WO1999019366A9 - Chelates of chitosans and alkaline-earth metal insoluble salts and the use thereof as medicaments useful in osteogenesis - Google Patents

Abstract

Amorphous or partially crystalline chelates of chitosans, functionalized at the nitrogen with polycarboxylic functions, with alkaline-earth metal insoluble salts. Said chelates are useful for the preparation of medicaments promoting the reparation of bone tissue.

Description

CHELATES OF CHITOSANS AND ALKALINE-EARTH METAL INSOLUBLE SALTS AND THE USE THEREOF AS MEDICAMENTS USEFUL IN OSTEOGENESIS

The present invention relates to chelates of chitosans with inorganic salts, in particular to amorphous or partially crystalline chelates consisting of chitosans functionalized at the nitrogen with polycarboxylic functions, linked to alkaline-earth metal insoluble salts, suitable for the preparation of medicaments useful to promote the reparative process of bone matrix and its mineralisation.

The mineralised tissues of vertebrates contain crystals of carbonated hydroxyapatite, which have grown in an organized way inside a collagen fibril structure. Vesicles containing calcium ions and phosphate ions are formed in the matrix, they come close to the collagen fibrils and give origin to crystals growing in the form of plates, 35x8x1.5 nm (tendon) [Erts et al., 1994] or 50x25x3 (bone) [Zhang and Gonsalveε, 1995]. Parent compounds are also in equilibrium with complexing agents .

Biological hydroxyapatites exhibit low crystallinity and lack stoichiometry due to the presence of ions absorbed or included in the crystal lattice [Bigi et al. , 1994] .

The inorganic component of these tissues is formed in the presence of various substances exerting inhibiting actions and structural alterations; for instance, hydroxyapatite is formed according to the reaction: 2CaHP0₄ + 2 Ca₄(P0₄)₂0 —>Ca₁₀(PO₄)₆(OH)₂ at 38°C which is effected by the influences of macromolecular species, e.g. albumin, ionic species, e.g. the hydrogen carbonate ion, and other species which produce important alterations, such as hydroxyapatite carbonation [Martin and Brown, 1994] .

Various recipes for the preparation of hydroxyapatite-based bone cements have been described by Driesεens et al., 1994, Akai, 1994 and Ito et al., 1994. However, the treatment of bone lesions with said cements is relatively naive, because hydroxyapatite is the ultimate product of the mineralisation process in vivo. The artificial hydroxyapatite granules are not valid surrogates of the nanocrystals generated in vivo; on the contrary, they slow down the cellular proliferation and can be considered foreign bodies. Porous hydroxyapatites and coated hydroxyapatites were developed to obviate to these drawbacks [Eggli et al., 1987].

Various cements containing chitosans have been proposed by Sumita, 1988, who mixed tricalciu phosphate and tetracalcium phosphate with chitosan acid solutions: the resulting material hardens within 12 minutes and the hardening is believed to take place following formation of hydroxyapatite [Lacont et al., 1996]. Takechi et al., 1996 also suggest chitosan or alginate to obtain a time- stable cement and ascribe the cement stability to the decrease in liquid-permeability carried out by the biopolymer. Analogously Ito, 1991, mixes hydroxyapatite with ZnO, CaO and chitosan. Ishhii mixes Ca₃(P0₄)₂ with chitosan. Hydroxyapatite capability of interacting with biopolymers is well documented in the chromatography field.

Wherever a cement is not strictly necessary for the medication of a bone defect, materials conceived in a totally different way might be more desirable. Bones being subject to a continuous restructuring by osteoblasts, resorbable hydroxyapatites have been developed as compounds that osteoblasts can restructure.

Such hydroxyapatites should be inorganic compounds comprising biopolymers intercalated in the crystal lattice [Messersmith and Stupp, 1992]. However, such materials are not at present available. Lohman, 1993, discusses the preparation of modified chitosans, giving some generic information on the aspect of NaCl, CaS0₄ and CaC0₃ crystals, obtained in the presence of biopolymers, but he does not characterise the resulting materials .

Chitin is often found in combination with CaCO in insects and arthropods.

While chitosan and many of its derivatives have chelating ability towards transition metal ions but not for calcium, [Muzzarelli and Tubertini, 1969], chitosans capable of chelating calcium have been prepared by functionalizing chitosan with polycarboxylic groups

[Muzzarelli and Delben, 1989]. For example, using the corresponding keto acids and chitosan, Muzzarelli and

Zattoni, 1986, prepared glutamate glucan and aspartate glucan, which have widely been characterized by Chiessi et al. [1992]. Furthermore, Rinaudo et al. [1994] and

Muzzarelli et al. [1985] have prepared highly carboxymethylated chitosans. Said chitosans have been described for the separation of metal ions and for research on their structure, but nothing is known in terms of their effect on the crystallization of sparingly soluble salts. Gruber [1996] suggests, for example, the reaction of cis-epoxysuccinic acid (oxirane-dicarboxylic acid) with a chitosan salt, to give N-[ ( 3 ' -hydroxy-2 ' , 3 ' -dicarboxy )ethyl]chitosan for cosmetic purposes, without discussing its chelating capacity. On the other hand, Inoue et al. [1996] synthesized a class of chitosans carrying chelating EDTA-type functional groups and used them for the separation of lanthanides and other metal ions. Inoue et al. [1995] discuss the selectivity of said chitosans towards some transition metals. Analogously, Ina and Nakamura [1992] proposed carboxymethylated chitosans for the preparation of detergents. No Author who described carboxylated chitosans ever disclosed bioinorganic compounds in terms of novel compositions of the matter; on the contrary, they only considered aspects of analytic chemistry or preparation of mixtures, see Menon et al. [1995], Rinaud et al. [1994] and Guibal et al. [1995].

The osteoinductive , osteoconductive , resorption capacities of chitin- and chitosan- based materials have already been reported by Muzzarelli et al. [1993] and Muzzarelli [1996]. Such capacities are not present in alginates, therefore no relationships between alginate- Ca chelates and the modified chitosan - alkaline-earth metal chelates of the invention could be foreseen.

It has now surprisingly been found that the well- known precipitation reactions with alkaline-earth metals, in particular calcium, when carried out in aqueous medium in the presence of a highly carboxylated chitosan give amorphous or only partially crystalline compounds, incorporating a biopolymer, and which can, in some cases, be isolated as water soluble materials. Summary of the invention

The present invention relates to amorphous or partially crystalline chelates consisting of highly carboxylated chitosans linked to alkaline-earth metal insoluble salts. A further object of the present invention is a process for the preparation of said chelates.

A still further object of the present invention is the use of said chelates for the preparation of medicaments useful in human and animal medicine, together with pharmaceutical compositions containing said chelates as active ingredients.

The present invention also relates to the use of said chelates for the cosmetic treatment of human body, as well as the cosmetic compositions containing them. The advantages provided by the present invention basically derive from:

1) Characteristics of homogeneity, solubility, malleability and hydrophilicity of the bioinorganic compounds isolated in the form of solid materials. 2) No or very poor crystallinity characteristics.

3) Biocompatibility , biodegradability and complete resorption.

4) Oεteoinductive , oεteointegration and accelerated mineralisation of the bone medicated with said bioinorganic materialε.

5) Simple preparation and iεolation processes. From the activity point of view, therefore, the product has both an organic and an inorganic component: the first one provides a carrier for the adhesion, migration and proliferation of the tisεue cells and a carrier for the correct regeneration of the extracellular bone matrix, the second one acts as a nucleation medium for the onset of the mineralisation procesε and aε a carrier for the ex novo formation of hydroxyapatite. Thiε pro oteε the healing proceεs and the regeneration when thiε cannot occur spontaneouεly , εuch aε is the caεe with defectε of wide size.

The present invention will be disclosed in further detail by the following Examples and Figures, in which:

Figure 1 shows the X-ray diffraction spectra for brushite (A), and for some chelates of the present invention (B) and (C), aε further illuεtrated below, in particular for DCMC-Ca-P chelateε;

Figure 2 εhowε the X-ray diffraction εpectra for some chelateε of the present invention (A) and (B), in particular for DCMC-Ca-F chelates.

According to the present invention, highly carboxylated chitosanε (in the following referred to aε functionalized chitosans, for sake of shortness) are known in literature and the preparation thereof is disclosed in a number of publications. According to a preferred embodiment of the invention, the functionalized chitosan is selected from dicarboxymethyl chitosan (obtained from glyoxylic acid), glutamate glucan (obtained from ketoglutaric acid), aspartate glucan (obtained from oxalacetic acid) and N-[(3'- hydroxy-2¹ , 3 ' -dicarboxy )ethyl]chitosan (obtained from oxiranedicarboxylic acid).

The present invention is not limited to specific alkaline-earth metal inεoluble εaltε. Exampleε of εaid εaltε are phoεphate, εulfate, oxalate, carbonate, hydrogen carbonate, fluoride of one of the variouε alkaline-earth metal cationε.

In a preferred embodiment, εaid salts are calcium saltε, particularly calcium phoεphate.

A moεt preferred chelate iε that of calcium phoεphate with a derivatized chitoεan in a 1:1 weight ratio.

The procesε for the preparation of the chelateε of the preεent invention compriεes the following stepε: diεsolution of the functionalized chitosan in a solution of a soluble salt, preferably the sodium salt, of the desired alkaline-earth metal anion; addition of a soluble salt of the desired alkaline- earth metal anion in equimolar amounts, to give the chelate with the alkaline-earth metal insoluble salt, and subsequent isolation.

Alternatively, the process according to the present invention compriseε mixing carboxylated chitoεan with an inεoluble εalt of the desired alkaline-earth salt.

In the exemplary case of a calcium chelate, the carboxylated chitosan is diεsolved in a solution of the selected sodium salt and a soluble calcium salt is added in equimolar amounts, to form an insoluble calcium salt. ■

The reaction conditionε will be εelected by thoεe εkilled in the art, depending on the functionalized chitoεan and the final alkaline-earth metal inεoluble salt . A most preferred embodiment of the invention provides the chelate of dicarboxymethyl chitoεan (in the following DCMC) with an inεoluble calcium εalt.

Table A εummarizeε macroεcopic observations for a number of anionε in water at the εpecified pH valueε, when reacted with calcium ionε, in the presence of DCMC.

TABLE A

Sodium salt Final pH Aspect of the system with DCMC with no DCMC

phosphate 6.1-6.2 clear, homogeneous precipitate sulfate 5.9-6.7 turbid clear, ho ogen, oxalate 5.8-6.6 turbid precipitate carbonate 8.3-9.0 turbid, no precip. precipitate hydrogen carbonate 7.2-9.0 turbid clear fluoride 6.2-6.9 precipitate with turbid clear supernatant

For selected molar ratios, specified below, it is possible to observe that, in the presence of DCMC, calcium phosphate does not precipitate. This behavior differs from the regular behaviors of calcium salts in the absence of the biopolymer.

When DCMC is reacted with calcium ions, the reaction results in products of various aspect, depending on the Ca/DCMC molar ratio, as shown in table

B below.

TABLE B

CALCIUM MOLAR RATIO OBSERVATIONS

(μ mols) Ca/DCMC

1.5 0.0125 No precipitate

2.0 0.0156 No precipitate

2.6 0.0208 Turbidity

5.2 0.0400 Precipitate

7.7 0.0588 Gelling precipitate

26 0.2000 Precipitate

52 0.4000 Non-homogeneouε precipitate

77 0.5882 Non-homogeneouε precipitate

128 Non-homogeneouε precipitate

Note :

DCMC concentration: 0.56% (128 μmolε) Ca acetate concentration: 1.0% Temperature 20"

In practice precipitation occurε for high ratios, whereas no precipitate is observed for low ratios; but for ratio 0.0588 a rigid gel forms, that takes the shape of the container and can be sliced with a knife. The

DCMC-Ca gel dissolveε upon contact with a disodium orthophosphate εolution, yielding a clear solution, with no pH changes observed (5.5).

The most favourable conditions for the formation of a water-soluble DCMC-calcium phosphate chelate (DCMC-Ca-

P) are illustrated in the following table C.

TABLE C

DCMC Ca/DCMC ratio Noteε μ ols grams mgra s molar weight

DCMC 0.56% so: 1. DCMC

2.6 0.1 0.56 51 9 precipit. similar to the

5.1 0.2 1.1 25 4.7 control

7.7 0.3 1.7 17 3 id.

10.0 0.4 2.2 13 2.4 id.

13.0 0.5 2.8 10 1.9 id.

15.0 0.6 3.3 7 1.6 id.

31 1.2 6.7 4 0.7 id.

54 2.1 11.8 2.4 0.4 clear

79 3.1 17.4 1.6 0.2 precipitate

94 3.7 20.7 1.4 0.25 id.

110 4.4 24.6 1.2 0.12 id.

130 5.0 28 1 0.18 id.

It can be observed that, for equimolar amountε of disodium hydrogen orthophosphate and calcium acetate, a

Ca/DCMC 2.4 molar ratio gives a homogeneous syεtem, from which a water-εoluble DCMC-Ca-P chelate can be recovered by freeze-drying. This product is soluble in a wide range of pH, but the polymer can easily be isolated from mother liquors with ammonium hydroxide (pH 8).

X-ray diffraction spectrometry.

As illustrated in Figure 1, spectra recorded for compounds described at lines 1-5 of table C, dried at

40 °C, showed a marked depression of the peaks typical of brushite (Figure 1A) at 20 valueε of 11.68, 20.96 and

29.28, which are, on the contrary, very intense in the control precipitate under the same experimental conditions and which correspond to the values of the crystallographic tables. Of the other peaks, only that at 4.64 20 is further affected by the DCMC concentration (Figure IB, Table C, sample of line 5).

The spectra showed that even small amounts of DCMC in the precipitation medium lead to almost amorphous DCMC-

Na₂HP0₄.2H₂0 combinations, in which no diffractometric peaks of brushite are present (Figure 1C, Table C, sample of line 8 ) .

Reflectivity Infrared Spectrometry. Analyεiε were carried out on εampleε prepared aε follows:

DCMC: a 0.4% DCMC solution was treated with glacial acetic acid diluted 1:3 to pH 6.09. The reεulting solution was treated with acetone and the precipitate was obtained by centrifugation, then freeze-dried. The resulting product was ground with KBr for analysis.

DCMC-P-Ca: was prepared treating a 0.4% DCMC solution (75.2 g) with Na₂HPO₄.2H₂₀ (1%, 18 ml), then with calcium acetate (1%, 21 ml). Part of this solution (20 ml) was treated with acetone and centrif ged. The freeze-dried compound was ground with KBr.

The spectra showed that the reaction with calcium phosphate induced a shift of the peaks from 1640 to 1590 cm^-1 and the lowering of the peaks at 1400 and 1150 cm^"

1. Remarkable is also the lowering of the two peaks at

540 and 470 cm^-1.

Elemental analysis

Preliminary elemental analysiε on the DCMC sample showed that the chemical functionalization introduces carbon and oxygen into the polyεaccharide with a reεulting N/C ratio for DCMC (0.115) lower than for plain chitoεan (0.182), as expected following carboxymethylation. The sum N+C+H for DCMC is lower than 40%, in that sodium and the increased oxygen should be considered, while for plain chitosan it is close to 60 %.

In the derivative containing calcium phosphate, the

N percent drops to less than one half (1.66) of the corresponding value for DCMC (3.53) and therefore the organic/inorganic ratio in the chelate is about 1:1 by weight. TABLE D

Elemental analyεiε data on chitoεan with a 0.20 acetylation degree and on itε derivativeε.

percent chitosan DCMC DCMC-P-Ca gr .ac. 0. 20 2496

N 8.26 3.53 1.66

C 44.71 30.59 21.68

H 4.90 3.51

N/C 0.182 0.115 0.076

Behavior of other sparingly soluble calcium salts.

The other sparingly soluble calcium salts tested aε described above are: sulfate, oxalate, carbonate, hydrogen carbonate and fluoride. No εolubilization phenomena εimilar to that with the phoεphate εalt have been observed, always obtaining a precipitate upon mixing the saltε. On the contrary, amorphouε or poorly cryεtalline saltε formed analogouεly. In particular, Figure 2 reportε the X-ray diffraction εpectra for the DCMC-calcium fluoride derivative (abbreviated DCMC-F- Ca). The precipitation of calcium fluoride starting from calcium acetate and sodium fluoride in the presence of DCMC results in products which, once dried at 55°C, become hard and yellowish, especially those isolated from media with higher DCMC content. In the X-ray diffraction spectra for these compounds, the characteristic peak for calcium fluoride <111> at 28,3 20 showed intensity inversely proportional to the DCMC content.

The chelates of the present invention promote the mineralisation of newly formed bone tissue, and are therefore useful for the preparation of medicaments for the treatment of pathological conditions of bone tisεue.

A εtudy was carried out on sheep to evaluate the activity of the chelates of the invention. Sheep were selected in view of histologic, physico-mechanical and phyεiopathological similarity to human femurs.

The day before surgery, 100 mg/kg of Cefazolin

(Cefamezin) were administered to the animalε. The animalε underwent εurgery after εtarving for 12 h. Following a premedication with ketamine (10 mg/Kg) and xylazine (0.2 mg/kg) general anaesthesia was induced with thipethane-natrium (8 mg/kg, 2.5 % solution) and maintained with a gas mixture of 0.5-1.0 % Halotane and N₂0+0₂ (1:1) in automatic ventilation. A bone defect was created in the femural epiphyεeε by means of a 6 mm drill. During surgery the drill holes were carefully rinsed with 0.9 % NaCl solutionε and cleaned out in order to remove abraded particles, reduce drilling temperature and avoid bone necrosiε. In the right leg the hole waε completely filled with imidazolyl-chitoεan or DCMC-Ca-P-chitosan, while in the other one it was left open to serve as control. The wounds were sutured atraumatically in two layers and disinfected. Animals were submitted to antibiotic therapy (25 mg/]cg cefazolin pro die) for 7 days, and were allowed to bear weights as tolerated. Neither intra- and post -operative nor general and local septic complications were observed. Animals were sacrificed 40 and 60 days after surgery. Femurs were removed and sawn to produce fragments for histologic examination. In sheep medicated with DCMC-Ca-P, macroscopic analysiε evidenced an irregular rimmed area εmaller than the εurgical defect, filled with a tissue without histoarchitectural characteristic of bone tisεue. In control femurε the hole had not changed much in εhape and εize since surgery and the area lacked in bone tisεue. Microεcopic analysis of treated legs clearly showed the difference between the reparative- reconstitutive bone tissue with or without chitosan. First of all the number of cells were higher in the treated legs than in the control ones, and their large size and star-εhape were εuggeεtive for activation. However, the most important factor was the presence of a wide osteogenic reaction (Figε. a, b) moving from the rim of the εurgical leεion toward the centre. Vaεcular endothelial cells synthesize and secrete an array of soluble mediators either conεtitutively or in reεponεe to induction stimuli such as injury or inflammation. Among these, it iε important to mention growth factors and cytokines: fibroblast growth factor (FGF), interleukin-1 (IL-1), interleukin-6 (IL-6), colony simulating factors (SCUFFS) of the G, GM and M subtypeε, arachidonic acid, proεtacyclin, and small peptides like endotelin-1. These regulatory compounds have been seen, in other studies, to control the recruitment, proliferation, differentiation, functioning, and/or survival of various cells including bone-forming osteoblaεts and bone- degrading osteoclaεtε. Lastly, endothelial cell release of other short- live mesεenger moleculeε, namely reactive oxygen εpecies and nitric oxide (NO), may help to control osteoclaεt activity. In fact, activated oxygen εpecieε εeem to enhance reεorption, whereaε NO haε been εhown to interfere with oεteoclast bone resorption and, furthermore, inhibitorε of NO εynthetaεe potentiate oεteoclaεtε bone-reεorption. The reεults illustrated in the present invention evidence the important role of DCM-chitosan in stimulating bone tiεsue reconεtitution and suggest that their action can be potentiated by chelation of calcium phosphate.

In a further embodiment of the invention, the chelates are useful in dentistry.

The avulsion of a dental unit and a cystiε in a 15- year old patient waε performed: the εpace left waε filled with freeze-dried DCMC-CaP. Radiographic obεervationε εhowed precocious formation of osseous trabeculae 15 days a.s.

As far as the industrial applications of the invention are concerned, an aspect thereof are pharmaceutical or cosmetic compositions containing an effective amount of one or more chelateε of the invention aε active ingredients, in admixture with conventional carriers and excipientε. Said compoεitionε are prepared in formε and according to procedures well- known to those skilled in the art, aε described, for example, in "Remington's Pharmaceutical Sciences Handbook", Mack Publishing Company, New York, U.S.A.

Doεageε and posology will be determined by the physician, depending on the type of the pathology to be treated, aε well aε on the conditionε of the patient and any other uεeful considerations. A further, εpecific embodiment of the preεent invention provideε medical articles in the forms of freeze-dried materials, powders, films, membranes, solutions, gels and malleable pastes. A further preferred embodiment provides the chelates of the invention as active ingredients in compositions for strengthening teeth enamel, εuch aε tooth-pastes, pastes and gels.

The chelateε of the invention are alεo uεeful for the treatment of hair and can εuitably be formulated in coεmetic compoεitionε .

The following exampleε further illuεtrate the invention.

Example 1: Preparation of DCMC-Ca chelate. A εerieε of solutions are prepared, so as to have a DCMC constant content and a calcium acetate increasing content, pH being 5.5. When the Ca/DCMC molar ratio is lower than 0.02, no precipitate is observed, whereas at 0.2 - 0.5 ratios precipitation occurs. For intermediate values 0.06-0.20 a rigid gel forms, that takes the shape of the container and can be sliced with a knife. The gel redisεolves when placed in a phosphate solution, yielding a clear solution. For valueε from 0.4 to 1.0 a non-homogeneouε precipitate is formed. Example 2: Study of the DCMC-Ca-P system.

When solutions containing both DCMC (at variable concentration) and Na₂HP0₄.2aq (at constant concentration) are treated with calcium acetate (at conεtant concentration), precipitation takeε place in all cases, except for Ca/DCMC molar ratios close to 2.4- 4.2, as shown in table C. Example 3: Preparation of the bioinorganic compound

DCMC-Ca-P.

The aqueouε εolution of DCMC (0.48%, 220 g) and of Na_?HP0₄.2aq (1.0%, 67 ml) is mixed with the calcium acetate one (1.0%, 72 ml). Referred to 1 g of DCMC (dry weight), the amounts are 0.6 g of sodium salt and 0.7 of calcium acetate. The resulting solution iε dialyzed and reeze-dried, to give a spongy material which becomes a gel when mixed with water or saline. Example 4: Preparation of the bioinorganic compound DCMC-Ca-F .

The formation of the chelate DCMC-Ca-F from NaF and calcium acetate εolutionε in the presence of DCMC yields products that, once dried, become hard and yellowish. In the X-ray diffraction spectra, the single (111) peak at 28.34 20 typical for CaF₂ showed intensity inversely proportional to the DCMC concentration.

Example 5: Preparation of the bioinorganic compound DCMC-Ca-carbonate . A constant amount of calcium acetate (1.0% solution, 3.2 ml = 164 micromols = 6.55 mg Ca) iε added to a mixture of DCMC (0.48% at pH 6 in variable amount, with 0.1 N ammonia) and of εodium carbonate (1.0%, 1.7 ml = 164 micromolε = 0.017 g). No formation of precipitate iε observed, although the solutions remain turbid, whereas the control (no DCMC) gives a precipitate . Bibliography

Akai, T. (1994). Jpn. Kokai Tokkyo Koho JP 06,105,901

Filed 29 09 92. CA 121.18134

Bigi, A., Foresti, E., Gregorini, R., Ripamonti, A., Roversi, N. and Shah, J. S. (1992). Calcif . Tissue

Int . , 50, 439-444.

Chieεεi, E., Paradoεεi, G. , Venanzi, M. and Piεpiεa, B.

(1992). J. Inorg. Bioche . , 46, 109-118.

Driessens, F.C., Boltong, M.G. , Bermudez, 0., Planell, J.A., Ginebra, M.P. and Fernandez, E. (1994). J.

Ma ter . Sci . : Ma ter . Med . , 5, 164-170.

Eggli, P.S., Mueller, W. and Schenk, R.K. (1987). In

Biomaterialε and Clinical Applications, eds . A.

Pizzoferrato, P.G. Marchetti, A. Ravaglioli and A.J.C. Lee. Elsevier, Amεterdam, pp. 53-62.

Erts, D. , Gathercole, L.J. and Atkins, E.D.T. (1994). J.

Mater . Sci . : Ma ter . Med . , 5, 200-206.

Gruber, J.W. (1996). In Advances in Chi tin Sciences, eds. A. Do ard, C. Jeuniaux, R.A.A. Muzzarelli and G. Roberts. J. Andre, Lyon, pp. 230-235.

Guibal, E., Jansson-Charrier , M., Saucedo, I. and

Cloirec, P. (1995). Langmuir, 11, 591-598.

Ina, Y.and Nakamura, K. (1992). Jpn. Kokai Tokkyo Koho

JP 04,149,202. Filed 15 10 90. Ina, Y. and Nakamura, K. (1992). Jpn. Kokai Tokkyo Koho

JP 04,149,203. Filed 15 10 90.

Inoue, K., Ohto, K., Yashizuka, K., Shinbaru, R. and

Kina, K. (1995). Bunεeki Kag, 44, 283-287.

Inoue K., Yamaguchi, T., Shinbaru, R. , Hirakawa, H. , Yoεhizuka, K., Ohto, K. (1996). In Advances in

Chi tin Sciences, edε . , A. Domard, C. Jeuniaux, R.A.A. Muzzarelli and G. Roberts. J. Andre, Lyon, pp. 271-278. Ishii, T. (1992). Jpn. Kokai Tokkyo Koho JP 04,242,658. Filed 28 12 50. CA 118.11794. Ito, M. , Miyazaki, A., Yamagishi, T., Yagaεaki, H., Haεhem, A. and Oεhida, Y. (1994). Biomed. Mater. Engin., 4, 439-449. Klokkevold, P.R., Vandemark, L., Kenney, E.B. and Bernard, G.W.(1996). J. Periodonticε, 67, 1170- 1175.

Lacout, J.L., Mejdoubi, E. and Hamad, M. (1996). J.

Mater. Sci.: Mater. Med., 7, 371,374. Lohman, D. (1993). Eur .Pat .Appl . 0563,013A2 Filed 18 03. Martin, R.I. and Brown, P.W. (1994). J. Mater. Sci. : Mater. Med., 5, 96-102.

Maruyama, M. and Ito, M. (1996). Biom. Mat. Res., 32,

527-532 Menon, P.R., Napper, S.A. and Mukherjee, D.P. (1995). Proc. South. Biomed. Eng. Conf. 14th., 95-97. CA 124:066505.

Meεsersmith, P.B. and Stupp, S.I. (1992). J. Mater.

Res., 7, 2599-2611. Muzzarelli, R.A.A., Biagini, G. Bellardini , M. , Simonelli, L., Castaldini, C. and Fratto, G. (1993). Bio aterials, 14, 39-43.

Muzzarelli, R.A.A., Tanfani, F., E anuelli, E. and Bolognini, L. (1985). Biotechnol. Bioengin. , 27, 1115-1121. Muzzarelli, R.A.A. and Zattoni, A. (1986). Int. J. Biol. Macromol., 8, 137-142.

Muzzarelli, R.A.A. and Tubertini, 0. (1969). Talanta., 16,1571-1579.

Muzzarelli, R.A.A., (1996). Eur. Pat. Spec. EP 0,512,095

Bl Filed 18 11 91

Rinaudo, M. , Desbrieres, J., Klein, J.M. and Mahler, B. Demande Brevet FR 2,721,933/Al Depot 30 06 94.

Rinaudo, M. , (1996). In Advances in Chitin Science, eds., A. Domard, C. Jeuniaux, R.A.A. Muzzarelli and

G.A.F. Roberts. Jaεcques Andre Publ . , Lyon, F. ISBN

2907922. Summita,M. (1988) . Eur .pat . appl . 0,323,632A1 Filed 28 12.

Takechi , M., Miyamoto, Y., Yshikawa, K., Yuasa, M. ,

Nagaya a, M. , Kon, M. and Aεaoka, K. (1996). J.

Mater. Sci. : Mater. Med., 7,317-322.

Zhang S. and Gonsalveε, K.E. (1995). J. Appl. Polym. Sci., 56, 687-695.

Claims

1. Amorphous or partially crystalline chelates consiεting of chitoεanε, functionalized at the nitrogen with polycarboxylic functionε, linked to alkaline-earth metal insoluble salts.

2. Chelates according to claim 1, wherein said insoluble salts are calcium salts.

3. Chelateε according to claim 2, wherein said insoluble salt is calcium phosphate.

4. Chelateε according to claim 3, wherein the weight ratio of calcium phoεphate to functionalized chitoεan is 1:1.

5. A process for the preparation of chelates of claims 1-4, which compriεes the following stepε: a) mixing of a functionalized chitoεan with a εoluble εalt of the deεired alkaline-earth metal anion; b) addition of a soluble salt of the deεired alkaline- earth metal to form the chelate with the alkaline- earth metal inεoluble εalt; c) iεolation of the chelate.

6. A proceεε according to claim 5, wherein the functionalized chitosan is selected from: dicarboxymethyl chitosan, gluta ate glucan, aspartate glucan, N- [ ( 3 ' -hydroxy-2 ' , 3 ' -dicarboxy )ethyl]chitoεan .

7. A proceεε according to claim 5 or 6 , wherein the εalt of εtep a) iε a εodium salt εelected from: phoεphate, sulfate, oxalate, carbonate, hydrogen carbonate, fluoride.

8. A process for the preparation of the chelates of claims 1-4, which compriεeε mixing the unctionalized chitosan with an insoluble εalt of the deεired alkaline- earth metal.

9. A process according to any one of claims 5-7 or to claim 8, wherein the molar ratio of alkaline-earth metal εalt to functionalized chitoεan iε 1:3.

10. The uεe of the chelateε of claimε 1-4 as carriers for medicaments or growth factors.

11. The use of the chelates of claimε 1-4 for the preparation of a medicament useful to promote the restructuring proceεs of the bone matrix and for the mineraliεation thereof.

12. Pharmaceutical or coεmetic co poεitionε comprising an effective amount of at least one chelate of claims 1- 4 as active ingredient, together with conventional carriers and/or excipients.

13. Medical and cosmetic articles containing the chelates of claims 1-4.

14. Medical articles according to claim 13, in the form of freeze-dried materials, powders, films, membranes, solutions, gels and malleable pasteε.