WO2002045718A1 - Utilisation de composes actifs capables de moduler la voie intracellulaire declenchee par le recepteur de dp dans des cellules de langerhans - Google Patents

Utilisation de composes actifs capables de moduler la voie intracellulaire declenchee par le recepteur de dp dans des cellules de langerhans Download PDF

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WO2002045718A1
WO2002045718A1 PCT/EP2001/014205 EP0114205W WO0245718A1 WO 2002045718 A1 WO2002045718 A1 WO 2002045718A1 EP 0114205 W EP0114205 W EP 0114205W WO 0245718 A1 WO0245718 A1 WO 0245718A1
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receptor
langerhans cells
cells
active compound
pgd
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PCT/EP2001/014205
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François TROTTEIN
Véronique ANGELI
Monique Capron
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Institut Pasteur De Lille
Institut National De La Sante Et De La Recherche Medicale (Inserm)
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Priority to AU2002229611A priority Critical patent/AU2002229611A1/en
Publication of WO2002045718A1 publication Critical patent/WO2002045718A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/52—Isomerases (5)
    • 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
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4166—1,3-Diazoles having oxo groups directly attached to the heterocyclic ring, e.g. phenytoin
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/472—Non-condensed isoquinolines, e.g. papaverine
    • 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
    • A61K31/557—Eicosanoids, e.g. leukotrienes or prostaglandins
    • A61K31/5575—Eicosanoids, e.g. leukotrienes or prostaglandins having a cyclopentane, e.g. prostaglandin E2, prostaglandin F2-alpha
    • 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
    • A61P17/06—Antipsoriatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection

Definitions

  • the invention relates to the use of active compounds capable of modulating the intracellular pathway triggered by the prostaglandin D 2 receptor (DP receptor) in Langerhans cells and/or dendritic cells and the subsequent emigration of Langerhans cells and/or dendritic cells, in the preparation of a medicine for modulating the immune response.
  • the invention relates to pharmaceutical compositions for topical application comprising prostaglandin D 2 (PGD 2 ), agonists or antagonists of the DP receptor.
  • composition for topical application to the skin and/or the mucosa refers to any composition, whose vehicle is appropriate for applying at the site of the disease for exertion of local action, preferably limited to the epidermis area of the skin and to the mucosa, including surface of the lung.
  • the invention is in the area of the topical treatment of a variety of cutaneous pathologies induced by or associated with an immune mucosal response.
  • na ⁇ ve T cells are activated by antigens presenting cells, i.e. Langerhans cells (LCs) and their derived activated form, namely the dendritic cells, in the draining lymph nodes (LNs).
  • LCs Langerhans cells
  • LNs draining lymph nodes
  • Langerhans cells Under normal, non-inflammatory conditions, Langerhans cells reside in the epidermis anchored to neighboring keratinocytes via homotypic E- cadherin interactions. In this environment, they display an immature phenotype characterized by high antigen uptake and processing abilities and poor T cell stimulatory function. However, in response to stimulation occuring during infection or topical application of allergens, Langerhans cells are activated and a proportion of activated Langerhans cells, migrate via afferent lymphatics to regional lymph nodes where they accumulate as immunostimulatory dendritic cells. Emigration of Langerhans cells from the epidermis may also be initiated via an antigen-independent manner, for instance by skin irritants or ultraviolet irradiation. During their migration,
  • MHC major histocompatibility complex
  • adhesion molecules such as ICAM-1 , VLA-6 (CD49f), CD40 and CD44 also play part in the migratory properties of Langerhans cells.
  • chemokines and their G protein-coupled receptors are also involved in
  • chemokine receptor, (CCR)7 is sharply up-regulated during Langerhans cells maturation and is particularly important to attract Langerhans cells into the lymph nodes.
  • Langerhans cells emigration is also associated to a rapid reduction in the expression of receptors for inflammatory chemokines such as CCR1.
  • Mechanisms that negatively regulate the emigration of epidermal Langerhans cells after activation have also been reported.
  • the anti-inflammatory cytokine IL-1 ra has been shown to block the binding of IL- 1 ⁇ to its receptor.
  • IL-4 and IL-10 may also act as regulators of Langerhans cells migration. It was recently shown that IL-4 interferes with the
  • the term “Langerhans cells” includes hereafter, not only the Langerhans cells which are located in the epidermis, but also the emigrated Langerhans cells in the lymph nodes which have been activated and which are also called “Dendritic cells” and the non-lymphoid dendritic cells located in peripheral tissues, for instance in the pulmonary epithelium.
  • Langerhans cells play different roles in the evolution of certain cutaneous pathologies and cutaneous inflammatory disorders. Indeed, emigration and/or activation of Langerhans cells have been first directly involved in hypersensitive response and IgE-mediated immune response in eczema, contact and atopic dermatitis. Langerhans cells may also play a role in the epidermotropism of T lymphocyte (2), in epidermotropic cutaneous T lymphoma such as mycosis fungo ⁇ des, and in cutaneous auto-immune diseases such as cutaneous lupus erythematous (3).
  • RLV Rausch Leukaemia virus
  • Immunomodifiers that block or stimulate the migration of Langerhans cells have been proposed in the treatment of those pathologies induced by or associated with an immune cutaneous response.
  • Imiquimod a topical immune response modifier
  • Imiquimod a topical immune response modifier
  • Imiquimod was known for its anti-viral and anti-tumour effects in animal models and has been approved for the topical treatment of external genital and perianal warts in humans (6).
  • the present invention results from the analysis of the effects of the helminth parasite Schistosoma mansoni, the causative agent for schistosomiasis, on the activation state and migratory abilities of Langerhans cells.
  • the inventors have shown that, following murine infection, schistosomula activate Langerhans cells but, surprisingly, impede their migration to the lymph nodes. They also identified that the inhibitory effect, which also occurs in a TNF -induced model of Langerhans cells migration, appears to be mediated by excreted/secreted lipophilic factors produced by parasite larvae, and particularly by prostaglandin D 2 (PGD 2 ).
  • PGD 2 is a naturally occurring prostano ⁇ d that shows various kinds of pharmacological activity such as platelet aggregation, bronchoconstriction, sleep induction, and fever under various physiological and pathological conditions.
  • PGD 2 is known to be the major cyclooxygenase product from arachidonic acid in the epidermis and dermis.
  • prostaglandin D 2 participates in the development and the modulation of the inflammatory response in the skin (7). Indeed, PGD 2 can produce vasodilatation in rat skin and elicit a wheal and erythema reaction in human skin by intradermal injection.
  • the inventors have now identified a novel function for PGD 2 in that it also participates in the control of Langerhans cells migration by feedback mechanism.
  • the invention results from the finding that modulation of the intracellular pathway triggered by the receptor of prostaglandin D 2 (DP receptor) in the Langerhans cell directly influences the capacity of emigration of the Langerhans cells.
  • the invention also results from the identification of a novel mechanism that may play a key role in the maintenance of Langerhans cells homeostasis in the skin.
  • the inventors have shown first, that prostaglandin D 2 induces a rearrangement of the F-actin cytoskeleton that is mediated by the pathway involving the synthesis of cyclic AMP (cAMP) and the activation of the protein kinase cAMP-dependent (PKA/cAMP pathway) triggered by the DP receptor and second, that the subsequent modification of the actin-based cytoskeleton may explain the retention of Langerhans cells in the epidermis.
  • cAMP cyclic AMP
  • PKA/cAMP pathway protein kinase cAMP-dependent pathway
  • the invention provides novel use of PGD 2 , agonists or antagonists of DP receptor for modulating the immune cutaneous response and more generally, of an active compound capable of modulating the intracellular pathway triggered by the DP receptor in Langerhans cells and the subsequent emigration of Langerhans cells, in the preparation of a medicine for modulating the immune cutaneous response.
  • the invention also provides a pharmaceutical composition for topical application to the skin and/or the mucosa of human or animals comprising an active compound capable of modulating the intracellular pathway triggered by the DP receptor in Langerhans cells and the subsequent emigration of Langerhans cells, said composition further comprising a pharmaceutically acceptable vehicle.
  • a pharmaceutical composition of the invention essentially consists of
  • a pharmaceutically acceptable vehicle appropriate for topical application to the skin and/or the mucosa.
  • immune cutaneous response refers to the migration of Langerhans cells to lymph nodes and the priming of cytotoxic CD8+ T and helper CD4+ T lymphocytes towards specific antigen via the activation of Langerhans cells. Modulation may be negative via blocking of the TNF ⁇ or IL1 ⁇ - induced emigration mechanism or positive via disinhibition of the blockade of the migration by specific inhibitors.
  • PGD 2 prostaglandin D 2
  • PGD 2 is a eicosanoid derived from arachidonic acid and synthesized through the so-called arachidonate oxygenation pathway by which molecular oxygen is incorporated into arachidonic acid.
  • PGD 2 is the major eicosanoid product of mast cells.
  • PGD 2 is the 9 ⁇ , 15S-dihydroxy-11-oxo-prosta-5Z,13E- dien-1-oic acid.
  • PGD 2 binds at least to DP receptor.
  • the enzyme responsible for PGD 2 synthesis is called PGD 2 -synthase which converts PGH 2 to PGD 2 .
  • the intracellular pathway triggered by the DP receptor is characterized in particular by intracellular cAMP synthesis and the subsequent increase of cAMP-dependent protein kinase A (PKA) activity, which consequently yields to the rearrangement of the F-actin cytoskeleton and retention of Langerhans cells in the epidermis.
  • PKA cAMP-dependent protein kinase A
  • Langerhans cells may be positive, i.e., characterized by an in vivo activation of the transduction pathway, after administration of a therapeutic amount of the pharmaceutical compositions or the medicine, or negative, i.e., characterized by inhibition of the transduction pathway, after administration of a therapeutic amount of the pharmaceutical compositions or the medicine.
  • Any compound that enables the modulation of the intracellular pathway triggered by the DP receptor and the subsequent emigration of Langerhans cells, can be used in the preparation of the pharmaceutical compositions or the medicine for modulating the immune cutaneous response.
  • Said compound can be a ligand to the DP receptor, such as PGD 2 , an agonist thereof or an antagonist.
  • an agonist of DP receptor is used.
  • the term "agonist of DP receptor” corresponds to functional and/or structural analogues of PGD2, which can bind to the DP receptor and activate its transduction pathway, preferably as efficiently as PGD 2 , or more efficiently.
  • Synthetic and natural prostanoid agonists of the DP receptor are well known in the Art.
  • An example of an agonist of DP receptor is the (4S)-(3-
  • an agonist of the DP receptor is selected among the prostanoid agonists of the DP receptor, such as ZK118182, RS- 93520, SQ27986, ZK 110841 or BW245C.
  • an antagonist of DP receptor is used.
  • the term "antagonist of DP receptor” corresponds to natural or synthetic molecules, which can bind to the DP receptor and block the activation of the DP receptor by PGD 2 or by an agonist of said receptor.
  • An example of an antagonist of DP receptor is the ( ⁇ )-3-benzyl-5-(6- carboxyhexyl)-1)(2-cyclohexyl-2-hydroxyethylamino) hydantoin, also known as BWA868C.
  • Another compound able to modulate the intracellular pathway triggered by DP receptor in Langerhans cell and the subsequent emigration of Langerhans cells can be an enzyme involved in cyclo-oxygenation pathway of acid arachidonic, and preferably PGD 2 -synthase which catalyses the reaction from PGH 2 to PGD 2 .
  • PGD 2 -synthase which catalyses the reaction from PGH 2 to PGD 2 .
  • the local delivery of PGD 2 -synthase can lead to in situ synthesis of PGD 2 .
  • the invention also concerns the use of PGD 2 -synthase or activators or inhibitors of PGD 2 -synthase in the preparation of a medicine for modulating the immune cutaneous response.
  • the invention also relates to pharmaceutical compositions for topical application to the skin and/or the mucosa which comprise or consists of (a) PGD 2 synthase or activator thereof or inhibitors of PGD 2 -synthase, and (b) a pharmaceutically acceptable vehicle.
  • a specific inhibitor of PGD 2 -synthase is SeCI 4 .
  • a compound able to modulate the intracellular pathway triggered by the DP receptor in
  • Langerhans cell and the subsequent emigration of Langerhans cells can also be any inhibitor or activator of cAMP synthesis and PKA activity in the Langerhans cells.
  • an activator of the cAMP synthesis is the adenylate cyclase activator forskolin.
  • an inhibitor of the activity is the PKA inhibitor H89, or ⁇ N [2-((p-Bromocinnamyl) amino) ethyl] - 5 - isoquinolinesulfonam.de, HCI ⁇ .
  • the modulation of the immune cutaneous response via modulators of the intracellular pathway triggered by the DP receptor in Langerhans cell can be achieved without producing significant systemic effects associated with the systemic administration of such compounds, most notably neuroleptic effects such as sleep induction and fever, the vasodilatation effects and the anti-inflammatory effects such as the inhibition of platelet aggregation which are some pharmacological actions of prostaglandin D 2 .
  • the active compound is comprised in the composition of the invention in an amount sufficient to deliver said active compound, when applied topically to a patient, in the absence of serious toxic effects and in an amount sufficient to obtain the desired therapeutic treatment.
  • the composition comprises the active compound in an amount from 0.005% to 5% by weight of the composition, preferably 0.001% to 1% and more preferably 0.001% to 0.1%.
  • concentration of active compound in the pharmaceutical composition will depend on different factors known to those skilled in the art, such as absorption or inactivation. It is to be noted that concentration values may also vary with the severity of the condition to be alleviated.
  • compositions for topical application comprise a pharmaceutical acceptable vehicle.
  • vehicle may act as a dilutant, dispersant or other vehicle facilitating the distributions of the active compounds when applied to the skin and/or the mucosa.
  • Any appropriate vehicle for topical application to the skin and/or the mucosa can be used and include liquid or solid emollients, solvents, humectants, and thickeners typically found in pharmaceutical formulations.
  • the composition can include a diluent such as saline solution, polar lipid compounds, polyethylene glycols, glycerine, butylene and propylene glycol or other synthetic solvents.
  • Preferred solvents used in the compositions for prostaglandin D 2 are ethanol, acetone, acetone/vegetal oil or 2-propanol.
  • vegetal oil might be olive oil.
  • Other solvents such as ethyl acetate, and other primary alcohols may also be present.
  • compositions of the invention may further comprise other active compounds which do not impair the desired action but supplement the desired action, such as antibacterial agents (benzyl alcohol or methyl parabenzens for example), antifungals, anti-inflammatories, antivirals (imiquimod or aciclovir for example), or immunosuppressive agents depending of the desired treatment.
  • active compounds such as antibacterial agents (benzyl alcohol or methyl parabenzens for example), antifungals, anti-inflammatories, antivirals (imiquimod or aciclovir for example), or immunosuppressive agents depending of the desired treatment.
  • a short chain diol or triol compound preferably serves as a penetration enhancer.
  • compositions of the invention may further comprise other active compounds which do not impair the desired action but supplement the desired action, such as antibacterial agents (benzyl alcohol or methyl parabenzens for example), antifungals, anti-inflammatories, antivirals (imiquimod or aciclovir for example), or immunosuppressive agents depending of the desired treatment.
  • active compounds such as antibacterial agents (benzyl alcohol or methyl parabenzens for example), antifungals, anti-inflammatories, antivirals (imiquimod or aciclovir for example), or immunosuppressive agents depending of the desired treatment.
  • composition does not contain any other compound which cooperates substantially to provide the desired pharmacologic and/or therapeutic effect.
  • compositions of the invention as above-defined may optionally contain adjunct components selected from the following: antioxidants such as ascorbic acid, humectants such as glycerols and sorbitol, chelating agents such as ethylenediaminetetraacetic acid, buffers such as lactic acid, acetates, citrates or phosphates, thickening agents such as xanthan gum, beeswax or polyethylene glycol, emollients such as mineral oil, lanolin and its derivatives, or squalene, opacifiers and stabilizers.
  • antioxidants such as ascorbic acid, humectants such as glycerols and sorbitol
  • chelating agents such as ethylenediaminetetraacetic acid
  • buffers such as lactic acid, acetates, citrates or phosphates
  • thickening agents such as xanthan gum, beeswax or polyethylene glycol
  • the pH of the composition is appropriate for topical application to the skin, and preferably comprised between pH 4 and 7.
  • Suitable formulations for topical application to the skin and/or the mucosa include lotions, ointments, creams or gels, slow- release transdermal patches and aerosols.
  • compositions and ointments are commercially available for dermatologic applications and are well known in the art.
  • the composition can be prepared with a vehicle that protects the active compound against rapid release, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are well known to those skilled in the art.
  • the compounds able to modulate the intracellular pathway triggered by the DP receptor and the subsequent emigration of Langerhans cells, as defined above, are used in the preparation of a pharmaceutical composition or a medicine for the treatment and/or the prevention of pathologies induced by or associated with an immune cutaneous response. Indeed, modulating the immune cutaneous response induced by or associated with the pathologies will enable to prevent, reduce or suppress (treat) the symptoms associated to said pathologies.
  • a pathology induced by an immune cutaneous response is a pathology which is mediated, amongst others, via the emigration and the activation of Langerhans cells.
  • a pathology associated with the modulation of an immune cutaneous response is a pathology which stimulates or inhibits the emigration and the activation of Langerhans cells.
  • topically applying a composition of the invention to the skin enables the prevention and/or the treatment of atopic dermatitis and contact dermatitis, i.e, examples of pathologies induced by an immune cutaneous response.
  • a composition according to the invention enables the inhibition of the migration of airway dendritic cells to the draining lymphe nodes. Accordingly, in a specific embodiment of the invention, a sufficient amount of a compound able to modulate the intracellular pathway triggered by the DP receptor and the subsequent emigration of Langerhans cells, is used in the preparation of a medicine for the treatment and/or the prevention of eczematous dermatitis, contact eczema, contact or irritant dermatitis, atopic dermatitis, psoriasis, drug eruptions, urticaria or asthma.
  • the one skilled in the Art will advantageously select the compounds of the invention capable of stimulating the intracellular pathway triggered by the DP receptor in order to block the migration of
  • a preferably used compound is selected among PGD 2 or agonists of DP receptor, PGD 2 -synthase, activators of PGD 2 synthase, activators of cAMP synthesis and/or of PKA activity, most preferably PGD 2 or an agonist thereof.
  • the active compounds are also used in the preparation of a medicine for the treatment of viral cutaneous pathologies such as those derived from neophasic lesions, human papillomavirus infection, HIV or RLV infections, skin cancers such as epidemo ⁇ d and epicellular carcinoma or melanoma, bacterial, fungal and other parasital cutaneous pathologies.
  • viral cutaneous pathologies such as those derived from neophasic lesions, human papillomavirus infection, HIV or RLV infections, skin cancers such as epidemo ⁇ d and epicellular carcinoma or melanoma, bacterial, fungal and other parasital cutaneous pathologies.
  • disinhibition of Langerhans cells migration is preferably sought.
  • the one skilled in the art will advantageously select the compounds of the invention capable of inhibiting the activation of the intracellular pathway triggered by the DP receptor in order to stimulate the migration of Langerhans cell to lymph nodes and promote immune cutaneous response.
  • antagonists of the DP receptor are preferably used in the preparation of the medicine, in order to antagonize the inhibitory effects that block migration of Langerhans cells caused by the pathogens, by the infected cells or by the abnormal cells.
  • an active compound may also be used in the preparation of a medicine for the treatment of epidermotropic cutaneous T lymphoma such as mycosis fungojdes or Sezary syndrom, and auto-immune diseases such as cutaneous lupus erythematous. In these pathologies, a reduction of the immune cutaneous response is sought.
  • the used compound is preferably selected among PGD 2 , agonists of DP receptor, PGD 2 -synthase, activators of PGD 2 -synthase, activators of PKA and of cAMP synthesis, most preferably
  • PGD 2 or an agonist thereof.
  • the compounds capable of modulating the intracellular pathway triggered by the DP receptor and the subsequent emigration of Langerhans cells may also be used to reduce or prevent skin graft rejection, especially after transplantation of skin allo-grafts.
  • the invention thus provides a use of an active compound capable of modulating the intracellular pathway triggered by the DP receptor and the subsequent emigration of Langerhans cells, to reduce or prevent skin allo-graft rejection.
  • said compound is preferably selected among PGD 2 , agonists of DP receptor, PGD 2 -synthase, activators of PGD 2 -synthase, activators of cAMP synthesis and of PKA, most preferably PGD 2 or an agonist thereof.
  • the medicine comprising a biologically active compound capable of modulating the intracellular pathway triggered by the DP receptor and the subsequent emigration of Langerhans cells and/or dendritic cells, is prepared for administration to mammals and specifically to humans. It may be administered via any suitable means provided that said biologically active compound can reach the Langerhans cells and/or dendritic cells. For example, it may be administered via oral route, intradermal or subcutaneous injection. One preferred route is via topical administration in a cream or a gel formulation or using a patch. It may also be delivered to the mucosa via oral or nasal administration from a metered dose inhaler or aerosols.
  • the invention further provides a method for modulating the immune response comprising a step of topically administering a compound capable of modulating the intracellular pathway triggered by the DP receptor and the subsequent emigration of Langerhans cells, said compound being more preferably, PGD 2 or an agonist or an antagonist of the DP receptor.
  • the topical administration might concern either the skin or a mucosal tissue such as buccal, nasal, vaginal or lung mucosa.
  • the invention also provides a method for treating or preventing a pathology induced by or associated with an immune cutaneous response, said pathology being preferably selected from the following: asthma, eczematous dermatitis, contact eczema, contact or irritant dermatitis, atopic dermatitis, psoriasis, drug eruptions, urticaria, viral cutaneous pathologies such as those derived from human papillomavirus infection, HIV or RLV infections, skin cancers such as epidemoid or epicellular carcinoma or melanoma, bacterial, fungal and other parasital cutaneous pathologies, epidermotropic cutaneous T lymphoma such as mycosis fungoides or Sezary Syndrom and auto-immune diseases such as cutaneous lupus erythematous, said method comprising a step of topically administering to the lesioned tissues a therapeutically efficient amount of a compound capable of modulating the intracellular pathway triggered by the DP receptor and the subsequent
  • the method comprises the step of topically administering to the mucosa of the lung via oral or nasal administration from a metered dose inhaler or aerosols, a sufficient amount of an agonist of the DP receptor for the prevention of asthma.
  • the invention further provides a method for preventing skin graft rejection comprising topically administering a compound capable of modulating the intracellular pathway triggered by the DP receptor and the subsequent emigration of Langerhans cells, said compound being preferably PGD 2 or an agonist, to the skin part surrounding the graft before and/or after the skin graft transplantation.
  • the following experimental part and figures show the results that reveal the new mechanism of prostaglandin D 2 in the control of Langerhans cells migration from the epidermis to the lymph node.
  • the results demonstrate for the first time that (i) the activation of the DP receptor mediates the inhibition of Langerhans cells migration; (ii) PGD 2 and agonist of PGD 2 specifically inhibit the TNF ⁇ -induced Langerhans cells migration through a cAMP dependent pathway ; (iii) PGD 2 induces a prolonged F-actin depolymerization in Langerhans cells through a cAMP/PKA pathway; (iv) the subsequent modification of the actin-based cytoskeleton may explain the retention of Langerhans cells in the epidermis and (v) antagonist of DP receptor restore the ability of Langerhans cells to migrate to the lymph nodes.
  • FIG. 1 Immunohistochemical staining of murine epidermal sheets after transcutaneous infection by S. mansoni.
  • Epidermal sheets were prepared either from non-infected or from S. mansoni- ' mfected mice and Langerhans cells were stained for MHC class II (A and B) or for CD86 (C and D).
  • the arrow indicates the "ghost" of parasite.
  • the isotype control mAb did not reveal any reactivity (not shown) (magnification, x 400).
  • FIG. 1 Effect of S. mansoni infection on the migration of epidermal Langerhans cells in vivo (A) and ex vivo (B).
  • A Epidermal sheets were prepared at different times after the infection (1 to 120 h) and the number of
  • Controls included epidermal sheets from naive mice and from mice exposed to water without parasites (non-infected). Results are expressed as means ⁇ SD and are representative of four independent experiments (n 7).
  • FIG. 3 Effect of S. mansoni infection on the TNF ⁇ -induced Langerhans cells migration in vivo.
  • mice 4 mice/time point) were i.d injected with 30 ⁇ l of PBS/BSA (carrier) containing or not 50 ng TNF ⁇ into both ear pinnae. Ears were removed 1 h later, epidermal sheets were prepared and the number of Langerhans cells/mm 2 were determined by immunohistochemistry.
  • PBS/BSA carrier
  • Langerhans cells were interdigitated among surrounding keratinocytes and still expressed E-cadherin (not shown).
  • FIG. 4 (A) RT-PCR analysis of mRNAs specific for pro- and anti- inflammatory cytokines in the epidermis of S. mansoni infected mice. Epidermal sheets were prepared from non-infected (0 h) or infected (1, 6, 24 and 120 h) mice, total RNA extracted and RT-PCR was carried out using the primers shown in Table 1. Representative results of three independent experiments are shown.
  • mice were treated with neutralizing anti-IL-10 or isotype-matched mAbs (lgG1).
  • TNF ⁇ was i.d injected 1 h before the analysis.
  • Significant differences are designated by * (p ⁇ 0.001 ).
  • FIG. 5 Effect of schistosomula excreted/secreted products on the TNF ⁇ - induced Langerhans cells migration in vivo.
  • FIG. 7 (A) Expression of mRNA for the DP receptor in total epidermal cells and in the Langerhans cells (XS52) and keratinocytes (Pam212) lines as assessed by RT-PCR. (B) Effect of the DP receptor antagonist BWA868C on Langerhans cells emigration after infection with S. mansoni. Fifteen mins before the infection, mice were injected i.d with increasing amounts of
  • FIG. 8 Effect of PGD 2 on cellular F-actin content in XS52. Cells were incubated for the indicated periods of time in the presence of PGD 2 (10 nM),
  • mice were i.d injected in the ear with BW245C (100 nM) or were treated by topical application with BW245C (1 ⁇ M) or vehicle (ethanol or acetone) alone.
  • Epidermal sheets were prepared 1 h after TNF ⁇ injection.
  • FIG. 10 Effect of the topical application of BW245C on contact hypersensitivity elicited by FITC.
  • A Mice were treated by topical application of BW245C (50 ⁇ M in acetone/vegetal oil) 15 min before and 5 h after FITC sensitization. Five days after sensitization, mice were challenged with FITC and 24h later, ear swelling was measured.
  • FIG. 11 Effect of the topical application of BW245C during the sensitization phase on the contact hypersensitivity elicited by oxazolone.
  • Mice received daily a topical application of oxazolone (0.2% in acetone) containing (A), or not ( ⁇ ), BW245C (50 ⁇ M) for 7 days.
  • A Every two days, desquamation, erythema and the presence of scabs were scored from 0 to 4, as explained in 1.10 hereinafter.
  • mice received topical application of acetone ( ⁇ ). The total of scores is determined for each group of mice.
  • B Skin thickness was measured every two days.
  • FIG. 13 Effect of BW245C in the pathology in a model of atopic dermatitis elicited by ovalbumine.
  • A Histological analysis of the skins. Skin sections from mice topically sensitized with ovalbumine and treated, or not, with
  • Figure 15. Effect of the topical application of BW245C on the TNF ⁇ -induced migration of Langerhans cells ex vivo.
  • FIG. 16 Flow cytometric staining pattern of MLN single cell suspensions, labelled with anti-mouse MHC II (PE) and anti-mouse CD11c (APC) Abs. Two clusters were distinguished in the MHCM + CD11c + quadrant. The animals received an intratracheal instillation of 10 mg/ml FITC-OVA or PBS-DMSO as a control. After 24 hours, only airway DCs acquire a strong FITC signal.
  • PE anti-mouse MHC II
  • APC anti-mouse CD11c
  • AH reagents were purchased from Sigma (St Quentin-Fallavier, France) unless otherwise notified. H89 was from Calbiochem (La Jolla, CA) and BW245C from Cayman Chemical (Ann Harbor, Ml). BWA868C was kindly donated by Dr S. Lister (Glaxo Wellcome, Greenford, UK).
  • the anti-l-A d /l-E d mAbs (clone M5 /114, rat lgG2b) and the anti-DEC-205 (NLDC-145, rat lgG2a) were kindly provided by Drs A. Ager (NIMR, London, UK) and D. Sacks (NIH, Bethesda, MA), respectively.
  • the FITC-conjugated anti-CD80 hamster IgG
  • anti-CD86 rat lgG2a
  • biotin-conjugated anti-CD11c hamster IgG
  • IgG mAbs the FITC-conjugated anti-human CD45 (mouse lgG1 ), phycoerythrin-conjugated anti- human CD1a (mouse lgG1) and cy-Chrome- conjugated anti-human HLA-DR (mouse lgG1 ) mAbs were purchased from Pharmingen (San Diego, CA). The following were used as secondary Abs: biotin-conjugated anti-rat and anti-FITC peroxydase-conjugated (Boehringer-
  • the biotinylated reagents were detected using ABC complex HRP (Dako Co., Carpinteria, CA).
  • the neutralizing anti- IL-10 mAb (clone JES052A5, rat lgG1) was from R&D systems (Abingdom, U.K) and the isotype control mAb from Caltag Laboratories (Burlingama, CA).
  • the Langerhans cells line XS52 was donated by Dr A. Takashima (University of Texas, Dallas, TX). XS52 has been established from mouse epidermis (11) and presents the phenotypic and functional features of Langerhans cells (12). XS52 was cultured in RPMI containing 10% (vol/vol) heat-inactivated FCS in the presence of 2 ng/ml GM-CSF (Biosource, Camarillo, CA) and
  • Mouse Pam212 keratinocytes (kind gift from Dr S. H. Yuspa, NCI, NIH) were cultured in Eagle's MEM complemented with 10% FCS and 0.05 mM CaCI 2 .
  • mice C57BL/6 mice (6- to 8- wk old) were purchased from Iffa-Credo (L'leysle, France).
  • the S. mansoni (Puerto Rican strain) life cycle was maintained in Biomphalaria glabrata snails as the intermediate host and the hamster Mesocricetus auratus as the definitive host.
  • Mechanically- transformed schistosomula and excreted/secreted (excreted/secreted) products from schistosomula (10 3 parasites/ml) were prepared as described (13).
  • the methanol/chloroform-extracted fraction from the schistosomula excreted/secreted products was obtained as reported (1 1 ).
  • mice were anesthetized with pentobarbital (30 mg/kg) (Sanofi, Libourne, France) and exposed to 250 cercariae by immersion of the ears for 25 min.
  • Recombinant murine TNF ⁇ (specific activity > 5 x 10 7 U/mg) (R&D systems) was reconstituted in sterile PBS containing 0.1 % (wt/vol) BSA as a carrier protein. Mice were intradermally (i.d) injected with 50 ng TNF ⁇ (30 ⁇ l) into both ear pinnae with 27 34 -gauge stainless steel needles. Epidermal sheets were analyzed 1 h after injection, a time previously shown to be optimal for
  • mice were treated with BW245C 15 min before TNF ⁇ injection. To do that, 30 ⁇ l of BW245C (100 nM) or DMSO (as control) was injected i.d or 10 ⁇ l of BW245C (1 ⁇ M in acetone/olive oil (4:1 , vol/vol) or ethanol (70% pure)) or DMSO was applied topically on each side of the ear.
  • mice with Abs Treatment of mice with Abs was as follows: one hour before infection, C57BL/6 mice were injected i.d with 40 ⁇ g of neutralizing anti-IL-10 or isotype-matched control mAb diluted in sterile PBS (final volume: 30 ⁇ l).
  • Epidermal sheets were fixed in paraformaldehyde (PFA, 2% in PBS) for 10 mins at room temperature, washed three times with PBS.
  • PFA paraformaldehyde
  • Epidermal sheets were then incubated with primary Abs for 90 mins and washed in PBS before adding either biotinylated conjugated goat anti-rat Ig or peroxydase-conjugated anti-FITC for an additional 30 mins.
  • sheets were developed with 3-amino 9- ethyl carbazol, washed three times in PBS and mounted onto glass slides in Immumount (Shandon, Pittsburg, PA) for immunohistochemical analysis. Langerhans cells were enumerated by counting MHC class II positive cells.
  • Epidermal sheets were prepared from each experimental group and for each sheet 10 random fields were examined. Cell frequency was converted to Langerhans cells/mm 2 and results were expressed as mean ⁇ SD. The statistical significance of differences between experimental groups was calculated using the Student's t test.
  • ears were rinsed in 70% ethanol and split into dorsal and ventral halves with forceps.
  • Biopsies were performed from healthy skin obtained after plastic surgery. 100 ⁇ l of BW245C (50 ⁇ M) or DMSO (negative control) was i.d administrated 15 min before TNF- ⁇ treatment as described above. Four skin explants, were floated, dermal side down, on 4 ml RPMI supplemented with 25 mM HEPES, 10 % FCS and gentamycin (50 ⁇ g/ml) (21 ). After 24 h incubation at 37°C in a 5% C0 2 incubator, the number of emigrated Langerhans cells from skin explants was determined by flow cytometry.
  • Langerhans cells were stained with FITC-conjugated anti-CD45, phycoerythrine-conjugated anti-CD1a and cy-Chrome anti-HLA-DR mAbs.
  • the total number of emigrated Langerhans cells was determined on a FACScalibur flow cytometer (Becton Dickinson). Data were analyzed using CellQuest software.
  • RNA from mice were excised, the epidermis were separated from the dermis and total RNA was isolated using TRIzol reagent (Life Technologies, Grand Island, NY). RNA from resting XS52 and Pam212 cells were isolated as described above. cDNA was synthesized from 1 ⁇ g of total RNA with random hexamer primers and Superscript reverse transcriptase (Life technologies) using standard procedures. PCR amplifications were performed with the primer pairs indicated in Table 1. Amplified products were subjected to 1% agarose gel electrophoresis and visualized by ethidium bromide staining. Table I Sequences of Primers Used for PCR Amplification of cDNA, Product sizes and PCR Cycle Numbers
  • IL-1 ra 5' 5'-CCTGCAAGATGCAAGCCTTCAGG 353 35 3' 5'-CAGCCTCTAGTGTTGTGCAGAGG
  • XS52 cells were harvested, washed and resuspended at a density of 10 6 cells/ml in HBSS containing 1 mM CaCI 2 , 0.5 mM MgCI 2 and 0.1% BSA after which the assay was performed as described (20). Briefly, after allowing the cells to equilibrate for 15 min at 37°C, stimulators were added for various periods of time, then the reaction was stopped by adding cold PBS containing 0.1% PFA. Cells were washed and stained for 30 min by incubation in a mixture of saponin (final concentration 0.1 %) and FITC- phalloidin (2.5 ⁇ g/ml). After washing, cells were subjected to FACS analysis (Becton Dickinson FACS). 1.10 Induction and elicitation of contact hypersensitivity responses.
  • mice were sensitized by painting 10 ⁇ l of a 0.5 % (wt/vol) solution of FITC prepared in acetone/dibutylphtalate (1 :1 , vol/vol) on the total surface of the left ear.
  • Ten ⁇ l of BW245 (50 ⁇ M) or DMSO (as a control) prepared in acetone/olive oil (4:1 , vol/vol) was applied topically on each side of the left ear 15 min before and 6 h after the sensitization or the challenge phase.
  • Challenge phase was elicited 5 days after the sensitization by painting the dorsal and ventral surface of the right ear with 10 ⁇ l of 0.5% FITC (15).
  • Ear thickness was measured using an engineers' micrometer (Mitutoyo, Kawasaki, Japan) 24 h after the challenge. Results are expressed as net ear swelling, which was calculated by substracting the thickness of the vehicle- treated ear from the thickness of the FITC-challenged ear.
  • SKH-1 mice were sensitized every day by painting 50 ⁇ l of oxazolone (0.2% in acetone) containing, or not, 50 ⁇ M BW245C on the back of the mice for 7 days (16,
  • mice 17) 1 As negative control, mice received acetone alone. Every 2 days, desquamation, erythema and the presence of scabs were scored (0 to 4). Skin thickness was also measured every 2 days. For the curative treatment, mice were sensitized with oxazolone every day for 10 days and from the day 11 to 21 , mice were treated, or not, with 50 ⁇ M BW245 in the presence of oxazolone.
  • mice Epicutaneous sensitization of mice was performed as described previously (18). Briefly, mice were shaved with an electric razor. 25 ⁇ l of a solution of
  • mice were bled and sera were collected one day after the series of three epicutaneous sensitizations.
  • the standard PharMingen protocol for sandwich ELISA was used to quantify the total amount of IgE in serum.
  • Rat anti-mouse IgE mAb (clone R35-72, Pharmingen) was used for coating the plates, and biotin-conjugated rat anti-mouse IgE mAb (clone R35-92, Pharmingen) was used for detection.
  • ovalbumine lgG1 measurement flat-bottom
  • specimens were obtained from patches areas on the skin 24h after the patch from the third sensitization was removed. Specimens were fixed in 4% buffered formalin and embedded in ImmunoHistoWax (ImmunoHistoWax, Interstiles sprl, Brussels, Belgium). Multiple 4- ⁇ m sections were stained with hematoxylin and eosin safran or blue toluidine.
  • FITC-OVA Fluorescein-conjugated ovalbumin
  • Molecular probes was diluted in PBS to a final concentration of 10 mg/ml.
  • mice were anesthetized by intraperitoneal injection of avertin (2.5 % in PBS) and 80 ⁇ l of FITC-OVA solution, containing or not BW245C (10 or 100 ⁇ M in DMSO), was administered intratracheally under direct vision through the opening vocal cords using a 18 G polyurethane catheter connected to the outlet of a micropipette as previously described (21). Control mice received 80 ⁇ l of PBS containing DMSO.
  • Lymph node (LN) digestion medium consisted of RPMI 1640, 5% (vol/vol) fetal calf serum (FCS) (Greiner), 1 mg/ml collagenase type 2 (Worthington Biochemical Corp.) and 2000 IU DNAse I.
  • FCS fetal calf serum
  • EDTA-treated FCS was prepared by passing FCS through a 0.2 ⁇ m filter and by adding 1 ml of a 0.1 mM EDTA solution for 10 ml of FCS.
  • FACS-EDTA buffer contained PBS without Ca 2+ and Mg 2+ , 0.1 % azide, 5% EDTA-treated FCS and 5 mM EDTA.
  • MLNs avertin and mediastinal LNs
  • FITC-OVA-instilled animals Due to photosensitivity of the FITC material, organs from FITC-OVA-instilled animals were protected from direct light throughout the manipulation.
  • MLNs were minced using iridectomy scissors and incubated for 30 min in the digestion medium in a humidified incubator according to a modified protocol (23). Organ fragments were resuspended in a fresh digestion medium and incubated for another 20 min. Samples were centrifuged and resuspended in Ca 2+ - and Mg 2+ -free PBS containing 10 mM EDTA for 5 min at room temperature.
  • the mAbs used to identify mouse DC populations were: PE-conjugated anti-MHC class II (MHC II, clone M5.114.15.2) and APC-conjugated anti-CD11c (Pharmingen). All staining were performed on ice in FACS-EDTA buffer. Cells were preincubated with anti-CD 16/CD32
  • ⁇ exhibited a more dendritic morphology with typical interdigiting cellular processes. Moreover, the MHC class II staining on Langerhans cells from infected epidermis was more intense. Immunolabelling with the Langerhans cells -specific Ab NLDC-145 confirmed the activated phenotype of Langerhans cells in infected skins. The expression of other surface markers known to be expressed by mature Langerhans cells was next analysed.
  • Langerhans cells After activation, Langerhans cells normally migrate from the epidermal site of antigen capture to the systemic lymph nodes. The migratory behavior of Langerhans cells after S. mansoni penetration has therefore been studied.
  • the frequency of MHC class II positive cells was determined in the epidermis at different times post-infection (1 to 120 h).
  • the density of MHC class II positive epidermal cells in naive and in non- infected mice ranged between 450 to 470 Langerhans cells/mm 2 .
  • the number of Langerhans cells/mm 2 remaining in the epidermis was not reduced 1 to 120 h after S. mansoni infection.
  • a complementary approach based on the spontaneous migration of Langerhans cells from skin explants cultured in vitro for 24 h was utilized (Fig. 2B).
  • the inventors have next investigated whether S. mansoni infection could alter Langerhans cells migration in a system known to promote a strong Langerhans cells departure to the systemic lymph nodes.
  • infected mice were injected into ear pinnae with TNF ⁇ and the capacity of Langerhans cells to emigrate from the skin was then assessed 1 h after injection.
  • TNF ⁇ caused approximately 54 % reduction in Langerhans cells frequency compared to control mice (carrier).
  • infection by S. mansoni inhibits the TNF ⁇ - induced Langerhans cells migration 6 and 24 h after infection.
  • TNF ⁇ treatment or not, with S. mansoni.
  • S. mansoni As observed in Fig. 3B, in non- infected mice, TNF ⁇ administration resulted in increased number of CD11c positive cells in the T cell area of the systemic lymph nodes whereas few CD11c positive cells were detected in the systemic lymph nodes from TNF ⁇ - treated S. a ⁇ son -infected mice.
  • 2.4 IL-10 is not sufficient to inhibit Langerhans cell migration in S. mansoni infected mice
  • schistosomula excreted/secreted products were i.d injected into the ear pinnae and the density of Langerhans cells remaining in the epidermis was determined 1 h after TNF ⁇ injection.
  • schistosomula excreted/secreted products had a strong inhibitory effect on the TNF ⁇ -induced Langerhans cells mobility.
  • the excreted/secreted products contain bioactive lipophilic compounds able to activate host cells (10). The lipophilic fraction from the excreted/secreted products on the TNF ⁇ -induced Langerhans cells departure was further tested. Compared to the control
  • DMSO DMSO
  • DP receptor is expressed on total epidermal cells.
  • Langerhans cells (XS52) and Keratinocytes (Pam212) lines were used.
  • Fig. 7A mRNAs for the DP receptor in total mouse epidermis as well as on the Langerhans cells and to a lesser extent, Keratinocytes lines, was detected.
  • mice were treated with the highly specific DP receptor antagonist BWA868C 15 min before the infection, six hours later, the Langerhans cells frequency was established in DMSO and in BWA868C-treated animals.
  • PGD 2 induced a transient rise in F-actin content (reached at 10 sec) followed by a rapid and sustained actin depolymerization.
  • XS52 were not responsive to PGE 2 .
  • the effect of the PKA on the PGD 2 -induced actin rearrangement was tested.
  • cells were pretreated with the potent PKA inhibitor H89 (or DMSO as a control) for 15 min prior to stimulation with PGD 2 .
  • inhibition of PKA exacerbates the transient increase of F-actin content and, in the second phase of the response, prevents the F-actin depolymerization.
  • BW245C The effect of the topical application of BW245C in the reduction of the cutaneous pathology in two different models of contact dermatitis was then investigated.
  • the therapeutical potency of BW245C treatment during the sensitization phase or during the challenge phase was also compared.
  • a murine model of contact hypersensitivity induced by the hapten fluoroscein isothiocynate (FITC) was used.
  • FITC hapten fluoroscein isothiocynate
  • mice treated topically with the DP receptor agonist BW245C (50 ⁇ M) developed a profoundly reduced contact hypersensitivity response (80% inhibition) compared to controls (Fig.10A).
  • Fig.10A mice treated topically with the DP receptor agonist BW245C
  • Fig.10B mice treated topically with the DP receptor agonist BW245C (50 ⁇ M) developed a profoundly reduced contact hypersensitivity response (80% inhibition) compared to controls.
  • Fig.10A show that modulating Langerhans cell migration with DP receptor agonists is able to prevent the symptoms associated to contact dermatitis.
  • the topical treatment of mice with BW245C during the challenge phase also reduces the ear swelling (30% inhibition compared to controls) (Fig. 10B).
  • mice displayed a strong eczematous reaction characterized by erythema, desquamation and the presence of scab. All these criteria were scored from 0 to 4 and results are expressed as the sum of all criteria.
  • the topical application of BW245C during the sensitization phase reduced the symptoms associated to the pathology compared to controls. This effect is also associated with a significant decrease of skin thickness (Fig. 11 B).
  • mice with BW245C during the challenge phase decreased the cutaneous pathology since the total of the scores and the skin thickness are reduced compared to controls (Fig.12). All together, these results show that the symptoms associated to contact dermatitis are prevented or reduced by BW245C treatment during the sensitization or the challenge phase, respectively.
  • a reduction of the immune response is sought.
  • the inventors used a murine model of dermatitis elicited by epicutaneous sensitization with the protein ovalbumin. Compared to controls, mice topically treated with BW245C developed a weak eczematous reaction as seen on skin sections stained with hematoxylin eosine safran (Fig.13A). Fig. 13B shows a significant reduction of the epidermis thickness in BW245C-treated mice compared to untreated mice.
  • mice treated with BW245C had a reduced number of infiltrating mast cells in the skin compared to controls (Fig. 13C).
  • the immune response in atopic dermatitis is associated with a type 2 immune response resulting in the induction of antigen specific lgG1 and IgE.
  • the antibody response was analysed by ELISA.
  • the topical application of BW245C induced a significant reduction in the ovalbumine specific lgG1 titer.
  • these mice also displayed a significant decrease in total IgE level compared to controls (Fig. 14B).
  • AH activation of the DP receptor triggered by topical application of BW245C during the sensitization phase reduces profoundly the Th2 immune response and consequently decreases the symptoms associated as skin thickness and mast cell infiltration.
  • the DP receptor agonist BW245C inhibits the TNF ⁇ -induced migration of Langerhans cell in human
  • MLNs were subjected to collagenase/DNAse/EDTA treatment, cell suspensions were stained using MHC II and CD11c Abs and examined by flow cytometry. The cells were analyzed on bivariate plots of MHC class II versus CD11c and examined for FITC positivity. Dead cells and debris were excluded using propidium iodide. As previously described (21 ), two main clusters could be distinguished in the MHCII + CD11 c + quadrant. One group of cells called airway DCs had a strong expression of MHCII and intermediate to high levels of CD11c.
  • the other group described as non airway DCs had intermediate expression of MHCII and high level expression of CD11c.
  • FITC positivity was detected in both cell groups.
  • the percentage of FITC-OVA + cells was higher in the airway DC population.
  • No FITC-OVA cells could be detected in the LNs of the control group mice instilled with PBS/DMSO. This experiment shows that lung DCs transport instilled FITC-OVA into the LNs that drain the lungs.
  • the inventors have identified the mechanisms that lead to the retention of Langerhans cells in the epidermis and attempted to identify the responsible factor(s).
  • IL-10 specific mRNA is strongly increased in S. ma ⁇ so/i -infected skin. Despite this, using IL-10 KO or anti-IL-10 treated mice, the inhibition of Langerhans cells migration in infected epidermis was still observed. Similarly, RT-PCR analysis suggests that the chemokine receptors CCR-1 and CCR-7 do not appear to be implicated in the herein described inhibitory effects. The hypothesis that factors released by parasites while penetrating the skin may inhibit the TNF ⁇ -induced signals that stimulate Langerhans cells migration was checked. It was found that injection of lipophilic factors from the schistosomula excreted/secreted products mimicked the inhibitory effects observed during infection.
  • PGD 2 but not PGE 2 nor the other major eicasonoids produced by schistosomula, specifically induces the retention of Langerhans cells in the skin after TNF ⁇ treatment. Furthermore, the inventors also demonstrate that PGD 2 specifically activates Langerhans cells by interacting with the AC- coupled DP receptor, and that the resulting signaling pathway interferes with the TNF ⁇ -induced signals implicated in Langerhans cells departure. This later finding is important since PGD 2 , as well as its metabolite 15d-PGJ2, can also activate the peroxisome proliferative-activated receptors, a family of nuclear receptors recently shown to inhibit the chemoattractant-induced migration of various cells.
  • topical administration of an agonist of the DP receptor to the mucosa of the lungs can block the migration of DC to the lymph nodes providing clear evidence of a possible use of topical formulations containing agonist of DP receptor in the prevention of asthma.
  • Schistosoma mansoni a comparative study of schistosomula transformed mechanically and by skin penetration. Electrophysiological responses to a wide range of substances. Parasitology 88: 477-489.

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Abstract

L'invention concerne l'utilisation de composés actifs capables de moduler la voie intercellulaire déclenchée par le récepteur de la prostaglandine D2 (récepteur de DP) dans des cellules de Langerhans et/ou des cellules dendritiques et l'émigration subséquente de cellules de Langerhans et/ou de cellules dendritiques, dans la préparation d'un médicament permettant de moduler la réponse cutanée immune. Plus précisément, l'invention concerne des compositions pharmaceutiques destinées à une application topique, ces compositions renfermant de la prostaglandine D2 (PGD2), des agonistes ou des antagonistes du récepteur de DP. L'invention se situe dans le domaine des traitements topiques d'une palette de pathologies induites par une réponse immune ou associées à celle-ci.
PCT/EP2001/014205 2000-12-08 2001-11-08 Utilisation de composes actifs capables de moduler la voie intracellulaire declenchee par le recepteur de dp dans des cellules de langerhans WO2002045718A1 (fr)

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