Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
  • A61K31/737—Sulfated polysaccharides, e.g. chondroitin sulfate, dermatan sulfate
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

• the invention relates to new uses of fucans for the repair of connective tissue damage, and in particular for the regulation of fibroblastic functions.
• Fibroblasts play an essential role in the balance and repair of connective tissue. They are in particular responsible for the renewal of the extracellular matrix, and their functions are modulated in return by the substances present in this matrix.
• the connective tissue is the framework for constant exchanges between all the cells involved in this process. These exchanges take place in particular via cytokines or soluble mediators transmitted by the extracellular matrix.
• the healing process begins, after the formation of a temporary matrix (red thrombus), by the recruitment of inflammatory cells (leukocytes, macrophages and polynuclear), which initiate a phase of destruction of the injured tissue.
• a temporary matrix red thrombus
• inflammatory cells leukocytes, macrophages and polynuclear
• inflammatory cells participate in the destruction: - by secreting matrix proteases such as collagenase (MMP8), leukocyte elastase, or cathepsin G), by releasing cytokines, and in particular
• IL1 interleukin 1
• MMP1 interstitial collagenase
• MMP9 gelatinase B
• MMP2 gelatinase A
• Certain pathologies are accompanied by a chronic inflammatory state of the connective tissue, where the balance between the phases of destruction, repair, and resolution is disrupted, which leads to defective reconstruction of the injured tissue.
• the periodontium represents all of the structures (gum, dento-alveolar ligament, alveolar bone and cementum) which support the tooth in its dental alveolus.
• Periodontopathies manifest themselves by inflammatory episodes of infectious origin, more or less localized, often recurrent, and at the end of which the periodontal tissue does not rebuild properly.
• Another therapeutic approach would be to improve the quality of the repair process, so that it can lead to complete regeneration.
• this approach in particular requires being able to stimulate the appropriate cell population at the right time, in order to orient cell proliferation, and thus control the processes of tissue reorganization.
• glycosaminoglycans enter into the composition of proteoglycans present at the cell / extracellular matrix interface, and play a role in the regulation of cellular functions. It is also known that glycosaminoglycans in soluble form, for example heparin or derivatives of dextran, can modify cellular functions through their interaction with different constituents of the extracellular matrix.
• heparin a stimulating effect on cell proliferation has been shown in the case of hamster pulmonary fibroblasts, bovine lens epithelial cells [ULRICH. Et al., Biochem. Biophys. Res. Commun., 139, p. 728-732, (1986)] or endothelial cells capillaries [(SUDLALTER et al., J. Biol. Chem., 264, p. 6892-6897, (1989)].
• heparin inhibits in a dose-dependent manner the proliferation of certain cell types: this anti-proliferative effect has been mainly studied in the case of smooth muscle cells (CML), for which the inhibition appears for concentrations of 1 ⁇ g / ml of heparin in culture media.
• CML smooth muscle cells
• Heparin opposes both MLC migration and proliferation, but does not affect re-endothelialization or the volume of connective tissue [CLO ES and CLO ES, Lab. Invest, 52, p. 611-615, (1985); CLOWES and CLOWES, Wax. Res., 58, p 839-845, (1986); CLOWES et al., J. Cell. Biol., 107, p. 1939-1945, (1988)].
• sclera fibroblasts [DEL VECCHIO et al., Invest. Ophthalmol. Screw. Sci. , 29, p. 1272-1276, (1988)], fibroblasts 3T3 (fibroblasts of mouse embryos retaining contact inhibition) [PAUL et al., Thromb. Res., 18, p. 883-888, (1980)], epithelial cells of the cervix of the spleen (LYONS-GIORADANO et al., Biochem. Biophys. Res. Commun., 148, p. 1264-1269, (1987)], human dermal fibroblasts
• FERRAO and MASON [Biochim. Biophys. Acta. 1180, 225-230, (1993)] studied the action of different polysaccharides on the proliferation of human dermal fibroblasts, and indicate that at concentrations of the order of 100 ⁇ g / ml, heparin, 1 heparan sulfate, pentosan polysulfate, and a fucoidan inhibit this proliferation, while chondroitin sulfate, dermatan sulfate, and hyaluronate have no effect. It is stated that the proliferation-inhibiting effect results. stimulation of the synthesis of type I collagen. On the other hand, inhibition of the synthesis of collagen I is observed when the polysaccharides are added to cultures which have reached confluence.
• polysaccharides on cellular functions is complex, and can vary according to the polysaccharide, the cell type, and the tissue concerned, as well as according to the concentration of polysaccharide used, and the state of the cells.
• Fucans are sulfated polysaccharides, used in the constitution of the cell walls of thalli of brown algae (Pheophyceae); they are also present in certain marine animals, such as sea urchins and sea cucumbers.
• Raw fucan, also called fucoidan, obtained by acid extraction from the cell walls of thalli of brown algae, consists of a population heterogeneous of molecules, which mainly includes polymers of
• Fucans have various biological activities: it has thus been shown that they have anticoagulant, antithrombotic activities [T. NISHINO and T. NAGUMO, Carbohydr. Res. 229, p. 355-362, (1992); EP 0403 377; S. COLLIEC et al. Thromb. Res. 64, p. 143-154 (1991);
• fucan includes both high molecular weight fucans and lower molecular weight preparations obtained therefrom.
• fucans have a different activity profile on fibroblastic functions than that of heparin. They found in particular, by testing these two polysaccharides under the same conditions, that while heparin inhibits both the proliferation of dermal fibroblasts and that of the gingival fibroblasts, the fucans activate the proliferation of dermal fibroblasts while inhibiting that gingival fibroblasts. In addition, the inventors have also found that the fucans modify the morphology of the dermal fibroblasts, which round off, while the gingival fibroblasts, on the contrary, retain a fibroblastic morphotype. The inventors have also found that fucans increase the amount of proteins in the cell layer, the activity of MMP-2 (gelatinase A), and inhibit leukocyte elastase.
• the subject of the present invention is the use of a fucan for obtaining a medicament which modulates the expression and / or the activity of fibroblast metalloproteinases, and in particular MMP-2, and which inhibits l leukocyte elastase.
• the fucans thus make it possible to control the proteolytic activity in the connective tissues, such as the dermal and gingival tissues, and in particular to limit the elastase activity, which has significant destructive potential vis-à-vis the macromolecular connective structures, while by promoting, on the contrary, the activity of proteases which participate in tissue reconstruction, such as MMP-2.
• said medicament can also be used to inhibit the proliferation of gingival fibroblasts and activate their collagen synthesis. It allows the treatment of periodontal pathologies, through an improvement of the resolution phase.
• the fucans make it possible to engage these gingival fibroblasts in the path of remodeling, necessary for any process of repair or tissue regeneration.
• said medicament can also be used to activate the proliferation of dermal fibroblasts, and their collagen synthesis. It thus allows the treatment of dermal lesions, by improving the recovery phase of the injured tissue.
• the medicaments obtained in accordance with the invention can be administered by the general route (oral or parenteral). They can also be administered locally, in the form of gels, creams, ointments, lotions, lozenges, mouthwashes, etc.
• They can also be administered in si tu via substrates, absorbable or non-absorbable devices, such as, for example, delayed release carriers, or slow disintegrating sponges.
• Fucans can also be used in cosmetology, as activators of fibroblast proliferation in the context of treatments for aesthetic purposes, for example anti-wrinkle treatments, or prevention of skin aging, etc.
• Fucan The fucan used is a fraction of average molar mass 20,00012,000 obtained from the brown marine alga Ascophylum nodosum, according to the method described in Patent EP 0 403 377. This fucan has a high rate of fucose (44 ⁇ 5 %), few uronic acids (7 ⁇ 3%), 28 ⁇ 3% of sulphate groups and no proteins.
• the fibroblasts used were obtained from explants of healthy dermal and gingival tissues from three different donors (aged 15 to 30 years).
• the culture protocol is the same for gingival fibroblasts and dermal fibroblasts. From the explant, the cells are cultured in a DMEM culture medium (DULBECCO'S Modified Eagle Medium) containing D-glucose at 1 g / 1 and GLUTAMAX at 0.862 g / 1, an antibiotic (penicillin- streptomycin 1000 U / ml), fungizone (amphotericin B at 250 U / ml; GIBCO BRL) and 20% fetal calf serum (SVF).
• DMEM culture medium DULBECCO'S Modified Eagle Medium
• the samples are taken out of the transport medium. Rinsed three times in the culture medium, they are finely cut into small pieces of about 1 mm. They are arranged so that the epithelial layer is oriented above and the connective layer is below, in contact with the 25 cm culture dish.
• the box is placed vertically in an incubator at 37 ° C (95% air, 5% CO2) for half an hour, so as to promote adhesion.
• the dish After adding a drop of medium to each tissue fragment, the dish is replaced in the incubator overnight.
• the medium is then changed every three days, and the explants are removed as soon as the fibroblasts adhere to the wall. When the fibroblasts have invaded the entire bottom of the dish, the primary culture is finished.
• DPBS DULBECCO'S Phosphate Buffer Saline
• the cells are distributed in 3 dishes, in DMEM (10 to 12 ml) at 10% FCS, and 1000 U / ml of penicillin-streptomycin.
• the medium is changed regularly until the new box is completely colonized.
• the confluent cells are trypsinized, suspended in DMEM at the rate of 7000 to 10000 cells / ml and then distributed in the wells of 24-well plates.
• the wells are completed with 10% FCS medium, and the seeded plates are replaced for two hours in the incubator to allow adhesion. Three hours after seeding, the media are replaced by a DMEM / 10% FCS medium (control group) or else DMEM / 10% CFS with 1, 10 or 100 ⁇ g / ml of the polysaccharide tested (fucan or heparin).
• the cells are counted each day until the 4th day.
• the percentage of proliferation is calculated by the relation:
• EXAMPLE 2 INFLUENCE OF FUCANE ON THE ACTIVITY OF MMP-2 (GELATINASE A) AND ON THE MORPHOLOGY OF FIBROBLASTS:
• the cells are seeded in 24-well plates (7,000 to 10,000 cells / ml) and cultured in the presence of DMEM / 10% FCS. At confluence, the medium is replaced by DMEM for the controls, or DMEM containing the different concentrations of fucan (1, 10, 100 ⁇ g / ml) for 24 hours. The media are then recovered, for the detection of the gelatinolytic activity of pro-MMP-2, and the fixed and stained cells (methanol / GIEMSA) are counted and analyzed by morphometry on a BIOCOM 200 station.
• the gelatinolytic activities present in the culture media are detected by zymography, after electrophoresis, under non-reducing conditions, in polyacrylamide-SDS gel + gelatin, then elimination of SDS.
• the results are quantified by semi-automatic image analysis on a BIOCOM 200 station. For each band appearing on the gel, the product of the gray density (D) is determined by the surface of the band (S). This product, related to the number of cells, makes it possible to appreciate and compare the different gelatinolytic activities.
• the equivalent diameter is the diameter of the smallest circle that entirely contains the cell. It defines the greatest length of the cell.
• the form factor is determined by the ratio 4 ⁇ S / P 2 , where S represents the surface and P the perimeter: when it tends towards zero, it translates an elongated structure; when it increases, it reflects a rounding of the shape.
• gingival and dermal fibroblasts react differently to fucan: Gingival fibroblasts: The surface as well as the diameter of these cells decrease, while their perimeter increases. These parameters make it possible to calculate a form factor tending towards zero, implying an elongated cell of fibroblastic type. Dermal fibroblasts: Under the influence of fucan these cells see their surface and their diameter increase while their perimeter remains constant. The form factor increases, which implies rounding cells. EXAMPLE 3 INFLUENCE OF FUCANE ON THE ACTIVITY OF LEUCOCYTA ELASTASE
• leukocyte elastase The activity of leukocyte elastase in the presence of fucan (1 ⁇ g / ml or 10 ⁇ g / ml) or heparin H108 (lII / ml or 10 IU / ml) is measured using the synthetic peptide as substrate:
• EXAMPLE 4 INFLUENCE OF FUCANE ON BIOSYNTHESIS OF FIBRILLARY COLLAGENS The biosynthesis of collagens is measured after incorporation of tritiated proline (H-Pro).
• the cells are cultured in DMEM / 10% FCS until confluence.
• the media are then replaced by DMEM containing ascorbic acid (50 ⁇ g / ml) and 3 H-Pro (25 ⁇ Ci / ml) for the controls, or this same medium supplemented with fucan at different concentrations (1, 10, 100 ⁇ g / ml), or else heparin H108 at a concentration of 400 ⁇ g / ml.
• the media and the cell layer are recovered.
• the specific extraction of proline and hydroxyproline radiolabelled by the method of ROJKIND and GONZALES, [Anal. Biochem. 57: 1-7 (1974)] allows the determination of the total collagen synthesis / total protein synthesis ratio.
• Tables Va gingival fibroblasts
• Vb dermal fibroblasts
• the cells are cultured as described above, in the presence of tritiated proline; fucan is added at a concentration of 100 ⁇ g / ml, or heparin H108 (average molar mass 21,00012,000, activity 173 IU / mg, marketed by CHOAY SANOFI) at a concentration of 400 ⁇ g / ml.
• differential intracellular and pericellular collagens are extracted by deoxycholate (which essentially extracts intracellular procollagen) and SDS (which dissolves the pericellular collagen accumulated in the extracellular matrix).

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• 1997
  • 1997-12-18 FR FR9716080A patent/FR2772618B1/fr not_active Expired - Fee Related
• 1998
  • 1998-12-17 ES ES98962487T patent/ES2212833T3/es not_active Expired - Lifetime
  • 1998-12-17 WO PCT/FR1998/002758 patent/WO1999032099A2/fr not_active Ceased
  • 1998-12-17 AU AU17649/99A patent/AU1764999A/en not_active Abandoned
  • 1998-12-17 JP JP2000525090A patent/JP4589528B2/ja not_active Expired - Fee Related
  • 1998-12-17 BR BR9813778-6A patent/BR9813778A/pt not_active Application Discontinuation
  • 1998-12-17 US US09/581,810 patent/US6559131B1/en not_active Expired - Fee Related
  • 1998-12-17 AT AT98962487T patent/ATE255900T1/de not_active IP Right Cessation
  • 1998-12-17 EP EP19980962487 patent/EP1039916B8/fr not_active Expired - Lifetime
  • 1998-12-17 DE DE69820480T patent/DE69820480T2/de not_active Expired - Lifetime

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