular crosslinking, i.e., the crosslinking reaction between two polymers and/or within one polymer can also be carried out in the presence of a car- bodiimide, i.e., by a carbodiimide-mediated reaction.

The crosslinked chitosan-chelating agent conjugates according to the present invention are obtainable by reacting chitosan with the chelating agents described above at a molar ratio of chitosan to chelating agent of less than about 1:20, in the presence of a carbodiimide as described above. The inter- and/or intramolecular crosslinked chitosan-conjugate can be used for the same purpose as the (uncrosslinked) chitosan- conjugate as described above, but may have different properties in terms of complexing activity, viscosity, gelling properties, drug-releasing (delivering) properties, etc.

The chitosan-conjugates or its crosslinked derivatives bearing the substructure defined above or in case of the crosslinked derivatives bearing a substructure containing more than one amide bond in the moiety stemming from the chelating agent will bind polyvalent cations, inhibit zinc proteases, and exhibit bioadhesive and gelling properties. It is possible to use the conjugates of the present invention in bioadhesive matrix systems, as gelling agents and complexing polymer for polyva- lent cations in pharmaceutical and cosmetic applications. The inhibition of protease by the chitosan-conjugates of the inven- tion and particularly of the chitosan-EDTA conjugates enables the administration of all kinds of pharmaceutically active agents such as peptide- and protein-containing drugs. The advantage is most significant in the administration of vaccines (antigens), DNA and RNA. The bioadhesive carrier matrix accord- ing to the present invention allows the oral administration of

active agents which can up to now only be administered par- enterally. The active agents are incorporated into the inven- tive matrix and are sufficiently protected against an enzymatic attack by, for instance, digestive enzymes and are released in a controlled manner, as described above.

The chitosan-conjugates according to the present invention, and in particular the chitosan-EDTA conjugates inhibit various digesting enzymes, such as zinc protease, they are capable of complexing polyvalent cations, they represent a readily hy- drated and strongly swelling gelling agent and they have bioad- hesive properties.

What has been described above in relation to EDTA as the chelating agent also applies in case that NTA or DTPA is em- ployed as the acidic chelating agent.

The invention is described by the following example without being limited thereto.

Example To a 1% (m/v) aqueous chitosan-HCl solution at pH 4-5 is added a 20fold molar excess of EDTA (moles of D-glucosamine units of chitosan to moles of EDTA) . Then, the pH value is adjusted to 6.0 with 5 N NaOH, and 1-ethyl-3-(3-dimethylaminopropyl)carbo- diimide hydrochloride [= EDAC] is added to a final concentra- tion of 0.1 M. The reaction mixture is stirred at room tempera- ture for 12 hours, then neutralized with 5 N NaOH. The purifi- cation of the coupling product is effected by exhausting dialy- sis against deionized water, 0.05 N NaOH, and again deionized water. After lyophilization, the conjugate can be stored at room temperature.

In the coupling reaction thus described, the fraction of re- sidual primary amine moieties at chitosan is 0.1% + 0.03% (average of three determination runs; t standard deviation). By using a more than 20fold excess of EDTA, the degree of coupling can be but marginally increased. Reaction mixtures containing a less than 20fold excess of EDTA result in lower degrees of coupling and the occurrence of cross-links since some EDTA molecules will react with more than one amine group of chito- san.