Use of a tetrapeptide with the formula N-Palmitoyl-Lys-Thr-Phe-Lys in the treatment of non-human animal atopic dermatitis

Abstract

The invention relates to a tetrapeptide of the formula N-Palmitoyl-Lys-Thr-Phe-Lys, for its use in the prevention and / or treatment of non-human animal atopic dermatitis, and to a veterinary topical composition comprising at least said tetrapeptide and at least one physiologically acceptable excipient, for its use in the prevention and / or treatment of non-human animal atopic dermatitis, in particular canine atopic dermatitis. Figure to be published with the abstract: 1

Description

Title of the invention: Use of a tetrapeptide of formula N-Palmitoyl-Lys-Thr-Phe-Lys in the treatment of non-human animal atopic dermatitis

[0001] The present invention is in the field of atopic dermatitis and in particular the prevention and / or treatment of non-human animal atopic dermatitis.

[0002] More specifically, the present invention relates to a tetrapeptide of formula N-Palmitoyl-Lys-Thr-Phe-Lys, for its use in the prevention and / or treatment of non-human animal atopic dermatitis (AD), particularly in companion animals such as dogs and cats, and to a composition comprising at least said tetrapeptide and at least one physiologically acceptable excipient, for its use in the prevention and / or treatment of non-human animal atopic dermatitis (AD), particularly in companion animals such as dogs and cats.

[0003] According to the Scientific Council of the French Society of Allergology, atopic dermatitis, also called atopic eczema, is a chronic inflammatory skin disease. In humans, it develops preferentially in infants and children, but can persist or even appear sometimes in adolescents and adults.

[0004] Atopic dermatitis also affects some companion animals such as canids and felids. The reported prevalence in companion animals varies, at least in part, due to the different populations studied, diagnostic methods, and geography (i.e., due to the different types and levels of allergens present in the environment). The prevalence has been reported in 12.5% ​​of cats with dermatological signs in a specialized university clinic versus slightly more than 1% of cats presented for skin conditions in general veterinary practice. AD affects approximately 10% of the global canine population. It is one of the most common causes of dermatological disorders in dogs.

[0005] According to the new definition of ICADA (“International Committee on Allergy Diseases of Animals”), AD is a “typically pruritic, hereditary, inflammatory skin disease, primarily mediated by T lymphocytes, involving an interaction between skin barrier abnormalities, sensitization to allergens and microbiotic dysbiosis”.

[0006] This incurable disease requires lifelong care, which can be complex due to its multimodal nature and the need for owner involvement. The age of onset is generally less than 3 years in dogs and cats.

[0007] Atopic pets are often pruritic. Erythema and secondary skin infections are common in dogs. Affected cats often present with miliary dermatitis or eosinophilic granuloma complex. Domestic animals may exhibit alopecia, excoriations due to scratching, or otitis externa (which may be the only manifestation in some dogs).

[0008] The most frequent complications of atopic dermatitis are colonization of skin lesions by Staphylococcus aureus, causing dysbiosis of the skin microbiome, or the herpes virus.

[0009] It is likely that several genes contribute to the emergence of the pathology. Recent studies have notably highlighted abnormalities affecting the gene encoding filaggrin and other molecules necessary for the formation of the stratum corneum and the resistance of the skin barrier.

[0010] This alteration of the skin's structure will also allow environmental allergens (pollen, dust, soaps, etc.) to penetrate the epidermis and stimulate the immune system. The latter will overreact to what it perceives as an attack, triggering eczema: redness, itching, inflammation, and oozing.

[0011] Like many chronic diseases that cannot be cured, atopic dermatitis in pets, such as dogs and cats, has a real impact on the quality of life of the animal, but also of everyone around it. Therefore, there is no treatment that can cure it: it is a lifelong genetic disease with a chronic course, characterized by periods of remission and relapse (flare-ups).

[0012] During a flare-up, treatment aims to reduce inflammation and itching, to relieve the animal and prevent secondary infection. The application of a topical corticosteroid, an immunomodulator, an antiseptic solution, possibly with antihistamine tablets as an additional treatment, can help the animal get through the attack.

[0013] Atopic dermatitis therefore requires drug treatment during flare-ups, but also the use of maintenance or comfort products between flare-ups. These maintenance and comfort products are generally topical skincare products. These are the products targeted by the present invention.

[0014] These topical treatments are intended to restore, nourish, hydrate, and soothe the skin. Depending on the condition of the animal's skin and coat, different treatments are generally prescribed by veterinarians: - Soothing and moisturizing treatments to relieve itching and reduce lesions on allergic skin, - Anti-seborrheic treatments to restore balance to oily and malodorous skin, or even dry skin with dandruff, - antiseptic treatments, to disinfect and cleanse skin infected by bacteria or yeasts.

[0015] Gentle shampoos based on moisturizing and soothing active ingredients such as lipids like ceramides and fatty acids, combined with other ingredients such as vitamins, moisturizing or soothing plant extracts, or ingredients that reduce the adhesion of microorganisms to the skin, are already available on the market. Examples include the products sold under the trade names Allermyl® Allercalm® and Allerderm® Spot-on by Virbac, and the shampoo sold under the trade name Douxo® S3 Calm by Céva Santé Animale.

[0016] However, there is still a need for topical formulations for pets suffering from atopic dermatitis or prone to developing atopic dermatitis, usable to prevent and / or treat non-human animal atopic dermatitis.

[0017] The invention described below makes it possible to meet this need.

[0018] The Inventors have demonstrated that a tetrapeptide with a specific formula, as defined below, reduces the overexpression of thymic stromal lymphopoietin (TSLP) and can thus be advantageously used in the prevention and / or treatment of non-human animal atopic dermatitis. TSLP is a cytokine produced by keratinocytes and mast cells. Its production is greatly increased during atopic dermatitis and promotes the initiation of a Th2-type inflammatory response by dendritic cells (Carmi-Levy I et al., Clin. Rev. Allerg. Immunok, 2011; 41: 245-253).TSLP thus plays an important role in the atopic march (from atopic dermatitis to asthma) since its levels are correlated with skin sensitization and asthma severity in mice, whereas this sensitization and its cutaneous and bronchial consequences are greatly reduced in mice whose gene encoding TSLP is inactivated (Leyva-Castillo JM et al., J. Invest. Dermatol., 2013; 133: 154-163).

[0019] The invention therefore relates first to a tetrapeptide of the formula N-Pahnitoyl-Lys-Thr-Phe-Lys (also called Pal-KTFK tetrapeptide), for its use as an active ingredient for the prevention and / or treatment of non-human animal atopic dermatitis, preferably in companion animals such as dogs and cats, and especially for the prevention and / or treatment of canine atopic dermatitis.

[0020] The tetrapeptide with the formula N-Palmitoyl-Lys-Thr-Phe-Lys therefore comprises a palmitoyl chain substitution at the N-terminus and no substitution at the C-terminus, and, in that order, the following amino acids: lysine (Lys), threonine (Thr), phenylalanine (Phe), and lysine. The tetrapeptide Pal-KTFK is a known compound, notably used in human cosmetics in the form of oil-wax-surfactant-water microemulsions. It is, in particular, present in the product sold under the trade name Crystalide® by the company Sederma. According to the International Nomenclature for Cosmetic Ingredients (INCI), the tetrapeptide present in the Crystalide® product promotes the synthesis of the transparent α-crystalline protein found in the lens (approximately 50% of its content), but which is also distributed in many tissues, including the skin.It can therefore be used to improve complexion and in compositions designed to combat the effects of aging in human cosmetics.

[0021] Its role in reducing TSLP overexpression had, however, never been mentioned in the literature, let alone highlighted.

[0022] According to the invention, the tetrapeptide Pal-KTFK can be implemented in a topical, cosmetic or dermatological composition.

[0023] Thus, the present invention has as its second object a veterinary topical composition comprising at least one physiologically acceptable excipient and, as an active ingredient, at least one tetrapeptide of formula N-Pahnitoyl-Lys-Thr-Phe-Lys, for its use for the prevention and / or treatment of non-human animal atopic dermatitis, preferably in companion animals such as dogs and cats, and particularly for the prevention and / or treatment of canine atopic dermatitis. DEFINITIONS

[0024] According to the invention, a topical composition is understood to be a cosmetic or dermatological composition intended to be applied to the skin, mucous membranes and coat of the animal.

[0025] For the purposes of the present invention, the prevention of atopic dermatitis includes the treatment of skin with an atopic (or pre-atopic) tendency.

[0026] Atopic (or pre-atopic) skin is an extremely fragile skin type. It is characterized by an impaired skin barrier that promotes the penetration of allergens, resulting in dry skin that is more vulnerable to external aggressors and a predisposition to allergies. Atopic (or pre-atopic) skin can easily develop atopic dermatitis.

[0027] According to the invention, a physiologically acceptable excipient is an excipient suitable for external topical administration, in particular an excipient that is dermatologically acceptable. These excipients, suitable for the formulation of a topical composition, are well known to those skilled in the art.

[0028] The expression "non-human animal" refers to the veterinary field, in particular companion animals such as dogs (canids) and cats (felids), and especially dogs. DETAILED DESCRIPTION

[0029] According to a particular embodiment of the invention, the topical composition may comprise at most about 10 ppm of said tetrapeptide.

[0030] According to a preferred embodiment, the content of said tetrapeptide within said composition is approximately 2 to 8 ppm, and even more preferably approximately 4 to 6 ppm relative to the total weight of said topical composition.

[0031] According to a particular embodiment, said tetrapeptide is used in the form of an emulsion, in particular an oil-in-water emulsion comprising one or more fatty acid triglycerides. As an example of such a tetrapeptide formulated as an emulsion, mention may be made of the product sold under the name Crystalide® by the company Sederma.

[0032] According to a particular embodiment of the invention, the topical composition further comprises calcium ions.

[0033] The presence of calcium ions in the topical composition usable according to the present invention is advantageous because these calcium ions contribute to the proper functioning of the skin's barrier function. Calcium ions serve as essential signaling molecules that regulate numerous cellular functions in keratinocytes, including proliferation and differentiation. Since keratinocytes require different calcium concentrations for various functions (low calcium for proliferation, high calcium for differentiation), an epidermal calcium gradient is formed. In the lower layers of the epidermis (basal layer and stratum spinosum), the calcium gradient is at its lowest but rises, reaching its peak in the granular layer, and then declines again towards the outermost layers of the stratum corneum, where keratinocytes reach their final stage of differentiation.This keratinocyte differentiation is mandatory for proper skin barrier function. In addition to driving keratinocyte differentiation, the calcium gradient plays a role in keratinocyte migration and wound healing. Taken together, the calcium gradient and signaling are essential for a healthy skin barrier and barrier homeostasis. Calcium gradient collapse... It disrupts skin homeostasis. In atopic dermatitis, calcium signaling is dysregulated and the calcium gradient is flattened. The loss of the epidermal calcium gradient may partly result from fewer calcium-sensitive receptors. This leads to significant dysregulation of proteins that constitute the skin barrier, including loricrin and filaggrin, thus resulting in reduced barrier function.

[0034] According to a particularly advantageous embodiment of the invention, calcium ions are encapsulated in a supramolecular structure comprising one or more concentric lipid bilayers enclosing aqueous compartments containing said calcium ions (vectorized calcium). These supramolecular structures are generally in the form of liposomes or lipid cones. In this type of structure, the lipid bilayers are formed by amphiphilic lipids, generally phospholipids, and the calcium ions are present in the aqueous compartments between the lipid bilayers. An example of vectorized calcium is the product sold under the trade name EpiCalsome® by Mibelle Biochemistry.

[0035] When calcium ions are present in the topical composition usable according to the invention, their content is preferably from 13 to 32 ppm approximately, and even more preferably from 19 to 25 ppm approximately, relative to the total weight of said composition.

[0036] In addition to the tetrapeptide as defined above, the topical composition usable according to the invention may further contain one or more additional active ingredients whose presence may enhance and / or complement the advantageous properties of the tetrapeptide as defined above in the prevention and / or treatment of atopic dermatitis. Such additional active ingredients may include, in particular, anti-pruritic and / or anti-neurogenic inflammation agents, agents that rebalance the cutaneous immune system, and agents that act on the dysbiosis of the cutaneous microbiome, including agents that inhibit the action of S. aureus or reduce its action.

[0037] Examples of anti-pruritic agents include, but are not limited to, saccharidic gums obtained by bacterial fermentation of sorbitol, such as the product sold under the trade name Biosaccharide Gum-2® by the company Solabia; certain plant extracts such as extracts of Boswellia serrata, Acmella oleracea, Aloe vera, oat (Avena saliva), burdock (Arctium iappa), calendula (Calendula), fumitory (Fumaria officinalis), witch hazel (Hamamelis virginiana), mallow (Malva sylvestris), lemon balm (Melissa officinalis), stinging nettle (Urtica dioica or Urtica urens), wild pansy (Viola tricolor), horsetail (Equisetum arvenses), and sarsaparilla (Smilax). aspera), white birch (Betula alba), heart pea (Cardiospermum halicacabum), etc., corn or oat starch hydrolysates, and oily actives such as peroxidized corn oil (Zea mays) and bisabolol.

[0038] As examples of cutaneous immune system rebalancing agents, non-limiting examples include unsubstituted [3-glucans such as the product sold under the trade name Plerasan Re-Balance ® by ROELMI HPC, as well as [3-glucans substituted with carboxymethyl groups such as the product sold under the trade name CM-Glucan Forte (Magnesium Carboxymethyl Beta-Glucan) by Mibelle Biochemistry.

[0039] Examples of active ingredients acting on skin microbiome dysbiosis include, but are not limited to, alpha-glucan oligosaccharides, Yacon tuber extract (Polymnia sonchifolia), Lactobacillus, rhamnose, glucose, glucuronic acid, glycolipids such as flocculosin, which is a low molecular weight glycolipid isolated from the yeast-like fungus Pseudozyma flocculosa, and biopeptides from yeasts, in particular biopeptides from the yeast Metschnikowia reukaufii, isolated from the Hoya camosa porcelain flower.

[0040] The quantity of additional active ingredients in the topical composition usable according to the invention may vary depending on the nature of the ingredient used and the desired effect. Those skilled in the art shall ensure that the presence of this additional active ingredient does not alter the intrinsic properties of the tetrapeptide of the formula defined above.

[0041] The topical composition usable according to the invention may be presented in the various galenic forms classically used for topical application in pets, i.e. in the form of liquid shampoos having different functions, in particular as a moisturizing or soothing shampoo, dry shampoos, no-rinse cleansing foam, lotion, in particular in the form of ear lotion in drops, spray, ointment, or single-dose solution in the form of a single-use pipette (in English "spot-on"), said galenic forms also containing usual physiologically acceptable excipients.

[0042] These forms of topical presentation can be prepared by techniques known to those skilled in the art.

[0043] Physiologically acceptable excipients may include, but are not limited to: - emulsifiers and co-emulsifiers such as sorbitan olivate, glyceryl stearate-citrate, coco-glucoside and glyceryl stearate, - consistency agents such as beeswax, glycol palmitate, and microcrystalline wax, - emollients such as silicone oils (such as dimethicone, caprylyl methicone, etc.), mineral oils (such as hydrogenated polyisobutene, paraffin oil, isohexadecane, etc.), isononyl isononanoate, lanolin, cetyl palmitate, palmitic acid, Ci0-Ci8 triglycerides, petroleum jelly (or petrolatum), vegetable oils such as sweet almond (Prunus dulcis), wheat germ (Triticum vulgare), and hemp (Cannabis saliva) oils, and vegetable butters such as cocoa (Theobroma cacao) and mango (Mangifera indica L.) butters, - Penetrating agents such as octyl dodecanol and propylene glycol, - humectants such as butylene glycol and polyethylene glycol and glycerin ethers such as glycereth-26, urea, - antioxidants such as vitamin C and its derivatives, vitamin E, vitamin F, vitamin PP (niacin and nicotinamide), - chelating agents such as phytic acid and ethylenediaminetetraacetic acid, - surfactants, particularly anionic ones, such as the disodium salt of glutamic acid amide and coco acid, sodium lauryl ether sulfate, and sodium C14.16 olefin sulfonate, - preservatives such as phenylethyl alcohol, hydroxyacetophenone, chlorphenesin, chlorhexidine digluconate, chlorhexidine, micronized silver and sodium benzoate, - gelling agents such as polymers, in particular polyacrylates (Carbomers), polyacrylamides, sclerotium gum, bentone, and cellulose derivatives such as hydroxyethylcellulose and chitosan derivatives, - pH modifying agents such as citric acid and sodium hydroxide, - solubilizers such as PEG 40 hydrogenated castor oil and ethoxylated sorbitan monolaurate (20 moles), - colorings such as beta-carotene, caramel, spirulina extract, orcanette root extract (Alkanna tinctoria), mica, iron oxide, etc.

[0044] -denaturing agents such as Petit Grain Bigarade essential oil (Citrus aurentium var. amara) to discourage licking.

[0045] The topical composition usable according to the invention is particularly well tolerated by the skin, exhibits no cytotoxicity, and its application to the skin, even for long periods, does not cause any systemic effects. It can be applied to the skin and / or mucous membranes, particularly to areas where it is needed, as well as to the animal's coat.

[0046] The duration of application will vary depending on the improvement of the skin in the treated areas. As an indication, the topical composition according to the invention is preferably applied between flare-ups, and the duration of treatment can vary from approximately 8 to 15 days.

[0047] Other features and advantages of the present invention will become apparent from the following examples and the accompanying figures in which:

[0048] [Fig. 1] is a schematic representation of a reconstructed skin model used in Example 1. EXAMPLES

[0049] The following products were used in the examples: - Tetrapeptide Pal-KTFC 6 ppm sold under the reference WGPALKTFKMBAL by the company Sederma; - Sodium dodecyl sulfate (reference L4509, Sigma) - Thapsigargine (reference T7459, Invitrogen TM) - ELISA kit for the assay of TSLP sold under the reference ELH-TSLP-1 by Raybiotech;

[0050] - Basal culture medium for culturing primary keratinocytes, free of components of animal origin, chemically defined and supplemented with a non-animal serum.

[0051] These ingredients were used as received from the manufacturers, without further purification.

[0052] EXAMPLE 1: Demonstration of the properties of the tetrapeptide Pal-KTFK for the prevention and / or treatment of atopic dermatitis

[0053] The study was conducted on a reconstruction of human epidermis. Human skin equivalents reconstructed in vitro closely mimic normal human skin. Since these models largely reproduce the barrier function properties of normal human skin, they can be used to screen for potential skin irritants and to study effects related to product efficacy.

[0054] There are many similarities between human atopic dermatitis and animal (non-human) atopic dermatitis, particularly canine atopic dermatitis. Indeed, the results of several transcriptomic studies performed on skin from humans or dogs with atopic dermatitis have been compared (Freudenberg et al., Human Disease. J. Invest. Dermatol., 2019, 139(4), 968-71). Overall, the authors showed a high degree of similarity in expression patterns between the two species. Pathways that appear to be overexpressed in both species are linked to the inflammatory response, the activation of T lymphocytes and leukocytes, and the cytokine response. Lipid homeostasis and metabolism are underexpressed in both species.The authors conclude that there is a relatively high degree of concordance between canine and human atopic dermatitis at the molecular level, thus validating this model for mechanistic and therapeutic studies. Therefore, the trials presented in this example were performed on the AltemaSensiSkin® human epidermis model. This model includes keratinocytes. normal human epidermal NHEK, one of the main cell types of the skin, cultured on polycarbonate inserts.

[0055] Figure 1 is a schematic profile (Fig. 1a) and vertical sectional (Fig. 1b) representation of the AltemaSensiSkin model used. This model (1) comprises at least one culture well (2) containing a basal culture medium (3) in which a culture insert (4) is placed. The insert has a membrane (5) at its base on which the reconstruction of the human epidermis (6) rests. The products to be tested (7) can be applied locally directly to the surface of the reconstruction of the human epidermis (6), and their irritant potential can be evaluated using a number of parameters, including cell viability, measurements of biological markers, or by studying the morphological components of the tissue.

[0056] The AltemaSensiSkin® model is derived from the AltemaSkin® model, which is an in vitro reconstruction of the human epidermis (RhE) from the company Cell Altemativ (adapted from the method of Poumay et al. (Arch. Dermatol. Res., 2004, 296: 203-211)). The AltemaSensiSkin® model is, in fact, an "immature" reconstructed AlternaSkin® epidermis designed to respect the morphology and properties of infant skin and / or sensitive skin, particularly the stratum corneum. It was used in this example as a model of atopic skin after incubation of the reconstructed skin with thapsigargin. Indeed, TSLP secretion is highly dependent on calcium ions. Treatment with thapsigargin (TG) promotes the depletion of intracellular calcium reserves and induces the secretion of TSLP, which is abundantly produced by keratinocytes in cases of atopic dermatitis. 1.2 Samples tested

[0057] The different samples tested are presented in Table 1 below:

[0058] [Table 1] Sample Description: Thapsigargin 1 pM (TG) Positive control for inflammation (atopic dermatitis) PAL-KTFK Tetrapeptide 3% Pal-KTFK in solution in a mixture of 75 pL 37% HCl and 5 mL SDS 2% dimethyl sulfoxy Positive control for the MTT test 1.3 MTT cytotoxicity test

[0059] The MTT test allows the measurement of cellular metabolic activity, an indicator of cell viability, proliferation, and toxicity. This colorimetric assay is based on the reduction of a yellow tetrazolium salt [3-(4,5-) bromide dimethylthiazol-2-yl)-2,5-diphenyltetrazolium or MTT] into purple formazan crystals by metabolically active cells.

[0060] The tetrapeptide Pal-KTFK 6 ppm was tested at a concentration of 3% on previously reconstructed epidermis sensitized with thapsigargin at 1 pM.

[0061] To this end, treatment with 1 pM thapsigargin was carried out in the basal culture medium of RhE (n=4 per sample) for 42 h at 37°C. After this treatment time, the culture medium containing 1 pM thapsigargin was replaced with fresh culture medium without thapsigargin. The test product (20 pL) was applied directly to the surface of the epidermis and then incubated at 37°C for 20 hours. A positive control (SDS 2%) was performed under the same conditions, as well as a negative control without a sample.

[0062] At the end of the incubation period, the tissues were placed in a 0.5 mg / mL MTT solution in the culture medium for 3 h, then extracted in 2 x 500 pL of isopropanol. Cell viability was assessed by spectrophotometry at 570 nm using a Tristar2® microplate reader sold by Berthold Technologies GmbH, and absorbance values ​​were recorded using ICE® software version 1.0.9.8. Statistical analysis was performed using Graph Pad Prism v5.03 software.

[0063] Each measurement was taken in triplicate. 1.4. TSLP Assay

[0064] Treatment with 1 pM thapsigargin was carried out in the basal culture medium of RhE (n = 4 per sample) for 48 hours at 37°C to induce TSLP secretion. After this treatment time, the culture medium with thapsigargin was replaced with fresh culture medium. The test product (20 pL) was then applied directly to the epidermal surface and incubated at 37°C for 20 hours. A positive thapsigargin control was performed under the same conditions for 42 hours with thapsigargin but without application of the test product for the following 20 hours.

[0065] A negative (untreated) control was carried out under the same conditions but without application of thapsigargin or product to be tested.

[0066] TSLP was measured using the ELH-TSLP-1 ELISA kit to determine the quantity produced by reconstructed epidermis after various thapsigargin treatments. For this purpose, cell supernatants were collected after the required treatment time (42 hours), and a quantitative assay was performed using the kit according to the manufacturer's recommendations. The procedure involves loading the samples and a range of standards into the wells of a duplicate 96-well microplate. The TSLP marker present in a sample is bound by the antibody immobilized at the bottom of the well. The wells are then washed and A biotinylated antibody is added. After washing, HRP-streptavidin is loaded into the wells. The wells are then washed again, and a substrate solution is added. A blue color develops, the intensity of which is proportional to the amount of TSLP marker bound. A developing solution changes the color from blue to yellow, and the color intensity is measured at a wavelength of 450 µm using a Tristar2® microplate reader sold by Berthold Technologies GmbH. Absorbance values ​​are recorded and analyzed using ICE vl.0.9.8 software. The result is interpreted as a sample / untreated ratio. A value greater than or equal to 2 indicates TSLP overexpression. 1.5 Results

[0067] The results of the cell viability test are presented in Table 2 below: [Tables2] Sample Cell Viability Viability (%)* Standard Deviation (%) Untreated Control 100 2.97 Positive Control (SDS 2%) 1.2 0.18 Thapsigargin Control 42 H 67.2 4.00 PAL-KTFK 3% 42 H 64.9 5.54

[0068] * : Average of 3 trials

[0069] The results presented in Table 2 show that prior treatment with thapsigargin leads to significant sensitization of the epidermis, indicative of a state of atopic dermatitis. Subsequent application of the PAL-KTFK peptide to the previously sensitized epidermis with thapsigargin does not result in a significant decrease in cell viability, which decreases from 67.2% to 64.9%, representing a reduction of only about 3.4%. Therefore, the PAL-KTFK tetrapeptide used according to the present invention does not exhibit a significant cytotoxic effect on the cells of the reconstructed epidermal model used in the test described in this example.

[0070] The results of the TSLP assay are presented in Table 3 below: [Tables3] Sample TSLP Concentration (pg / ml) Untreated Control 0.29 Thapsigargine Control 2.44 Thapsigargin + tetrapeptide PAL-KTFK 6 ppm at 3% 1.30

[0071] The results presented in Table 3 show that contact of the reconstructed epidermis with thapsigargin leads to overexpression of the cytokine TSLP, involved in the phenomenon of atopic dermatitis (8.4 for the untreated control). Treatment of the reconstructed epidermis for 20 hours with the tetrapeptide PAL-KTFK according to the invention results in a significant decrease in TSLP overexpression compared to the thapsigargin control (-1.14 pg / mL), i.e., by a factor of 1.87.

[0072] All of these results demonstrate that the tetrapeptide Pal-KTFK has advantageous properties for use in the prevention and / or treatment of atopic dermatitis. EXAMPLE 2: Preparing a shampoo

[0073] In this example, a veterinary shampoo for the treatment of atopic dermatitis and having the following composition was prepared: PHASE A: - Demineralized water 58.09% - Crosspolymer of acrylates and Cio-C3o alkyl acrylate 1.0% PHASE B: - Sodium lauryl sulfate in aqueous solution sold under the trade name TEXAPON NSO UP by BASF 25.0% - Mixture of sodium coco amphoatheta, demineralized water and sodium chloride sold under the trade name DEHYTON MC by BASF... 8.0% - Tetrapeptide PAL-KTFK formulated as an emulsion, sold under the trade name Crystalide ® by the company Sederma 1.0% PHASE C: - Phenoxyethanol 0.6% - Chlorphenesin 0.25% - Demineralized water 5.0%

[0074] The elements of phase B were mixed under low stirring, then phase B was added to phase A.

[0075] The elements of phase C were mixed, phase C was added to the mixture of phases A and B.

[0076] The pH of the shampoo thus obtained was adjusted between 5 and 6 using a 32% aqueous sodium hydroxide solution.

[0077] EXAMPLE 3: Preparation of a liquid composition in spray form

[0078] In this example, a liquid composition in the form of a spray for veterinary use in the treatment of atopic dermatitis and having the following composition was prepared:

[0079] PHASE A: - Demineralized water 88.10% - Mixture of microcrystalline cellulose, cellulose gum and xanthan gum marketed under the trade name VIVAPUR CS TEX by the company JRS Rettenmaier 1.0% PHASE B: - Tetrapeptide PAL-KTFK formulated as an emulsion, sold under the trade name Crystalide ® by Sederma 3.0% - Mixture of hydrogenated phospholipids, ethylene glycol, glycerin, calcium chloride dihydrate and demineralized water sold under the trade name Epicalsome ® by Mibelle Biochemistry 2.0% PHASE C: - Demineralized water 5.0% - Phenoxyethanol 0.6% - Chlorphenesin 0.2%

[0080] Phase A was prepared by dissolving the ingredient Vivapur CS TEX in demineralized water under agitation until a homogeneous mixture was obtained.

[0081] Phase B was obtained by mixing the different ingredients under agitation.

[0082] Phases A and B were then mixed under stirring, and then phase C was incorporated into the mixture of phases A and B under stirring.

[0083] This composition was then packaged in a container equipped with a device allowing it to be sprayed in the form of a spray. EXAMPLE 4#: Preparing milk

[0084] In this example, a veterinary milk for the treatment of atopic dermatitis and having the following composition was prepared: PHASE A: - A mixture of cetearyl alcohol and behentrimonium methosulfate marketed under the trade name VARISOFT BTMS by the company EVONIK 2.0% - Mixture of dicaprylic ether and tocopherol marketed under the trade name CETIOL OE by BASF 2.5 PHASE B: - Demineralized water 88.97% - Hydroxypropyl starch phosphate 0.5% - Chlorphenesin 0.28% PHASE C: - Phenoxyethanol 0.6% PHASE D: - Tetrapeptide PAL-KTFK formulated as an emulsion, sold under the trade name Crystalide® by Sederma 3.0% - Mixture of hydrogenated phospholipids, ethylene glycol, glycerin, calcium chloride dihydrate and demineralized water sold under the trade name Epicalsome ® by the company Mibelle Biochemistry 2.0% PHASE E: 50% aqueous citric acid solution 0.15% The components of phase A were weighed and then stirred at 75°C. The components of phase B were weighed and then stirred at 75°C.

[0085] The emulsion was made by pouring phase B into phase A under vigorous stirring, then cooled to a temperature of about 50°C before introducing phase C. The components of phase D were then successively introduced into the mixture thus obtained after it had been cooled to a temperature of about 35°C.

[0086] The pH of this lotion was adjusted to a value between 5 and 6, using a 50% citric acid solution.

Claims

Demands

1. Tetrapeptide of formula N-Palmitoyl-Lys-Thr-Phe-Lys, for its use as an active ingredient for the prevention and / or treatment of non-human animal atopic dermatitis, in particular canine atopic dermatitis.

2. Veterinary topical composition comprising at least one physiologically acceptable excipient and, as an active ingredient, at least one tetrapeptide of formula N-palmitoyl-Lys-Thr-Phe-Lys, for use in the prevention and / or treatment of human atopic dermatitis, in particular canine atopic dermatitis.

3. Veterinary topical composition for use according to claim 2, characterized in that the content of said tetrapeptide within said composition is 2 to 8 ppm, relative to the total weight of said topical composition.

4. Veterinary topical composition for use according to claim 2 or 3, characterized in that said tetrapeptide is used in the form of an emulsion.

5. Veterinary topical composition for use according to claim 4, characterized in that said emulsion is an oil-in-water emulsion comprising one or more fatty acid triglycerides.

6. Veterinary topical composition for use according to any one of claims 2 to 5, characterized in that said composition further comprises calcium ions.

7. Veterinary topical composition for use according to any one of claims 2 to 6, characterized in that said calcium ions are encapsulated in a supramolecular structure comprising one or more concentric lipid bilayers enclosing between them aqueous compartments comprising said calcium ions.

8. Veterinary topical composition for use according to claim 6 or 7, characterized in that the calcium ion content is 13 to 32 ppm relative to the total weight of said composition.

9. Veterinary topical composition for use according to any one of claims 2 to 8, characterized in that said composition further comprises one or more additional active ingredients selected from anti-pruritic actives, actives rebalancing of the cutaneous immune system and active ingredients acting on dysbiosis of the cutaneous microbiome.

10. Veterinary topical composition for use according to claim 9, characterized in that the anti-pruritic actives are selected from saccharide gums obtained by bacterial fermentation of sorbitol, plant extracts of Boswellia serrata, Acmella Oleracea, Aloe vera, oat (Avena sativa), burdock (Arctium iappa), calendula or marigold (Calendula). fumitory (Fumaria officinalis), witch hazel (Hamamelis virginiana), mallow (Malva sylvestris), lemon balm (Melissa officinalis), common or stinging nettle (Urtica dioica or Urtica urens), wild pansy (Viola tricolore), field horsetail (Equisetum arvenses), sarsaparilla (Smilax aspera), white birch (Betula alba), heart pea (Cardiospermum halicacabum), hydrolysates of corn or oat starch, and oily actives selected from peroxidized corn oil (Zea mays) and bisabolol.

11. Veterinary topical composition for use according to claim 9, characterized in that the cutaneous immune system rebalancing actives are selected from unsubstituted [3-glucans and [3-glucans substituted with carboxymethyl groups.

12. Veterinary topical composition for use according to claim 9, characterized in that the active ingredients acting on skin microbiome dysbiosis are selected from alpha-glucan oligosaccharides, Yacon tuber extract (Polymnia sonchifolia), Lactobacillus, rhamnose, glucose, glucuronic acid, glycolipids and biopeptides from the yeast Metschnikowia reukaufii, isolated from the Hoya camosa porcelain flower.

13. Veterinary topical composition for use according to any one of claims 2 to 12, characterized in that it is in the form of liquid shampoo, dry shampoo, no-rinse cleansing foams, lotion, spray, ointment, or single-dose solution in the form of a single-use pipette.

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