FR2718024A1 - Drug and pharmaceutical composition for the treatment of inflammation. - Google Patents

Abstract

The use of at least one polymer or biopolymer known as HBGFPP, which is capable of specifically protecting growth factors of families FGF and beta-TGF from trypsin damage, and does not significantly inhibit coagulation, for preparing a drug for treating inflammation, is disclosed.

Description

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  The present invention relates to the use of polymers or biopolymers for the preparation of a

  medicine for the treatment of inflammation.

  It is furthermore related to a composition containing these polymers and intended for such treatment. Inflammation is a particularly complex manifestation that develops as a result of spontaneous or traumatic tissue injury. Characterized by a change in capillary permeability, it is accompanied by infiltration of blood components, molecular and cellular, which is generally manifested by erythema and edema. Many chemical mediators are released or activated during this process in which elements of the blood, especially white blood cells, accumulate and release different lytic activities. Many

  substances are used as anti-inflammatory agents.

  They can roughly be classified as agents

steroidal and nonsteroidal.

  Growth factors belonging to the family of Heparin Binding Growth Factors (HBGF) are naturally released in the case of injury or tissue trauma either directly by these tissues in their constituent cells, from the elements of the extracellular matrix which represents a site of natural storage of these molecules, either by the circulating cells and in particular the cells of the inflammation. The inflammatory response is accompanied by a local increase in growth factor concentrations, some of which

  belong to the HBGF family.

  The sucrose sulfate ester and its aluminum salt sucralfate are products described and used as anti-inflammatory agents (US Patent No. 3,432,489) and in various combinations and pharmaceutical compositions described in a series of patents (US 4,975,281, 4,885 .281,

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  US 5,013,557, US 5,164,379, US 5,196,405, US 5,240,710 and DK

102,488 and DK 505,588).

  Other compounds have been described as anti-inflammatory agents: suramin, an organic component sulphonated by LAROCCA R et al. (US 7479817), compositions containing low molecular weight mixtures of heparin by COHEN IR et al. (WO9219249), heparin fragments exhibiting inhibitory effects of the complement system by EKREH P et al. (WO9202232), products derived by chemical modifications of glycosylated proteins by MIYAZAKI K et al. (EP454898), proteoglycans

  heparan sulfate as the syndecan by BERNFIELD M et al.

  (WO9305167), oligosaccharides derived from dermatans

  sulfates by BIANCHINI P et al. (W09305075).

  The synthesis of another family of polymers, called CMDBS (Carboxy Methyl Dextran Benzylamine Sulfonate) has been described in patent FR 2 461 724 as well as in US Pat. No. 4,740,594. Some of these polymers mimic heparin and can be used as replacement products for plasma heparin, thanks to their anticoagulant properties

and anticomplement.

  Among all the CMDBS polymers, some mimic another property of heparin which consists in stabilization, protection and potentiation in vitro of the biological activity of the growth factors of the FGF family. (Tardieu et al, Journal of Cellular

  Physiology, 1992,150 pages 194 to 203).

  The patent FR 2,644,066 describes the use of certain CMDBS associated with FGF for the healing of the skin and the cornea. Experiments were performed by causing a cutaneous injury with a 6 mm diameter punch in the rat. In this example, the CMDBS associated with FGF 2 provides a net effect on speed and

  quality of skin repair.

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  It follows from the analysis of the state of the art that polymers of the CMDBS type have already been used in combination with growth factors on certain

  lesions of a specific type of tissue, the skin tissue.

  Due to the unpredictability of the therapeutic effects of a given molecule, it was not clear that these polymers, alone, and not associated with growth factors, could have an effect on other tissues than

those of the skin.

  Indeed, it is well known that the different tissues of the human or animal body have both structural and functional specificities that make it impossible to predict the effect of the molecule, known for its effect on scarring of the cutaneous tissue, on the skin. tissues of

  various origins subject to inflammation.

  Similarly, it is well known that it is impossible to predict the in vivo activity of a molecule on a particular tissue from results obtained in vitro on a particular tissue.

specific experimental model.

  Surprisingly, it has been found, according to the invention, that certain polymers defined as belonging to the class of HBGFPP and in particular CMDBS have an effect on the intensity of the inflammatory reaction which they decrease very markedly while by accelerating the repair, regeneration and restoration of damaged tissue. Has an increase in speed and quality of healing of lesions of the tissues of the digestive tract, skin tissues, bone tissue, HBGFPP

  combine anti-inflammatory effects.

  It has also been shown that very low doses of

  these polymers make it possible to obtain therapeutic effects.

  The present invention relates to a use of at least one polymer or a biopolymer, referred to as HBGFPP, specifically protecting the growth factors of the

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  FGF and TGF beta families of tryptic degradation and not significantly inhibiting coagulation, for the manufacture of a drug for the treatment of

inflammations of various tissues.

  Such a polymer particularly exhibits anti-coagulant activity of less than 50 international units per mg of polymer measured according to Maillet et al (Mol Immunol, 1988, 915-923). Advantageously, it potentiates FGFs in vitro. Preferably, it does not substantially activate the complement system, that is to say it has an anti-complement activity greater than 0.5 μg for the CH 50

  (according to Mauzac et al., Biomaterials, 6, 61-63, 1985).

  Advantageously, said polymer or biopolymer is a polysaccharide which can be composed mainly of residues

glucose.

  It may also include glucosamine and / or uronic acid residues, particularly in the form of dimer

glucosamine-uronic acid.

  Particularly preferred polysaccharides are substituted dextrans, glycosaminoglycans optionally associated with a lipid, a peptide or a protide or sulphates

of these polymers.

  Such a polymer advantageously has a molecular weight of

  at least 10 kDa and preferably about 40

kDa.

  The present invention further relates to a

  pharmaceutical composition containing these polymers.

  The polymers and / or biopolymers can be selected from natural substances which can then optionally be modified by addition of appropriate chemical groups, or be obtained entirely by synthesis. These natural, semi-synthetic or fully synthetic polymers are then selected on the basis of their ability to interact specifically with several growth factors in particular

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  those of the family of FGF and TGF beta. They can also be selected for their ability to protect this or these factors against proteolytic degradations, and to act as inhibitors of proteinase activities involved in the inflammatory process such as leukocyte elastase,

  and plasmin for example, and for their anti-inflammatory activity.

  coagulating. These polymers are designated under the generic name of HBGFPP (heparin binding growth factor protectors and promoters). Two prototypes of these polymers or bio-polymers are given as examples as well as the processes and

  criteria for selecting these polymers.

  The first example of HBGFPP belongs to the family of CMDBS which are known products, namely functionalized biospecific dextrans substituted with carboxymethyl, benzylamide and benzylamine sulfonate residues. These polymers illustrate the production of HBGFPP from naturally occurring products (dextrans) which are subsequently chemically substituted. The second example describes the selection of completely natural products such as glycosaminoglycans

  sulphates purified from tissue extracts.

  These two examples illustrate the ability of these HBGFPPs to interact, stabilize, protect and potentiate the growth factors of FGF and TGF beta families and their use in a pharmaceutical composition for the treatment of inflammations of

all origins.

  The various examples described in this invention show that in addition to the clean healing effects of CMDBS on different types of regeneration or tissue healing, the intensity of the local inflammatory reaction

is greatly diminished.

  In the present application, treatment is understood to mean any curative or preventive operation carried out for the prophylaxis or resorption of inflammations

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  to intervene in particular proteinase activities such as leukocyte elastase, plasmin, metalloproteinases in addition to the stimulating effects of regeneration and

tissue healing.

  The following examples illustrate a decrease in the intensity of the inflammatory reaction observed in injured tissues treated with an effective fraction of HBGFPP, for example CMDBS combined with one or more compatible and pharmaceutically acceptable vehicles with the type of lesion. The pharmaceutical composition may also be associated with agents such as, for example, antibacterials, antifungals, vitamins or the like, antiseptics, analgesics, sun-protective agents and other anti-inflammatory agents or any other types of compounds. associated for their specific effects and administered in any acceptable pharmaceutical gallenic forms and compatible with the type of

treatment envisaged.

  Advantageously, such a composition is designed to be directly absorbed orally, deposited on the lesion or injected if it is directly accessible particularly in subcutaneous lesions, buccal or rectal or during surgical procedures by depositing or injecting tissues. The unit dose is 10 to 2500 μg CMDBS or HBGFPP per ml or cm 2 of tissue to be treated in a volume adapted to the specificity of the pathology. The dose of CMDBS or HBGFP product used corresponds, depending on the surface of the wound, to a fraction of a starting solution of 10 to 2500 μg per milliliter (which for local application on common wounds involves a removal rarely greater than a few hundred microliters of the solution) this dose being applied once or twice per 24 hours. The sucralfate compositions used are from 0.1 to 5% (according to US Patent No. 5,196,405)

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  less than 10 times higher than those described in the present invention and the effects described in all the examples of this US Pat. No. 5 196405 are obtained from compositions 50

milligrams per milliliter.

  The vehicle may be physiological saline or buffers such as PBS containing 0.15 molar NaCl or any other kind of compatible and non-irritating solution for the damaged tissue. Formulations making it possible to obtain pasty or gel or aerosol solutions according to the standard techniques known to those skilled in the art may be

  proposed depending on the type and accessibility of the lesion.

  The fields of application of these compounds correspond therapeutically or prophylactically to the pathologies associated with the activities of elastase and / or

plasmin.

  The main pathologies associated with an elastase-type activity cover the following main areas: - skin lesions including the lips, the oral cavity, the gum (periodontal diseases), the vaginal mucosa for example and the anal surfaces for inflammatory superficial eruptions , pruritic, erythematous, acne or rosaceae like inflammation of seborrhea or pustular type, boils, abscesses; - bacterial, fungal, viral infections of the skin such as candida or herpes, - acute or chronic dermatitis in response to various agents such as sunburn or superficial burns, inflammatory reactions in response to foreign bodies such as dressings, catheters and hemorrhoids; - perianal vulvitis or pruritus; - the usual pathologies and disorders of the otorhinolaryngological and pulmonary arteries such as sinusitis or

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  allergic rhinitis, acute or chronic, otitis, pulmonary inflammatory reactions, oedemas, emphysema, fibrosis, side reactions & bronchitis, asthma; - ocular pathologies related to disorders of inflammation of allergenic beauty products, conjunctivitis, punctate keratitis, ulcers; - diseases related to immunological or viral disorders such as arthritis and rheumatoid arthritis, synovitis, gout and various forms of arthritic inflammation, lupus erythematosus, anaphylaxis, bacterial septicemia, AIDS; parasitic diseases such as toxoplasmosis, cytomegalovirus, malaria, polyomelitis, etc .; the treatment of inflammatory reactions of organs such as glomerulonephritis, for example for the kidney, or those associated with surgical procedures, which may or may not involve the implantation of materials foreign to the body; - Vascular pathologies with atherosclerotic or aging-related aneurysms

  like that of arteriocléromatous type.

  The main pathologies associated with plasmin-type activities also cover areas of

  tumor progression and induced complications.

  The invention will be illustrated, without being limited in any way by the following examples, in which:

  Figure i shows the formula of CMDBS.

  Figure 2 illustrates the potentiation of the biological activity of FGF1 (2a) and FGF2 (2b) by heparin, mesoglycan and sulodexide. The measurement of the biological activity is carried out on CCL39 cells by measuring the increase of the incorporation of tritiated thymidine into

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  the dose of FGF1 and FGF2 added alone or in the presence of 20 μg of heparin, 10 μg of mesoglycan, or

10 μg of sulodexide.

  Figures 3 and 4 illustrate the protective effect of heparin, mesoglycan and sulodexide against thermal degradation of FGF1 (3) and FGF2 (4). FGF samples are incubated alone or in the presence of 20 μg of heparin, 10 μg of mesoglycan or 10 μg of sulodexide at 20 ° C (a) and 37 ° C (b) for 1, 7, 15, 30 μg. days. The measurement of the biological activity presented on the abscissa corresponds to the values of the stimulation units (ED50) of the incorporation of

  tritiated thymidine in CCL39 cells.

  Figure 5a illustrates the protective effect of heparin, mesoglycan and sulodexide against proteolytic degradation of 125I-FGF1. Proteolytic digestion was performed at 37C and the samples were separated by 18% polyacrylamide gel electrophoresis. The gels are dried and autoradiographed. The first lane contains I-FGF1 alone, in the second lane (lane 2) the 125I-FGF1 is incubated in the presence of trypsin and heparin (lane 3),

  mesoglycan (lane 4) or sulodexide (lane 5).

  Figure 5b illustrates the protective effect of heparin, mesoglycan and sulodexide against proteolytic degradation of 125I-FGF2. The layout of the tracks is

  identical to that presented for 125I-FGF1 in 5a.

  FIGS. 6A and 6B show, respectively, osseous tissues presenting an inflammation treated with a collagen dressing soaked with saline (6A), and

  the same dressing soaked with CMDBS (6B).

  FIGS. 7A to 7C, on the one hand, and 7D to 7F, on the other hand, respectively represent sections of skin wounds treated with collagen soaked in CMDBS and collagen.

  soaked with a physiological solution.

  Figure 8 illustrates the expression of metallo-

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  proteinases at J = 5 (lanes 1 to 11) and J = 6 (lanes 12 to 17) in healthy tissues (lanes 1 and 12), scar tissue treated with CMDBS (lanes 2 to 4 and 13 to 15), and scar tissue treated with physiological solution (lanes 5-11, 16 and 17). EXAMPLE 1 Preparation and Selection of CMDBS a) Preparation of CMDBS The CMDBS are dextrans substituted with carboxymethyl, benzylamide and benzylamide sulfonate groups. The method of synthesis of CMDBS can be that described by M.MAUZAC and J.JOSEFONVICZ in Biomaterials

1984,5,301-304.

  According to this process, the carboxyl methyl dextran (CMD) is prepared from dextran by substitution of some glycosylated units with carboxylic groups on the

carbon in position 5 or 6.

  In a second step, the benzylamide is coupled to the carboxylic groups to form carboxymethyl benzylamide dextran (or CMBD). Finally some aromatic rings of benzylamide are sulfonated to result in carboxymethyl

  dextran benzylamide sulfonate or CMDBS.

  The sodium salts of these derivatives are ultrafiltered, lyophilized and dissolved in the appropriate buffer prior to use. The general formula of CMDBS is represented on the

figure 1.

  CMDBS have a statistical distribution of the different substituents. Percentages for each type of

  CMDBS are determined by conventional methods.

  b) Selection of CMDBS i: Protection and stabilization tests of FGFs During the synthesis of CMDBS it is possible to control the substitution rate of each group by modifying the conditions of the substitution reaction 2718024. The control of parameters such as temperature, reaction time, relative concentrations of

  constituents and the number of substitution reaction etc ...

  allow to obtain a very large number of substituted polymers. The substitution of the carboxymethyl hydroxyls on the carbons at the 5 and 6 positions makes it possible to obtain carboxymethylation levels ranging from 0 to 200% (100% for each of the carbons in position 5 and 6). The carboxymethyl group can in turn be partially or fully used for the binding of benzylamide. The benzylamide groups may be partially or fully used for sulfonation. The functionalized substituted dextrans used according to the invention are among those

  specially described in French Patent No. 2,461,724.

  In addition to the ability to stabilize and protect growth factors of the FGF family as described in Tardieu et al. J. Cell.Physio.1992 150 p 194-203; and in French Patent No. 2,461,724; the selected CMDBS must be able to interact with at least one member of the TGF beta family growth factor family according to an evaluation method described below and protect TGFs

beta against proteolysis.

  ii: Evaluation of the interaction capacities between CMDBS and the

  growth factors of the TGF beta family.

  In order to measure the ability of some CMDBS to interact with members of the TGF beta family and through this interaction to protect TGF beta, a screening test was established. This test consists in measuring the ability of the selected CMDBS to allow the TGF beta to keep its activity

  despite protease treatment.

  In the example below, the CMDBS used is batch 26.2 defined by a substitution rate of 110% of carboxymethyl units, 3.6% of benzylamide units and 36.5% of sulfonate units and has an anticoagulant activity of 4%.

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  IU / mg (International Units). The anti-complement activity of this lot is 1.1 μg of CH 50, measured according to Mauzac et al.

(previously mentioned).

  The heparin used as a control comes from Sanofi institutions (Choay Institute) and has an anticoagulant activity of 175 IU / mg. The TFG beta 1 is prepared from human platelets according to the protocol described in numerous publications and commonly used by patients. those skilled in the art, for example, in Growth Factors and their Receptors 1992, Vol. i PP 419-472 by A. Roberts and M. Sporn edited by A. Roberts and M. Sporn and published by Springer Verlag Berlin. The TGF beta biological activity assay used in this example is that of growth inhibition of CCL64 cells (from the American Tissue Culture Collection). This inhibition is measured by the ability of TGF b to inhibit the incorporation of tritiated Thymidine in a dose-dependent manner in these FGF growth factor-stimulated CCL64 cells or by fetal calf serum according to the protocol described by Van Zolen in Progress in Growth Factor Resarch, 1990.2 p. 131 to 152. TGF beta is used in two doses, one corresponding to the 50% inhibition capacity of tritiated thymidine incorporation (defined as the unit of inhibitory activity).

  the other, corresponding to the inhibition capacity of 100%.

  In this example, the values obtained are 250 μg of TGF beta to obtain the unit of inhibition activity on CCL64 cells cultured in 1 ml of culture medium. The% inhibition is obtained with lng of TGF beta in 1 ml of

culture centre.

  A 50ng sample of TGF beta in phosphate buffered saline containing 0.1% bovine serum albumin (from the company SIGMA in Saint Louis USA) is incubated alone, or combined with either 5000 μg of CMDBS or 5000 μg

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  heparin, with or without 500 μg of trypsin. The final volume of the incubated solution is adjusted to 1 ml and the incubation is carried out at 37VC for a variable time (10 minutes in

the example described in Table 1).

  Samples of a volume of 20 μl of each of the incubation reactions are taken and added to the CCL64 cells grown in 24-well plates each containing one milliliter of culture medium according to the protocol described by E. Zohlen mentioned above. In these conditions

  the final concentration of TGF beta per well is lng / ml.

  Table I summarizes the results obtained under various conditions and shows the protective effect of CMDBS. Thus, after incubation at 37 ° C., 75% of the biological activity of TGF beta is still present, whereas heparin which can nevertheless bind to TGF beta (Mac Caffrey et al., J. of Cell Physiology , 1992, vol.52, 430-440) does not protect TGF beta against this proteolytic degradation (less than 20% of biological activity remains). It should be noted that in the case of FGFs, heparin provides protection against trypsin-induced proteolysis (Tardieu et al., Journal of Cellular

Physiology, 1992, 150: 194-203).

  It has been verified that the CMDBS has no inhibitory power on the activity of trypsin (TableAU2). Thus, 10 μg of trypsin were incubated either with a substrate (S.87 supplied by the company Serbio, Paris and used according to the recommendations of this supplier) or with this substrate and a trypsin inhibitor such as that derived from soybean ( as the Soyabean trypsin inhibitor or STI from Sigma) these incubations being made in the absence or in the presence of varying amounts of CMDBS (lot AM26). The enzymatic activity of trypsin was measured by spectrophotometric absorption of the transformation product of S 87 into

function of the incubation time.

  Example 2: Selection of other HBGFPP:

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  Two commercial preparations of proteoglycosaminogly-

  cane and glycosaminoglycans were selected according to their ability to interact with the growth factors of

  family of FGFs as well as those of the TGF beta family.

  Mesoglycan and sulodexide were provided by Sigma Chemical Co., St. Louis MO USA. Their

  properties are summarized in Table 3.

  The cells used in this example are CCL39 cells that come from the American Tissue Culture Collection. The conditions of culture and tests of biological activity measurement of FGFs are the same as those

  described in the publication Tardieu et al. J.Cell.Physiol.

  1992. The FGF growth factors used are the forms

recombinant FGF1 and FGF 2.

  a) Effect of mesoqlycan and sulodexide on activity

biological FGFs in vitro.

  In these experiments FGF1 or 2 is used at a dose corresponding to the effective dose (denoted ED50) to induce stimulation of the biological activity of 50% of the dose inducing maximum stimulation. The biological activity is measured by the ability to induce an increase in the incorporation of tritiated thymidine into the cells according to the protocols widely described in numerous publications including that of Tardieu et al mentioned

  previously and also in French Patent No. 2,644,066.

  In this example the ED50 is 5 ng / ml for FGF1 and 3 ng / ml for FGF 2, measured values experimentally (Figs.2a and 2b). The same FGF dose stimulation experiment is performed in the presence of 10 μg / ml Mesoglycanee or Sulodexide or 20 μg / ml Heparin. FIG. 2 shows that under these conditions the ED50 becomes 0.4 ng / ml and 0.2 ng / ml respectively for FGF1 and FGF2 in the presence of these doses of mesoglycan or heparin. In addition to this ability to potentiate the biological activity of FGFs

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  HBGFPPs protect FGFs against thermal damage as well as inactivation induced by the proteolytic action of trypsin (Figs.4 and 5). In the same way, these HBGFPPs protect FGF1 and 2 against inactivation induced by the proteolytic activity of trypsin (Figs.5a

and 5b).

  b) Protective effects of mesoqlycan, sulodexine, dextran, dextran sulfate and sucrase against TGFbeta. Several other compounds have been evaluated: dextran sulfate (Sigma Chemical, molecular weight 40,000, dextran used for the synthesis of CMDBS (also from Sigma) of sucrase or sucrose octasulfate (supplied by D. Bar Shalom, BUKH Company). MEDIC, Denmark) Some of these compounds have been chosen because they protect and stabilize FGFs such as sucrase confers on U.S. Patent No. 5202311 or Dextran Sulphate Sulfurates in Japanese Patent No. 138907/88). Dextran is the one used for the synthesis of the

CMDBS AM26.

  The experiment for protecting the biological activity of TGFbeta was performed in the same way as with the CMDBS as described in Example 1 ii. The incubation mixture contains 50 ng TGF beta (in 0.1% bovine serum albumin) and trypsin (500 μg). Mesoglycanee or Sulodexide or dextran sulfate or dextran or sucrase

  are used at the dose of 5000 pg.

  The biological activity of TGFbeta is measured as described above after a 50-fold dilution and using

CCL64 cells.

  The results are shown in Table 4.

  These results illustrate that with the exception of some

  CMDBS capable of meeting the two selection criteria

  For FGF and TGFbeta alone among the other compounds tested the mesoglycan exhibits significant protective activity

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for TGFbeta.

  EXAMPLE 3 In Vitro Inhibitory Effects of CMDBS on

  activity of leukocyte elastase and on plasmin.

  The inhibition powers of different CMDBS and their intermediate compounds from their synthesis, have been

  established for leukocyte elastase and plasmin.

  Purified leukocyte elastase was obtained from Elastin Products Co (Owenville, Mo., USA) and plasmin from

SIGMA.

  The inhibition of enzymatic activities by these different compounds is carried out at 37 ° C. in a thermostated bath. The enzymes in question are dissolved in 100 mM Tris-HCl buffer, pH8 for elastase and pH 7.4 for plasmin, in the presence of 0.02% sodium azide and 0.01% Triton X100. for plasmin. Concentrations of

  substrates and those of enzymes are: 0.10 mM MeO-Suc-Ala

  Ala-Pro-Val-pNA (paranitroanilide) for elastase at 8.3 nM and 0.20 mM dVal-Leu-dLys-pNA for plasmin at 77 nM. For

  each of the conditions is established IC50.

  Table 5 gives the results obtained in which the AM6 batch corresponds to a 40,000 kD dextran T40. Lot EM5 corresponds to a 10,000 kD dextran T10. Intermediate synthetic products are listed according to the above abbreviations indexed by a number which specifies the number

  of each of the substitution reactions.

  The IC50 values demonstrate that CMDBS have non-competitive hyperbolic-like inhibitory effects on leukocyte elastase activity comparable to those of heparin, one of the best inhibitors of this activity (Ki of the order of 1 nM). In addition, and unlike heparin, CMDBS exert inhibitory effects on plasmin. EXAMPLE 4 Anti-Inflammatory Effect of CMDBS in Bone Tissue

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  In order to study the anti-inflammatory effect of CMDBS in bone tissue, a model of regeneration of calvaria bone in rabbits was used. After making a cut of this bone in its half thickness, the inflammatory reaction in the granulation tissue in the presence or in

absence of CMDBS.

  White adult rabbits of the New Zealand breed having finished their bone growth and weighing 3 & 4 kg are divided into 2 groups of 8 rabbits of both sexes. These rabbits were anesthetized by intraperitoneal injection of ketamine hydrochloride (100O mg / kg) and acepromazine (100O mg / kg). A paramedian frontoparietal incision was made to access the calvaria. Four bone defects of 5 mm in diameter and 3 mm in depth were made using a bur. During the operation, the operated side was constantly irrigated to avoid thermal accidents. The two cephalic regions have not been filled and therefore serve as control while the left and right posterior sides are filled with collagen (hemostatic collagen microfilar (Pangen) from laboratories Fournier Dijon, France) or impregnated collagen overnight in a solution of CMDBS at 100 μg / ml. Appropriate haemostasis was performed prior to suturing the wound. The wounds are closed with nylon suture and the operated side is covered with a sterile dressing. The animals were

  convalescence and no post-treatment complications

  operative procedure was observed. The animals were euthanized on days 3, 4, 8 and during the 12 weeks following the intervention. All animals received identical treatment and served for their own control. After macroscopic examination of the site of the operation, the skulls are decalcified and embedded in paraffin. The operated side was studied under a microscope to be cultured. After the models are decalcified and placed in paraffin,

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  Sagittal sections (5 mm thick) of the zones corresponding to the defects thus created were cut with a microtome and stained with hematoxylin and eosin. These sections are studied under the microscope and the defects were 100 times magnified, using a 2.5 x 2.5 mm grid standardized by 10 divisions of 0.25 mm on each side. These measurements were then reported on a graph to compare the results according to the different groups treated. In this work, the CMDBS, lot AM26, whose content in methyl carboxylic acid was 110%, was used.

  benzylamide of 2.5% and sulfonated benzylamide of 36.5%.

  The specific anti-coagulant activity of this CMDBS is 4 IU / mg compared to standard heparin (sample no. 108, Sanofi-Choay Recherches, Gentilly, France) which has an activity

anticoagulant of 173 IU / mg.

  FIGS. 6A and 6B show the difference of the inflammatory reaction observed after 3 weeks in the granulation tissue corresponding to the filling of the bone defect

performed experimentally.

  In FIG. 6A, which corresponds to the collagen dressing soaked only with physiological saline, the inflammatory reaction is very strong; the granulation tissue contains a large number of inflammatory cells and few

osteoblastic cells.

  On the other hand, the same treatment with a collagen impregnated with the CMDBS solution made this inflammatory reaction regress very clearly, favoring the repair of the defect.

  bone and thus demonstrating the anti-inflammatory effect of CMDBS.

  EXAMPLE 5: Anti-inflammatory effect of CMDBS on the gastric mucosa. Principle The oral administration of 1 ml of absolute ethanol in the rat causes within one hour the appearance of

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  hemorrhagic lesions of the gastric mucosa.

  Methods Groups of 6 male Sprague Dawley rats, approximately g, were fasted 24 hours before the test. On the day of the test, 1 ml is administered orally and the animals are sacrificed 1 h 30 after. The stomach is removed, opened according to

large curvature and rinsed.

  The involvement of the mucosa is listed according to the following scale. 0: normal mucosa 1: small superficial bleeding pink streaks 2: red, short and wide superficial haemorrhagic streaks 4: red-black, long and wide superficial haemorrhagic striations: perforation Treatment: CMDBS was dissolved in distilled water, PGE2

  in ethanol at 10% and then diluted in saline.

  The treatments are done orally, i hour before

  ethanol and 45 minutes later under a volume of 5 ml / kg.

Expression of results

  For each batch of animals, average values +/-

  e.s.m. alteration scores were calculated. They are presented in the table below. The statistical comparison is made using White's non-parametric test, compared to the control group with a difference

significant noted p S 0.01.

  Results As illustrated by these results, the administration of absolute ethanol induces in the majority of animals the appearance of large confluent hemorrhagic streaks. In control animals the alteration score is 3.75 +/- 0.17.

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  The CMDBS administered orally at a dose of 100 μg / kg (twice daily) reduced the severity of the lesions by 56% (p> 0.01) within 24 hours. At higher doses the cytoprotective activity is not found. CMDBS has comparable effects to prostaglandin E2 as a protective agent of the gastric mucosa against irritating topical agents that induce a local intense inflammatory response (reduction

63% of lesions).

  EXAMPLE 6: Anti-inflammatory effect on induced colitis

by TNB in the rat.

  Principle: Intrarectal administration of trinitrobenzene sulfonic acid (TNB) causes chronic colitis in rats, associated with severe mucosal lesions produced by an acute inflammatory reaction with release of mediators (PAF, interleukins). , eicosanoids), and accompanied by significant quantifiable edema Evans blue. Methods. Groups of 6 male Sprague Dawley rats, approximately g, are placed in young water 24 hours before the test. On the day of the test, the animals are anesthetized with pentobarbital (30 mg / kg) intraperitoneally. 20 mg of TNB dissolved in 0.25 ml of 50% ethanol are instilled into the colon at 6 cm from the anus. The animals are put back in their cage after

  Wake up with food and drink at will.

  4 days later, the animals are sacrificed and the colon is removed. 30 minutes before sacrifice, I ml of Evans Blue is administered by the venous route to evaluate the edema accompanying the colitis according to the following protocol: The colon is put in a tube containing 9 ml of formamide. After centrifugation, the supernatant is removed and assayed

  spectrometric is performed at 620nm wavelength.

Treatment:

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  CMDBS was dissolved in 0.5% carboxymethylcellulose. The treatments were carried out intrarectally in a volume of 1 ml / kg, 4 hours before TNB once per week.

  day until the sacrifice of the animal.

Results

  For each batch of animals, average values +/-

  e.s.m. alteration scores were calculated. They are presented in Table 7. The statistical comparison is carried out using the "t" variance test with respect to the control group with a significant difference noted p S 0.05.

  In this example, the CMDBS has an anti-

  Inflammatory, as shown by enumeration of inflammatory cells revealed by Evans blue staining. 300 mg of CMDBS per kg halves this coloration (p S

0.05).

  EXAMPLE 7 Effect on cutaneous inflammation in the rat

  A. Animals The experiments were carried out on Hairless male OFA-hr / hr Ico, EOPS rats, free of specific pathogenic organisms, aged.ia.EOPS (free of specific pathogenic organisms); from 9 to 10 weeks (250-300 gr) (IFFA CREDO, France). These rats are characterized by a very underdeveloped hair system due to a recessive gene hr, defined by the term "Hairless". Upon receipt, the rats are housed and housed according to the guidelines of the Economic Community

  European, decree of April 1986, n 86/609 / CEE.

B. Operating procedure

  Rats are anesthetized by intravenous injection

  peritoneal sodium pentobarbital (0.1 ml / 100 g for healthy rats and 0.05 ml / 100 g for diabetic rats). Three dermoepidermal skin defects are made on both sides of the spine using a 6 mm diameter punch. Cattle collagen compresses

* 22 2718024

  inactivated (PANGEN, Fournier) are die-cut, soaked with the test solution or with a solution of PBS.ia.PBS; for 2 hours at room temperature before being deposited in each wound. The whole is then covered with an occlusive dressing (OPSITE) keeping the collagen hydrated, then an elastoplast preventing the rat to get rid of his dressing. The rats are placed in individual cages with water and food at will. Each

  Experimental condition groups batches of 6 to 10 animals.

  The scar process is considered at varying times

  expressed in days (Jx) after the date of the transaction (OJ).

  C. Treatment of Diseases Wounds of healthy rats are treated with collagen dressings soaked with physiological solution

  whether or not containing CMDBS-AM6 at 50 μg / ml.

  D. Dana Samples Batches of control animals (without CMDBS) or treated (with CMDBS) are sacrificed by injection of an excess of pentobarbital sodium. The contours of the wound are plotted on a layer, then the wound is taken using the same punch of the same size as that used at OJ and kept at -80'C. For the histological study, the pieces are removed by providing an excess of 5 mm of uninjured skin around the original contours and then placed in a fixator

  whose composition varies according to the study envisaged.

  E. Histological study The tissue samples are placed for 24 to 48 hours in an excess of fixative (4% paraformaldehyde, 0.1% glutaraldehyde, 5% sucrose, 100mM PBS, pH7) and then gradually dehydrated before being included in Paraplast Plus (Prolabo). 5 mm sections are made with a paraffin microtome (Reichert-Yung) and stained with

trichrome Masson (GABE, 1988).

  The results of the histological observations are presented in FIGS. 7A to 7F, which report this histological study of cutaneous cicatrization on day 6 postoperatively,

  (coloring of the Trichrome de Masson cups).

  Figures 7A-7C show wounds treated with CMDBS (50mg / ml) and Figures 7D-7F represent wounds treated with the vehicle alone. These figures show an inequality in the quality of the granulation tissue. There is a more mature appearance of the scar (Fig.A, x4) compared to the control (Fig.D, x4). An enlargement (x25) of the epidermal region shows a reconstitution of it in

both cases (Fig.7B and 7E x25).

  The difference lies in the maturation of the granulation tissue: the wound treated (Fig.7Cx25) with CMDBS shows the presence of fibroblasts (arrow) which begins to orient and which produce a larger matrix than in the wound witness. Inflammatory cells in wounds treated with CMDBS are limited in number compared to the control (Fig.7F x25) which shows

  the persistence of important inflammatory phenomena.

  EXAMPLE 8 Demonstration of Matrix Metallodroteinase Activities Extracted from Cutaneous Tissues During Healing. Skin samples are taken according to the same protocol and the same experimental conditions as

  those presented in the previous example.

  Wound treatment The wounds of healthy rats are treated with collagen dressings soaked in a saline solution

  whether or not it contains CMDBS-AM6 at 50 mg / ml.

  Matrix metalloproteinases (MMPs) and, more particularly, 92 kD collagenases (type V collagenase) and 72 kD collagenases (type IV collagenase) are highlighted.

  thanks to the zymogram technique.

  The skin taken at OJ is called control (C). The

24 2718024

  Sampling done on day x (Jx) at the same location according to the original contours is called wound (P). C and P are

  always treated at the same time and according to the same protocol.

  Each sample is sprayed for one minute in a liquid nitrogen grinder (Bioblock). The powder is weighed and mixed in a 100 mM phosphate buffer pH7.4, 1M NaCl (1 ml

  per 100 mg of powder). The whole is homogenized with the Ultra-

  turax for 30 seconds, left 1 hour at 4'C then

  centrifuged at 43700g (Beckman J2-21) at 4 ° C for 30 minutes.

  The volume of the supernatant is measured then aliquoted before being

  kept at -80'C. The pellet, weighed, is stored at -80 ° C.

  The extracts obtained from crushed skin skin or not (25 mg of non-reducing protein, unheated buffer), are deposited on 10% polyacrylamide gels containing 1 mg / ml of gelatin (Sigma, ref G2500). After migration in Laemmli buffer, the proteins

  deposited on gels are renatured with a solution of Tris-

  100 mM HCl, pH 7.4 containing 2.5% triton × 100 (2 times 30 minutes). The Triton is removed by two washings in 100 mM Tris-HCl buffer. The electrophoresis gels are incubated at 37 ° C. for 48 hours in 100 mM Tris-HCl buffer containing 10 mM CaCl 2, 0.001% NaN 3, 0.0015% Brij 35, 1 mM ZnCl 2. After carefully removing the incubation buffer, the gels are stained for 20 minutes with shaking with a solution of Coomasie Blue R250 (5 mg / ml) containing acetic acid (10%), propanol-2 (30%). ), then discolored (10% acetic acid, 40% methanol) until the bands are visible. The gels are photographed. To ensure that the different bands of digestion obtained are due to MMPs, the samples are incubated in the presence of specific inhibitors; Phenyl methyl sulfonyl fluoride or PMSF (2 mM final) inhibitor of serine endopeptidases, ethylene diamine tetracetic acid or EDTA (20 mM final) inhibitor of metalloendopeptidases, N-ethyl maleimide or NEM (2 mM

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  final) Figure 8 shows the expression of Matrix Metalloproteinases detected in the wound extracts as a function of the treatment or not with the CMDBS and as a function of the time of the cicatrization. mg of protein obtained from the extracts of shredded skin or skin scrap on D5 and D6 postoperatively are deposited on a 10% polyacrylamide gel containing as substrate 1 mg / ml of gelatin. Tracks 1.12 represent healthy skin that serves as an internal migration tipple. Lanes 2, 3, 4 and 13, 14, 15 represent extracts obtained from scars treated with CMDBS at 50 mg / ml (6 different rats). Lanes 5 to 11 and 16, 17 represent the corresponding witnesses. Each track

represents a different rat.

  Two types of collagenase are found in healthy skin: The shape of 72 kDa and its activated form the 68 kDa. On the other hand, on days 5 and 6, in wounds treated or not treated with CMDBS, 92 kDa is present in an identical manner. The difference lies in the expression levels of the 72 kDa and its activated form, the 68 kDa. At day 5, in the control wounds, 72 kDa and 68 kDa were very significantly expressed compared to wounds treated with CMDBS. In addition, there is the appearance of low molecular weight species that do not exist in wounds treated with CMDBS. At J6, this difference is confirmed since for the same protein deposition as at J5, the activity of collagenases is such that the tracks 16 and 17 appear in the form of two white traces. This activity does not exist in rats

processed at CMDBS.

  CMDBS thus modulate the expression levels of MMPs either directly or indirectly through their role

  inhibitor on plasmin which is one of their activators.

TABLE 1

  Protective effects of CMDBS and heparin against degradation of TGFbeta by trypsin incubation mixture at 37 C for 10 min inhibitory activity and containing milliliter by thymidine incorporation indication: CMDBS or Heparin (5000 Ig): tritiated in CCL64 (after iTGF (50 ng): Trypsin® (500 μg) dilution of the incubation mixture

50 times.

  Incubation buffer alone O CMDBS (5000 μg) 0 Heparin (5000 μg) 0 Trypsin (1000 μg) O TGF beta (50 μg) 100 8 TGF + CMDBS (AM26 batch) 100 8TGF + Heparin 100 3TGF + Trypsin 5 OTGF + CMIDBS + Trypsin 7.5 ITGF + Heparin + Trypsin IO

TABLE 2

  Noninhibitory effect of CMDBS against trypsin Ilrvpsinc (10ug / ml) + S87 1.00 Trypsin + S87 + 5ug / ml 100

GMDB3S

Tr psinc + S87 + 50, ug / ml 100

(CMI) T-S

ryp.çinc + Sg7 + 500ug / ml 100 (.MrnS

TABLE 3:

  Oriqin, anticoagulant activity and partial composition of Mesoqlycan and Sulodexide (supplier information Sulodexide Mesoglycan Origin duodenum pig aorta Activity 50-70 IU / mg <50 IU / mg anticoagulant Chemical composition Dermatan sulfate 20 - 35% 25 - 60% Chondroitin 2 -7% 3-15% Sulfate Heparan sulfate + +

TABLE 4

  Protection of TGFbeta with Various Polymers 9 TG Fbcta 100% TGF beta + trypsin 0 TGF beta + mcsoglycanc 100 TGF beta + mcsoglycanc + trypsin 50 TGFbeta + sulodcxin 100 TFG bcia + sulodcxinc + trypsinc 20 TGF beta + Dextran 100 TGF beta + Dcxtranc + trypsinc 0 TGF bcta + Dxxtran Sulfate 100 TGF beta + Dcxtranc Sulfate + trypsinc 0 TG F beta + Sucrasc 100 TGF beta + Sucrasc + trypsinc 0

TABLE 5

  Inhibition of Elastase and Plasmin Activities Tested Compounds Leukocyte Elastase Plasmin IC50 in gg / ml IC 50 in g / ml CMDBS lot AM6 2.2 1.5

T40> 100> 100

CMDBS Lot EM5 10 7

T10 CMD2B 50 53

T10 5CMD1B> 100> 100

T103CMD> 100> 100

T10O> 100> 100

Heparin 1.8 Lipo-heparin 0.5

TABLE 6

  Treatment Doses in pg / kg Scores Variations PO Cookies - 3.75 +, / 0.17

PGE2 200 1.4 / 0.35 -63%

1.6 + / _ 0.25 -56%

CMDBS 300 3.2., 0.33-16%

1000 3,1.0,35 -17%

TABLE 7

  Treatments Doses Concentration Numbers of Blue Evans rats in gg / g of colon Controls 5 0.17., 003

TNB 0857.01I

018./ 014_

  9g / kg 6 0.54 +, 0.16 CMDTS 300 g / kg 6_ 0.45 +, 0.12

32 2718024

Claims (13)

  1. Use of at least one polymer or biopolymer, termed HBGFPP, specifically protecting growth factors of FGF and TGFbeta families from tryptic degradation and not significantly inhibiting coagulation, for the manufacture of a   drug for the treatment of inflammations.   2. Use according to claim 1, characterized in that the polymer or biopolymer has an anticoagulant activity less than 50 international units per mg of polymer.   3. Use according to one of claims 1 and 2,   characterized in that said polymer exhibits anticomplement activity.   4. Use according to one of claims 1 & 3,   characterized in that said polymer does not activate   substantially not the complement system.   5. Use according to one of claims 1 to 4,   characterized in that said polymer or biopolymer is a polysaccharide.   6. Use according to claim 5, characterized in that said polysaccharide is mainly composed of glucose residues.   7. Use according to claim 5, characterized in that the polysaccharide comprises glucosamine residues and / or uronic acid.   8. Use according to claim 7, characterized   in that the polysaccharide comprises glucosamine dimers uronic acid.   9. Use according to one of claims 1 to 5,   characterized in that said polysaccharide is a substituted dextran. 10. Use according to claim 9, characterized   in that said polysaccharide is a CMDBS. 33 2718024   11. Use according to one of claims 1 to 5,   characterized in that said polysaccharide is a glycosaminoglycan optionally associated with a lipid, a   peptide or a protide, or a sulfate of one of these compounds.   12. Pharmaceutical composition for the treatment of inflammations containing at least one polymer as defined   in one of claims I to 11 in association with the   less a pharmacologically acceptable excipient.   13. Composition according to claim 12, characterized in that it contains between about 10 and 2500 μg of polymer   or biopolymer / ml of composition.