US20080131472A1 - Sulfated Depolymerized Derivatives of Exopolysaccharides (Eps) from Mesophilic Marine Bacteria, Method for Preparing Same, and Uses Thereof in Tissue Regeneration - Google Patents

Sulfated Depolymerized Derivatives of Exopolysaccharides (Eps) from Mesophilic Marine Bacteria, Method for Preparing Same, and Uses Thereof in Tissue Regeneration Download PDF

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US20080131472A1
US20080131472A1 US11/629,579 US62957905A US2008131472A1 US 20080131472 A1 US20080131472 A1 US 20080131472A1 US 62957905 A US62957905 A US 62957905A US 2008131472 A1 US2008131472 A1 US 2008131472A1
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low
sulfated polysaccharide
polysaccharide derivative
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Karim Senni
Farida Gueniche
Myriam Yousfi
Florence Fioretti
Gaston-Jacques Godeau
Sylvia Colliec-Jouault
Jacqueline Ratiskol
Corinne Sinquin
Gerard Raguenes
Anthony Courtois
Jean Guezennec
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Institut Francais de Recherche pour lExploitation de la Mer (IFREMER)
Universite Paris 5 Rene Descartes
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Universite Paris 5 Rene Descartes
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Definitions

  • the present invention relates to polysaccharide derivatives obtained from native polysaccharides of a specific type, called exopolysaccharides (EPSs) that are excreted by various strains of mesophilic marine bacteria from a deep hydrothermal environment.
  • EPSs exopolysaccharides
  • the present invention relates to low-molecular-weight highly sulfated polysaccharide derivatives that can be obtained by free-radical depolymerization and sulfation of exopolysaccharides.
  • heparin a sulfated polysaccharide
  • the commercially available heparins are currently extracted from porcine intestinal mucosa.
  • pathogenic agents for example, prion.
  • the identification of novel polysaccharides from origins other than animal origins appears to be a particularly promising line of research.
  • a screening of samples derived from the hydrothermal environment of the ocean depths has made it possible to isolate a large variety of mesophilic bacterial strains capable of producing native EPSs at atmospheric pressure and at ambient temperature.
  • European patent EP 975 791 describes the strain Vibrio diabolicus , the native EPS HE 800 and the use thereof as a medicament, including, in particular, as a retroviral, antitumor and antithrombotic agent.
  • the EPS HE 800 of 800 000 g/mol, is not sulfated; it consists of approximately 30% by weight of amino sugar, 32% by weight of acid monosaccharides and 1% by weight of neutral monosaccharides; its protein content is close to 1% by weight.
  • European patent application No. 1 296 695 describes the use of the EPS HE 800 in its native form as a material for facilitating bone healing (Zanchetta et al., Calcif Tissue Int., 2003, 72: 74-79).
  • a second native EPS called GY 785 and produced by the bacterium Alteromonas infernos , has also been identified and described in French patent No. 2 755 142.
  • the native EPS GY 785 consists of a heterogeneous population of polysaccharide chains having an average molar mass of greater than 10 6 g/mol.
  • the native EPS GY 785 is slightly sulfated (amount of sulfate less than 10% by weight); it consists of 57% by weight of neutral monosaccharides (predominantly glucose and galactose) and 42% by weight of acidic monosaccharides (glucuronic acid and galacturonic acid); it does not comprise any amino sugars or acetate, lactate, pyruvate and succinate substituents; its protein content is approximately 4% by weight (Guezennec J., Ind. Microb. Biotech., 2002, 29: 204-208).
  • a third native EPS, called ST 716 and produced by the bacterium Alteromonas macleodii subsp. fijiensis has also been identified and described in European patent EP 1171625 (Rougeaux H. et al., Carbohydr. Res., 1998, 312: 53-59).
  • the native EPS ST 716 is slightly sulfated (its amount of sulfate is approximately 5% by weight); it consists of 40% by weight of neutral monosaccharides and 40% of acidic monosaccharides; its protein content is 2 to 4% by weight.
  • EPS HYD 721 produced by a Pseudoalteromonas (Rougeaux H. et al., Carbohydr. Res., 1999, 315: 273-285 and Raguenes G. et al., 1997), the EPS HYD 657 (Cambon-Bonavita M. et al., J. Applied Microbiol, 2002, 93: 310-315) and the EPS MS 907 (Raguenes G. et al., Curr. Microbiol, 2003, 46: 448-52).
  • a subject of the present invention is sulfated polysaccharide derivatives that come from the treatment of native EPSs excreted by mesophilic bacterial strains from a deep hydrothermal environment and that are of pharmaceutical or cosmetic value or of value in tissue engineering.
  • the sulfated polysaccharide derivatives of native exopolysaccharides (EPSs) excreted by mesophilic marine bacteria from a deep hydrothermal environment according to the invention can be obtained by means of the method comprising the following steps:
  • the native EPS can be used in a liquid form, i.e. as it is excreted by the bacteria into the culture medium.
  • the culture medium is centrifuged and only the supernatant containing the native EPS and free of bacterial debris is conserved.
  • the native EPS can be collected by any suitable technique known to those skilled in the art, in particular by membrane ultrafiltration, and can then optionally be lyophilized as it is or in the form of an addition salt.
  • the step consisting of free-radical depolymerization of the native EPS is preferably carried out by addition of a solution of an oxidizing agent to a reaction mixture comprising the native EPS, preferably in the presence of a metal catalyst.
  • the oxidizing agent is preferably chosen from peroxides, in particular hydrogen peroxide, and peracids, in particular peracetic acid and 3-chloroperbenzoic acid.
  • the addition is preferably carried out continuously and with stirring for a period of between 30 minutes and 10 hours.
  • Reaction mixture is preferably maintained at a pH of between 6 and 8, for example by continuous addition of a basifying agent such as sodium hydroxide, and at a temperature of between approximately 30 and 70° C. throughout the duration of the free-radical depolymerization reaction.
  • the native EPS is present in the reaction mixture at a concentration of between approximately 2 and 10 mg/ml of reaction mixture.
  • the oxidizing agent is a solution of hydrogen peroxide.
  • H 2 O 2 preferably having a concentration of between approximately 0.1% and 0.5% by weight, preferably of the order of 0.1% to 0.2% by weight, which is added at a flow rate of V1/1000 to V1/10 ml/minute, preferably V1/50 and V1/500 ml/minute, very preferably of the order of V1/100 ml/minute, V1 being the volume of the reaction medium containing a marine exopolysaccharide (EPS) to which a solution of hydrogen peroxide is added.
  • EPS marine exopolysaccharide
  • the metal catalysts that can be used during the depolymerization step are preferably chosen from Cu ++ , Fe ++ and Cr +++ ions and the Cr 2 O 7 2 ⁇ anion, as described in particular in patent application EP 0 221 977.
  • the metal catalyst is present in the reaction mixture at a concentration of between approximately 10 ⁇ 3 M and 10 ⁇ 1 M, and preferably at a concentration of between approximately 0.001 and 0.05 M.
  • the free-radical depolymerization process in accordance with the invention and as described above makes it possible to obtain, in a single step, without fractionation by stearic exclusion chromatography, and with a good yield, homogeneous, low-molecular-weight polysaccharide derivatives.
  • low-molecular-weight polysaccharide derivatives is intended to mean derivatives with a molecular weight of less than or equal to 100 000 g/mol, preferably between 5000 and 50 000 g/mol, and more preferably less than or equal to 25 000 g/mol.
  • the process comprises a step consisting of reduction of the polysaccharide derivatives obtained, using a reducing agent, so as to stabilize the chains, the reducing ends of which are very reactive, and in particular so as to avoid chain hydrolysis by the “peeling” reaction.
  • a reducing agent so as to stabilize the chains, the reducing ends of which are very reactive, and in particular so as to avoid chain hydrolysis by the “peeling” reaction.
  • the nature of the reducing agents that can be used to this effect is not essential. It may in particular be sodium borohydride.
  • the metal catalyst used for the depolymerization can be eliminated at the end of the depolymerization reaction, and in the embodiment in which a reduction step is carried out, at the end of the reduction, by ion exchange chromatography, preferably a weak cation exchange resin passivated beforehand, or by treatment with EDTA (ethylenediaminetetraacetic acid).
  • ion exchange chromatography preferably a weak cation exchange resin passivated beforehand, or by treatment with EDTA (ethylenediaminetetraacetic acid).
  • a step consisting of N-deacetylation of the polysaccharide derivatives comprising N-acetylated hexosamines and obtained at the end of the free-radical depolymerization step and/or at the end of the reduction step is carried out prior to the sulfation step.
  • This N-deacetylation step is carried out according to a protocol adapted from Zou et al. (Carbohyd. Res., 1998, 309: 297-301).
  • the N-deacetylation step is carried out by addition, to the reaction mixture comprising the polysaccharide derivatives, of a solution of sodium borohydride, with stirring.
  • a basifying agent preferably sodium hydroxide
  • the mechanism of basic hydrolysis of an amide in a basic medium, and preferably in the presence of sodium hydroxide, is shown schematically below:
  • reaction medium After reaction for one hour, the reaction medium is neutralized by continuous addition of acetic acid until a pH of 5 is obtained.
  • the polysaccharide derivatives obtained can be recovered by membrane ultrafiltration and then can optionally be lyophilized.
  • the N-deacetylation step is carried out on the polysaccharide derivatives originating from the depolymerization of native EPSs excreted by hydrothermal mesophilic marine bacteria of the Vibrio genus, preferably HE 800.
  • the native EPSs excreted by said bacteria of the Vibrio genus are characterized in that they contain N-acetylated hexosamines.
  • polysaccharide derivatives resulting from the depolymerization and/or from the reduction and/or from the N-deacetylation can, if necessary, be recovered by any suitable technique well known to those skilled in the art, such as, for example, by membrane ultrafiltration, and then can optionally be lyophilized as they are or in the form of an addition salt with a weak or strong base, that may, for example, be chosen from pyridine, triethylamine, tributylamine, tetrabutylammonium hydroxide and sodium hydroxide.
  • This lyophilized salt may, for example, be prepared by elution of an aqueous solution of the polysaccharide derivatives at a concentration of between 1 and 8 mg/ml on an ion exchange resin column such as, for example, those sold under the name Dowex® by the company Dow Chemical.
  • the eluate is collected as long as the pH remains acid, for example less than 5, then the pH is subsequently adjusted to approximately 6.5 with the desired base as defined above.
  • the polysaccharide derivatives in the form of a salt are then ultrafiltered and lyophilized.
  • the lyophilized polysaccharide derivatives are preferably dissolved in an anhydrous solvent at the beginning of the sulfation step; this solvent is preferably chosen from dimethylformamide (DMF), dimethyl sulfoxide (DMSO) and/or formamide.
  • the amount of polysaccharide derivatives present in the anhydrous solvent may be between approximately 1 and 10 mg/ml, preferably between approximately 1 and 5 mg/ml, and even more preferentially this amount is approximately 2.5 mg/ml.
  • the dissolution of the EPS in the anhydrous solvent is preferably carried out, with stirring, at ambient temperature for approximately 1 to 2 hours and then at a temperature of between 40 and 50° C., preferably at a temperature of approximately 45° C. for approximately 2 hours under argon with molecular sieve.
  • the chemical sulfation agent(s) used during the sulfation step can be added to the depolymerized and/or reduced and/or N-deacetylated EPSs that are in lyophilized form or in the form of a solution.
  • the sulfation agents are preferably chosen from complexes of pyridine sulfate (free or coupled to a polymer), of dimethylformamide sulfate, triethylamine sulfate and of trimethylamine sulfate.
  • the chemical sulfation agent(s) is (are) added to the solution of polysaccharide derivatives in a weight amount preferably representing from approximately 4 to 6 times, and even more preferably approximately 5 times, the mass of polysaccharide derivatives in solution.
  • the chemical sulfation reaction is then preferably carried out with stirring for a period of between approximately 2 and 24 hours depending on the desired degree of sulfation. When the desired degree of sulfation is reached, the sulfation reaction is stopped after cooling of the reaction medium:
  • the solution of sulfated polysaccharide derivatives is preferably dialyzed in order to remove the various salts, and then lyophilized.
  • sulfated polysaccharide derivatives is intended to mean polysaccharide derivatives that have been subjected to a chemical sulfation treatment and comprise sulfate groups, irrespective of whether or not they have sulfate groups before this sulfation treatment.
  • the low-molecular-weight sulfated polysaccharide derivatives in accordance with the invention have a molecular weight of less than or equal to 100 000 g/mol, preferably of between 5000 and 50 000 g/mol, a polydispersity index of less than 5, preferably of between 1.5 and 4, and a degree of sulfate group substitution of between 10% and 45% by weight, and preferably of between 20% and 40% by weight, inclusive.
  • the low-molecular-weight sulfated polysaccharide derivatives in accordance with the invention have a molecular weight of less than or equal to 25 000 g/mol, a polydispersity index of less than 2, and a degree of sulfate-group substitution of between 10% and 45% by weight, and preferably of between 20% and 40% by weight, inclusive.
  • the low-molecular-weight sulfated polysaccharide derivatives in accordance with the invention are obtained by treatment of native EPSs excreted by mesophilic marine bacteria of hydrothermal origin preferably belonging to the Alteromonas or Vibrio genus.
  • the bacteria of the Alteromonas genus are selected from the strains GY 785, HYD 657, HYD 721, HYD 1545, HYD 1644, ST 716 and MS 907.
  • the invention relates to low-molecular-weight sulfated polysaccharide derivatives obtained from native EPSs excreted by bacteria of the Alteromonas genus, said native EPSs having a neutral monosaccharide content of from 20% to 70%, preferably from 30% to 60%, and more preferably from 38% to 57% by weight.
  • the invention also relates to low-molecular-weight sulfated polysaccharide derivatives obtained from native EPSs excreted by bacteria of the Alteromonas genus, said native EPSs having an acidic monosaccharide content of from 5% to 60%, preferably of between 6% and 50%, and more preferably of between 8% and 42% by weight.
  • the invention also relates to low-molecular-weight sulfated polysaccharide derivatives obtained from native EPSs excreted by bacteria of the Alteromonas genus, said native EPSs having an amino sugar content of from 0% to 1% by weight in their side composition.
  • the low-molecular-weight sulfated polysaccharide derivatives of the invention are obtained from native EPSs excreted by bacteria of the Alteromonas genus, said native EPSs having an side composition comprising:
  • the low-molecular-weight sulfated polysaccharide derivatives of the invention are obtained from native EPSs excreted by bacteria of the Vibrio genus, preferably by the bacterial strain HE 800.
  • the native EPSs excreted by bacteria of the Vibrio genus are not sulfated.
  • the invention relates to low-molecular-weight sulfated polysaccharide derivatives obtained from native EPSs excreted by bacteria of the Vibrio genus, said native EPSs having a neutral monosaccharide content of from 0% to 5%, preferably from 0% to 1% by weight.
  • the invention relates to low-molecular-weight sulfated polysaccharide derivatives obtained from native EPSs excreted by bacteria of the Vibrio genus, said native EPSs having an acidic monosaccharide content of from 20% to 50%, preferably from 25% to 40%, and more preferably from 30% to 32% by weight.
  • the invention relates to low-molecular-weight sulfated polysaccharide derivatives obtained from native EPSs excreted by bacteria of the Vibrio genus, said native EPSs having an amino sugar content of from 20% to 50%, preferably from 25% to 40%, and more preferably from 30% to 35% by weight.
  • the invention relates to low-molecular-weight sulfated polysaccharide derivatives obtained from native EPSs excreted by bacteria of the Vibrio genus, said native EPSs having an N-acetylated group content of from 0% to 15%, preferably from 4% to 8%, and more preferably from 5% to 6% by weight.
  • the low-molecular-weight sulfated polysaccharide derivatives in accordance with the invention are characterized in that they are obtained from native EPSs excreted by bacteria of the Vibrio genus, said native EPSs having an side composition comprising:
  • the invention relates to low-molecular-weight sulfated polysaccharide derivatives obtained from native EPSs that have a protein content of from 0% to 15%, preferably from 0% to 5%, and more preferably from 0% to 1% by weight.
  • the low-molecular-weight sulfated polysaccharide derivatives in accordance with the invention can be used as wound-healing agents for connective tissues, and are in particular capable of stimulating fibroblast proliferation, of inhibiting the secretion of pro-inflammatory cytokines or soluble mediators by fibroblasts, of inhibiting the secretion of matrix metalloproteases by fibroblasts, of inhibiting the conventional complement pathway, of stimulating the proliferation of fibroblasts to the detriment of myofibroblasts, and of selectively stimulating the proliferation of medullary cells intended to become mesenchymal cells to the detriment of other subpopulations in a heterogeneous population of cells.
  • Fibroblasts have a central role in the process of wound healing of a damaged tissue with a view to the formation of a replacement tissue so as to restore the functionality thereof.
  • the wound-healing process takes place in 3 phases during which the fibroblasts cause the regeneration processes to progress according to chronological sequences that are precise but interlinked with one another:
  • the first inflammatory and vascular wound-healing phase is characterized by the release of a large number of growth factors, of cytokines and of proteases and the migration of inflammatory cells, fibroblasts and vascular cells at the level of lesion.
  • the influx of inflammatory cells and the production of cytokines induce the production of hydrolases such as serine proteases or matrix metalloproteases by the fibroblasts.
  • hydrolases such as serine proteases or matrix metalloproteases
  • the tissue reconstruction phase (proliferative phase or granulation tissue) is reflected by the loss of tissue substances that has occurred when there is a lesion being made up with an extracellular matrix that is relatively unorganized and richly vascularized.
  • the fibroblasts proliferate rapidly under the effect of growth factors. These fibroblasts perform a remarkable job of reconst ruction by secreting extracellular matrix components such as glycosaminoglycans (GAGs), fibronectin and collagen. Some of these fibroblasts acquires a myofibroblastic phenotype, expressing in particular smooth muscle ⁇ -actin.
  • the cicatricial tissue retracts by virtue of the contractile capacities of the myofibroblasts.
  • Tissue remodeling is a dynamic balance between the synthesis of the extracellular matrix and the degradation thereof. When this balance is in equilibrium, wound healing is normal. However, when the balance leans for a lengthy period toward extracellular matrix synthesis, the development of a fibrosis is witnessed. When the balance leans toward excessive degradation of the extracellular matrix, an inflammatory pathology takes hold.
  • the present invention relates to the use of the low-molecular-weight sulfated polysaccharide derivatives in accordance with the invention, as wound-healing agents for connective tissues, in particular dermal and gingival connective tissues.
  • the low-molecular-weight sulfated polysaccharide derivatives of the invention have fibroblast proliferation-activating properties, it is therefore particularly advantageous to use them as an agent capable of stimulating fibroblast proliferation, or as a regenerating agent for the preparation of a pharmaceutical composition with wound-healing activity, said composition making it possible in particular to promote the reconstruction and the remodeling of connective tissues, in particular of dermal and gingival connective tissues.
  • low-molecular-weight sulfated polysaccharide derivatives in accordance with the invention can be used as agents capable of inhibiting the secretion of pro-inflammatory cytokines or soluble mediators by connective tissue fibroblasts, including in particular interleukin 1 ⁇ (IL-1 ⁇ ) and/or TNF- ⁇ (tumor necrosis factor).
  • IL-1 ⁇ interleukin 1 ⁇
  • TNF- ⁇ tumor necrosis factor
  • Certain chronic inflammatory pathologies such as periodontitis, chronic ulcers, delayed wound healing or rheumatoid arthritis, are accompanied by an excessive and uncontrolled degradation of matrix macromolecules. These pathologies are very often associated with the deleterious secretion of cytokines, in particular of pro-inflammatory cytokines, and with persistent activation of complement, resulting, inter alia, in the prejudicial overproduction of chemoattractant anaphylatoxins. In these inflammatory pathologies, the excessive production of pro-inflammatory cytokines disturbs the physiological cellular and tissue functions, including in particular cell migration, cell proliferation, and the expression, set n and activation of certain proteases such matrix metalloproteases (MMPs).
  • MMPs matrix metalloproteases
  • Mmps involved in matrix protein degradation is not generally constitutive and is induced by pro-inflammatory cytokines such as IL-1 ⁇ and/or TNF- ⁇ and/or growth factor weight sulfated polyse derivatives with the invention can be used as e of inhibiting ne secretior from metal as active tissue gival connectiv issue.C.
  • tives according to the invention are particularly effective for inhibiting secretion of gelatinase A (MMP-2)
  • MMP-2 gelatinase A
  • the inhibition of tgrated on are as IL-15.
  • Complement is a component of innate immunity (spontaneous activation in response to an attack) which is defined as a complex set of soluble or membrane proteins. When there is an inflammatory response, these proteins become activated in a series of proteolytic chain reactions that generates peptides with biological activities. Complement activation, which is rapid and localized, is subject to various particularly effective mechanisms of control. However, some of these control mechanisms are disturbed in inflammatory pathologies, such as autoimmune pathologies, leading to persistent complement activation. Surprisingly and unexpectedly, the inventors have demonstrated the use of the low-molecular-weight sulfated polysaccharide derivatives of the invention as agents capable of inhibiting the conventional complement pathway.
  • the low-molecular-weight sulfated polysaccharide derivatives of the invention are used as anti-inflammatory agents for the preparation of a pharmaceutical composition for treating inflammatory pathologies in which complement is activated, in particular autoimmune pathologies, for instance pemphigus.
  • the low-molecular-weight sulfated polysaccharide derivatives of the invention as anti-inflammatory agents for the preparation of a pharmaceutical composition for in particular treating inflammatory pathologies of connective tissues, in particular of dermal and gingival connective tissues, for instance periodontitis, chronic ulcerations or delayed wound healing.
  • the low-molecular-weight sulfated polysaccharide derivatives of the invention are used as anti-inflammatory agents for the preparation of a pharmaceutical composition for treating inflammatory pathologies affecting dermal and gingival connective tissues, in particular autoimmune, infectious or neoplastic pathologies, for instance sarcomas.
  • pathological fibroses appears to follow a route similar to that followed during tissue regeneration.
  • the normal control of the cellular functions that occur during tissue regeneration processes is disturbed.
  • imbalances in the cellular component can be reflected, inter alia, by an uncontrolled influx of inflammatory cells that maintain the tissue degradation and/or by the persistence of cell subpopulations such as myofibroblasts, leading to fibrotic pathologies.
  • myofibroblasts appear transiently during normal wound-healing processes, this cell subpopulation persists when tissue regeneration becomes pathological.
  • the sulfated polysaccharide derivatives in accordance with the invention can be used as agents capable of stimulating the proliferation of fibroblasts to the detriment of myofibroblasts in connective tissues, in particular dermal and gingival connective tissues. More particularly, the sulfated polysaccharide derivatives in accordance with the invention can be used as agents capable of stimulating the proliferation of fibroblasts to the detriment of myofibroblasts in two-dimensional cell cultures or in reconstructed dermal and gingival connective tissues.
  • sulfated polysaccharide derivatives in accordance with the invention makes it possible to stimulate the proliferation of fibroblasts responsible for tissue homeostasis while at the same time controlling the persistence of myofibroblasts, two cellular events originating from the stimulation of the nonpathological process of tissue regeneration.
  • the fibroblast subpopulation Given their property of selecting the fibroblast subpopulation to the detriment of the myofibroblast subpopulation, it is therefore particularly advantageous to use the low-molecular-weight sulfated polysaccharide derivatives as defined above, as anti-fibrotic agents for the preparation of a pharmaceutical composition, said composition making it possible, in particular, to prevent or treat hypertrophic wound-healing processes or fibrotic pathologies of connective tissues, in particular dermal and gingival connective tissues.
  • the medullary cells intended to become mesenchymal cells are adult stem cells capable of developing into differentiated mesenchymal cells depending on the tissue in which they find themselves, in particular into fibroblasts, chondrocytes, osteoblasts, adipocytes and muscle cells having specialized morphological characteristics and functions.
  • the inventors have demonstrated that the low-molecular-weight sulfated polysaccharide derivatives in accordance with the invention are capable of selectively stimulating the proliferation of medullary cells intended to become mesenchymal cells to the detriment of other subpopulations in a heterogeneous population of cells.
  • This selective proliferative effect is all the more advantageous since medullary cells intended to become mesenchymal cells are rare since they are particularly difficult to obtain and to maintain in culture. It is therefore advantageous to complement cell culture media intended for the obtaining and maintaining of medullary cells intended to become mesenchymal cells, in the context of tissue or cell therapy.
  • the sulfated polysaccharide derivatives in accordance with the invention therefore allow a selection-amplification by proliferation of medullary cells intended to become mesenchymal cells.
  • medullary cells intended to become mesenchymal cells represent a source of pluripotent cells that can be used in tissue therapy for transplant purposes in humans, insofar as they are capable of differentiating into fibroblasts, chondrocytes, osteoblasts, adipocytes and muscle cells depending on the tissue in which they are implanted.
  • Each of the pharmaceutical compositions or medicaments containing the low-molecular-weight sulfated polysaccharide derivatives obtained in accordance with the invention can also be used in combination with one or more growth factors present in the pharmaceutical composition or present in a different pharmaceutical composition that will then be administered separately, i.e. before, simultaneously, or after the administration of the pharmaceutical composition containing the sulfated polysaccharide derivatives.
  • growth factors are in particular chosen from FGFs (fibroblast growth factors), TGF ⁇ s, BMPs (bone morphogenic proteins) and CTGF (connective tissue growth factor).
  • the low-molecular-weight sulfated polysaccharide derivatives as defined above can be used for the preparation of a pharmaceutical composition with wound-healing and/or antifibrotic and/or anti-inflammatory activity.
  • the pharmaceutical compositions or medicaments of the invention are intended to be administered via the appropriate route.
  • the pharmaceutical composition or the medicament of the invention is preferably in an injectable form, in which the sulfated polysaccharide derivatives have a molecular weight of between 5000 and 50 000 g/mol, preferably less than or equal to 25 000 g/mol, a polydispersity index of between 1.5 and 5, preferably less than or equal to 2, and a degree of sulfate group substitution of between 10% and 45%, and preferably between 20% and 40%, inclusive.
  • the pharmaceutical, cosmetic or dermatological compositions can be administered locally and can be in the form of a gel, a cream, an ointment, an emulsion or a solution.
  • They can also be used in situ by means of substrates, of medical devices that are resorbable or nonresorbable, such as, for example, delayed-release supports, slowly disintegrating sponges, or surgical implants.
  • FIG. 1 is an infrared spectrum of the polysaccharide derivatives HE 800 that have undergone a free-radical depolymerization before (HE 800 DR) and after the N-deacetylation reaction (HE 800 DRN);
  • FIG. 2 represents the infrared spectra of the HE 800 derivative that has undergone a free-radical depolymerization (derivative HE 800 DR) or of the N-deacetylated and sulfated HE 800 derivative (HE 800 DRNS);
  • FIG. 4 is a set of 6 photographs a, b, c, d, e and f. This figure relates to an immunodetection test, in a dermal fibroblast culture, of ⁇ -actin filaments that are characteristic of the myofibroblast subpopulation;
  • FIG. 5 is a graph which reflects a result obtained by zymography, and shows the effect of HE 800 DRNS on the secretion of MMP-2 by fibroblasts in culture: FIG. 5 shows that the secretion of MMP-2 is inhibited;
  • FIG. 6 is a set of two photographs, a and b, which show the effect of a derivative in accordance with the invention on the secretion of a matrix protease, stromelysin (MMP-3).
  • MMP-3 matrix protease
  • the protein content was determined according to the BCA (bicinchoninic acid) method described by Wiehelman K. et al. ( Anal. Biochem. 1988, 175: 231-237).
  • the neutral monosaccharide content was determined by the Tillmans and Philippi method ( Analyt. Chem., 1929, 28: 350) modified by Rimington ( Biochem. J., 1931, 25: 1062-1071).
  • the uronic acid (GlcA) content was established using a modification of the m-hydroxydiphenyl-H 2 SO 4 method (Filisetti-Cozzi and Carpitta, Anal. Biochem., 1991, 197: 157-162) and using glucuronic acid as standard. Interference from neutral hexoses was avoided by using potassium sulfamate and carrying out controls comprising all the reagents with the exception of the m-hydroxydiphenyl.
  • the neutral monosaccharide and acidic monosaccharide contents were determined by gas chromatography.
  • the analysis of the glycoside residues in the form of trimethylsilylated derivatives was carried out according to the method of Kamerling et al. ( Biochem. J., 1975, 151: 491-495) and modified by Montreuil et al. (Glycoproteins In: Carbohydrate analysis, a practical approach, 1986, Chaplin M. F. and Kennedy J. F. (eds), IRL Press, Oxford, 143-204).
  • hexosamine and N-acetylhexosamine (GalNAc and GlcNAC) content is determined by the method of Belcher et al. ( Analyst, 1954, 79: 201-208) adapted from that of Elson and Morgan ( Biochem J., 1933, 27: 1824-1828) and using N-acetylglucosamine and glucosamine as standards.
  • the amount of free sulfates is quantified by ion exchange chromatography on a Dionex® DX-500 system connected to a conductimeter, and according to the method described by the manufacturer Dionex. The result obtained makes it possible to calculate the amount of sulfates really bound to the EPS derivative, which is equal to the amount of total sulfates (obtained by elemental analysis) minus the amount of free sulfates (obtained by ion exchange chromatography).
  • HE 800 DR corresponds to the low-molecular-weight polysaccharide derivative
  • HE 800 DRS corresponds to the low-molecular-weight sulfated polysaccharide derivative
  • HE 800 DRNS corresponds to the low-molecular-weight N-deacetylated and sulfated polysaccharide derivative.
  • the low-molecular-weight sulfated polysaccharide derivatives in accordance with the invention are obtained: (i) for the DRS series, by applying a first step consisting of free-radical depolymerization (DR) and a sulfation step (S), and (ii) for the DRNS series, by applying a first step consisting of free-radical depolymerization (DR) followed by an N-deacetylation step (N) and a sulfation step (S).
  • Hydrogen peroxide (H 2 O 2 ) is then added to the reactor at a flow rate of 1 ml/min using a peristaltic pump.
  • the hydrogen peroxide is prepared extemporaneously from a concentrated solution.
  • the solution containing the polysaccharide derivative is filtered through a büchner funnel equipped with filters made of 3 ⁇ m glass micro-fibers.
  • the residual copper is then eliminated by passing the solution containing the polysaccharide derivative over a ChelexTM resin, with a capacity of 0.4 meq/ml.
  • the solution containing the polysaccharide derivative percolates, at a flow rate of 4 to 5 ml/min, through a column (25 ⁇ 400 mm) of 200 ml of resin passivated beforehand.
  • the solution containing the polysaccharide derivative has a basic pH of 10.
  • the solution containing the polysaccharide derivative is ultrafiltered through a Pellicon 2 ultrafiltration system (Millipore) equipped with a Pall membrane of 1000 g/mol.
  • the conductivity of the solution (4 to 5 mS) is measured throughout the diafiltration.
  • the solution is brought back to a neutral pH. It is then concentrated then lyophilized (CIRP lyophilizer). Once lyophilized, the polysaccharide derivative obtained is characterized.
  • the polysaccharide HE 800 DR is N-deacetylated.
  • the process for N-deacetylation of the HE 800 derivative is carried out on large amounts of product.
  • EPS HE 800 DR 259 mg of EPS HE 800 DR are solubilized in 10 ml of water and placed in a round-bottomed flask with magnetic stirring. 263 mg of NaBH 4 are solubilized in 1.25 ml of water and then added to the solution of EPS HE 800 DR. When the temperature of the mixture reaches 80° C., 1.25 ml of 10 N NaOH are added. The final concentration of the solution is then 1 N with respect to sodium hydroxide and 2% with respect to NaBH 4 , for a total volume of 12.5 ml.
  • EPS EPS are solubilized in 20 ml of H 2 O.
  • the polysaccharide derivative is placed in the H+ form by elution on a Dowex resin column.
  • the elution is carried out with water, and the eluate is collected as long as the pH remains acidic, preferably less than 5.
  • the pH is immediately adjusted to 6.5 with the desired base (pyridine, triethylamine, tributylamine, sodium hydroxide).
  • the polysaccharide derivative in the form of a salt is then lyophilized.
  • the polysaccharide derivative in the form of a salt is dissolved in 100 ml of anhydrous DMF with gentle stirring (250 rpm) for 2 hours at ambient temperature, and then for 2 hours at a temperature of 45° C.
  • reaction medium i.e. 5 times the mass of the polysaccharide.
  • the temperature of the mixture is then maintained at 45° C. for 2 hours with stirring.
  • the reaction is terminated by adding 40 ml of water and sodium hydroxide in order to obtain a pH at 9.
  • the reaction mixture is then dialyzed in water with a dialysis bag having a cutoff threshold of 3500 Da.
  • the solution containing the sulfated EPS is filtered through filters of 2.7 ⁇ m and 0.7 ⁇ m and ultrafiltered through a 1000 g/mol membrane and then lyophilized 2 .
  • HPSEC high performance steric exclusion chromatography
  • the column was calibrated with polysaccharide standards as follows: pullulans: 758 000-5900 g/mol (Polymer Laboratories, Interchim), noncommercial standard polysaccharides: 4000; 3000 and 1500 g/mol; melezitose: 522 g/mol (FLUKA), sucrose: 342 g/mol; glucose: 180 g/mol (SIGMA).
  • pullulans 758 000-5900 g/mol
  • FLUKA melezitose
  • sucrose 342 g/mol
  • glucose 180 g/mol
  • the neutral monosaccharide content was determined by the method of Tillmans and Philippi ( Analyt. Chem., 1929, 28, 350-) modified by Rimington ( Biochem. J., 1931, 25: 1062-1071).
  • the uronic acid (GlcA) content was established using a modification of the m-hydroxydiphenyl-H 2 SO 4 method (Filisetti-Cozzi and Carpitta, Anal. Biochem., 1991, 197: 157-162) and using glucuronic acid as standard. Interference from neutral hexoses was avoided by using potassium sulfamate and carrying out controls comprising all the reagents with the exception of the m-hydroxydiphenyl.
  • hexosamine and N-acetylhexosamine (GalNAc and GlcNAc) content is determined by the method of Belcher et al. ( Analyst, 1954, 79: 201-208) adapted from that of Elson and Morgan ( Biochem J., 1933, 27: 1824-1828) and using N-acetylglucosamine and glucosamine as standard.
  • the amount of free sulfates is quantified by ion exchange chromatography on a Dionex® DX-500 system connected to a conductimeter and according to the method described by the manufacturer Dionex.
  • the result obtained makes it possible to calculate the amount of sulfates really bound to the EPS derivative, which is equal to the amount of total sulfates (obtained by elemental analysis) minus the amount of free sulfates (obtained by ion exchange chromatography).
  • FT-IR Fourier transform infrared spectroscopy
  • Composition of an HE 800 DR Derivative Characteristics HE 800 DR derivative Neutral monosaccharides 0 (g/100 g) 1 Acidic monosaccharides (g/100 g) 1 40 Hexosamines (g/100 g) 1 40 Total —SO 3 Na (g/100 g) 2 0 Mc (g/mol) 4 15 000 Mw (g/mol) 4 29 000 Mn (g/mol) 4 13 000 I (Mw/Mn) 4 2.2 1 Colorimetric assays. 2 Assay by elemental analysis. 3 Assay by ion exchange chromatography. 4 Determined by HPSEC chromatography in pullulan equivalents.
  • Composition of an EPS HE 800 DRS Derivative Characteristics HE 800 DRS derivative Neutral monosaccharides 0 (g/100 g) 1 Acidic monosaccharides (g/100 g) 1 30 Hexosamines (g/100 g) 1 30 Total —SO 3 Na (g/100 g) 2 25 Mc (g/mol) 4 4800 Mw (g/mol) 4 5800 Mn (g/mol) 4 4500 I (Mw/Mn) 4 1.3 1 Colorimetric assays. 2 Assay by elemental analysis. 3 Assay by ion exchange chromatography. 4 Determined by HPSEC chromatography in pullulan equivalents.
  • Composition of an EPS HE 800 DRNS Derivative Characteristics HE 800 DRNS derivative Neutral monosaccharides 0 (g/100 g) 1 Acidic monosaccharides (g/100 g) 1 20 Hexosamines (g/100 g) 1 20 Total —SO 3 Na (g/100 g) 2 34 Mc (g/mol) 4 22 000 Mw (g/mol) 4 27 000 Mn (g/mol) 4 19 000 I (Mw/Mn) 4 1.4 1 Colorimetric assays. 2 Assay by elemental analysis. 3 Assay by ion exchange chromatography. 4 Determined by HPSEC chromatography in pullulan equivalents.
  • FIG. 1 shows the infrared spectra of the polysaccharide derivatives, before (HE 800 DR) and after the N-deacetylation reaction (HE 800 DRN).
  • the FT-IR spectra in FIG. 1 were recorded on a Bruker Vector 22 spectrophotometer (resolution of 4 cm ⁇ 1 ).
  • 2 mg of EPS HE 800 derivative were treated with 200 mg of KBr during the N-deacetylation step.
  • the analysis of the infrared spectra of the derivatives, before and after the N-deacetylation step shows, at the frequency of 1550 cm ⁇ 1 , the loss of an absorption band characteristic of N-acetylated groups ( FIG. 1 ).
  • FIG. 2 shows the infrared (IR) spectra of the non-sulfated HE 800 polysaccharide derivative (HE 800 DR) and of the N-deacetylated and sulfated HE 800 poly-saccharide derivative (HE 800 DRNS). The appearance of the bands corresponding to the presence of a sulfate ester (1250, 820 and 600 cm ⁇ 1 ) is observed for the polysaccharide derivative HE 800 DRNS.
  • IR infrared
  • GY 785 DR corresponds to the low-molecular-weight polysaccharide derivative obtained after a depolymerization step.
  • GY 785 DRS corresponds to the low-molecular-weight polysaccharide derivative obtained after a depolymerization step followed by a sulfation step.
  • sulfated EPS GY 785 obtained above in the preceding step were dissolved in 95 ml of water. After dissolution, 2 ml of a catalytic solution containing 36 mg of copper acetate monohydrate (10 ⁇ 3 M) were added. The temperature of the reactor is then brought to 60° C. and the pH is adjusted to 7.5 by the addition of 1 M sodium hydroxide. A 0.115% (v/v) solution of hydrogen peroxide was then added at a flow rate of 1 ml per minute, and the pH was regulated at around 7.5 by the addition of 1 M sodium hydroxide. The reaction was stopped after 1 hour.
  • the reduction is carried out at the end of depolymerization by the addition to the reactor of sodium borohydride (270 mg of NaBH 4 dissolved in 10 ml of water). The reduction is carried out with stirring for 2 hours at ambient temperature. The reduction is stopped by the addition of 10 N acetic acid, which makes it possible to eliminate the excess NaBH 4 remaining in the form of hydrogen gas that is given off.
  • the solution was then filtered through a Büchner funnel with filters made of glass microfibers (porosity 3 ⁇ m). The filtered solution was eluted on a CHELEX® 20 column (BIORAD) in order to eliminate the residual copper. The decontaminated solution was then ultra-filtered through a cassette (cutoff threshold 1000 Da) and then lyophilized.
  • the solution containing the sulfated EPS GY 785 was frozen and lyophilized.
  • GY 785 DR derivative Neutral monosaccharides 40 (g/100 g) 1 Acidic monosaccharides (g/100 g) 1 15 Hexosamines (g/100 g) 1 0 Total —SO 3 Na (g/100 g) 2 10 Mc (g/mol) 4 16 000 Mw (g/mol) 4 40 000 Mn (g/mol) 4 13 000 I (Mw/Mn) 4 3 1 Colorimetric assays. 2 Assay by elemental analysis. 3 Assay by ion exchange chromatography. 4 Determined by HPSEC chromatography in pullulan equivalents.
  • GY 785 DRS Derivative Characteristics GY 785 DRS derivative Neutral monosaccharides 20 (g/100 g) 1 Acidic monosaccharides (g/100 g) 1 10 Hexosamines (g/100 g) 1 0 Total —SO 3 Na (g/100 g) 2 45 Mc (g/mol) 4 23 000 Mw (g/mol) 4 29 000 Mn (g/mol) 4 21 000 I (Mw/Mn) 4 1.4 1 Colorimetric assays. 2 Assay by elemental analysis. 3 Assay by ion exchange chromatography. 4 Determined by HPSEC chromatography in pullulan equivalents.
  • HYD 721 DR corresponds to the low-molecular-weight polysaccharide derivative obtained after a depolymerization step
  • HYD 721 DRS corresponds to the low-molecular-weight polysaccharide derivative sulfated by chemical sulfation according to the invention.
  • the sulfated HYD 721 polysaccharide derivative is obtained by carrying out the process described in example 2. However, since the native EPS HYD 721 does not contain N-acetylated hexosamines, the process for preparing the sulfated derivative does not comprise an N-deacetylation step.
  • HYD 721 DR Derivative Characteristics HYD 721 DR derivative Neutral monosaccharides 68 (g/100 g) 1 Acidic monosaccharides (g/100 g) 1 17 Hexosamines (g/100 g) 1 0 Total —SO 3 Na (g/100 g) 2 11 Mc (g/mol) 4 10 000 Mw (g/mol) 4 12 000 Mn (g/mol) 4 7000 I (Mw/Mn) 4 1.7 1 Colorimetric assays. 2 Assay by elemental analysis. 3 Assay by ion exchange chromatography. 4 Determined by HPSEC chromatography in pullulan equivalents.
  • HYD 721 DRS Derivative Characteristics HYD 721 DRS derivative Neutral monosaccharides 35 (g/100 g) 1 Acidic monosaccharides (g/100 g) 1 5 Hexosamines (g/100 g) 1 0 Total —SO 3 Na (g/100 g) 2 43 Mc (g/mol) 4 17 500 Mw (g/mol) 4 20 000 Mn (g/mol) 4 10 000 I (Mw/Mn) 4 2 1 Colorimetric assays. 2 Assay by elemental analysis. 3 Assay by ion exchange chromatography. 4 Determined by HPSEC chromatography in pullulan equivalents.
  • the sulfated polysaccharide derivatives used in the proliferation assays were prepared and characterized according to the protocols of examples 2 and 3.
  • the cells are seeded in two culture dishes at a rate of 10 000 cells per well, in Dulbecco's MEM Glutamax I culture medium containing 100 U/ml of penicillin, 100 ⁇ g/ml of streptomycin, 2 ⁇ g/ml of fungizone and supplemented with 10% of fetal calf serum (FCS).
  • FCS fetal calf serum
  • the culture medium is replaced with a culture medium that may or may not be supplemented with various concentrations of a low-molecular-weight sulfated polysaccharide derivative: (i) the EPS GY 785 (GY 785 DRS) or (ii) the EPS HE 800 (HE 800 DRNS) HE 800.
  • the cells are then counted after 2, 4, 7 and 10 days of culture.
  • the controls correspond to cell cultures in the absence of derivatives in accordance with the invention (*).
  • the sulfated polysaccharide derivatives in accordance with the invention are capable of stimulating the proliferation of dermal and gingival fibroblasts in two-dimensional culture.
  • Reconstructed or latticed connective tissues consist of acid-soluble collagen type I fibers which, after neutralization, polymerize and form a gel containing fibroblasts.
  • the preparation of a lattice is carried out under cold conditions in order to have better control of the polymerization of the collagen fibers.
  • This lattice under the influence of the cells will undergo numerous rearrangements that are noticeable in particular by virtue of its retraction, which is observed during the first two weeks of culture.
  • This type of model makes it possible to study the behavior of the cells within an extracellular environment closer to the physiological environment than simple culturing on a dish.
  • FIG. 3 reveals that the low-molecular-weight sulfated polysaccharide derivative GY 785 DRS in accordance with the invention is capable, at the concentration of 10 ⁇ g/ml, of stimulating the proliferation of fibro-blasts in reconstructed connective tissues.
  • the cells are seeded into culture dishes at a rate of 10 000 cells per well, in Dulbecco's MEM Glutamax I culture medium containing 100 U/ml of penicillin, 100 ⁇ g/ml of streptomycin, 2 ⁇ g/ml of fungizone and supplemented with 10% of fetal calf serum (FCS).
  • FCS fetal calf serum
  • the culture medium is replaced with a culture medium that may or may not be supplemented with various concentrations of a sulfated derivative obtained from the EPS GY 785 (GY 785 DRS).
  • the cultures are then fixed with alcohol after 2, 4, 7 and 10 days of culture, and the immunodetection on these cell cultures is carried out as follows:
  • the fixed cells are re-permeabilized in 70% ethanol (20 min), and then rehydrated in PBS (10 min). Endogenous peroxidases are blocked with a methanol (30%), H 2 O 2 (0.3%) solution. This process is followed by rinsing with PBS (2 min) then blocking of the nonspecific antigenic sites with a PBS/1% skimmed milk solution (1 h). The cultures are then incubated with a primary antibody (mouse IgG) directed against human ⁇ -actin (1/30; 50 min) and then rinsed with PBS (3 ⁇ 10 min).
  • a primary antibody mouse IgG
  • the cells are then incubated, in the dark for 60 min, with a biotinylated anti-mouse IgG antibody (1/200), rinsed with PBS (3 ⁇ 10 min), and then incubated with peroxidase-coupled streptavidin (1/200).
  • the visualization of the peroxidase activity with 3,3′-diaminobenzidine is carried out in a Tris/HCl buffer (100 mM, pH 7.2-7.4) containing 0.1% of H 2 O 2 (15 min, in the dark).
  • the peroxidase activity reveals a brown fibrillar material that corresponds to the ⁇ -actin microfilaments in the cytoplasm of the positive cells.
  • the products used come from the company DAKO under the name DAKOImmuno-detection.
  • FIG. 4 demonstrates that the low-molecular-weight sulfated polysaccharide derivative GY 785 DRS in accordance with the invention is capable of stimulating the proliferation of fibroblasts to the detriment of myofibroblasts.
  • Photographs a, c and e correspond to cultures supplemented with 10 ⁇ g/ml of sulfated derivative, in which the dominant population is made up of fibroblasts to the detriment of myofibroblasts.
  • Photographs b, d and f correspond to control cultures without sulfated derivative, in which numerous myofibroblasts are visible.
  • control cultures not treated with the derivatives in accordance with the invention comprise a large number of myofibroblasts ( ⁇ -actin-positive), while fibroblasts not expressing ⁇ -actin form the dominant cell type in the treated cultures.
  • the cells are seeded into culture dishes at a rate of 40 000 cells per well and are brought to confluence in Dulbecco's MEM Glutamax I culture medium containing 100 U/ml of penicillin, 100 ⁇ g/ml of streptomycin, 2 ⁇ g/ml of fungizone and supplemented with fetal calf serum (FCS).
  • FCS fetal calf serum
  • the culture medium is replaced with medium that does not contain any FCS and may or may not be supplemented with IL-1 ⁇ at a final concentration of 100 U/ml, in the presence or absence of sulfated derivatives.
  • the culture media are sampled after 48 hours, in order to study the MMP secretion by the fibroblasts, by zymography or by Western blotting.
  • the metalloprotease secretion is detected and quantified by zymography. It is a particularly sensitive method based on SDS-PAGE electrophoresis carried out under reducing conditions.
  • the gel is then incubated at 37° C. in an incubation buffer (0.1 M Tris/HCl, pH 7.4, 30 mM CaCl 2 , 0.001% NaN 3 , 0.0015% Brij, 0.1 ⁇ M ZnCl 2 ).
  • the bands illustrating the MMP-2 metalloprotease activity appear destained.
  • the shade of gray and the surface area of these bands are quantified on an image analyzer, the [(shade of gray ⁇ surface area)/number of cells] ratio allows a quantification of the gelatino-lytic activities and comparison between the control cultures and the cultures containing the exopolysaccharide.
  • the graph in FIG. 5 shows that the MMP-2 secretion is inhibited when the fibroblasts are cultured in the presence of low-molecular-weight sulfated polysaccharide derivatives, in the presence or absence of IL1 ⁇ .
  • FIG. 6 shows that the addition of HE 800 DRNS greatly decreases the secretion of MMP-3 by the fibroblasts, whether this secretion is baseline (photograph a) or induced by an inflammatory cytokine IL-1 ⁇ (photo-graph b).
  • Photograph (a) shows confluent fibroblasts incubated for 48 h in a serum-free medium (lane 1), and in a serum-free medium containing 10 ⁇ g/ml of EPS derivative (lane 2);
  • photograph (b) shows confluent fibroblasts incubated for 48 h in a serum-free medium containing 100 U/ml of IL-1 ⁇ (lane 1), and in a serum-free medium containing 10 ⁇ g/ml of EPS derivative and 100 U/ml of IL-1 ⁇ (lane 2).
  • the sulfated polysaccharide derivatives in accordance with the invention are capable of inhibiting the secretion of matrix proteases, gelatinase A (MMP-2) and stromelysin 1 (MMP-3), by fibroblasts in two-dimensional cultures.
  • MMP-2 matrix proteases
  • MMP-3 stromelysin 1
  • Complement can be activated by 3 different activation pathways: the conventional pathway, the alternative pathway and the mannose-binding lectin (MBL) pathway resulting, via different mechanisms, in the formation of enzymes having identical substrates: the C3/C5 convertases capable of activating C3 and C5.
  • the activation reactions take place as a cascade: one component acquires an enzymatic activity that induces the activation of the next component, and so on.
  • the activation of the conventional pathway occurs when the C1 complex interacts with antigen-antibody complexes or immune aggregates containing IgGs or IgMs.
  • the system used to study the conventional pathway is based on the complement activation by an immune complex consisting of rabbit antibody-coated sheep red blood cells.
  • the rabbit antibodies having recognized as foreign the red blood cells, in the presence of human serum, trigger mainly the activation of the conventional pathway.
  • This activation leads to the formation of C3 convertase, which then triggers the activation of the alternative pathway.
  • the activation of these two pathways results in the formation of a membrane attack complex at the surface of the red blood cells.
  • This complex induces rupturing of the red blood cells and the release of hemoglobin.
  • the activation of the complement system is measured by assaying the amount of hemoglobin released, by measuring the spectrophotometric absorption at 414 nm.
  • the activator (sheep red blood cells) is also used as a revealer of the activation by virtue of the hemoglobin released during the cell lysis.
  • the dilution of the human serum is adjusted for a given amount of blood cells, such that 50% of the cells are lysed.
  • the antibody-coated red blood cells are incubated in the absence (control) and in the presence of various sulfated polysaccharide derivatives.
  • the amount of hemoglobin released decreases (decrease in the absorption at 414 nm), reflecting a decrease in the number of red blood cells lysed and an inhibitory effect of the sulfated polysaccharide derivatives on the activation of the conventional complement pathway.
  • the low-molecular-weight sulfated polysaccharide derivatives in accordance with the invention are capable of inhibiting the conventional complement pathway.
  • Stromal cells are obtained from ground bone marrow material, and this ground material is centrifuged so as to recover a cell pellet.
  • the cells are resuspended in culture medium made up, for 500 ml, of medium containing 10% of horse serum, 10% of fetal calf serum, 200 nM L-glutamine (4 ml), 100 ⁇ MEM vitamin (Gibco brl) (4 ml), 7.5% Na 2 HCO 3 (4 ml), 50 ⁇ essential amino acids with L-glutamine (Gibco brl) (4 ml), 100 ⁇ sodium pyruvate (4 ml), 100 ⁇ nonessential amino acids (Gibco brl) (1.6 ml), the whole being diluted in a 1 ⁇ McCoy's medium (Gibco brl).
  • the proliferation assays are carried out under the same conditions as those described for the proliferation assays on the cultures of human gingival and dermal fibroblasts.
  • the cell countings are carried out after 2, 4, 7 and 10 days of culture.
  • the immunodetections directed against certain phenotypic markers showed that these cells do not express a marker characteristic of macrophages, CD68; a minority of these cells express a leukocyte marker, CD45, but, on the other hand, they express a cyto-skeletal protein characteristic of mesenchymal cells, vimentin.
  • some of these cells are capable of expressing, without stimulation, collagen type I and collagen type III, which are characteristic of mesenchymal cells in culture.
  • the proliferation of the medullary cells intended to become mesenchymal cells is greatly stimulated in the presence of the EPS GY 785 DRS.

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US20110150795A1 (en) * 2008-04-15 2011-06-23 Innovactiv Inc. Cosmetic compositions comprising exopolysaccharides derived from microbial mats, and use thereof
CN103260395A (zh) * 2010-08-31 2013-08-21 法国海洋勘探研究所 用具有抗菌和愈合性质的成膜包衣包被的核及其获得方法
US20130302261A1 (en) * 2010-11-30 2013-11-14 Polymaris Biotechnology Exopolysaccharide for treatment or care of skin, mucous membranes, hair or nails
US20130336937A1 (en) * 2011-03-18 2013-12-19 Institut National de la Sante et de Ia Recherche Medicale Chondrogenic differentiation media and methods for inducing chondrogenic differentiation of cells
CN116731221A (zh) * 2023-06-26 2023-09-12 程晓琳 一种多糖衍生物及其在制药中的用途

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WO2010086696A1 (fr) * 2009-01-28 2010-08-05 Therapol Polysaccharides sulfatés de faible masse moléculaire comme candidats pour une thérapie anti-angiogénique
FR2953217B1 (fr) 2009-11-27 2012-10-05 Ifremer Procede de depolymerisation de polysaccharides par broyage mecanique
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JP6100451B2 (ja) * 2011-01-18 2017-03-22 ヒノキ新薬株式会社 皮膚外用剤
ES2424294B1 (es) * 2012-03-22 2014-07-21 Lipotec, S.A. Exopolisacárido para el tratamiento y/o cuidado de la piel, mucosas, cabello y/o uñas
EP3146971A1 (fr) * 2015-09-28 2017-03-29 Institut Francais de Recherche pour l'Exploitation de la Mer (IFREMER) Dérivé d'exopolysaccharide bactérien marin anti-métastatique et leurs utilisations
WO2017118979A1 (fr) * 2016-01-07 2017-07-13 B. G. Negev Technologies And Applications Ltd., At Ben-Gurion University Compositions et méthodes pour générer des réponses immunotolérantes
JP2019530663A (ja) 2016-09-01 2019-10-24 インスティトゥート デ ビオロジア モレクラル エ セルラル−イーベーエミセーＩｎｓｔｉｔｕｔｏ Ｄｅ Ｂｉｏｌｏｇｉａ Ｍｏｌｅｃｕｌａｒ Ｅ Ｃｅｌｕｌａｒ − Ｉｂｍｃ シアノバクテリア細胞外ポリマー、組成物およびそれらの使用
AU2021301259A1 (en) * 2020-07-02 2023-02-02 Fondation Sanfilippo Suisse Marine bacterial exopolysaccharide derivatives and uses thereof in the treatment of mucopolysaccharidoses
EP4112060A1 (fr) 2021-07-01 2023-01-04 Institut Francais de Recherche pour l'Exploitation de la Mer (IFREMER) Dérivés d'exopolysaccharide he800 de faible poids moléculaire ayant des propriétés anticancéreuses et leurs utilisations

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US20110150795A1 (en) * 2008-04-15 2011-06-23 Innovactiv Inc. Cosmetic compositions comprising exopolysaccharides derived from microbial mats, and use thereof
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CN116731221A (zh) * 2023-06-26 2023-09-12 程晓琳 一种多糖衍生物及其在制药中的用途

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