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EP2178538A2 - Anti-inflammatory dermatological composition comprising corticosteroids and hyaluronate fragments, and uses thereof - Google Patents

Anti-inflammatory dermatological composition comprising corticosteroids and hyaluronate fragments, and uses thereof

Info

Publication number
EP2178538A2
EP2178538A2 EP08774984A EP08774984A EP2178538A2 EP 2178538 A2 EP2178538 A2 EP 2178538A2 EP 08774984 A EP08774984 A EP 08774984A EP 08774984 A EP08774984 A EP 08774984A EP 2178538 A2 EP2178538 A2 EP 2178538A2
Authority
EP
European Patent Office
Prior art keywords
composition according
fragments
kda
hyaluronate
molecular weight
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP08774984A
Other languages
German (de)
French (fr)
Inventor
Christine Chaumont
Jean-Hilaire Saurat
Gürkan KAYAN
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Pierre Fabre Dermo Cosmetique SA
Original Assignee
Pierre Fabre Dermo Cosmetique SA
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Filing date
Publication date
Application filed by Pierre Fabre Dermo Cosmetique SA filed Critical Pierre Fabre Dermo Cosmetique SA
Publication of EP2178538A2 publication Critical patent/EP2178538A2/en
Withdrawn legal-status Critical Current

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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
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/57—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone
    • A61K31/573—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone substituted in position 21, e.g. cortisone, dexamethasone, prednisone or aldosterone
    • 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
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/57—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone
    • 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
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin
    • 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
    • A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
    • A61K31/728—Hyaluronic acid
    • 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
    • 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
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to a dermatological composition comprising corticosteroids and hyaluronate fragments, as well as its uses.
  • Hyaluronate is the major component of the extracellular matrix and is found in significant amounts in the skin.
  • HA is a linear glycosaminoglycan nonsulphate composed of repetitive units of D-glucuronic acid and N-acetyl-D-glucosamine (Tammi R., Agren UM., Tuhkanen AL., Tammi M. Hyaluronan metabolism in skin. Prog Histochem & Cytochem 29: 1-81, 1994).
  • HA is synthesized primarily by dermal fibroblasts and epidermal keratinocytes (Tammi R., Agren UM., Tuhkanen AL., Tammi M. Hyaluronan metabolism in skin, Prog Histochem & Cytochem 29: 1-81, 1994 ). With its residues bearing a negative charge, the HA acts as a water pump to maintain the viscoelasticity of the skin. HA has a major role in controlling the diffusion of food, hormones, vitamins and inorganic salts of connective tissue and in cleaning up metabolic waste that can induce inflammatory reactions. With age, the amount of HA and its level of polymerization decrease, resulting in a decrease in the amount of water retained in the connective tissue.
  • HA exists as a high molecular weight polymer (600-1000 kDa).
  • the physiological degradation of HA in the skin is done by (i) internalization by keratinocytes via CD44 and (ii) intracellular fragmentation into smaller fragments by hyaluronidases.
  • the fragmented HA is released by the keratinocytes, passes the basement membrane and is released directly into the lymphatic vessels (Tammi R., Agren UM, Tuhkanen AL, Tammi M. Hyaluronan metabolism in skin, Prog Histochem & Cytochem 29: 1- 81.1994).
  • HA In inflammatory conditions, the accumulation of low molecular weight forms of HA has been demonstrated.
  • platelet chemotactic factors such as fibrin stimulate the influx and activation of fibroblasts that degrade HA by hyaluronidase secretion resulting in high tissue concentrations of small HA fragments.
  • the generation of these small fragments of HA is also done by a variety of other mechanisms such as depolymerization by reactive oxygen species released by granulocytes or in irradiated skin by ultraviolet light, or de novo synthesis of fragments of low molecular weight.
  • HA high and low molecular weight HA may have different biological effects on cells and tissues (McKee CM, Penno MB, Cowman M., Burdick MD, Strieter RM, Bao CI Noble PW).
  • Hyaluronan (HA) fragments induce chemokine gene expression in alveolar macrophages. The role of HA size and CD44.J Clin Invest 98: 2403-2413, 1996.
  • Termer CC Hennies 1, Voith U., Ahrens T., Weiss JM.
  • CD44 the principal receptor of HA, is a polymorphic transmembrane glycoprotein that has several isoforms generated by alternative splicing and post-translational modifications.
  • CD44 two major functions of CD44 in murine skin are (i) regulation of keratinocyte proliferation in response to extracellular stimuli and (ii) maintenance of local homeostasis of HA (Kaya G., Rodriguez L. , Jorcano JL., Vassalli P., Stamenkovic I. Selective suppression of CD44 in keratinocytes of mice bearing an antisense CD44 transgene driven by a tissue-specific promoter disrupts hyaluronate metabolism in the skin and irregular keratinocyte proliferation. Genes Dev 11: 996-1007, 1997). A decrease in epidermal CD44 expression in patients with Sclero-atrophic lichen was also observed.
  • the authors of the present invention have surprisingly found that it is possible to prevent the occurrence of atrophy of the skin by the concomitant use of a corticosteroid and fragments of HA of molecular weight between 20 and 50OkDa.
  • these HA fragments should also inhibit the other effects of corticosteroids, whose main therapeutic effect is the anti-inflammatory effect.
  • the present invention allows somehow dissociation of the therapeutic effect of the major side effect of topical corticosteroids. It therefore allows the use of a single topical preparation composed of the combination of HA fragments and a corticosteroid.
  • dissociation of the therapeutic effect and the side effect therefore means reducing or even eliminating the atrophying properties of the corticosteroid while preserving its anti-inflammatory effect.
  • the invention therefore relates more precisely to an anti-inflammatory dermatological composition intended for topical administration, characterized in that it comprises 0.005 to 0.1%, preferably 0.01 to 0.05% by weight of a corticoid and 0.1 to 1%, preferably 0.5 to 1% by weight of hyaluronate fragments of average molecular weight between 20 and 500 kDa, preferably between 20 and 375 kDa, more preferably between 20 and 150 kDa.
  • anti-inflammatory is intended to mean the conventional inhibition of topical manifestations such as redness, edema, vesicles, pain and pruritus, which are induced by a large number of patients. pathologies at the level of the skin and which are attenuated by the application of topical corticosteroids.
  • the term "potentiator” is intended to mean avoiding the main side effect of topical corticosteroid such as cutaneous atrophy, while obtaining a better anti-inflammatory effect than that which would be obtained with the same amount of topical corticosteroid alone, or the same anti-inflammatory effect as that which would be obtained with a smaller amount of corticosteroid.
  • the hyaluronate fragments of the present invention are capable of being obtained by heat treatment at a temperature above 100 ° C. of high molecular weight sodium hyaluronate fibers.
  • the hyaluronate fragments are also capable of being obtained by ultrasonic treatment of high molecular weight sodium hyaluronate fibers for 10 to 90 minutes, advantageously 45 minutes, at 400W and at 4 ° C., followed by filtration on gel, advantageously on Sephacryl S-400 gel.
  • the composition according to the invention advantageously comprises 0.05% by weight of a corticoid and 0.5% by weight of hyaluronate fragments.
  • composition according to the invention advantageously comprises 0.01% by weight of a corticoid and 1% by weight of hyaluronate fragments.
  • the corticoid may advantageously be chosen from alclometasone dipropionate, amcinonide, beclometasone dipropionate, betamethasone benzoate, betamethasone dipropionate, betamethasone valerate, budesonide, clobetasol propionate and clobetasol butyrate.
  • the corticosteroid is advantageously clobetasol propionate.
  • the invention also relates to a pharmaceutical composition comprising a composition as defined above and one or more pharmaceutically acceptable excipients.
  • the pharmaceutical composition according to the invention advantageously comprises a pharmaceutically acceptable emollient base.
  • the term “emollient base” refers to any cosmetic product which contributes to relaxing the tissues, calming inflammation and softening the skin.
  • the pharmaceutical composition according to the invention also advantageously comprises other dermato excipients logically acceptable for its presentation in the form of cream, balm, gel, spray, ointment, lotion, film-forming solution, transdermal system, for example a patch, mousse, shampoo.
  • the subject of the invention is also a composition according to any one of the preceding claims as a medicament, advantageously intended to treat inflammatory dermatoses which are commonly listed as indications of topical corticosteroids, and more particularly those which are localized on the fragile zones. such as the face, areas where the side effects of topical corticosteroids are particularly marked. Indeed, thanks to the dissociation of the therapeutic effect and the major side effect of topical corticosteroids, and to the potentiation of the anti-inflammatory effect, these fragile areas can be treated at a lower level. risk.
  • the subject of the invention is also a combination product comprising, on the one hand, a corticoid in the form of cream and, on the other hand, hyaluronate fragments of average molecular weight between 20 and 500 kDa, advantageously between 20 and 375 kDa, more preferably between 20 and 150 kDa, also in cream form, for separate dermatological use, simultaneous or spread over time, in inflammatory dermatitis therapy.
  • Hairless SKH1 mice received 2 times daily for 5 days a topical treatment on the back with a steroid (0.05% clobetasol propionate or 0.1% desonide) with or without HA fragments (molecular weight between 20%). and 500 kDa, and obtained by the method comprising the sonication and filtration steps described above). These fragments will be designated in the following examples by HAF.
  • the dermal and epidermal atrophy and the cutaneous hyaluronate concentration were respectively determined by the measurement of the dermis-epidermal thickness by optical microscopy and by ELISA.
  • TPA-Induced Inflammation in Mouse Ear Skin inflammation was induced by topical application of 0.005% TPA (12-o-tetradecanoylphorbol-13-acetate) in acetone to the ears of C57B1 / 6 mice; the control animals received the same volume of acetone.
  • Clobetasol propionate (0.05%) and HAF (1%) were dissolved in 100 ⁇ l of vehicle, and were applied together with TPA for 4 days; the control animals received the same vehicle volume.
  • Inflammation was determined by measuring the thickness of the ears by a clip and dermal-epidermal thickness in light microscopy and by the assay of myeloperoxidase activity. The animals were sacrificed 24 hours after the last application. Biopsies of 6 mm were taken, frozen in liquid nitrogen and then stored at -70 ° C. until the day of the analysis. The rest of the tissue was fixed with formalin and analyzed by immunohistology.
  • Myeloperoxidase activity was determined in the supernatant of homogenates of ear biopsies.
  • the biopsies immersed in 1.5 ml of 50 mM sodium phosphate buffer pH 6.0 containing 0.5% of hexadecyltrimethylammonium bromide (HTAB), were milled for 45 seconds at 0 ° C. in a Polytron PT 1200 homogenizer.
  • the enzymatic activity of myeloperoxidase was determined according to the method of Bradley et al. modified for the use of the photometric plate reader.
  • the following reagents were added in wells of 96-well plates: 50 ⁇ l of supernatant, 50 ⁇ l of phosphate buffer + HTAB, 50 ⁇ l of o-dianisidine 0.68 mg / ml dissolved in water; the reaction was initiated by the addition of 0.003% hydrogen peroxide prepared extemporaneously. The density optical was measured at 450 nm. The enzymatic activity was compared to that of the ears biopsies treated only with TPA. The expression of CD44, CD44v3 and pro-HB-EGF was analyzed by immunohistochemistry and western blotting according to methods already described (PLoS Med 3 (12): e493, 2006).
  • the epidermal and cutaneous thicknesses were measured by an ocular micrometer. Ten measurements were performed per mouse. The results are summarized in Table 1 below.
  • the prevention index is the ratio between control treated with clobetasol propionate (PC) alone and PC + HAF composition.
  • Figure 1 shows histological sections of the dermis and epidermis of hematoxylin-eosin stained mice.
  • the epidermal thickness was measured by an ocular micrometer after treatment at different concentrations of desonide. Ten measurements were performed per mouse. The results are shown in Table 2.
  • the prevention index is the ratio, at a given corticoid concentration, between the control treated with the corticosteroid alone and with the corticosteroid + HAF composition. Table 2
  • the epidermal thickness was measured by an ocular micrometer after treatment with different corticosteroids. Ten measurements were performed per mouse. The results are collated in Table 3.
  • the prevention index is the ratio between control treated with corticosteroid alone and the corresponding composition including HAF. Table 3.
  • HAFs prevent epidermal atrophy induced by various topical corticosteroids (CS).
  • Untreated hyaluronic acid, fragments obtained by the action of hyaluronidase, as well as HAF were compared for their preventive effects.
  • the epidermal thickness was measured by an ocular micrometer. Ten measurements were performed per mouse. The results are shown in Table 4.
  • the prevention index is the ratio between the control treated with the corticosteroid alone and each corticosteroid composition + HAF. Table 4
  • hyaluronidase-prepared fragments do not prevent epidermal atrophy induced by clobetasol propionate or desonide.
  • Figure 2 is an immunohistochemical analysis of mouse sections by anti-CD44. It shows that HAFs restore and enhance the expression of CD44 in mouse skin treated with clobetasol propionate.
  • Figure 3 is a Western blot analysis of mouse skin protein extracts with an anti-CD44v3 antibody. It shows that the fragments of HAF restore and increase the expression of CD44v3 in mouse skin treated with desonide and thus the potentiating effect of HAF.
  • Figure 4 is a western blot analysis of mouse skin protein extracts with 25 kDa anti-pro-HB-EGF antibody, A representing vehicle, B, clobetasol propionate and C clobetasol propionate + HAF. It shows that HAFs restore and enhance expression of pro-HB-EGF in mouse skin treated with clobetasol propionate.
  • the epidermal inflammation induced by the application of Phorbol TPA ester was measured after application of TPA, then after treatment with clobetasol propionate, and after treatment with the composition comprising clobetasol propionate and HAF. Ten measurements were performed per mouse. The results are summarized in Table 5 below.
  • the anti-inflammation index is the ratio between the control treated with TPA, and the composition TPA + PC or TPA + PC + HAF.
  • the dermal inflammation induced by the application of Phorbol TPA ester was measured after application of TPA, then after treatment with clobetasol propionate, and after treatment with the composition comprising clobetasol propionate and HAF. Ten measurements were performed per mouse. The results are collated in Table 6 below.
  • the anti-inflammation index is the ratio between the control treated with TPA, and the association TPA + PC or TPA + PC + HAF.
  • the dermal cellularity induced by the application of Phorbol TPA ester was measured after application of TPA, then after treatment with clobetasol propionate, and after treatment with the composition comprising clobetasol propionate and HAF. Ten measurements were performed per mouse. The results are shown in Table 7.
  • the anti-inflammation index is the ratio between the control treated with TPA, and the association TPA + PC or TPA + PC + HAF.
  • the cutaneous myeloperoxidase activity induced by the application of Phorbol TPA ester was measured, after application of TPA, then after treatment with clobetasol propionate, and after treatment with the composition comprising clobetasol propionate and HAF. Ten measurements were performed per mouse. The results are summarized in Table 8.
  • the anti-inflammation index is the ratio between the control treated with TPA, and the association TPA + PC or TPA + PC + HAF.
  • HAFs do not inhibit the anti-inflammatory effect of clobetasol propionate but, on the contrary, potentiate its anti-inflammatory effect.

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  • Pain & Pain Management (AREA)
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Abstract

Anti-inflammatory dermatological composition for topical administration, characterized in that it comprises 0.005% to 0.1%, preferably 0.01% to 0.05% by weight of a corticosteroid and 0.1% to 1%, preferably 0.5% to 1% by weight of hyaluronate fragments with an average molecular weight of between 20 and 500 kDa, preferably between 20 and 375 kDa, more preferentially between 20 and 150 kDa.

Description

COMPOSITION DERMATOLOGIQUE ANTI-INFLAMMATOIRE ANTI-INFLAMMATORY DERMATOLOGICAL COMPOSITION
COMPRENANT DES CORTICOÏDES ET DES FRAGMENTS DECOMPRISING CORTICOIDS AND FRAGMENTS OF
HYALURONATE, ET SES UTILISATIONS.HYALURONATE, AND USES THEREOF
La présente invention a pour objet une composition dermatologique comprenant des corticoïdes et des fragments de hyaluronate, ainsi que ses utilisations.The present invention relates to a dermatological composition comprising corticosteroids and hyaluronate fragments, as well as its uses.
Le hyaluronate (HA) est la composante majeure de la matrice extracellulaire et se trouve en quantités importantes dans la peau. Le HA est un glycosaminoglycan linéaire nonsulphate composé d'unités répétitives de D-acide glucuronique et de N- acétyl-D-glucosamine (Tammi R., Agren UM., Tuhkanen AL., Tammi M. Hyaluronan metabolism in skin. Prog Histochem & Cytochem 29:1-81, 1994).Hyaluronate (HA) is the major component of the extracellular matrix and is found in significant amounts in the skin. HA is a linear glycosaminoglycan nonsulphate composed of repetitive units of D-glucuronic acid and N-acetyl-D-glucosamine (Tammi R., Agren UM., Tuhkanen AL., Tammi M. Hyaluronan metabolism in skin. Prog Histochem & Cytochem 29: 1-81, 1994).
Dans la peau normale, le HA est synthétisé essentiellement par les fïbroblastes dermiques et les kératinocytes épidermiques (Tammi R., Agren UM., Tuhkanen AL., Tammi M. Hyaluronan metabolism in skin. Prog Histochem & Cytochem 29:1-81, 1994). Grâce à ses résidus portant une charge négative, le HA joue le rôle d'une pompe à eau permettant de maintenir la viscoélasticité de la peau. Le HA a un rôle principal dans le contrôle de la diffusion des aliments, des hormones, des vitamines et des sels inorganiques du tissu conjonctif et dans le nettoyage des déchets métaboliques pouvant induire des réactions inflammatoires. Avec l'âge, la quantité de HA et son niveau de polymérisation diminuent, résultant en une diminution de la quantité d'eau retenue dans le tissu conjonctif. La peau subit alors un processus de vieillissement qui aboutit à une augmentation de la fïbrose et à une baisse de la teneur en fibres élastiques. Dans la peau normale, le HA existe comme un polymère de haut poids moléculaire (600 - 1 000 kDa). La dégradation physiologique du HA dans la peau se fait par (i) l'internalisation par les kératinocytes via le CD44 et (ii) la fragmentation intracellulaire en fragments de taille plus faible par les hyaluronidases. Le HA fragmenté est relâché par les kératinocytes, passe la membrane basale et est libéré directement dans les vaisseaux lymphatiques (Tammi R., Agren UM., Tuhkanen AL., Tammi M. Hyaluronan metabolism in skin. Prog Histochem & Cytochem 29:1- 81,1994). Dans des conditions inflammatoires, l'accumulation des formes de bas poids moléculaire de HA a été démontrée. Pendant l'inflammation, des facteurs chimiotactiques plaquettaires comme de la fibrine stimulent l'afflux et l'activation des fîbroblastes qui dégradent le HA par sécrétion de hyaluronidase résultant en des concentrations tissulaires élevées de petits fragments de HA. La génération de ces petits fragment du HA se fait aussi par une variété d'autres mécanismes comme la dépolymérisation par les espèces réactives d'oxygène relâchées par les granulocytes ou dans la peau irradiée par les ultraviolets, ou la synthèse de novo de fragments de bas poids moléculaire. Plusieurs études ont suggéré que le HA de haut et bas poids moléculaire peut avoir des effets biologiques différents sur les cellules et les tissus (McKee CM., Penno MB., Cowman M., Burdick MD., Strieter RM.I Bao CI Noble PW. Hyaluronan (HA) fragments induce chemokine gène expression in alveolar macrophages. The rôle of HA size and CD44. J Clin Invest 98:2403-2413, 1996; Termeer CC, Hennies 1, Voith U., Ahrens T., Weiss JM., Prehm P., Simon JC. Oligosaccharides of hyaluronan are potent activators of dendritic cells. J Immunol 165:1863-1870, 2000; Fitzgerald KA., Bowie AG., Skeffmgton BS., O'Neill LA. Ras, protein kinase C zêta, and I kappa B kinases 1 and 2 are downstream effectors of CD44 during the activation of NF-kappa B by hyaluronic acid fragments in T-24 carcinoma cells. J Immunol 164:2053-2063, 2000). II a été démontré (FR 04 00826) que le HA non sulfaté hydrolyse en des fragments de poids moléculaires compris entre 50 et 750 kDa possède une activité biologique sur la peau, notamment une augmentation du renouvellement épidermique, de l'expression du CD44 épidermique et du dépôt de matrice extracellulaire, qui est amplifiée lorsque ces fragments sont associés à un rétinoïde. Le CD44, le récepteur principal du HA, est une glycoprotéine transmembranaire polymorphique qui a plusieurs isoformes générées par l'épissage alternatif et les modifications post-traductionnelles. Il a été démontré que deux fonctions majeures du CD44 dans la peau murine sont (i) la régulation de la prolifération keratinocytaire en réponse à des stimuli extracellulaires et (ii) le maintien de l'homéostase locale du HA (Kaya G., Rodriguez L, Jorcano JL., Vassalli P., Stamenkovic I. Sélective suppression of CD44 in keratinocytes of mice bearing an antisense CD44 transgene driven by a tissue-specifîc promoter disrupts hyaluronate metabolism in the skin and impairs keratinocyte prolifération. Gènes Dev 11 :996-1007, 1997). Une diminution de l'expression du CD44 épidermique chez les patients atteints de lichen scléro-atrophique a également été observée. Cette diminution est potentiellement responsable de la déposition dermique du HA et de l'atrophie épidermique dans cette maladie (Kaya G., Augsburger E., Stamenkovic L, Saurat JH. Decrease in epidermal CD44 expression as a potential mechanism for abnormal hyaluronate accumulation in superfïcial dermis in lichen sclerosus et atrophicus. J InvestDermatol 115:1054-1058, 2000). Il a été récemment démontré (i) que la réponse proliférative in vitro et in vivo des kératinocytes induite par les fragments de HA de taille intermédiaire suit une voie dépendante du CD44 et requiert la présence de heparin-binding epidermal growth factor (HB-EGF), erbBl, et des metalloproteinases matricielles, et (ii) que les fragments de HA de taille intermédiaire pourraient constituer une base pour le développement de nouvelles thérapies de l'atrophie cutanée humaine (Kaya G., Tran C, Sorg O., Hotz R., Grand D., Carraux P., Didierjean L., Stamenkovic L, Saurat J.-H. Hyaluronate fragments reverse skin atrophy by a CD44-dependent mechanism. PLoS Med 3 (12) : e493, 2006).In normal skin, HA is synthesized primarily by dermal fibroblasts and epidermal keratinocytes (Tammi R., Agren UM., Tuhkanen AL., Tammi M. Hyaluronan metabolism in skin, Prog Histochem & Cytochem 29: 1-81, 1994 ). With its residues bearing a negative charge, the HA acts as a water pump to maintain the viscoelasticity of the skin. HA has a major role in controlling the diffusion of food, hormones, vitamins and inorganic salts of connective tissue and in cleaning up metabolic waste that can induce inflammatory reactions. With age, the amount of HA and its level of polymerization decrease, resulting in a decrease in the amount of water retained in the connective tissue. The skin then undergoes an aging process which results in an increase in fibrosis and a decrease in the content of elastic fibers. In normal skin, HA exists as a high molecular weight polymer (600-1000 kDa). The physiological degradation of HA in the skin is done by (i) internalization by keratinocytes via CD44 and (ii) intracellular fragmentation into smaller fragments by hyaluronidases. The fragmented HA is released by the keratinocytes, passes the basement membrane and is released directly into the lymphatic vessels (Tammi R., Agren UM, Tuhkanen AL, Tammi M. Hyaluronan metabolism in skin, Prog Histochem & Cytochem 29: 1- 81.1994). In inflammatory conditions, the accumulation of low molecular weight forms of HA has been demonstrated. During inflammation, platelet chemotactic factors such as fibrin stimulate the influx and activation of fibroblasts that degrade HA by hyaluronidase secretion resulting in high tissue concentrations of small HA fragments. The generation of these small fragments of HA is also done by a variety of other mechanisms such as depolymerization by reactive oxygen species released by granulocytes or in irradiated skin by ultraviolet light, or de novo synthesis of fragments of low molecular weight. Several studies have suggested that high and low molecular weight HA may have different biological effects on cells and tissues (McKee CM, Penno MB, Cowman M., Burdick MD, Strieter RM, Bao CI Noble PW). Hyaluronan (HA) fragments induce chemokine gene expression in alveolar macrophages.The role of HA size and CD44.J Clin Invest 98: 2403-2413, 1996. Termer CC, Hennies 1, Voith U., Ahrens T., Weiss JM. , Prehm P., Simon JC, Oligosaccharides of Hyaluronan are Potent Activators of Dendritic Cells Immunol 165: 1863-1870, 2000, Fitzgerald KA, Bowie AG, Skeffmgton BS, O'Neill LA, Ras, Protein Kinase C zeta, and I kappa B kinases 1 and 2 are downstream effects of CD44 during the activation of NF-kappa B by hyaluronic acid fragments in T-24 carcinoma cells (Immunol 164: 2053-2063, 2000). It has been demonstrated (FR 04 00826) that the non-sulphated HA hydrolyzed to molecular weight fragments of between 50 and 750 kDa has a biological activity on the skin, in particular an increase in epidermal renewal, the expression of epidermal CD44 and extracellular matrix deposition, which is amplified when these fragments are associated with a retinoid. CD44, the principal receptor of HA, is a polymorphic transmembrane glycoprotein that has several isoforms generated by alternative splicing and post-translational modifications. It has been shown that two major functions of CD44 in murine skin are (i) regulation of keratinocyte proliferation in response to extracellular stimuli and (ii) maintenance of local homeostasis of HA (Kaya G., Rodriguez L. , Jorcano JL., Vassalli P., Stamenkovic I. Selective suppression of CD44 in keratinocytes of mice bearing an antisense CD44 transgene driven by a tissue-specific promoter disrupts hyaluronate metabolism in the skin and irregular keratinocyte proliferation. Genes Dev 11: 996-1007, 1997). A decrease in epidermal CD44 expression in patients with Sclero-atrophic lichen was also observed. This decrease is potentially responsible for the dermal deposition of HA and epidermal atrophy in this disease (Kaya G., Augsburger E., Stamenkovic L, Saurat JH, Decrease in epidermal CD44 expression as a potential mechanism for abnormal hyaluronate accumulation in superficial dermis in lichen sclerosus and atrophicus J InvestDermatol 115: 1054-1058, 2000). It has been recently demonstrated that (i) the in vitro and in vivo proliferative response of keratinocytes induced by intermediate size HA fragments follows a CD44 dependent pathway and requires the presence of heparin-binding epidermal growth factor (HB-EGF) , erbBl, and matrix metalloproteinases, and (ii) that intermediate-sized HA fragments could provide a basis for the development of novel therapies for human cutaneous atrophy (Kaya G., Tran C, Sorg O., Hotz R , Grand D., Carraux P., Didierjean L., Stamenkovic L., Saurat J.-H. Hyaluronate fragments reverse skin atrophy by a CD44-dependent mechanism.PLoS Med 3 (12): e493, 2006).
Il a par ailleurs été récemment démontré que contrairement aux fragments de petites tailles (1 - 50 kDa) ou grandes tailles (400 - 1 000 kDa), les fragments de HA de taille intermédiaire induisent une hyperplasie épidermique significative et une prolifération kératinocytaire, une augmentation de l'expression de CD44 et de HA épidermiques et dermiques, ainsi qu'une altération de la structure dermique et une augmentation de sa cellularité chez les souris SKHl hairless et DBA/1. Il a enfin également été démontré que le rétinaldehyde prévient l'atrophie épidermique induite par un corticostéroïde, le propionate de clobétasol, chez les souris SKHl hairless (Kaya G., Tran C, Sorg O., Grand D., Hotz R., Carraux P., Didierjean L., Saurat J.- H. Prévention of corticosteroid-induced skin atrophy by rétinaldehyde in mouse. JEADV 19: 124, 2005).It has also been recently demonstrated that unlike small fragments (1-50 kDa) or large sizes (400-1000 kDa), intermediate-sized fragments of HA induce significant epidermal hyperplasia and keratinocyte proliferation, an increase the expression of epidermal and dermal CD44 and HA, as well as an alteration of the dermal structure and an increase in its cellularity in SKH1 hairless and DBA / 1 mice. Finally, retinaldehyde has also been shown to prevent corticosteroid-induced epidermal atrophy, clobetasol propionate, in hairless SKH1 mice (Kaya G., Tran C., Sorg O., Grand D., Hotz R., Carraux). P., Didierjean L., Saurat J.- H. Prevention of corticosteroid-induced skin atrophy by retinaldehyde in mouse, JEADV 19: 124, 2005).
Il avait été observé précédemment que des fragments de HA de taille intermédiaire permettaient la réparation d'une atrophie déjà établie, due au vieillissement, et aggravée par l'usage de corticoïdes utilisés par voie systémique au long cours (Kaya G., Tran C, Sorg O., Hotz R., Grand D., Carraux P., Didierjean L., Stamenkovic L, Saurat J.-H. Hyaluronate fragments reverse skin atrophy by a CD44-dependent mechanism. PLoS Med 3 (12) : e493, 2006 et brevet FR 04 00826).It has previously been observed that fragments of intermediate-sized HA allow the repair of an already established atrophy, due to aging, and aggravated by the use of corticosteroids used systemically in the long term (Kaya G., Tran C, Sorg O., Hotz R., Grand D., Carraux P., Didierjean L., Stamenkovic L, Saurat J.-H. Hyaluronate fragments reverse skin atrophy by a CD44-dependent mechanism. PLoS Med 3 (12): e493, 2006 and patent FR 04,00826).
Les auteurs de la présente invention ont découvert, de manière surprenante, qu'il est possible de prévenir la survenue de l'atrophie de la peau par l'utilisation concomitante d'un corticoïde et de fragments de HA de poids moléculaire compris entre 20 et 50OkDa.The authors of the present invention have surprisingly found that it is possible to prevent the occurrence of atrophy of the skin by the concomitant use of a corticosteroid and fragments of HA of molecular weight between 20 and 50OkDa.
Dès lors qu'ils exercent une inhibition de l'effet atrophiant des corticoïdes, ces fragments de HA devraient également inhiber les autres effets des corticoïdes, dont le principal effet thérapeutique recherché, à savoir l'effet anti-inflammatoire.Since they exert an inhibition of the atrophying effect of corticosteroids, these HA fragments should also inhibit the other effects of corticosteroids, whose main therapeutic effect is the anti-inflammatory effect.
Les auteurs de la présente invention ont découvert, de manière surprenante, qu'au contraire, l'utilisation concomitante d'un corticoïde et de ces fragments de HA n'annule pas l'effet anti-inflammatoire du corticoïde.The authors of the present invention have discovered, surprisingly, that on the contrary, the concomitant use of a corticosteroid and these fragments of HA does not cancel the anti-inflammatory effect of the corticoid.
Ainsi la présente invention permet en quelque sorte la dissociation de l'effet thérapeutique de l'effet secondaire majeur des corticoïdes topiques. Elle permet donc l'utilisation d'une seule préparation topique composée de l'association des fragments de HA et d'un corticoïde.Thus the present invention allows somehow dissociation of the therapeutic effect of the major side effect of topical corticosteroids. It therefore allows the use of a single topical preparation composed of the combination of HA fragments and a corticosteroid.
On entend donc par "dissociation de l'effet thérapeutique et de l'effet secondaire" le fait de diminuer, voire supprimer, les propriétés atrophiantes du corticoïde tout en préservant son effet anti-inflammatoire.The term "dissociation of the therapeutic effect and the side effect" therefore means reducing or even eliminating the atrophying properties of the corticosteroid while preserving its anti-inflammatory effect.
De plus, de manière encore plus surprenante, une potentialisation de l'effet thérapeutique anti-inflammatoire a même été observée.In addition, even more surprisingly, potentiation of the anti-inflammatory therapeutic effect has even been observed.
L'invention concerne donc plus précisément une composition dermatologique anti-inflammatoire destinée à une administration topique, caractérisée en ce qu'elle comprend 0,005 à 0,1 %, de préférence 0,01 à 0,05 % en poids d'un corticoïde et 0,1 à 1%, de préférence 0,5 à 1 % en poids de fragments de hyaluronate de poids moléculaire moyen compris entre 20 et 500 kDa, de préférence entre 20 et 375 kDa, plus préférentiellement entre 20 et 150 kDa.The invention therefore relates more precisely to an anti-inflammatory dermatological composition intended for topical administration, characterized in that it comprises 0.005 to 0.1%, preferably 0.01 to 0.05% by weight of a corticoid and 0.1 to 1%, preferably 0.5 to 1% by weight of hyaluronate fragments of average molecular weight between 20 and 500 kDa, preferably between 20 and 375 kDa, more preferably between 20 and 150 kDa.
On entend qualifier par "anti-inflammatoire", au sens de la présente invention, le fait d'inhiber par voie topique les manifestations classiques telles que rougeur, œdème, vésicules, douleur et prurit, qui sont induits par un grand nombre de pathologies au niveau de la peau et qui sont atténuées par l'application de corticoïdes topiques.For the purposes of the present invention, the term "anti-inflammatory" is intended to mean the conventional inhibition of topical manifestations such as redness, edema, vesicles, pain and pruritus, which are induced by a large number of patients. pathologies at the level of the skin and which are attenuated by the application of topical corticosteroids.
On entend qualifier par "potentialisateur", au sens de la présente invention, le fait d'éviter l'effet secondaire principal du corticoïde topique tel que l'atrophie cutanée, tout en obtenant un meilleur effet anti-inflammatoire que celui qui serait obtenu avec la même quantité de corticoïde topique seul, ou le même effet antiinflammatoire que celui qui serait obtenu avec une quantité de corticoïde plus faible.For the purpose of the present invention, the term "potentiator" is intended to mean avoiding the main side effect of topical corticosteroid such as cutaneous atrophy, while obtaining a better anti-inflammatory effect than that which would be obtained with the same amount of topical corticosteroid alone, or the same anti-inflammatory effect as that which would be obtained with a smaller amount of corticosteroid.
Les fragments de hyaluronate de la présente invention sont susceptibles d'être obtenus par traitement thermique à une température supérieure à 1000C de fibres de hyaluronate de sodium de haut poids moléculaire.The hyaluronate fragments of the present invention are capable of being obtained by heat treatment at a temperature above 100 ° C. of high molecular weight sodium hyaluronate fibers.
Les fragments de hyaluronate sont également susceptibles d'être obtenus par traitement aux ultrasons de fibres de hyaluronate de sodium de haut poids moléculaire, pendant 10 à 90 minutes, avantageusement 45 minutes, à 400W et à 4° C, suivi d'une filtration sur gel, avantageusement sur gel Sephacryl S-400. La composition selon l'invention comprend avantageusement 0,05% en poids d'un corticoïde et 0,5% en poids de fragments de hyaluronate.The hyaluronate fragments are also capable of being obtained by ultrasonic treatment of high molecular weight sodium hyaluronate fibers for 10 to 90 minutes, advantageously 45 minutes, at 400W and at 4 ° C., followed by filtration on gel, advantageously on Sephacryl S-400 gel. The composition according to the invention advantageously comprises 0.05% by weight of a corticoid and 0.5% by weight of hyaluronate fragments.
La composition selon l'invention comprend avantageusement 0,01% en poids d'un corticoïde et 1% en poids de fragments de hyaluronate.The composition according to the invention advantageously comprises 0.01% by weight of a corticoid and 1% by weight of hyaluronate fragments.
Le corticoïde peut être avantageusement choisi parmi le dipropionate d'alclométasone, l'amcinonide, le dipropionate de béclométasone, le benzoate de bétaméthasone, le dipropionate de bétaméthasone, le valérate de bétaméthasone, le budésonide, le propionate de clobétasol, le butyrate de clobétasol, le désonide, la désoximétasone, la dexaméthasone, le diacétate dédiflorasone, le valérate de diflucortolone, la flurandrénolone, l'acétate de fluprednidène, le fluocortolone, le butyle de fluocortine, le fluocinonide, l'acétonide de fluocinolone, l'acétonide de fluclorolone, le pyvalate de flumétasone, le chlorhydrate de feudiline, la flumétholone, l'halcinonide, l'hydrocortisone, l'acétate d'hydrocortisone, le butyrate d'hydrocortisone, le valérate d'hydrocortisone, l'acétate de méthylprednisolone, le furoate de mométasone, la méthylprednisolone, la prednisolone, l'acétonide de triamcinolone ainsi que leurs mélanges.The corticoid may advantageously be chosen from alclometasone dipropionate, amcinonide, beclometasone dipropionate, betamethasone benzoate, betamethasone dipropionate, betamethasone valerate, budesonide, clobetasol propionate and clobetasol butyrate. desonide, deoximetasone, dexamethasone, diacetate dediflorasone, diflucortolone valerate, flurandrenolone, fluprednidene acetate, fluocortolone, fluocortin butyl, fluocinonide, fluocinolone acetonide, fluclorolone acetonide, flumetasone pyvalate, feudiline hydrochloride, flumetholone, halcinonide, hydrocortisone, hydrocortisone acetate, hydrocortisone butyrate, hydrocortisone valerate, methylprednisolone acetate, mometasone furoate , methylprednisolone, prednisolone, triamcinolone acetonide and mixtures thereof.
Le corticoïde est avantageusement le propionate de clobétasol. L'invention a également pour objet une composition pharmaceutique comprenant une composition telle que définie ci-dessus et un ou plusieurs excipients pharmaceutiquement acceptables.The corticosteroid is advantageously clobetasol propionate. The invention also relates to a pharmaceutical composition comprising a composition as defined above and one or more pharmaceutically acceptable excipients.
La composition pharmaceutique selon l'invention comprend avantageusement une base émolliente pharmaceutiquement acceptable.The pharmaceutical composition according to the invention advantageously comprises a pharmaceutically acceptable emollient base.
On entend par "base émolliente", au sens de la présente invention, tout produit cosmétique qui contribue à relâcher les tissus, à calmer l'inflammation et à adoucir la peau.For the purposes of the present invention, the term "emollient base" refers to any cosmetic product which contributes to relaxing the tissues, calming inflammation and softening the skin.
La composition pharmaceutique selon l'invention comprend également avantageusement d'autres excipients dermato logiquement acceptables pour sa présentation sous forme de crème, baume, gel, spray, pommade, lotion, solution filmogène, système transdermique, par exemple un patch, mousse, shampooing.The pharmaceutical composition according to the invention also advantageously comprises other dermato excipients logically acceptable for its presentation in the form of cream, balm, gel, spray, ointment, lotion, film-forming solution, transdermal system, for example a patch, mousse, shampoo.
L'invention a également pour objet une composition selon l'une quelconque des revendications précédentes à titre de médicament, avantageusement destiné à traiter les dermatoses inflammatoires qui sont communément répertoriées comme indications des corticoïdes topiques, et plus particulièrement celles qui sont localisées sur les zones fragiles telles que le visage, zones où les effets secondaires des corticoïdes topiques sont particulièrement marqués. En effet grâce d'une part à la dissociation de l'effet thérapeutique et de l'effet secondaire majeur des corticoïdes topiques, et d'autre part à la potentialisation de l'effet anti-inflammatoire, ces zones fragiles pourront être traitées à moindre risque.The subject of the invention is also a composition according to any one of the preceding claims as a medicament, advantageously intended to treat inflammatory dermatoses which are commonly listed as indications of topical corticosteroids, and more particularly those which are localized on the fragile zones. such as the face, areas where the side effects of topical corticosteroids are particularly marked. Indeed, thanks to the dissociation of the therapeutic effect and the major side effect of topical corticosteroids, and to the potentiation of the anti-inflammatory effect, these fragile areas can be treated at a lower level. risk.
L'invention a également pour objet un produit de combinaison comprenant d'une part un corticoïde sous forme de crème, et d'autre part des fragments de hyaluronate de poids moléculaire moyen compris entre 20 et 500 kDa, avantageusement entre 20 et 375 kDa, plus avantageusement entre 20 et 150 kDa, également sous forme de crème, pour une utilisation dermatologique séparée, simultanée ou étalée dans le temps, en thérapie des dermatoses inflammatoires.The subject of the invention is also a combination product comprising, on the one hand, a corticoid in the form of cream and, on the other hand, hyaluronate fragments of average molecular weight between 20 and 500 kDa, advantageously between 20 and 375 kDa, more preferably between 20 and 150 kDa, also in cream form, for separate dermatological use, simultaneous or spread over time, in inflammatory dermatitis therapy.
Les fragments de hyaluronate sont susceptibles d'être obtenus par l'un ou l'autre des procédés décrits ci-dessus. L'invention va maintenant être illustrée de façon non limitative par les exemples qui suivent. MATERIEL ET METHODESThe hyaluronate fragments are obtainable by any of the methods described above. The invention will now be illustrated in a nonlimiting manner by the following examples. MATERIAL AND METHODS
Protocole d'atrophie cutanéeSkin atrophy protocol
Des souris SKHl hairless ont reçu 2 fois par jour pendant 5 jours un traitement topique sur le dos avec un stéroïde (propionate de clobétasol 0,05% ou désonide 0,1%) avec ou sans fragments de HA (de poids moléculaire compris entre 20 et 500 kDa, et obtenus par le procédé comportant les étapes de traitement aux ultrasons et filtration décrit ci-dessus). Ces fragments seront désignés dans les exemples qui suivent par HAF. L'atrophie dermique et épidermique et la concentration de hyaluronate cutané ont été déterminées respectivement par la mesure de l'épaisseur derme-épiderme en microscopie optique et par ELISA. Inflammation induite par TPA dans l'oreille de souris L'inflammation cutanée a été induite par application topique de TPA (12-o- tetradecanoylphorbol- 13 -acétate) 0,005% dans l'acétone, sur les oreilles de souris C57B1/6; les animaux contrôles ont reçu le même volume d'acétone. Le propionate de clobétasol (0,05%) et les HAF (1%) ont été dissous dans 100 μl de véhicule, et ont été appliqués conjointement au TPA pendant 4 jours; les animaux contrôles ont reçu le même volume de véhicule. L'inflammation a été déterminée par la mesure de l'épaisseur des oreilles par un clip et de l'épaisseur derme-épiderme en microscopie optique et par le dosage de l'activité myéloperoxydase. Les animaux ont été sacrifiés 24 h après la dernière application. Des biopsies de 6 mm ont été prélevées, congelées dans l'azote liquide, puis stockées à -700C jusqu'au jour de l'analyse. Le reste du tissu a été fixé au formol et analysé par immunohistologie.Hairless SKH1 mice received 2 times daily for 5 days a topical treatment on the back with a steroid (0.05% clobetasol propionate or 0.1% desonide) with or without HA fragments (molecular weight between 20%). and 500 kDa, and obtained by the method comprising the sonication and filtration steps described above). These fragments will be designated in the following examples by HAF. The dermal and epidermal atrophy and the cutaneous hyaluronate concentration were respectively determined by the measurement of the dermis-epidermal thickness by optical microscopy and by ELISA. TPA-Induced Inflammation in Mouse Ear Skin inflammation was induced by topical application of 0.005% TPA (12-o-tetradecanoylphorbol-13-acetate) in acetone to the ears of C57B1 / 6 mice; the control animals received the same volume of acetone. Clobetasol propionate (0.05%) and HAF (1%) were dissolved in 100 μl of vehicle, and were applied together with TPA for 4 days; the control animals received the same vehicle volume. Inflammation was determined by measuring the thickness of the ears by a clip and dermal-epidermal thickness in light microscopy and by the assay of myeloperoxidase activity. The animals were sacrificed 24 hours after the last application. Biopsies of 6 mm were taken, frozen in liquid nitrogen and then stored at -70 ° C. until the day of the analysis. The rest of the tissue was fixed with formalin and analyzed by immunohistology.
L'activité myéloperoxydase a été déterminée dans le surnageant des homogénats des biopsies d'oreilles. Les biopsies, immergées dans 1,5 ml de tampon phosphate de sodium 50 mM pH 6,0 contenant 0,5% de bromure d'hexadécyltriméthylammonium (HTAB), ont été broyées 45 secondes à 00C dans un homogéniseur Polytron PT 1200. L'activité enzymatique de la myéloperoxydase a été déterminée selon la méthode de Bradley et al. modifiée pour l'utilisation du lecteur photométrique de plaques. Les réactifs suivants ont été ajoutés dans des puits de plaques à 96 puits : 50 μl de surnageant, 50 μl de tampon phosphate + HTAB, 50 μl de o-dianisidine 0,68 mg/ml dissoute dans l'eau; la réaction a été initiée par l'adjonction de peroxyde d'hydrogène 0,003% préparé extemporanément. La densité optique a été mesurée à 450 nm. L'activité enzymatique a été comparée à celle des biopsies des oreilles traitées seulement au TPA. L'expression du CD44, CD44v3 et pro-HB-EGF a été analysée par immunohistochimie et western blotting selon des méthodes déjà décrites (PLoS Med 3 (12) : e493, 2006).Myeloperoxidase activity was determined in the supernatant of homogenates of ear biopsies. The biopsies, immersed in 1.5 ml of 50 mM sodium phosphate buffer pH 6.0 containing 0.5% of hexadecyltrimethylammonium bromide (HTAB), were milled for 45 seconds at 0 ° C. in a Polytron PT 1200 homogenizer. The enzymatic activity of myeloperoxidase was determined according to the method of Bradley et al. modified for the use of the photometric plate reader. The following reagents were added in wells of 96-well plates: 50 μl of supernatant, 50 μl of phosphate buffer + HTAB, 50 μl of o-dianisidine 0.68 mg / ml dissolved in water; the reaction was initiated by the addition of 0.003% hydrogen peroxide prepared extemporaneously. The density optical was measured at 450 nm. The enzymatic activity was compared to that of the ears biopsies treated only with TPA. The expression of CD44, CD44v3 and pro-HB-EGF was analyzed by immunohistochemistry and western blotting according to methods already described (PLoS Med 3 (12): e493, 2006).
RESULTATSRESULTS
Les épaisseurs épidermique et cutanée (distance entre la couche granuleuse et les glandes sudorales) ont été mesurées par un micromètre oculaire. Dix mesures ont été pratiquées par souris. Les résultats sont rassemblés dans le tableau 1 ci-dessous. L'index de prévention est le rapport entre le contrôle traité par le propionate de clobétasol (PC) seul et la composition PC + HAF.The epidermal and cutaneous thicknesses (distance between the granular layer and the sweat glands) were measured by an ocular micrometer. Ten measurements were performed per mouse. The results are summarized in Table 1 below. The prevention index is the ratio between control treated with clobetasol propionate (PC) alone and PC + HAF composition.
Tableau 1Table 1
La figure 1 montre des coupes histologiques du derme et de l'épiderme de souris colorés par hématoxyline-éosine.Figure 1 shows histological sections of the dermis and epidermis of hematoxylin-eosin stained mice.
Ces résultats démontrent que les HAF préviennent l'atrophie cutanée induite par le propionate de clobétasol (PC).These results demonstrate that HAFs prevent skin atrophy induced by clobetasol propionate (PC).
L'épaisseur épidermique a été mesurée par un micromètre oculaire après traitement à différentes concentrations en désonide. Dix mesures ont été pratiquées par souris. Les résultats sont rassemblés dans le tableau 2. L'index de prévention est le rapport, à une concentration en corticoïde déterminée, entre le contrôle traité par le corticoïde seul et par la composition corticoïde + HAF. Tableau 2The epidermal thickness was measured by an ocular micrometer after treatment at different concentrations of desonide. Ten measurements were performed per mouse. The results are shown in Table 2. The prevention index is the ratio, at a given corticoid concentration, between the control treated with the corticosteroid alone and with the corticosteroid + HAF composition. Table 2
Ces résultats démontrent donc que les HAF préviennent l'atrophie épidermique induite par le désonide de façon dose dépendante.These results therefore demonstrate that HAFs prevent epidermal atrophy induced by desonide in a dose-dependent manner.
L'épaisseur épidermique a été mesurée par un micromètre oculaire après traitement avec différents corticoïdes. Dix mesures ont été pratiquées par souris. Les résultats sont rassemblés dans le tableau 3. L'index de prévention est le rapport entre le contrôle traité par le corticoïde seul et la composition correspondante comprenant les HAF. Tableau 3.The epidermal thickness was measured by an ocular micrometer after treatment with different corticosteroids. Ten measurements were performed per mouse. The results are collated in Table 3. The prevention index is the ratio between control treated with corticosteroid alone and the corresponding composition including HAF. Table 3.
Ces résultats démontrent donc que les HAF préviennent l'atrophie épidermique induite par différents corticostéroïdes topiques (CS). L'acide hyaluronique non traité, les fragments obtenus par action de la hyaluronidase, ainsi que les HAF ont été comparés pour leurs effets préventifs. L'épaisseur épidermique a été mesurée par un micromètre oculaire. Dix mesures ont été pratiquées par souris. Les résultats sont rassemblés dans le tableau 4. L'index de prévention est le rapport entre le contrôle traité par le corticoïde seul et chaque composition corticoïde + HAF. Tableau 4These results therefore demonstrate that HAFs prevent epidermal atrophy induced by various topical corticosteroids (CS). Untreated hyaluronic acid, fragments obtained by the action of hyaluronidase, as well as HAF were compared for their preventive effects. The epidermal thickness was measured by an ocular micrometer. Ten measurements were performed per mouse. The results are shown in Table 4. The prevention index is the ratio between the control treated with the corticosteroid alone and each corticosteroid composition + HAF. Table 4
Contrairement aux HAF, les fragments préparés par l'action de hyaluronidase, ainsi que l'acide hyaluronique non traité, ne préviennent pas l'atrophie épidermique induite par le propionate de clobétasol ou le désonide.In contrast to HAF, hyaluronidase-prepared fragments, as well as untreated hyaluronic acid, do not prevent epidermal atrophy induced by clobetasol propionate or desonide.
La figure 2 est une analyse immunohistochimique des coupes de souris par anti-CD44. Elle montre que les HAF restaurent et augmentent l'expression du CD44 dans la peau de souris traitée par le propionate de clobétasol.Figure 2 is an immunohistochemical analysis of mouse sections by anti-CD44. It shows that HAFs restore and enhance the expression of CD44 in mouse skin treated with clobetasol propionate.
La figure 3 est une analyse par western blot des extraits de protéines de la peau de souris avec un anticorps anti-CD44v3. Elle montre que les fragments de HAF restaurent et augmentent l'expression du CD44v3 dans la peau de souris traitée par le désonide et donc l'effet potentialisateur des HAF. La figure 4 est une analyse par western blot des extraits de protéines de la peau de souris avec un anticorps anti-pro-HB-EGF de 25 kDa, A représentant le véhicule, B, le propionate de clobétasol et C le propionate de clobétasol + HAF. Elle montre que les HAF restaurent et augmentent l'expression du pro-HB-EGF dans la peau de souris traitée par le propionate de clobétasol.Figure 3 is a Western blot analysis of mouse skin protein extracts with an anti-CD44v3 antibody. It shows that the fragments of HAF restore and increase the expression of CD44v3 in mouse skin treated with desonide and thus the potentiating effect of HAF. Figure 4 is a western blot analysis of mouse skin protein extracts with 25 kDa anti-pro-HB-EGF antibody, A representing vehicle, B, clobetasol propionate and C clobetasol propionate + HAF. It shows that HAFs restore and enhance expression of pro-HB-EGF in mouse skin treated with clobetasol propionate.
L'inflammation épidermique induite par l'application d'ester de Phorbol TPA a été mesurée, après application de TPA, puis après traitement au propionate de clobétasol, et après traitement avec la composition comprenant du propionate de clobétasol et des HAF. Dix mesures ont été pratiquées par souris. Les résultats sont rassemblés dans le tableau 5 ci-dessous. L'index d' anti-inflammation est le rapport entre le contrôle traité par TPA, et par la composition TPA + PC ou TPA + PC + HAF.The epidermal inflammation induced by the application of Phorbol TPA ester was measured after application of TPA, then after treatment with clobetasol propionate, and after treatment with the composition comprising clobetasol propionate and HAF. Ten measurements were performed per mouse. The results are summarized in Table 5 below. The anti-inflammation index is the ratio between the control treated with TPA, and the composition TPA + PC or TPA + PC + HAF.
Tableau 5Table 5
L'inflammation dermique induite par l'application d'ester de Phorbol TPA a été mesurée, après application de TPA, puis après traitement au propionate de clobétasol, et après traitement avec la composition comprenant du propionate de clobétasol et des HAF. Dix mesures ont été pratiquées par souris. Les résultats sont rassemblés dans le tableau 6 ci-dessous. L'index d'anti-inflammation est le rapport entre le contrôle traité par TPA, et par l'association TPA + PC ou TPA + PC + HAF. Tableau 6The dermal inflammation induced by the application of Phorbol TPA ester was measured after application of TPA, then after treatment with clobetasol propionate, and after treatment with the composition comprising clobetasol propionate and HAF. Ten measurements were performed per mouse. The results are collated in Table 6 below. The anti-inflammation index is the ratio between the control treated with TPA, and the association TPA + PC or TPA + PC + HAF. Table 6
La cellularité dermique induite par l'application d'ester de Phorbol TPA a été mesurée, après application de TPA, puis après traitement au propionate de clobétasol, et après traitement avec la composition comprenant du propionate de clobétasol et des HAF. Dix mesures ont été pratiquées par souris. Les résultats sont rassemblés dans le tableau 7. L'index d'anti-inflammation est le rapport entre le contrôle traité par TPA, et par l'association TPA + PC ou TPA + PC + HAF.The dermal cellularity induced by the application of Phorbol TPA ester was measured after application of TPA, then after treatment with clobetasol propionate, and after treatment with the composition comprising clobetasol propionate and HAF. Ten measurements were performed per mouse. The results are shown in Table 7. The anti-inflammation index is the ratio between the control treated with TPA, and the association TPA + PC or TPA + PC + HAF.
Tableau 7Table 7
L'activité myéloperoxydase cutanée induite par l'application d'ester de Phorbol TPA a été mesurée, après application de TPA, puis après traitement au propionate de clobétasol, et après traitement avec la composition comprenant du propionate de clobétasol et des HAF. Dix mesures ont été pratiquées par souris. Les résultats sont rassemblés dans le tableau 8. L'index d'anti-inflammation est le rapport entre le contrôle traité par TPA, et par l'association TPA + PC ou TPA + PC + HAF.The cutaneous myeloperoxidase activity induced by the application of Phorbol TPA ester was measured, after application of TPA, then after treatment with clobetasol propionate, and after treatment with the composition comprising clobetasol propionate and HAF. Ten measurements were performed per mouse. The results are summarized in Table 8. The anti-inflammation index is the ratio between the control treated with TPA, and the association TPA + PC or TPA + PC + HAF.
Tableau 8Table 8
Les HAF n'inhibent pas l'effet anti-inflammatoire du propionate de clobétasol mais, au contraire, potentialisent son effet anti-inflammatoire. Les figures 5 et 6, qui représentent des coupes histologiques colorées par van Gieson élastine et par Sirius red, ont permis de mettre en évidence que les HAF protègent le réseau élastique et le collagène dermique de la destruction par le propionate de clobétasol. HAFs do not inhibit the anti-inflammatory effect of clobetasol propionate but, on the contrary, potentiate its anti-inflammatory effect. Figures 5 and 6, which represent histological sections stained by van Gieson elastin and Sirius red, have made it possible to demonstrate that HAFs protect the elastic network and dermal collagen from destruction by clobetasol propionate.

Claims

REVENDICATIONS
1. Composition dermatologique anti-inflammatoire destinée à une administration topique, caractérisée en ce qu'elle comprend 0,005 à 0,1 %, de préférence 0,01 à 0,05 % en poids d'un corticoïde et 0,1 à 1%, de préférence 0,5 à 1 % en poids de fragments de hyaluronate de poids moléculaire moyen compris entre 20 et 500 kDa, de préférence entre 20 et 375 kDa, plus préférentiellement entre 20 et 150 kDa, lesdits fragments de hyaluronate étant susceptibles d'être obtenus par traitement thermique à une température supérieure à 1000C de fibres de hyaluronate de sodium de haut poids moléculaire, ou par traitement aux ultrasons de fibres de hyaluronate de sodium de haut poids moléculaire, pendant 10 à 90 minutes, de préférence 45 minutes, à 400W et à 4° C, suivi d'une fïltration sur gel.1. Anti-inflammatory dermatological composition intended for topical administration, characterized in that it comprises 0.005 to 0.1%, preferably 0.01 to 0.05% by weight of a corticoid and 0.1 to 1% preferably 0.5 to 1% by weight of hyaluronate fragments of average molecular weight between 20 and 500 kDa, preferably between 20 and 375 kDa, more preferably between 20 and 150 kDa, said hyaluronate fragments being capable of be obtained by heat treatment at a temperature above 100 0 C of high molecular weight sodium hyaluronate fibers, or by ultrasonic treatment of high molecular weight sodium hyaluronate fibers, for 10 to 90 minutes, preferably 45 minutes at 400W and 4 ° C, followed by gel filtration.
2. Composition selon la revendication 1, caractérisée en ce qu'elle comprend 0,05% en poids d'un corticoïde et 0,5% en poids de fragments de hyaluronate.2. Composition according to claim 1, characterized in that it comprises 0.05% by weight of a corticosteroid and 0.5% by weight of hyaluronate fragments.
3. Composition selon la revendication 1, caractérisée en ce qu'elle comprend 0,01% en poids d'un corticoïde et 1% en poids de fragments de hyaluronate. 3. Composition according to claim 1, characterized in that it comprises 0.01% by weight of a corticosteroid and 1% by weight of hyaluronate fragments.
4. Composition selon l'une quelconque des revendications précédentes, caractérisée en ce que le corticoïde est choisi parmi le dipropionate d'alclométasone, l'amcinonide, le dipropionate de béclométasone, le benzoate de bétaméthasone, le dipropionate de bétaméthasone, le valérate de bétaméthasone, le budésonide, le propionate de clobétasol, le butyrate de clobétasol, le désonide, la désoximétasone, la dexaméthasone, le diacétate dédiflorasone, le valérate de diflucortolone, la flurandrénolone, l'acétate de fluprednidène, le fluocortolone, le butyle de fluocortine, le fluocinonide, l'acétonide de fluocinolone, l'acétonide de fluclorolone, le pyvalate de flumétasone, le chlorhydrate de feudiline, la flumétholone, l'halcinonide, l'hydrocortisone, l'acétate d'hydrocortisone, le butyrate d'hydrocortisone, le valérate d'hydrocortisone, l'acétate de méthylprednisolone, le furoate de mométasone, la méthylprednisolone, la prednisolone, l'acétonide de triamcinolone ainsi que leurs mélanges. 4. Composition according to any one of the preceding claims, characterized in that the corticoid is chosen from alclometasone dipropionate, amcinonide, beclometasone dipropionate, betamethasone benzoate, betamethasone dipropionate, betamethasone valerate. , budesonide, clobetasol propionate, clobetasol butyrate, desonide, deoximetasone, dexamethasone, diacetate dediflorasone, diflucortolone valerate, flurandrenolone, fluprednidene acetate, fluocortolone, fluocortin butyl, fluocinonide, fluocinolone acetonide, fluclorolone acetonide, flumetasone pyvalate, feudiline hydrochloride, flumetholone, halcinonide, hydrocortisone, hydrocortisone acetate, hydrocortisone butyrate, valerate of hydrocortisone, methylprednisolone acetate, mometasone furoate, methylprednisolone, prednisolone, triam acetonide cinolone as well as their mixtures.
5. Composition selon l'une quelconque des revendications précédentes, caractérisée en ce que le corticoïde est le propionate de clobétasol.5. Composition according to any one of the preceding claims, characterized in that the corticosteroid is clobetasol propionate.
6. Composition pharmaceutique, caractérisée en ce qu'elle comprend une composition selon l'une quelconque des revendications précédentes et un ou plusieurs excipients pharmaceutiquement acceptables.6. Pharmaceutical composition, characterized in that it comprises a composition according to any one of the preceding claims and one or more pharmaceutically acceptable excipients.
7. Composition pharmaceutique selon la revendication 6, caractérisée en ce qu'elle comprend une base émolliente pharmaceutiquement acceptable.7. Pharmaceutical composition according to claim 6, characterized in that it comprises a pharmaceutically acceptable emollient base.
8. Composition pharmaceutique selon l'une quelconque des revendications 6 ou 7, caractérisée en ce qu'elle comprend d'autres excipients dermatologiquement acceptables pour sa présentation sous forme de crème, baume, gel, spray, pommade, lotion, solution fîlmogène, système transdermique, mousse, shampooing.8. Pharmaceutical composition according to any one of claims 6 or 7, characterized in that it comprises other excipients dermatologically acceptable for its presentation in the form of cream, balm, gel, spray, ointment, lotion, fluorescent solution, system transdermal, foam, shampoo.
9. Composition selon l'une quelconque des revendications précédentes à titre de médicament. 9. A composition according to any one of the preceding claims as a medicament.
10. Composition selon la revendication précédente à titre de médicament destiné à traiter les dermatoses inflammatoires.10. Composition according to the preceding claim as a medicament for treating inflammatory dermatoses.
11. Produit de combinaison comprenant d'une part un corticoïde sous forme de crème, et d'autre part des fragments de hyaluronate de poids moléculaire moyen compris entre 20 et 500 kDa, préférentiellement entre 20 et 375 kDa, plus préférentiellement entre 20 et 150 kDa, également sous forme de crème, pour une utilisation dermatologique séparée, simultanée ou étalée dans le temps, en thérapie des dermatoses inflammatoires, lesdits fragments de hyaluronate étant susceptibles d'être obtenus par traitement thermique à une température supérieure à 1000C de fibres de hyaluronate de sodium de haut poids moléculaire, ou par traitement aux ultrasons de fibres de hyaluronate de sodium de haut poids moléculaire, pendant 10 à 90 minutes, de préférence 45 minutes, à 400W et à 4° C, suivi d'une fïltration sur gel. 11. Combination product comprising firstly a corticoid in the form of cream, and secondly hyaluronate fragments of average molecular weight between 20 and 500 kDa, preferably between 20 and 375 kDa, more preferably between 20 and 150 kDa, also in cream form, for separate dermatological use, simultaneous or spread over time, in inflammatory dermatitis therapy, said hyaluronate fragments being capable of being obtained by heat treatment at a temperature above 100 ° C. of fibers of high molecular weight sodium hyaluronate, or by ultrasonic treatment of high molecular weight sodium hyaluronate fibers, for 10 to 90 minutes, preferably 45 minutes, at 400W and 4 ° C, followed by filtration on gel.
EP08774984A 2007-07-16 2008-07-10 Anti-inflammatory dermatological composition comprising corticosteroids and hyaluronate fragments, and uses thereof Withdrawn EP2178538A2 (en)

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