"NON THROMBOGENIC POLYMERS WITH COMPATIBILITY WITH ORGANIC FLUIDS AND TISSUES" Technical Field The present invention relates to new polymer materials destined to come in contact with biological tissues and fluids, and the process for their preparation. Said materials are characterized in that they have molecules of a polysaccharide constituted by repetitive disaccharidic unities formed by a uronic acid and an hexosamine (for example heparins, heparans, dermatans or mixtures thereof) covalently bonded to the surface of a basic polymer.

Background Art It is known the anti-thrombotic activity of the glycosaminoglycanes (for example heparins, heparans, dermatans and mixtures thereof), that makes them fit to come in contact with biological fluids or tissues. These compounds are constituted by disaccharidic repetitive unities containing a residue of uronic acid and a residue of hexosamine.

The glycosaminoglycanes prevalent repetitive unities are represented as follows:

Wherein HS represents the heparansulphate wherein n normally varies between 10 and 30; Ch-6S represents chondroitine 6 sulphate (chondroitine A) wherein n normally varies between 10 and 50; Ch-4S represents chondroitine 4 sulphate (chondroitine C) wherein n normally varies between 10 and 50; HEP represents heparin wherein n normally varies between 10 and 20 and lastly DeS represents dermatan (chondroitine B) wherein n normally varies between 10 and 30.

The groups N-acetyl or N-sulphate of the residues of hexosamine can be transformed in amino groups through reactions of N-desulphation or N- deacetylation and the sulphate groups of the uronic acids through desulphation reactions can give rise to epoxy groups.

It is known the use of glycosaminoglycanes, heparin in particular, as agents to be supported on the surface of polymer materials in order to improve their compatibility with organic tissues or fluids. For example in US 4,987,181 depolymerized heparin is covalently bonded to an olefin copolymer. The resulting product is then used as adhesive film to be applied on polymeric surfaces that

must come in contact with organic fluids or tissues. In such process the depolymerization is useful to create the functional group necessary to the formation of the covalent bond, but it also gives rise to the decrease of some biological activities, such as the anti coagulant activity. In US 5,541,305 it is obtained a material compatible with blood by heparinizing a polymer having quaternary amino groups with alkali salts of heparin sulphate groups.

These products main problem is that, as there is not a strong bond between the polysaccharides and the polymer that forms the structure of the material, a release of the polysaccharidic portion takes often place in a short time.

Thus, the object of the present invention are new materials treated with polysaccharides with high anti-thrombotic and anti-inflammatory characteristics, that can come in contact with organic fluids or tissues.

Another object of the present invention is supplying a simple process for the production of said materials.

Disclosure of the Invention The polymer materials object of the present invention having anti-inflammatory, non thrombogenic surfaces with an enhanced compatibility with organic fluids and tissues, are characterized in that they have polysaccharides molecules covalently bonded to the surface of a basic polymer.

Said covalent bond can be obtained by reacting amino groups obtained on the functionalities N-acetyl and N-sulphate of the residues of hexosamine and/or epoxy groups obtained on the residues of uronic acid or they can be transformed in other groups reactive to the surface of the basic polymer through reaction with a compound of formula L-roi wherein RI is selected from the group comprising hydrogen linear or branched Cl-C20 alkyl, C4-C20 cycloalkyl, C6-C20 aryl, C7-C20 arylalkyl; L represents a group reactive towards the basic polymer and towards said functionalized glycosamino or uronic acid residues and it is selected from the group comprising: NHR', Si (OR') 3.

The covalent bond is preferably formed through an amination reaction with ammonia or primary amines of the epoxy groups obtained on the uronic acid residues; more preferably the amination reaction is made with ammonia.

In alternative the polysaccharides can be covalently bonded to the basic polymer through the use of spacers of general formula T-R"-T wherein: R"is selected from the group comprising: linear or branched Cl-C20 alkylene, C4-C20 cycloalkylene, C6-C20 arylene, C7-C20 arylalkylene; T is selected from the group comprising: NRIZ, Si (OR') 3, COR1", OR', X, wherein R111 is selected from the group comprising: Cl, Br, I.

T can also represent a group from which through simple reactions it is possible to obtain one of the groups described, in case the compound T-RII-T does not prove to be stable. For example compounds of formula NH2- (CHZ) nCHO wherein n varies from 2 to 20 do not prove to be stable as they would tend to polymerize, it is possible anyway to use compounds of formula NH2- (CH2) nCH (OEt) 2 wherein the aldehydic group is present as an acetal and can be restored through hydrolysis.

Examples of groups T-rut are: diaminobutane, diaminepentane, diaminehexane, diamineheptane, 1,4 (aminoethyl)-benzene, 1,4 diaminebenzene, 1-amine-4- trimetoxysilyl-butane, 1-amine-4-trimetoxysilyl-pentane, 4- methyl (trimetoxysilyl)-benzylamine, ethylene glycol, 1,6 dihydroxihexane, 1,4 bis bromomethylbenzene.

Preferably in case it is used polysaccharides functionalized with epoxy groups, the spacers used are compounds of general formula NH2- (CH2) n-NH2 wherein n varies from 1 to 12, more preferably from 3 to 8.

The polymer materials object of the present invention are obtained through a process comprising the following steps: a) formation of a free amino functionality on the residues of hexosamine, or formation of an epoxy functionality through desulphation of the uronic acid; b) optional treatment of the compound obtained in step a) with a compound L-Rl or with a compound T-Rll-T wherein the compound to be used is selected according to the functionalities present on the polysaccharide and on the basic polymer; for example the epoxy functionality can be

transformed in an amino functionality through an amination reaction with ammonia or with a primary amine; c) treatment of the basic polymer with a solution of the reaction product of step a) or step b).

Preferably the process comprises the following steps: a) introduction of an epoxy functionality on positions 2,3 of the uronic acid; b) treatment of the epoxide obtained by step a) with ammonia or with a primary amine; c) treatment of the basic polymer with a solution of the reaction product of step b).

Preferably in step b) ammonia is used.

In case spacers are used, preferred compounds are those of general formula NH2- (CH2) n-NH2 wherein n varies from 1 to 12, preferably from 3 to 8.

An alternative process to obtain the polymer materials object of the present invention is characterized by the following steps: a) formation of a free amino functionality on the residues of hexosamine, or formation of an epoxy functionality through desulphation of the uronic acid; b) reaction of a compound of general formula L-Rl or T-Rli-T wherein L, RI, T and Rll have akeady been described, with the surface of the basic polymer c) treatment of the reaction product of step b) with a solution of the reaction product of step a).

Preferably in step a) it is introduced an epoxy functionality and in step b) it is used compounds of general formula NH2- (CH2) n-NH2 wherein n varies from 1 to 12, preferably from 3 to 8.

Another alternative process to obtain the polymer materials object of the present invention is characterized in that the epoxy groups on the residue of the uronic acid are produced in the presence of the functionalized polymer. Such a process can be described through the following steps:

a) reaction of a compound of formula L-RI or T-R"-T wherein L, RI, T and R"have already been described, with the surface of the basic polymer b) treatment of the reaction product of step a) with a solution with a pH comprised between 9 and 11 containing the polysaccharide.

It is preferably used a solution of NaOH having a pH between 9.5 and 10.5 and in step a) it is used compounds of general formula NH2- (CH2) n-NH2 wherein n varies from 1 to 12, preferably from 3 to 8.

The skilled man can select the reagents and the reaction conditions to obtain the desired product in the various steps. For example to obtain the epoxidation of the residues of uronic acid, the reaction times range from 10 minutes to 5 hours while the temperature varies from 20 to 70°C at a pH comprised between 9 and 11. The polysaccharides reaction with the polymer surface can be realized by making flow a solution of the modified polysaccharides on the surface of the polymer that is going to be functionalized. Though the temperature is not a prerequisite characteristic for the realization of the present invention, it preferably varies between 10°C and 80°C, more preferably between 40°C and 70°C, in a time that varies between 10 minutes and 3 days.

In an epoxidation N-desulphation, N-deacetylation reaction, the quantity of groups that are reacted in comparison with the total quantity of reactive groups is indicated as a percentage. The used functionalization percentage influences whether and how the polysaccharides are bonded to the basic polymer, but it also influences the biological activity of the obtained polymer material. For example in heparin the preferred percentage of groups N-acetyl that reacted is comprised between 25 and 100%; the preferred percentage of groups that are reacted in the N-desulphation and epoxidation reactions is comprised between 5 and 60%, more preferably 10-40%.

Preferably used polysaccharides are glycosaminoglycanes (heparin, heparans, dermatans). More preferably it is used glycosaminoglycanes with medium molecular weights (Mw) comprised between 6000 and 30000 daltons, even more preferably comprised between 10000 and 18000 daltons.

As a basic polymer it can be used any type of polymer that owns functional groups, for example functionalized polyolefins, acrylic polymers, polycarbonates, polysilicates, polyvinylchloride, silicones, polysaccharides; it is preferably used polyvinylchlorides, silicones and functionalized polyolefins.

The materials obtained with the present invention show such an enhanced anti thrombogenic and anti-inflammatory activity, that it is possible to use these materials in contact with organic fluids and tissues.

The present invention is now more deeply described through examples that must be considered explanatory but that are not intended to limit in any way the invention itself.

EXAMPLES EXAMPLE 1 Preparation of epoxy heparins: general procedure 300 mg of heparin is dissolved in 10 ml of H20, brought to the temperature of 65°C and it is added 10 ml of a solution containing 800mg/1 of NaOH. The reaction time depends on the quantity of epoxy groups that it is intended to form (15 minutes to obtain the reaction of the 10% of the reactive groups, 30 minutes for the 20% and 3.5 hours for the 100%). At the end of the time the reaction is stopped by cooling it at the temperature of 10°C and neutralizing it with a solution of diluted HC1. The product is then purified through one or a combination of the methods listed below: -gel filtration; -dialysis; -ethanol precipitation: The product is characterized through spectroscopy TAM EXAMPLE 2 Preparation of heparins derivatized with Diaminobutane (HEP-DAB)

1.5 g of epoxy heparin at 10% prepared according to example 1 is dissolved in 7.5 ml of H2O. To the solution 0.240 g of diaminobutane (DAB) is added. After 48h at room temperature, the reaction is stopped, NaCl is added to the system up to a concentration 1M, then the polysaccharide derivative is precipitated with ethanol (EtOH). The ratio H2O/EtOH used is 1: 4. After the precipitation the sample is filtered, the precipitate is centrifuged at 4°C; the precipitation and the centrifugation are repeated till there are not any more traces of DAB in the supernatant.

EXAMPLE 3 Preparation of heparins derivatized with Diaminobutane in a single step In an alternative form of heparins derivatization with diaminobutane (DAB) this last is bonded to the polysaccharidic chain in the same reaction ambient that brings to the formation of the epoxide.

A solution containing 40 g/1 of NaOH and the DAB is added to a solution of HEP 40 g/1 brought to the temperature of 60°C. The used molar ratio HEP (PM=13500)/DAB was 1: 2. At the end of the 15 minutes the sample is cooled and brought to neutrality with HC1 4%, ETOH is added in a ratio 1: 4 v/v and it is left settling for 24 h at the temperature of 4°C. The precipitate is centrifuged at 4°C, the precipitation and the centrifugation are repeated till there are not any more traces of DAB in the supernatant.

EXAMPLE 4 Preparation of aminoheparins: general procedure It was prepared an heparinic derivative having an amino group directly bonded in position 2 of the residue of uronic acid.

125 mg of epoxy heparin obtained according to the general procedure of example 1 is dialyzed in dialysis tubes with a 6000-8000 Da cut-off against H20 for 16 hours. At the end of the dialysis the sample is concentrated through evaporation at a reduced pressure up to a volume of 2 ml, then 5.5 ml of a solution of NH3 at 32% (17 M) is added at a temperature of 10°C. The so obtained solution is put under stirring at room temperature for 50 hours, after which the excess of NH3 is eliminated by evaporating it at a reduced pressure.

EXAMPLE 5 Preparation of N-Desulphated heparins Heparin sodium salt is brought to acid form by using"Amberlite IR-120 (H)" by Carlo Erba, as an ionic exchange resin, the eluted is neutralized with pyridine, forming in this way the relevant salt, and brought to dryness.

Heparin pyridinic salt (10 g) is dissolved in a mixture of dimethylsulphoxide (DMSO) containing 5% of MeOH (1 1), then the mixture is warmed at 50°C for 1.5 h. In these conditions it is obtained a completely desulphated heparin; intermediate degrees of desulphation are obtained by using shorter times.

After the purification the product is characterized by 13C-NMR.

EXAMPLE 6 Preparation of N-Deacetylated heparins 25 g of heparin is dissolved in 1 1 of NaOH 0.1 M and warmed at 50°C. The deacetylation is complete after 8 h, also in this case the product is characterized by <BR> <BR> 13C-NMR.<BR> <BR> <BR> <BR> <P>DERIVATIZATION OF THE POLYMER MATERIALS EXAMPLE 7 Reaction with PVC of heparin derivatized with diaminobutane Example 7a) 557 mg of heparin derivatized with diaminobutane at 10% obtained as described in example 2 is dissolved in 50 ml of H20; the solution is fluxed inside a PVC tube (10 cm, =0.4 cm) at room temperature for 24 h. At the end the PVC is washed with NaCI 1M solutions; the quantity of heparin bonded to the support is calculated through the biological activity.

Example 7b) 500 mg of heparin derivatized with diaminobutane at 10% obtained as described in example 2 is dissolved in 50 ml of H20; the solution is fluxed inside a PVC tube (10 cm, rua=0.4 cm) at 50°C for 24 h. At the end the PVC is washed with NaCl 1M solutions.

EXAMPLE 8 Reaction of aminoheparins with PVC Example 8a) 50 mg of aminoheparin derivatized at 10% obtained according to the procedure described in example 4 is dissolved in 10 ml of H2O; the solution is

fluxed inside a PVC tube (10 cm, 0=0.4 cm) at room temperature for 24 h. At the end the PVC is washed with NaCl 1M solutions.

Example 8b) 75 mg of aminoheparin derivatized at 100% obtained according to the procedure described in example 4 is dissolved in 10 ml of H20; the solution is fluxed inside a PVC tube (10 cm, rua=0.4 cm) at room temperature for 24 h. At the end the PVC is washed with NaCl 1M solutions.

EXAMPLE 9 Reaction of aminoheparins with silicone 1.3 g of aminoheparin derivatized at 10% prepared according to the procedure described in example 4 is dissolved in 25 ml of H20; the solution is fluxed inside a silicone tube (10 cm, =0.5 cm) at room temperature for 24 h. At the end the silicone is washed with NaCl 1M solutions.

EXAMPLE 10 Functionalization of PVC with diaminobutane Example 10a) 63 mg of diaminobutane is dissolved in 10 ml of H20. The solution is fluxed inside a 20 cm PVC tube (0=0.4 cm) for 5 h at room temperature, obtaining tubes with a final DAB concentration equal to 10 mg/cm.

Example lOb) 126 mg of diaminobutane is dissolved in 10 ml of HzO. The solution is fluxed inside a 20 cm PVC tube (=0.4 cm) for 5 h at room temperature. It is obtained tubes with a final DAB concentration equal to 20 mg/cm.

EXAMPLE 11 Functionalization of silicone with basic groups 1.76 g of diamenutane is dissolved in 40 ml of H20. The solution is fluxed inside a 20 cm silicone tube (0=0.5 cm) for 5 h at room temperature.

EXAMPLE 12 Reaction of epoxy-heparins with PVC functionalized with diaminobutane Epoxy-heparins are prepared in the presence of the functionalized basic polymer.

Example 12a) 195 mg of heparin is dissolved in 10 ml of a NaOH aqueous solution with pH 10 at a temperature of 60°C, then it is fluxed, through a

peristaltic pump in a 10 cm tube obtained in example 10 a) for 30 minutes. At the end of the time the tube is washed with H20 and subsequently characterized.

Example 12b) example 12a) is repeated using 400 mg of heparin Example 12c) example 12a) is repeated using 120 mg of heparin and the tube obtained in example lOb) Example 12d) example 12c) is repeated using 240 mg of heparin EXAMPLE 13 Reaction of epoxy-heparins with silicone functionalized with diaminobutane Epoxy-heparins were prepared in the presence of the silicone tube previously derivatized.

400 mg of heparin is dissolved in 20 ml of a NaOH aqueous solution with pH 10 at a temperature of 60°C, then it is fluxed for 30 minutes through a peristaltic pump in 10 cm of a tube obtained in example 11. At the end of the time the tube is washed with H20 and subsequently characterized.

CHARACTERIZATION OF THE OBTAINED MATERIALS Test of anti-thrombotic activity The anti-thrombotic activity was determined by using a commercial kit (method STACHRON DS). 1 cm of tube for every sample to be analyzed is immersed in 200 ul of physiological solution (NaCl 0.9%) to which it is subsequently added at intervals of 2', 200 Ill of a solution containing factor HC II, 200 al of Trombina solution, 200 ul of the chromogenic substrate solution and 200 ul of a aqueous CH3COOH solution at 10%, in order to stop the reaction, stirring after every step.

The test is realized at a temperature of 37°C. The absorptions are registered at 405 nm using as a reference a solution that does not contain the cm of tube and does not contain heparin. The blanks are realized by adding the CH3COOH solution before adding factor HC II.

The inhibition percentage per cm of tube is determined by using the following equation <BR> <BR> <BR> <BR> <BR> <BR> AbsCampAbsRif- <BR> %I / cm =n#x100 AbsRif The values obtained from the various samples are reported in the following table: Example % I/cm Example % I/cm 7a) 46 12a) 21 7b) 80 12b) 39 8a) 40 12c) 47 8b) 41 12d) 63 9 85 13 65