WO2008090583A1

WO2008090583A1 - Phosphated derivatives of polysaccharides and uses thereof

Info

Publication number: WO2008090583A1
Application number: PCT/IT2008/000032
Authority: WO
Inventors: Agnese Magnani; Marco Consumi; Claudio Rossi; Giuseppe Greco
Original assignee: Universita' Degli Studi Di Siena
Priority date: 2007-01-22
Filing date: 2008-01-22
Publication date: 2008-07-31

Abstract

The present invention relates to phosphated products with polysaccharide basis, in particular: hyaluronic acid (HA), carboxymethylcellulose (CMC) and alginic acid (AA) and to their use.

Description

PHOSPHATED DERIVATIVES OF POLYSACCHARIDES AND USES THEREOF

_*****_*

TECHNICAL FIELD OF THE INVENTION

The present invention relates to phosphated products with polysaccharide basis, in particular: hyaluronic acid (HA), carboxymethylcellulose (CMC)~ and alginic acid (AA) and to their use. STATE OF THE ART

Among the most commonly used compounds in the biomedical field are polysaccharides of natural origin, used both as they are and chemically modified. The list of their applications is very long, for they are widely used as cellular scaffolds, coatings for biomedical devices, controlled release of drugs, etc. These macromolecules are used for their high biocompatibility and their role in mediating the material-cell interaction and immunological recognition. Consequently, the development of synthetic and/or semi-synthetic polyelectrolytes, that mimic the biological characteristics of natural polyelectrolytes or that have new chemical, physical and above all biological properties, has been ongoing for a long time. Among polyelectrolytes of biological origin are some polysaccharides, e.g. hyaluronic acid, which play a fundamental role in cell-substrate interactions. Hyaluronic acid is widely used in ophthalmology, in surgery, in tissue engineering and in the treatment of osteoarthritis [1-3]. The greatest limitation associated to its application as such pertains to its enzymatic degradation by hyaluronidase which, reducing its lifetime, considerably reduces its biological and pharmacological activity [4]. The sulfation of hyaluronic acid [5] has led to the formation of a product that is soluble in aqueous solutions, harder to degrade than the native polysaccharide, but whose degradation products can be toxic for cells. Phosphated derivatives, instead, should not exhibit any toxic effect because the degradation products of hyaluronic acid, like phosphate groups, are not toxic for the body.

In some known reactions, through reactants containing phosphate groups, polymeric gels are obtained [6-13], or phosphated mono-, oligo- and polysaccharides [14], but hitherto it was impossible to obtain phosphated polysaccharides soluble in water and in physiological solutions that have distinct biological and pharmacological characteristics and, in the specific case of hyaluronic acid, an enhanced resistance to degradation by hyaluronidase and hence longer-lasting biological and pharmacological activity.

Carboxymethylcellulose is also used in many biomedical applications, in particular in the formulation of preparations for oral and topical administration, given its ability to increase viscosity [15] and in the ophthalmic field [16-19]. Recently, it has been used in formulations for oral administration as a substitute for diuretics in hepatic and cardiac oedema and in preparations able to prevent the formation of post-operative adhesions [20] and epidural scars [21]. The sodium salt of carboxymethylcellulose is widely used as a stabiliser for tablets and emulsions [22,23]. In ophthalmology it is used as a viscoelastic substance during surgeries and laser therapy. Lastly, alginic acid is widely used in the pharmaceutical and biomedical field [24], for instance, in gastroenterology for the treatment of oesophageal reflux [25, 27], in dermatology for the treatment of wounds [28], in pharmacology as a system for the controlled release of drugs [29-32] and as an agent able to englobe cells and biomolecules [33,34]. In the present invention, the phosphation of alcoholic oxidryls, present in the polysaccharide chain, through the use of an appropriate phosphating reagent (diagram 1), leads to the formation of new derivatives with well defined chemical- physical and biological characteristics, different from those of the starting polysaccharides.

Diagram 1 : Phosphation Reaction

S = Polysaccharide repetitive unit

The polyelectrolytes that can be used as substrate for this invention are polysaccharides in general and in particular the sodic salts of hyaluronic acid, of carboxymethylcellulose and of alginic acid. The biopolymers obtained have characteristics of cellular biocompatibility, have no toxic effects on cells, and, in the case of hyaluronic acid, they have higher resistance to enzymatic degradation. These biopolymers can be used both as they are and mixed with other components for biomedical applications in the pharmaceutical, ophthalmologic, surgical, dermatological and cosmetic sector. Having these biopolymers available, it is possible to develop new hydrogels to be used in the biomedical-health care and pharmaceutical sector, with particular regard to the ophthalmologic and surgical sectors. They may also be used in cell growth processes, in systems for the controlled release of drugs, in anti-adhesions, in permanent, temporary and bioabsorbable biomedical implants. The new biopolymers can thus be available in the form of gels, creams, microspheres, tissues, and other formulations, depending on their intended therapeutic uses. These biopolymers can also be used in coating processes, imparting new biological characteristics to the surface of materials used as supports. These new biopolymers can be used in aqueous solution as biologically active molecules for application in the biological and biochemical field. DISCLOSURE OF THE INVENTION

Therefore, the present invention relates to a phosphated derivative of a polysaccharide characterised in that it is resistant to enzymatic degradation and/or it is not toxic. Preferably, the phosphated derivative has a phosphation degree between 1% and 34%. More preferably, the polysaccharide of the phosphated derivative is hyaluronic acid. Preferably, hyaluronic acid has a molecular weight between 10,000 and 50,000 Dalton. More preferably, hyaluronic acid has a molecular weight between 50,000 and 250,000 Dalton. Yet more preferably, hyaluronic acid has a molecular weight between 250,000 and 750,000 Dalton. More preferably still, hyaluronic acid has a molecular weight between 750,000 and 1 ,250,000 Dalton. In one embodiment, the polysaccharide is carboxymethylcellulose. Preferably, carboxymethylcellulose has a molecular weight between 90,000 and 2,000,000 Dalton.

In another embodiment, the polysaccharide is sodium alginate. Preferably, sodium alginate has a molecular weight between 50,000 and 250,000 Dalton. An object of the present invention is the phosphated derivative as described above for medical use.

An additional object of the invention is the use of the phosphated derivative of the invention for the preparation of a medicament. Preferably, the medicament has an activity comprised in the group of: anticoagulant, antiaggregant, osteoinductive, osteoregenerative, anti-inflammatory, cell growth stimulant, anti-post-operative adhesion and anti-hypertrophic scarring.

Another object of the present invention is the use of the phosphated derivative of the invention for the coating of a biomedical object. Preferably, the biomedical object is comprised in the group of: catheter, tube, probe, soft tissue prosthesis, animal origin prosthesis, artificial tendon, bone prostheses, contact lenses, syringe, surgical instrument, filtration system, laboratory instrument, container for cell cultures or for the regeneration of cells and tissues, support for peptides, proteins, antibodies. More preferably, the biomedical object is used in ophthalmology, dermatology, otorhinolaryngology, odontology, gynaecology, urology, surgery. Yet more preferably, surgery comprises osteoarticular, nervous, anastomotic, viscoelastic, ophthalmic, oncological, plastic, aesthetic, otorhinolaryngologic, abdominal-pelvic, urogynecologic, cardiovascular surgery. An object of the present invention is a pharmaceutical composition comprising a pharmaceutically effective and acceptable amount of the derivative of the invention. Preferably, the pharmaceutical composition is in gel, lotion, microsphere or membrane form. More preferably, the pharmaceutical composition has a controlled release. The procedures described herein can be applied to different salts of polysaccharides, without compromising the reaction, which is independent of the counter-ion of the polysaccharide.

These methods can be modified in various ways. Such modifications are not be construed as diverging from the spirit and from the perspectives of the invention and all those modifications which would be readily apparent to those skilled in the art are included within the scope of the present invention. The present invention shall now be described in some non limiting examples thereof, with particular reference to the following figures:

Figure 1: Infra-red (IR) spectra of the polysaccharides HA, CMC and of the respective phosphated derivatives. A) phosphated HA [molar ratio, polysaccharide/ trisodium trimetaphosphate (STMP, =1:10], B) native HA; C) phosphated CMC [polysaccharide/STMP molar ratio = 1 :10] D) native CMC.

Figure 2: IR spectra of the AA polysaccharides and respective phosphated derivative, A) phosphated AA [polysaccharide/STMP molar ratio = 1:10], B) native AA. Figure 3: Dispersion curves of the relaxation rate Ri, (obtained from measuring 1/T₁ of water) as a function of the applied magnetic field (ωo). K HA 1 :10, * HA 1 :5, *HA1 :1 , +HA.

Figure 4: Result of the cytotoxicity test on commercial mouse fibroblasts (ATCC NCTC L929) of phosphated hyaluronic acid compounds, with various degrees of phosphation. The values are the result of the average obtained on 5 replicates of the same sample, observing 5 different zones of each Petri dish for each sample. The data show that the samples of phosphated HA, regardless of the degree of phosphation, have not toxic effect on cells. The number of cells present in phosphated HA samples is comparable to those measured in the negative control, non cytotoxic PS and significantly higher than the one measured in the positive control, cytotoxic PVC.

Figure 5: Absorbance values measured for phosphated derivatives of HA A) obtained according to example 2) at different times (1 , 3, 12 and 24 hours) of digestion with the hyaluronidase enzyme, according to their phosphation percentages. MATERIALS AND METHODS

Reagents

The following reagents were used: hyaluronic acid (CAS n. 9067-32-7 ; MDL no.

MFCD00875848 ; Merck 13, 4776), carboxymethylcellulose (CAS n. 9004-32-4; MDL no. MFCD00081472), alginic acid (CAS no. 9005-38-3 ; MDL no. MFCD00081310;

Merck 13,238)

The reagent used for polysaccharide phosphation is trisodium trimetaphosphate

(STMP) (CAS no. 7785-84-4), a compound that is commonly used as a thickener for food substances [35-38]. For all polysaccharides of the invention, the reaction takes place as shown in diagram 1 , but on oxidrylic groups in different position, depending on the structure of the polymer.

The phosphating reaction was conducted both in aqueous solution (protocol a) and in a mixture of dimethyl sulfoxide/water (DMSO/H₂O) (protocol b). In both cases, a controlled phosphation varying the molar ratios between polysaccharides and phosphating agent was performed.

Protocol a)

The phosphation reaction is conducted adding, under agitation, the phosphating reagent to an aqueous solution, alkaline (pH=13) and thermostated at 25°C, of the sodic salt of the polysaccharide. The phosphating reagent is added in variable molar ratio according to the desired degree of phosphation (Table 1).

Table 1. Polysaccharide/STMP molar ratios used in the polysaccharide phosphation reaction.

The reaction mixture is kept under agitation at a pH value of 13 by successive additions of a solution of NaOH 1.0 M for 20 minutes. At the end, the pH is brought to neutrality by small successive additions of a 1.0 M HCI solution. The phosphated product is precipitated in acetone. The precipitate is then filtered and purified by dialysis, then dried or lyophilised. Protocol b)

Protocol b) differs from protocol a) only in the initial step, where the polysaccharide is first dispersed in an organic solvent (dimethyl sulfoxide) and subsequently to the mixture thus obtained is added bidistilled water and a solution of NaOH 1.0 M to bring the reaction mixture to pH=13. From this point onwards, the procedure is the same as in protocol a).

The two protocols a) and b) yield phosphated polysaccharides in which it is possible to modulate the degree of phosphation of the polysaccharide which, in the case of hyaluronic acid, also leads to a lower or higher resistance to biodegradation. The reaction of the polysaccharide in DMSO/water mixture can lead both to the formation of a phosphated derivative of the polysaccharide, and to a reticulation of the polysaccharide itself with formation of an insoluble hydrogel, depending on conditions of addition of the reagents and on the mixing rate. The phosphation reaction in all cases enables to phosphate the polysaccharide chain specifically and homogeneously. The pH conditions at which the reaction occurs do not lead to the hydrolysis of the polysaccharide in the reaction times used, as y

demonstrated in the literature [39] and verified through NMR relaxometric analysis (see the "chemical characterisation" section).

Some non limiting examples of the present invention are provided below: EXAMPLE 1 : Phosphating sodium hyaluronate in homogeneous phase protocol a)

1.0 g of the sodic salt of hyaluronic acid was solubilised in 50.0 cm³ of bidistilled water, the solution was mixed under agitation with a solution of NaOH obtained solubilising 40.0 g of base in 50.0 cm³ of bidistilled water. After 3 minutes of energetic agitation, the 23% m/v STMP solution in bidistilled water was slowly added until reaching the desired molar quantity of phosphating agent (table 1).

The reaction was left at 25 ⁰C under agitation for 20 minutes, whereupon a 1.0 M HCI solution was added until the reaction mixture was neutralised. The phosphated product was then precipitated in acetone, filtered, purified by dialysis (until the complete elimination of the residual reagents and of the internal products of the reaction) and stove dried, or lyophilised.

EXAMPLE 2: Phosphating sodium hyaluronate in heterogeneous phase Protocol b)

1.0 g of the sodic salt of hyaluronic acid was suspended in 25.0 cm³ of dimethyl sulfoxide for 6 hours, then 25.0 cm³ of bidistilled water were added and the mixture was vigorously agitated for 1 hour; subsequently, under agitation, a solution of NaOH, obtained solubilising 40.0 g of base in 50.0 cm³ of bidistilled water, was added. After 3 minutes of energetic agitation, the 23% m/v STMP solution in bidistilled water was added slowly until obtaining the desired molar quantity of phosphating agent (table 1). The reaction was left at 25 ⁰C under agitation for 20 minutes, after which the 1.0 M HCI solution was added until the reaction mixture was neutralised.

The phosphated product was then precipitated in acetone, filtered, purified by dialysis (until the complete elimination of the residual reagents and of the internal products of the reaction) and stove dried, or lyophilised.

EXAMPLE 3: Phosphation of the sodic salt of carboxymethylcellulose in homogeneous phase

Protocol a)

1.0 g of the sodic salt of carboxymethylcellulose acid was solubilised in 50.0 cm³ of bidistilled water, the solution was mixed under agitation with a solution of NaOH obtained solubilising 40.0 g of base in 50.0 cm³ of bidistilled water. After 3 minutes of energetic agitation, the 23% m/v STMP solution in bidistilled water was slowly added until reaching the desired molar quantity of phosphating agent (Table 1). The reaction was left at 25°C under agitation for 20 minutes, after which a 1.0 M HCI solution was added until the reaction mixture was neutralised.

The phosphated product was then precipitated in acetone, filtered, purified by dialysis (until the complete elimination of the residual reagents and of the internal products of the reaction) and stove dried, or lyophilised. EXAMPLE 4: Phosphation of the sodic salt of carboxymethylcellulose in heterogeneous phase Protocol b)

1.0 g of the sodic salt of carboxymethylcellulose was suspended in 25.0 cm³ of dimethyl sulfoxide for 6 hours, then 25.0 cm³ of bidistilled water were added and the mixture was vigorously agitated for 1 hour; subsequently, under agitation, a solution of NaOH, obtained solubilising 40.0 g of base in 50.0 cm³ of bidistilled water, was added. After 3 minutes of energetic agitation, the 23% m/v STMP solution in bidistilled water was added slowly until obtaining the desired molar quantity of phosphating agent (table 1). The reaction was left at 25 ⁰C under agitation for 20 minutes, after which the 1.0 M HCI solution was added until the reaction mixture was neutralised.

The phosphated product was then precipitated in acetone, filtered, purified by dialysis (until the complete removal of the residual reagents and of the internal products of the reaction) and stove dried, or lyophilised. EXAMPLE 5: Phosphation of the sodic salt of alginic acid in homogeneous phase Protocol a)

1.0 g of the sodic salt of alginic acid was solubilised in 50.0 cm³ of bidistilled water, the solution was mixed under agitation with a solution of NaOH obtained solubilising 40.0 g of base in 50.0 cm³ of bidistilled water. After 3 minutes of energetic agitation, was slowly added the 23% m/v STMP solution in bidistilled water until reaching the desired molar quantity of phosphating agent (table 1). The reaction was left at 25 ⁰C under agitation for 20 minutes, after which a 1.0 HCI M solution was added until the reaction mixture was neutralised. The phosphated product was then precipitated in acetone, filtered, purified by dialysis (until the complete removal of the residual reagents and of the internal products of the reaction) and stove dried, or lyophilised.

EXAMPLE 6: Phosphation of the sodic salt of alginic acid in heterogeneous phase Protocol b) 1.0 g of the sodic salt of alginic acid was suspended in 25.0 cm³ of dimethyl sulfoxide for 6 hours, then 25.0 cm³ of bidistilled water were added and the mixture was vigorously agitated for 1 hour; subsequently, under agitation, a solution of NaOH, obtained solubilising 40.0 g of base in 50.0 cm³ of bidistilled water, was added. After 3 minutes of energetic agitation, the 23% m/v STMP solution in bidistilled water was added slowly until obtaining the desired molar quantity of phosphating agent (table

1). The reaction was left at 25 ⁰C under agitation for 20 minutes, after which the 1.0

M HCI solution was added until the reaction mixture was neutralised.

The phosphated product was then precipitated in acetone, filtered, purified by dialysis (until the complete removal of the residual reagents and of the internal products of the reaction) and stove dried, or lyophilised.

Chemical characterisation

Infrared Analysis

The purified and dried compounds were analysed by infrared spectroscopy using a FT-IR spectrometer (Thermo Nicolet 5700 model) equipped with ATR (Attenuated

Total Reflection) accessory with a germanium crystal.

The experimental conditions were the following: number of scans: 256; resolution: 4.0 cm^"1; laser energy: 0.46 W; apodisation: Happ-Genzel; background correction: no.

The infrared spectra of the compounds before and after the modification are shown in

Figures 1 , 2. The main wave numbers observed in the spectra are shown in Table 2 together with the related attributions. Table 2. Main wave numbers observed in the IR spectra of the native and phosphated polysaccharides and their related attribution.

For each polysaccharide, the product with the highest degree of phosphation is shown.

The most significant wave number, which attest that phosphation has occurred, are those that fall within the 1350-1250 crrT¹ region, relating to the stretching of the P=O group and in the 1100-990 cm^"1 region relating to the stretching of the C-O-P group.

As shown, the aforesaid bands are present in all the spectra of the phosphated derivatives, albeit with different intensities because of the different degree of phosphation.

The most. characteristic bands of the native polysaccharides of the invention (which are obviously also found in the spectra of the phosphated polysaccharides) are those related to the vibrational modes of the amidic group (stretching of the C=O at 1648 cm^'1 and bending of the N-H at 1546 cm^"1) present in the spectrum of hyaluronic acid 4 and of the carboxylated group (asymmetric stretching at 1610 cm^"1 and symmetric stretching at 1403 cm^'1) present in the spectra of all three polysaccharides. In the spectra of the native and phosphated polysaccharides, moreover, a broad band is evident at 3500 cm^"1, due to the vibrations of the oxidrylic groups, whose intensity diminishes in the case of derivatives with high degree of phosphation.

Determination of the degree of phosphation

For each phosphated polysaccharide, the degree of phosphation was determined, expressed as the percentage of phosphated disaccharidic unit, considering that each disaccharidic unit contains a phosphate group.

Chart 2 shows, as an example, the disaccharidic unit of the sodic salt of monophosphated hyaluronic acid.

Chart 2 - Disaccharidic unit of monophosphated hyaluronic acid.

The degree of phosphation was determined in the following way:

A known quantity of phosphated polysaccharide was weighed exactly and destroyed in nitric acid heat-concentrated for 10 minutes. The product deriving from the destruction was diluted in a known quantity of distilled water and the content of phosphate groups was determined spectrophotometrically using a commercial kit

(Test Aquaquant® Merck KgaA - Darmstadt - Germany ). The results for each analysed sample are shown in Table 3. Table 3 - Values of the percentage of phosphation of the HA, CMC, AA phosphated polysaccharides as a function of the polysaccharide/phosphating agent molar ratio (STMP)

NMR Relaxometric analysis (¹H field-cycling)

The NMR relaxometric analysis (¹H field-cycling) was conducted on 3 samples of phosphated hyaluronic acid with different degrees of phosphating (HA1 :1 ; HA1 :5 HA1 :10). Comparing the data obtained with those relating to the unmodified polysaccharide (HA) it was possible to obtain information on the molecular weight and hence on the degree of phosphation of the three phosphated derivatives. The relaxometric data also provided the demonstration that the experimental conditions, drastic as they are, do not lead to deterioration of the polysaccharide or to a fragmentation of its chain. Figure 3 shows the dispersion curve of the relaxation rate Ri extrapolated from the measurement of 1/Ti of the water in relation to the applied magnetic field. The samples were prepared dissolving the polysaccharide in distilled water to obtain solutions whose polysaccharidic concentration was 15 mg/ml. In an unconfined polymeric system, the degree of relaxation should have a frequency of (1/Ti ^∞ ω^"1/3) linked to the molecular weight of the polymer itself. As shown in Figure 3, the values of 1/T₁ increase going from the native polymer towards the polymer with a higher degree of phosphation. This information shows that there is an increase in molecular weight, due to the introduction of the phosphate groups, and that the experimental conditions do not lead to a destruction of the polymeric chain. Biological characterisation Cytotoxicity Test Fibroblast Cultures

Cytotoxicity was evaluated using commercial mouse fibroblasts (ATCC NCTC L929) cultivated in polystyrene flasks with MEM (Eagle's Minimal Essential Medium) with addition of 10% (v/v) bovine fetal serum (Sigma Chemicals Co., USA), 20OmM L- glutamine (Sigma Chemicals Co., USA) and gentamicin 5 mg/ml of (Sigma Chemicals Co., USA). The fibroblasts were incubated at 37⁰C in atmosphere containing 5% of CO₂ until reaching the confluence of the cell monolayer. The cells were detached using a 0.25% v/v solution of sterile trypsin in 0.05% EDTA (Sigma Chemicals Co. USA) and suspended in medium. Test Execution In each well of a cell culture plate, 0.1 ml of a suspension containing 8 x10⁴ cells/ml were placed. The plate was then incubated at 37 ⁰C in 5% CO2 enriched atmosphere. Subsequently, to each well, containing a cell monolayer, was added 1.0 ml of MEM containing 5.0 mg/ml of the phosphated polysaccharide. Each solution was sterilised by filtration (Corning 0.22 micron filters). The plates were left in incubator for 24 hours. At the end, the cells were fixed with glutaraldehyde and marked with trypan blue to enable to count them by means of an optical microscope. The polystyrene surface (PS) of the culture plates constituted the negative control, whilst disks of polyvinyl chloride (PVC) stabilised with tin were used as positive control, in accordance with ISO standards (ISO-10993-5). The results are shown in Figure 4 and in Table 4. For each sample, 5 replications were repeated. Table 4 - cytotoxicity tests of the phosphated polysaccharides on commercial mouse fibroblasts (ATCC NCTC L929). The values are the results of the average obtained on 5 replication of the same sample, observing 5 different zones of each Petri dish for each sample.

No. of cells

Molar ratio

Sample (Average on 5 Standard deviation replications)

1:0.5 48 3

HA 1:1 46 4

Example 1 1:5 43 3

1:8 42 7

1:10 42 7

1:0.5 47 2

HA 1:1 46 4

Example 2 1:5 44 3

1 :8 40 6

1:10 42 2

1:1 42 2

CMC

Example 3 1:5 41 2

1:10 38 2 CMC ^{1 : 1 40 2}

Example 4 1:5 41 2

1:10 39 2

AA ^{' 1 : 1 43 5}

Example 5 1:5 40 4

1:10 41 6

Example 6 1:5 40 2

1:10 40 2 n Con ^tro il 1 (N. eg fative) 'PS 5.4 5

(Positive) PVC 2 1

The values in Table 4 and in Figure 4 clearly show that the samples of phosphated HA, phosphated CMC and phosphated AA have no toxic effect on the cells. The number of cells present in the aforesaid samples is comparable to those measured in the negative control, non cytotoxic PS, and significantly higher than the number measured in the positive control, cytotoxic PVC. Enzymatic Degradation

The enzymatic degradation was conducted according to the method by Ola M. Saad et al. [40] and making minimal variations thereto. Briefly, in ammonium acetate buffer at physiological pH, were prepared solutions of the phosphated derivatives each containing 15.0 micrograms of polymer in 1.0 ml of buffer solution. The solutions were placed in a bath, thermostated at 37⁰C. Subsequently, to each solution was added a known quantity of hyaluronidase (Sigma) to digest the hyaluronic acid and obtain uronic acid. The sodic salt of the hyaluronic acid was used as a control. For each sample was added an excess of hyaluronidase enzyme and the quantity of uronic acid, produced as a function of the digestion time, was determined by spectrophotometry, by conducting the carbazole assay [41 ,42]. Briefly, 100.0 microlitres of the solution originating from the degradation were added to a solution containing concentrated sulphuric acid and sodium tetraborate decahydrate. The resulting solution was heated to 100⁰C for 10 minutes. Subsequently, a 0.125% w/v carbazole solution in absolute ethanol was added and the mixture was heated for 15 more minutes at 100⁰C. The final absorbance of the solution at 530 nm was determined through a UV-visible spectrophotometer (Perkin-Elmer Lambda 25 model). For each sample, 3 replications were carried out.

Figures 5A and 5B show the absorbance values of the carbazole assay versus the percentage of phosphation of the polysaccharide at different digestion times (1 to 24 hours). The phosphated compounds of sodium hyaluronate synthesised according to Example 1 and Example 2, described above, were compared to non phosphated sodium hyaluronate (control).

As shown in Figures 5A and 5B, all phosphated derivatives of the hyaluronate have considerable resistance to hydrolysis, in terms of uronic acid production, by hyaluronidase. The control, i.e. the unmodified hyaluronic acid, is highly degraded after 3 hours of digestion and, even extending the digestion time to 24 hours, no significant increase in uronic acid in solution is observed. This allows to assume that during the initial hours the digestion is nearly complete, as confirmed by the literature and by the technical specifications provided by the manufacturer of the enzyme. With regard to the phosphated compounds, none of them reaches a level of degradation equivalent to the control during the first 24 hours; moreover, it is readily apparent that, increasing the degree of phosphation, resistance to degradation increases so much that the most phosphated polysaccharide after 24 hours of digestion is degraded by less than half the control. BIBLIOGRAPHY [1] In Hyaluronan, Kennedy J. F., Phillips G.O., Williams P.A., Eds., Woodhead

Publishing, Cambridge 2000.

[2] The Chemistry, Biology and Medical Applications of Hyaluronan and its

Derivatives (Laurent TC, ed) Wenner-Gren International Series, VoI 72, Portland Press, London (1998)

[3] Intra-articular hyaluronans: A review of product-specific safety profiles

Seminars in Arthritis and Rheumatism, Volume 32, Issue 5, April 2003, Pages 296-

309 Max I. Hamburger, Sharad Lakhanpal, Pekka A. Mooar and David Oster

[4] Lepperdinger G.; Fehrer C; Reitinger S.; In Chemistry and Biology of Hyaluronan; Gargu H. G., Hales CA, Eds; Elsevier ,2004, p. 71 - 82.

[5] 6,051 ,701 Cialdi, Magnani et al. April 18, 2000

[6] Hideaki T.; Kiyomi Y.; Shuji S.; Sep. Purif. Technol., In Press, Corrected Proof, ,

Available online 18 January 2006.

[7] Nuttelman, C.R.; Danielle, S.W.; Benoit, M.; Tripodi, C; Kristi, S. A. Biomaterials, 2006, 27, 1377-1386.

[8] Gliko-Kabir, I.; Yagen, B.; Penhasi, A.; Rubinstein, A. J. Control. Release 2000,

63,121-127

[9] Patent n.6,991 ,648 Machy , et al. January 31 , 2006

[10] Patent n.6, 946,443 Blanchat , et al. September 20, 2005 [11] Patent n.6, 846,497 Okoniewska , et al. January 25, 2005

[12] Patent n.4,369,308 Trubiano January 18, 1983

[13] Patent n.6410519 Gruskin et al. June 25, 2002

[14] Kitano, T.; Zerwekh, J. E.; Edwards, M.L.; Spine 1991, 16, 820-823.

[15] Japanese Patent JP19930339085, Mitsutomo et al., November 22, 1993 [16] Wade, A. and Weller, P. (1994) Handbook of Pharmaceutical Excipients,

American Pharmaceutical Association, Washington, USA and Pharmaceutical Press,

London, UK

[17] Patent n.4,819,617 Goldberg , et al. April 11 , 1989 [18] G., van Santvliet, L., Verschuren, E. Ludwig, A. Pharmazie 1996, 51, 357-362

[20] Heidrick, G.W.; Pippitt, CH. Jr; Morgan, M.A.; Thurnau, G.R.; J. Reprod. Med.

1994, 39, 575-8.

[21] Kitano, T.; Zerwekh, J. E.; Edwards, M.L.; Spine 1991 , 16, 820-823.

[22] Oza, K.P. et al. J. Disper. Sci. Technol. 1986, 7, 543-561. [23] Sebert, P. et al. Int. J. Pharm. 1994, 106, 103-108

[24] Hanks CT. In: Craig RG, editor. Restorative dental materials, 9th ed. St. Louis:

Mosby, 1993.

[25] Zentilin, P.; Dulbecco, P.; Savarino, E.; Parodi, A.; liritano, E.; Biliardi, C;

Reglioni, S.; Vigneti, S.; Savarino, V.; Aliment. Pharmacol. Ther. 2005, 21,29-34. [26] Matthew, I. R.; Browne, R.M.; Frame, J.W.; Millar, B. G. Biomaterials 1995, 16 ,

275-278.

[27] Atala, A.; Kim, W.; Paige, K.T.; Vacanti, C.A.; Retik, A.B.; J. Urol. 1994, 152 ,

641-643.

[28] Van Der Weyden, E.A.; Br. J. Community Nurs. 2005 , S21 , S24, S26-7. [29] Dai, C; Wang ,B.; Zhao, H. Colloids Surf. B Biointerfaces 2005, 41, 117-20

[30] Patent n.6,027,748 Conte , et al. February 22, 2000

[31] Vautier C. and Couvreur P. In Handbook of Pharmaceutical Controlled Release

Technology Donald L. W., Eds.; Marcel Dekker, New York, 2000;

[32] Wong, W.T.; Chan, L.W.; Kho, S.B.; Heng, P.W.S.; J. Control. ReI. 2002, 84, 99- 114 ^

[33] Murano, E.; J. Appl. lchthyol. 1998, 14, 245-249

[34] O. Smidsrod and G. Skjak-Braek. Trends Biotechnol. 1990, 8 (3), 71-78; L. Shapiro and S. Cohen, Biomaterials 1997, 18 (8), 583-590 [35] Patent n.6,936,136 Shannon , et al. August 30, 2005 stmp [36 ]Patent n.6,933,349 Chen , et al. August 23, 2005 stmp [37] Patent n.6,899,913 Buwalda , et al. May 31 , 2005 stmp [38] Patent n.6,890,579 Buwalda , et al. May 10, 2005 stmp

[39] Ghosh, S.; Kobal.l.; Zanette.D.; Reed.W. F. Macromolecules 1993,26, 4685- 4693. [40] Ola M. Saad, Richard A. Myers, D. Laird Castleton and Julie A. Leary, Analytical Biochemistry, 2005, 344 (2), 232-239. [41] T. Bitter and H. M. Muir. Anal. Biochem. 1962, 4, 330-334 [42] E.L. Hedberg, CK. Shih, LA Solchaga, A.I. Caplan and A.G. Mikos, J. Control. Release 2004, 100, 257-266.

Claims

1- A phosphated derivative of a polysaccharide, characterised in that it is resistant to enzymatic degradation and/or it is not toxic.

2- The phosphated derivative according to claim 1 , having a degree of phosphation between 1% and 34%.

3- The phosphated derivative according to claim 1 or 2 wherein the polysaccharide is hyaluronic acid.

4- The phosphated derivative according to claim 3, wherein the hyaluronic acid has a molecular weight between 10,000 and 50,000 Dalton. 5- The phosphated derivative according to claim 3, wherein the hyaluronic acid has a molecular weight between 50,000 and 250,000 Dalton.

6- The phosphated derivative according to claim 3, wherein the hyaluronic acid has a molecular weight between 250,000 and 750,000 Dalton.

7- The phosphated derivative according to claim 3, wherein the hyaluronic acid has a molecular weight between 750,000 and 1 ,250,000 Dalton.

8- The phosphated derivative according to claim 1 or 2 wherein the polysaccharide is carboxymethylcellulose.

9- The phosphated derivative according to claim 8, wherein the carboxymethylcellulose has a molecular weight between 90,000 and 2,000,000 Dalton:

10- The phosphated derivative according to claim 1 or 2 wherein the polysaccharide is sodium alginate.

11- The phosphated derivative according to claim 10, wherein the sodium alginate has a molecular weight between 50,000 and 250,000 Dalton. 12- The phosphated derivative according to one of the previous claims, for medical use.

13- Use of the phosphated derivative according to one of the previous claims for the preparation of a medicament. 14- Use according to claim 13, wherein the medicament has an activity selected from the group of: anticoagulant, antiaggregant, osteoinductive, osteoregenerative, anti-inflammatory, cell growth stimulant, anti-post-operative adhesion and anti- hypertrophic scarring.

15- Use of the phosphated derivative according to claims 1-12 for coating a biomedical object.

16- Use according to claim 15, wherein the biomedical object is selected from the group of: catheter, tube, probe, soft tissue prosthesis, animal origin prosthesis, artificial tendon, bone prostheses, contact lenses, syringe, surgical instrument, filtration system, laboratory instrument, container for cell cultures or for the regeneration of cells and tissues, support for peptides, proteins, antibodies.

17- Use according to claim 16, wherein the biomedical object is used in ophthalmology, dermatology, otorhinolaryngology, odontology, gynaecology, urology, surgery.

18- Use according to claim 17, wherein the surgery comprises osteoarticular, nervous, anastomotic, viscoelastic, ophthalmic, oncological, plastic, aesthetic, otorhinolaryngologic, abdominal-pelvic, urogynecologic, cardiovascular surgery. 19- A pharmaceutical composition comprising a pharmaceutically effective and acceptable amount of the derivative according to claims 1-12 . 0- The pharmaceutical composition according to claim 19, being in the form of gel, cream, microsphere or membrane. - The pharmaceutical composition according to claim 19-20 having a controlled

release.