Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
  • A61K36/18—Magnoliophyta (angiosperms)
  • A61K36/185—Magnoliopsida (dicotyledons)
  • A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K8/00—Cosmetics or similar toiletry preparations
  • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
  • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
  • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
  • A61K8/645—Proteins of vegetable origin; Derivatives or degradation products thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K8/00—Cosmetics or similar toiletry preparations
  • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
  • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
  • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
• • 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
  • A61P17/00—Drugs for dermatological disorders
  • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
• • 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
  • A61P17/16—Emollients or protectives, e.g. against radiation
• • 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
  • A61P17/18—Antioxidants, e.g. antiradicals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P39/00—General protective or antinoxious agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P39/00—General protective or antinoxious agents
  • A61P39/06—Free radical scavengers or antioxidants
• • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/14—Vasoprotectives; Antihaemorrhoidals; Drugs for varicose therapy; Capillary stabilisers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
  • A61Q11/00—Preparations for care of the teeth, of the oral cavity or of dentures; Dentifrices, e.g. toothpastes; Mouth rinses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
  • A61Q19/00—Preparations for care of the skin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
  • A61Q19/00—Preparations for care of the skin
  • A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
  • A61Q19/00—Preparations for care of the skin
  • A61Q19/08—Anti-ageing preparations
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
  • A61K2800/74—Biological properties of particular ingredients
  • A61K2800/78—Enzyme modulators, e.g. Enzyme agonists
  • A61K2800/782—Enzyme inhibitors; Enzyme antagonists
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
  • A61Q19/00—Preparations for care of the skin
  • A61Q19/007—Preparations for dry skin

Definitions

• Lupine peptide extract and pharmaceutical or cosmetic or nutraceutical composition comprising such an extract
• the present invention relates to a new peptide extract having anti-metalloprotease activity, in particular anti-collagenase and anti-gelatinase. It also relates to pharmaceutical, cosmetic or nutraceutical compositions comprising such an extract, in particular a pharmaceutical composition intended to treat inflammatory diseases, such as osteoarthritis, periodontosis, or ulcers or cosmetic compositions intended to combat actinic aging or not or accelerated by external aggressions (tobacco, pollution,)
• the pharmaceutical or cosmetic or nutraceutical composition is also intended to treat pathological or unsightly neoangiogenesis (proliferation of vessels) (psoriasis, tumors, erythosis, rosacea, rosacea, local treatment with irritants such as retinoic acid), scarring defects, burns or dental enamel attack (Ch M. Lapiere, Skin biology course - COBIP INSERM U 346, Lyon 1999)
• Metalloproteases are a family of zinc and calcium-dependent endopeptidases which have the combined property of degrading the various components of the connective tissue matrices (Thesis by S Charvat - Metalloproteinases and epidermis, pages 101-1 13 no. 248-98, 1998 , Lyon I)
• Collagenase fibrinase
• gelatinase denatured collagen, gelatin ex MMP2, MMP9
• stromelysins fibronectin, proteoglycan ex MMP3, MMP10
• Metalloproteases are particularly involved in the healing process by eliminating damaged tissue
• MMPs can act in an anarchic way and lead to significant lesions if their activity is not controlled Furthermore, it is known that metalloproteases are implicated in certain biological disorders such as inflammatory diseases, in particular osteoarthritis, periodontosis (H BIRKEDAL-HANSEN et al, Cntical Reviews in Oral Biology and Medicme, 4 (2) 197 250 ( 1993)) or in the aging process, notably linked to the action of solar radiation (MARTIN RIEGER, Allured's Cosmetics & Toilet ⁇ es®, vol 1 14, No 1 / January 1999 or GJ FISHER et al, The New England Journal of Medicine, vol 337, n ° 20 pp 1419-1428, “Pathophysiology of premature skin aging induced by ultraviolet light” and GJ FISHER et al, the Society for Inveshgative Dermatology, Inc 1998, pp 61-68 “Molecular mechanisms of photoagmg and its prevention by retinoic acid: ultraviolet irradi
• the object of the present invention is to provide a new broad spectrum inhibitor of metalloproteases of the collagenase or gelatinase type allowing the treatment of humans or mammals suffering from a condition or a disease linked to excessive or pathological degradation of the collagen or another supporting extracellular macroprotein or any other disease linked to an excess of expression of these proteolytic enzymes.
• the subject of the invention is a peptide extract of lupine (lupine) characterized in that it has an activity of inhibiting metalloproteases, in particular collagenases or gelatinases.
• the genus sweet white lupine (lupinus albus) is cited in particular, such as the Ares variety with low alkaloid content.
• the subject of the invention is in particular a new peptide extract of lupine (lupine) characterized in that it has an activity of inhibiting the purified collagenase of Clostridium histolycum on the DQ-gelatin, in particular greater than 50% at
• this lupine peptide extract is depleted in lipids. It advantageously comprises at least 50%, preferably at least 70%, preferably at least 80% of peptide.
• peptides are obtained by hydrolysis of the protein fraction of lupine.
• the hydrolysis can be carried out by any suitable means, in particular an enzymatic hydrolysis.
• a process for the preparation of such a lupine peptide extract comprises the following steps: preparation of a defatted and ground lupine cake or micronized lupine (lipid) meal, extraction of soluble protein and bone fraction or precipitation at pH acid (4 or 5) depending on the isoelectric point, - optionally separation of the protein fraction, hydrolysis of the protein fraction and recovery, optionally after filtration, of the protein extract.
• the invention also relates to the protein extract capable of being obtained by the above process.
• the invention includes the lipid lupine flours, the peptide extracts also comprising the sugars.
• the protein extract has the following amino acid composition (percentage by weight relative to the total weight of amino acids).
• the subject of the invention is also a pharmaceutical or cosmetic or nutraceutical composition
• a pharmaceutical or cosmetic or nutraceutical composition comprising a peptide extract as described above, optionally an appropriate inert vehicle, physiologically acceptable.
• a pharmaceutical or dermocosmetic and nutraceutical composition is intended in particular for the treatment of humans or mammals suffering from a condition or a disease linked to an excessive destruction of collagen and / or an excessive destruction of the supporting tissues.
• Such a composition can be used as a preventive or curative.
• osteoarthritis we cite for example osteoarthritis, periodontal diseases, diseases linked to skin lesions, inflammatory diseases, tumor or pathological neangiogenesis (erythrosis, rosacea, telangectasia, rosacea, psoriasis ...) , scarring, ulcers, burns, bulbous diseases, and attack of tooth enamel.
• the invention also relates to cosmetic compositions for the treatment of skin lesions due to aging such as lesions caused by the action of solar radiation (in English photoaging), the intrinsic deleterious effects of the skin, the deleterious effects of tobacco.
• the pharmaceutical, dermocosmetic or cosmetic compositions are, according to a variant, in the form of a formulation for topical application.
• the subject of the invention is therefore a cosmetic treatment method comprising the application of such a composition to the skin surface of an individual.
• the peptide extract according to the invention can also be incorporated or formulated in a polymeric vehicle or a delivery system for topical or local use, as in the case of the treatment of a periodontal disease, to be delivered directly into the periodontal pocket .
• the pharmaceutical compositions are in the form of a formulation for oral administration.
• compositions can generally be formulated in the form of tablets, capsules, ointment.
• This step comprises an aqueous solubilization of the soluble fraction at alkaline pH followed by a separation of the insolubles:
• the proteins are extracted at pH 9.0 (pH adjusted by adding sodium hydroxide) with a flour / water ratio equal to 1/10 (w / w).
• the solution is incubated with stirring at room temperature for one hour.
• the insoluble part of the cake is then separated from the soluble part by wringing.
• the cake obtained is washed.
• the soluble fraction containing the proteins and the soluble sugars is diafiltered on an ultrafiltration module with a cutoff threshold of 10.
• the ultrafiltration retentate containing the proteins is adjusted to the concentration of 100 g / 1 then hydrolysis at pH 8.0 in the presence of Alcalase® (NOVO NORDISK) at 55 ° C for approximately 3 h.
• the enzyme is denatured by hydrolysis for 15 min at 85 ° C.
• the peptides obtained are purified by diafiltration on an ultrafiltration module with a cutoff threshold of 10,000 daltons.
• the solution obtained is then nanofiltered in order to desalt (elimination of sodium chloride) and to concentrate the peptide fraction.
• the solution of peptides is finally discolored at using 3% activated carbon (1 hour at 50 ° C), the carbon being removed by filtration
• the solution is microfiltered (0.2 ⁇ m) in a sterile manner and then distributed in sterile containers at a concentration of 10% in the presence of preservatives
• the lupine C peptide extract is obtained according to the process described, used to obtain extract A, with the difference that the purification steps of ultrafiltration and discoloration are omitted.
• the anti-collagenase activity was measured in vitro in a biochemical model of the screening type based on the use of a purified collagenase and the substrate of the latter, gelatin conjugated to fluorescein (kit EnzChek TM Gelatinase / Collagenase, MOLECULAR PROBES) Collagenase purified from Clostridium histolyticum was supplied in the EnzChek TM Gelatinase / Collagenase kit (MOLECULAR PROBES) This enzyme has a dual functionality on collagen IV (epidermis / dermal basement membrane) and gelatin DQ-gelatin purified from pigskin and conjugated with fluorescein was supplied in the EnzChek TM Gelatinase / Collagenase kit (MOLECULAR PROBES)
• the reaction buffer consisting of 0.05 M Tris-HCl, 0.15 M NaCl, 5 mM CaCl 2 , 0.2 mM sodium azide (pH 7.6), was provided in the EnzChek TM Gelatinase / Collagenase kit (MOLECULAR PROBES)
• the peptide extract was dissolved in the reaction buffer. It was tested at 0.004, 0.02; 0.04; 0.2 and 0.4% (w / v).
• test extracts were incubated with DQ-gelatin at 1 mg / ml and collagenase at 0.2 IU / ml for 1 hour, 2 hours and 24 hours at room temperature.
• enzyme-free samples were incubated in the presence of DQ-gelatin and in the absence of collagenase
• control group and treated groups were compared by a factor analysis of variance (ANOVA 1, p ⁇ 0.05), followed by a Dunnett test. The effect of the extracts was thus compared with the one obtained in the control group
• the protein extract tested between 0.004 and 0.4% exhibited dose-dependent anti-gelatinase / collagenase activity.
• the peptide extract tested between 0.01 and 0.5% exhibits anti-gelatinase and collagenase activity dose dependent and time dependent
• Skin aging is characterized, among other things, by a modification of the mechanical skin properties of the skin, following a degradation of the collagen fibers of the dermis and other macroproteins. This degradation involves endogenous collagenases (Grymes, R.A., Kronberger A. and Bauer E.A. - Collagenases in disease - In "Connective Tissue Diseases of the skin” Eds. Lapière CM. And Krieg T., 1993, 69-85). G. Fischer and J.
• the anti-collagenase activity of a test product can be studied in vitro in an organotypic model of human skin.
• the principle of the test is as follows: an application of purified collagenase on the section is accompanied by degradation of the endogenous collagen fibers. The collagen fibers are then colored by Masson's trichrome. The digestion of endogenous collagen fibers by purified collagenase is evaluated qualitatively by morphological observation and quantitatively by image analysis. A product exhibiting anti-collagenase activity will partially or completely preserve the integrity of the collagen fibers brought into contact with the enzyme.
• test products were stored at + 4 ° C until the time of use.
• the phosphoramidon used as a reference product, came from SIGMA.
• the purified collagenase (type III, fraction A) came from SIGMA.
• the incubation medium for human skin sections hereinafter called
• vehicle was 0.15 M Tris HCI buffer, pH 7.5 containing 0.01 M chloride calcium
• the reagents, of analytical quality, came from CARLO ERBN GIBCO or SIGMA, unless otherwise indicated
• the skin sections were made from an operational waste collected after an abdominal plasty
• the subject was a 30 year old woman Skin explants of 4 cm in diameter were made They were placed on a hege support and frozen at -80 ° C
• Cross sections 6 ⁇ m thick were made with a cryomicrotome They were fixed on glass slides and kept hydrated with the vehicle during the test The test samples were all taken up in ethanol before being diluted in the test buffer
• Buffer extract ethanol (0, 1%, v / v or 1%, v / v), extracted enzyme (0.01, 0, 1 and 1%, w / v),
• Control enzyme buffer ethanol (0.1%, v / v), enzyme, Control ethanol (without enzyme) buffer, ethanol (0.1% or 1%, v / v),
• Buffer ref product, ethanol, enzyme, phosphoramidon 10 3 M The dilutions of the test products, of ethanol and of the reference product were deposited on the skin sections, at the rate of 100 ⁇ l per section, and pre- incubated for ten minutes at 37 ° C. Strips of filter paper (0.16 cm 2 of surface) soaked in vehicle alone (control without enzyme) or containing collagenase at 50 international units (IU) / ml (enzyme control) were then placed on the sections The slides were placed in a humid chamber at 37 ° C for three hours
• the activity of collagenase in the absence and in the presence of the test products, of ethanol or of the reference product was evaluated by image analysis.
• the image of the colored sections was digitized on a video screen, the image analysis software (IMAGENIA 2000, BIOCOM ® ) was used to calculate the area occupied by intact collagen fibers The results were expressed as a percentage of intact collagen fibers by optical field
• the percentage of inhibition is calculated by the following formula (% collagen fibers Product Ref - (% collagen fibers) Enzyme control x 100 (% collagen fibers) Ethanol control - (% collagen fibers) Enzyme control
• the percentage of inhibition is calculated by the following formula (% collagen fibers Product Ref- (% collagen fibers Control enzyme x 100 (% collagen fibers) Ethanol witness - (% collagen fibers) Enzyme witness
• the percentage of inhibition is calculated by the following formula (% collagen fibers) Product Ref- (% collagen fibers) Control enzyme x 100 (% collagen fibers) Ethanol control - (% collagen fibers) Enzyme control.
• the anti-collagenase activity of the extract at different concentrations has been studied in an organotypic model of human skin.
• Ethanol used as an intermediate diluent for the test products, was tested at 0, 1 and 1% (v / v). It had no effect on the breakdown of collagen fibers by collagenase.
• the peptide extract A exhibited, at low concentrations, a significant anti-collagenase activity.
• the anti-MMP-2 and anti-MMP-9 activity of the test products was measured in vitro in a biochemical model of the type screening based on the use of a purified human MMP-2 and a recombinant human MMP-9 and the substrate of the latter, gelatin conjugated to fluorescein (kit EnzChek TM Gelatinase / Collagenase, MOLECULAR PROBES)
• the MMP-2 purified from human fibrosarcoma came from BOEHRINGER MANNHEIM.
• the recombinant human MMP-9 came from R&D SYSTEMS.
• DQ-gelatin purified from porcine skin and conjugated to fluorescein was provided in the EnzChek TM Gelatinase / Collagenase kit (MOLECULAR PROBES).
• the reaction buffer for studying the activity of MMP-2 (TpRl) consisted of 50 mM Tris-HCl, 0.05% (w / v) of Triton XlOO and 5 mM CaCl 2 , pH 7.5.
• the reaction buffer for studying the activity of MMP-9 (TpR2) consisted of 50 mM Tris-HCl, 0.05% (w / v) of Brij 35 and 5 mM CaCl 2 , pH 7.4.
• the 1,10-phenanthroline was dissolved in the TpRl and TpR2 reaction buffers. It was tested at 8 and 80 ⁇ g / ml.
• Peptide extracts A, B and C were dissolved in the TpRl and TpR2 reaction buffers. They were tested at 0.01; 0, 1 and 1% (w / v).
• MMP-2 Before its use, MMP-2 was activated by an incubation of 30 minutes at 37 ° C in the presence of APMA diluted to 2.5 mM in TpR1 buffer.
• test products or of the reference product were incubated with DQ-gelatin, diluted to 25 ⁇ g / ml, and activated MMP-2, diluted to 1.25 ⁇ g ml, for 24 hours at 37 °. vs.
• a control corresponding to the “MMP-2 + DQ-gelatin” mixture, was incubated in parallel.
• test products or of the reference product were incubated with DQ-gelatin, diluted to 25 ⁇ g / ml, and MMP-9, diluted to 0.25 ⁇ g / ml, for 24 hours at 37 ° C.
• enzyme-free samples For each experimental condition, samples, hereinafter called “enzyme-free samples”, were incubated in the presence of DQ-gelatin and in the absence of MMP-9. These samples were used to measure the interference of the test products with the effect evaluation method (fluorimetry).
• control group and treated groups were compared by a factor analysis of variance (ANOVA 1, p ⁇ 0.05), followed by a Dunnett test
• Phenanthroline tested at 8 and 80 ⁇ g / ml, inhibits the activity of MMP-2 by 32 and 73% respectively. This expected result validates the test
• Phenanthroline tested at 8 and 80 ⁇ g / ml, inhibits the activity of MMP-9 by 80 and 76% respectively. This expected result validates the test.
• the cells were pre-incubated for 1 hour at 37 ° C. in the presence of the reference products and of the test product. Then, the cells were irradiated with a single dose of 10 J / cm in UVA, in the presence of the product to be tested and the reference products
• the cells were incubated for 48 h at 37 ° C, always in the presence of the product to be tested and of reference products.
• the determination of the various MMPs is carried out in the culture media using specific ELISA kits (Amersham )
• the 10 ⁇ M retinoic acid + EGF 10 ng / ml mixture was used for its TIMP1 induction capacities (TIMP1, Tissue Inhibitor of MMP, physiological inhibitor of MMPs)
• a stock solution containing 10% (w / v) of lupine peptides was prepared in deionized water. From this solution, dilutions were made in the fibroblast culture medium. The lupine peptide extract was tested at 0.5, 1 and 2% (v / v)
• 1,10-phenantroline tested at 80 ⁇ g / ml, inhibited by 99% the secretion of MMP-1, by 92% the secretion of MMP-9 and by 97% the secretion of MMP-3 by the irradiated fibroblasts
• the 10 ⁇ M retinoic acid + EGF 10 ng / ml mixture inhibited MMP-1 secretion by 58%, MMP-9 secretion by 67% and MMP-3 secretion by 44% by irradiated fibroblasts.
• the lupine peptide extract tested at 0.5, 1 and 2% (v / v), respectively decreased by 96, 97 and 97% the amount of MMP-1 secreted by fibroblasts (p ⁇ 0.05)
• the lupine peptide extract tested at 0.5, 1 and 2%, decreased the amount of MMP-3 secreted by fibroblasts by 97, 100 and 98% respectively (p ⁇ 0.05)
• the lupine peptide extract exhibits significant inhibitory properties with regard to the production of MMP-1, -9 and -3 by human dermal fibroblasts irradiated with UVA VII - Examples of formula for topical use:
• the percentages are expressed in total weight of the composition.
• the lupine peptide extract is used in the form of an aqueous solution at 10% by weight according to the invention or of a lyophilized powder called peptide extract in powder form.
• Soy extract (soy glycine) 0.1 to 10.0
• Lupine peptide extract (10% aqueous solution) 0.1 to 10.0
• Titanium dioxide 1.0 to 3.0
• Candelilla wax (Euphorbia Cerifora) 1.0 to 3.0
• Rice starch (Oriza Sativa) 1.0 to 3.0
• Caprylic / capric triglycerides 0.5 to 5.0
• PEG- 100 Stearate 0.5 to 5.0
• Cetareth-20 0, 1 to 0.5
• Pentaerythrityl tetraoctanoate 3.0 to 15.0 Isodecyl neopentanoate 3.0 to 15.0
• Disodium EDTA 0, 1 to 0.5
• Lupine peptide extract (10% aqueous solution) 0.1 to 10.0
• Salicylic acid 0.1 to 10.0
• Titanium dioxide 0.01 to 5.0
• Cetaryl Alcohol 0, 1 to 5.0
• Cetyl Palmitate 0, 1 to 5.0
• Cocoglycerides 0, 1 to 5.0
• Disodium EDTA 0, 1 to 0.5
• Citric acid 0, 1 to 0.5 4. Anti-redness cream for dry to very dry skin
• Lupine peptide extract (10% aqueous solution) 0.1 to 10.0
• Titanium dioxide 1.0 to 10.0
• Caprylic / capric triglycerides 1.0 to 5.0
• Disodium EDTA 0, 1 to 0.3
• the percentages are expressed in total weight of the composition.
• the lupine peptide extract is used in the form of an aqueous solution at 10% by weight according to the invention or of a lyophilized powder called peptide extract in powder form.
• peptide extract 10% aqueous solution + antiseptic (Triclosan) + antiplaque (Gantrez S97BF)
• the percentages are expressed in total weight of the composition.
• the lupine peptide extract is used in the form of an aqueous solution at 10% by weight according to the invention or of a lyophilized powder called peptide extract in powder form.

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• 1999
  • 1999-04-19 FR FR9904875A patent/FR2792202B1/fr not_active Expired - Lifetime
• 2000
  • 2000-04-18 WO PCT/FR2000/001007 patent/WO2000062789A1/fr not_active Ceased
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  • 2000-04-18 EP EP00920831A patent/EP1171143B9/fr not_active Expired - Lifetime
  • 2000-04-18 DE DE60015112T patent/DE60015112T8/de active Active
  • 2000-04-18 PT PT00920831T patent/PT1171143E/pt unknown
  • 2000-04-18 KR KR1020017013229A patent/KR100818010B1/ko not_active Expired - Fee Related
  • 2000-04-18 ES ES00920831T patent/ES2228500T3/es not_active Expired - Lifetime
  • 2000-04-18 AT AT00920831T patent/ATE279934T1/de not_active IP Right Cessation
• 2003
  • 2003-10-03 US US10/678,884 patent/US7029713B2/en not_active Expired - Lifetime
• 2004
  • 2004-11-12 US US10/985,988 patent/US7638149B2/en not_active Expired - Fee Related

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