EA040422B1 - THE USE OF PROBIOTICS IN THE TREATMENT AND/OR PREVENTION OF ADOPIC DERMATITIS - Google Patents

Description

Technical field

The present invention relates to a probiotic composition and the use of said composition for the treatment and/or prevention of atopic dermatitis. Therefore, the present invention can be considered to be included in the field of medicine, in particular in the treatment of skin diseases.

Background of the Invention

Atopic dermatitis (AD) is a chronic pruritic inflammatory skin disease characterized by the development of eczema lesions with a characteristic distribution pattern that affects patients with skin hyperreactivity to various environmental factors that are harmless to non-atopic patients. Atopic patients often have a personal or family history of asthma or allergic rhinitis or AD and have elevated serum IgE levels. Atopic dermatitis most commonly affects children, but may persist and/or may begin in adolescence or adulthood.

The prevalence of AD is 4 to 20% of the population. There are large differences between rural and urban environments, with the latter having a higher incidence. The incidence of AD is likely to increase for the following reasons: Western lifestyle, increasing maternal age, environmental pollution, maternal smoking, and reduced breastfeeding. Forty-five percent (45%) of children develop AD in the first six months of life and 85% in the first five years. When the disease begins in children under two years of age, 20% have persistent symptoms by age seven.

While atopic dermatitis presents clinically as eczema in both acute and chronic conditions, the disease has considerable variability in

1) the range of clinical manifestations;

2) their genetic basis; And

3) pathogenetic mechanisms underlying clinical manifestations.

One of the most common hypotheses to explain the origin of atopic dermatitis suggests that it is one of a number of manifestations affecting the skin in this case, a systemic process involving other organs and systems of the human body, giving rise to a number of symptoms, such as asthma, food allergies and allergic rhinitis among others. In fact, a high percentage of patients with these diseases have elevated peripheral blood levels of IgE and eosinophils, although the origin and clinical implications are unclear.

With regard to pathophysiological mechanisms, it is assumed that the main role in its origin is played by the immune response. Indeed, recent evidence points to the presence of skin dendritic cells (antigen-presenting cells) and the regulation of Th2 lymphocyte-mediated inflammatory-immune signals in the origin of AD. The data indicate that the pathophysiological origin of AD can be found in these immunological mechanisms, including both antigen-presenting skin dendritic cells and Th2 lymphocyte-mediated immune regulation and inflammatory signals. The source of the stimulus that triggers the immunological cascade that occurs in AD is currently unknown.

The disease has a wide range of clinical manifestations, ranging from minor forms such as hand eczema to more severe forms such as erythrodermic rash. There are no specific pathological or laboratory-type changes, so the diagnosis of AD is clinical and the standard diagnosis meets the Hanifin-Reik criteria (Hanifin J.M. et al., Acta Derm Venereol, 1980, 92:44-7).

The clinical characteristics of AD may vary with age and are included in the diagnostic criteria; they include itching, eczematous lesions, and scratching lesions.

Itching. One of the most important and natural manifestations of atopy. Atopic pruritus is intense and usually causes flare-ups. Itching causes patients to injure themselves by scratching. Although the cause of itching is not well understood, it appears to be related to the release of inflammatory mediators and cytokines.

Eczema. Eczematous lesions can be acute or chronic. Acute lesions are characterized by macules, papules, and erythematous, vesicular, exudative, and very pruritic plaques. Repeated scratching and abrasions lead to chronic lesions characterized by marked thickening of the skin accompanied by lichenification and the obvious presence of dry fibrous papules.

Lichenification. Characteristically lichenified skin lesions are observed, consisting of ill-defined plaques with skin thickening with pronounced skin folds and lines.

Prurigo. Small papules with an inconspicuous vesicle and severe chafing, which are the result of vigorous scratching.

exfoliative dermatitis. Cases of extensive atopy may present clinically as generalized exfoliative dermatitis included in the differential diagnosis of erythroderma.

In addition to establishing a diagnosis, the severity must be assessed or quantified. To this end, various methods and criteria have been developed for assessing severity and monitoring response to treatment in clinical trials and observational studies, and even in clinical practice where the goal is to represent patient progress with greater accuracy than a description provided by the subjective perception of the patient, caregiver, or clinician. In this regard, the most reasonable and used method is the so-called SCORAD index (Scale of atopic dermatitis), developed by the European Task Force on Atopic Dermatitis in 1993 (European Task Force on Atopic Dermatitis. Severity scoring of atopic dermatitis: the SCORAD index, Dermatology , 1993, 186:23-31). It has been proven to be the best method for assessing the severity of atopic dermatitis in comparative studies, and is also the most widely validated in terms of validity, reproducibility, sensitivity, and acceptability. SCORAD is a scoring system that takes into account the extent and intensity of the five major AD lesions (erythema, edema/papule, oozing/crusting, excoriation, and lichenification) and symptoms (itching and sleep loss).

There is some discrepancy in the SCORAD index values, which defines different severity levels. Some Spanish groups consider mild, moderate, or severe to be SCORAD scores of <25, 25-40, or >40, respectively, while other authors, based on correlations with laboratory tests, use scores of <20, 20-40, or >40, corresponding to definition of mild, moderate and severe symptoms.

There is a consensus regarding the treatment of AD, aimed, firstly, at preventing itching and eliminating the inflammatory lesion and, secondly, at preventing relapses (Munoz F., 2002, JANO, 1432:52-7; Boguniewitcz M., Schmid- Grendelmeier P., Leung DYM, 2006, J. Allergy Clin. Inmunol., 118:40-3). H1 antihistamines are prescribed to prevent itching and scratching, although their effectiveness has not been clearly demonstrated. The main goal of treating eczema is to control inflammatory lesions, which also helps control itching. Therefore, depending on the severity of the lesion and the patient's condition, topical corticosteroids of various strengths are used. The drugs with apparently better results are the traditional N-antihistamines that cause sedation (hydroxyzine, clemastine, dexchlorpheniramine) and the new generation N-antihistamines (dexfenfluramine, loratadine, cetirizine and derivatives), which are less sedative but sometimes show results which are not statistically significantly different from placebo (Boguniewitcz M. et al., 2006, Quoted ad supra).

The adverse effects of topical corticosteroids create limitations for use. The most common are adverse reactions at the site of application. With periocular application, the following usually appear: skin atrophy, petechiae, atrophic striae, hypertrichosis, depigmentation, telangiectasia, folliculitis and glaucoma. Less often, more serious systemic adverse effects are described, such as hypothalamic-pituitary-adrenal system suppression, growth retardation, or cushingoid.

The lack of response in some patients to corticosteroids and their adverse effects have led to the development of new drugs to treat inflammation. Among the best studied are topical calcineurin inhibitors (tacrolimus and pimecrolimus) Leung DYM, Bleder T., 2003, Lancet, 361:151-60; Munoz, F., 2002, JANO, 1432:52-7). However, there are still doubts about the safety of these drugs in long-term treatment. In fact, the catalog of products containing tacrolimus or pimecrolimus for the topical treatment of atopic dermatitis indicates that they should only be used in children over two years of age who are not recommended or have previously failed with topical corticosteroids.

The use of probiotics for the treatment of AD has been studied in recent years in a limited number of pilot studies, and results vary depending on the probiotic used and the age of the patients in the study (Soo-Pk Kim et al., 2014, Ann Allergy Asthma Immunol., 113:217e226; Gerasimov S. V. et al., 2010, Am J. Clin. Dermatol., 11:351e361; Farid R. et al., 2011, Iran J. Pediatr. 21:225e230). The most recent studies, including a meta-analysis of previous publications, demonstrate the beneficial effect of the use of probiotics under certain conditions related to age, bacterial strain or combination of probiotics and the dose used (Farid R. et al., 2011, Cited ad supra; Van del Aa L.B. et al., 2010, Clin. Exp. Allergy, 40:795e804 Boyle R. J. et al., 2008, Cochrane Database Syst Rev., 4:CD006135 Lee J. et al., 2008, J. Allegy Clin. Immunol., 121:116). The probiotics most studied for the treatment of atopic dermatitis and the prevention of new outbreaks of the disease belong to the genera Bifidobacterium and Lactobacillus, as they seem to give the best results (Lee J. et al., 2008, Quoted ad supra).

There is a well-known medical need to develop quality therapies that are safe and effective for the long-term management of AD, as atopic dermatitis affects a significant portion of the population, markedly affecting quality of life, which can be affected by medical complications, etc. Topical corticosteroids are considered to be very effective treatments, but they carry a high risk of adverse effects. The inclusion of novel alternatives to topical corticosteroids (such as calcineurin inhibitors) responds to this

- 2 040422 needs, although the risk of adverse effects of these new drugs has not yet been determined. On this basis, the prior art indicates that there is a need to develop new compositions useful in the treatment of AD that are effective and do not have the typical adverse effects of drugs used to date.

Detailed disclosure of the present invention

The present inventors have found that administration of a probiotic composition that modifies the intestinal microbiota of an individual suffering from atopic dermatitis, together with conventional treatment, surprisingly provides remission and/or improvement in the development of atopic dermatitis flares in the individual. As shown in the examples accompanying the present description, when a probiotic composition (comprising microorganisms of the genera Bifidobacterium and Lactobacillus) was administered along with conventional long-term treatment to patients with outbreaks of atopic dermatitis, an improvement in the SCORAD index of 82.36% was observed, while an improvement in this indicator in cases of receiving usual long-term treatment, but without the probiotic composition was 28.4%; differences were considered clinically and statistically significant. In addition, the improvement caused by the probiotic composition occurred from the first month of administration, maintaining or increasing the differences in subsequent analysis during 2 and 3 months of treatment. In addition, another advantage of treating AD with the probiotic composition of the present invention is that the use of corticosteroids in patients treated with said probiotic composition was lower than in the placebo group.

In addition to this beneficial effect on disease progression in patients with atopic dermatitis flare, it was also observed that three months after cessation of treatment with the above probiotic composition, individuals who were treated with said composition had fewer flare-ups and had longer disease-free periods than in those patients who did not receive treatment.

Thus, the usefulness of the probiotic composition described herein for the treatment and/or prevention of atopic dermatitis has been demonstrated.

The inventors have developed a number of aspects of the invention, which will be described in detail below.

The composition of the probiotic composition according to the present invention and its use in the treatment of atopic dermatitis (AD).

In one aspect, the present invention relates to a probiotic composition that contains the microorganisms Bifidobacterium animalis subsp. lactis (hereinafter B. lactis), Bifidobacterium longum and Lactobacillus casei.

In the present invention, the term probiotic composition means a composition containing at least one microorganism that, when ingested, interacts with the metabolism of the individual and has a beneficial effect on him. In the present invention, the probiotic composition contains the microorganisms B. lactis, B. longum and L. casei, hereinafter referred to as the probiotic composition according to the present invention. B. lactis is a bacterium commonly used as a probiotic found primarily in yogurt and other dairy products, including infant formula.

Scientific classification of B. lactis. Kingdom: Bacteria, phylum: Firmicutes, class: Actinobacteria, order: Bifidobacteria, family: Bifidobacteriaceae, genus: Bifidobacterium, species: Bifidobacterium animalis, subsp. lactis. L. casei is a bacterium commonly used as a probiotic found primarily in yogurt and other dairy products, including infant formula.

Scientific classification of L. casei. Kingdom: Bacteria, phylum: Firmicutes, class: Bacilli, order: Lactobacillales, family: Lactobacillaceae, genus: Lactobacillus, species: Lactobacillus casei.

In addition, B. longum is a gram-negative, catalase-negative round bacterium found in the gastrointestinal tract, where it produces lactic acid.

Scientific classification B. longum. Kingdom: Bacteria, phylum: Firmicutes, class: Actinobacteria, order: Bifidobacteria, family: Bifidobacteriaceae, genus: Bifidobacterium, species: Bifidobacterium longum.

In a particular embodiment, the probiotic composition according to the present invention comprises B. lactis CECT 8145 (and/or strains derived therefrom), B. longum CECT 7347 (and/or strains derived therefrom) and/or L. casei CECT 9104 (and/or or strains derived from it).

B. lactis strain CECT 8145 was isolated from the faeces of a healthy breastfed infant under three (3) months of age. This strain was deposited on 14 May 2012 under the Treaty of Budapest with the Spanish Type Culture Collection as International Depository Authority (located at Building 3 CUE, Parc Cientific Universitat de Valencia, C/ Catedratico Agustin Escardino, 9, 46980, Paterna (Valencia ), SPAIN). Assigned deposit number SEST 8145.

L. casei strain CECT 9104 was isolated from the faeces of a healthy breastfed infant under three (3) months of age. This strain was deposited on February 25, 2016 in accordance with the Budapest Treaty in the Spanish Type Culture Collection as an International Depository Authority (located in Building 3 CUE, Parc Cientific Universitat de Valencia, C/ Catedratico Agustin

- 3 040422

Escardino, 9, 46980, Paterna (Valencia), SPAIN). Assigned deposit number SEST 9104.

CECT 7347 B. longum strain was isolated from the faeces of a healthy breastfed infant under three (3) months of age and was deposited on 20 December 2007 in accordance with the Budapest Treaty with the Spanish Type Culture Collection as an International Depository Authority (located in the Building 3 CUE, Parc Cientific Universitat de Valencia, C/ Catedratico Agustin Escardino, 9, 46980, Paterna (Valencia), SPAIN). The assigned deposit number was CEST 7347.

The present invention also contemplates microorganisms or bacteria derived from microorganisms B. lactis, B. longum and L. casei (or their respective strains B. lactis CECT 8145, L. casei CECT 9104 and B. longum CECT 7347) and which can be part of the probiotic composition according to the present invention, since they retain the ability to reduce and/or improve the development of atopic dermatitis in people suffering from this pathology. Examples of strains or microorganisms derived from strains included in the probiotic composition according to the present invention may be mutants and genetically modified organisms that show variations in their genome compared to the genome of the strains of the present invention, but which do not affect the ability of strains to reduce and /or improve the development of atopic dermatitis in an individual. Strains derived from B. lactis, B. longum and L. casei (or from B. lactis strains CECT 8145, L. casei CECT 9104 and B. longum CECT 7347) can be obtained naturally or specifically by mutagenesis as known in this areas, for example, but not limited to, by growing the parental strain in the presence of mutagenic agents or stressors, or by genetic engineering aimed at modifying, deleting and/or inserting certain genes. Thus, as stated above, the present invention also contemplates genetically modified organisms derived from B. lactis, B. longum and L. casei (or from B. lactis strains CECT 8145,

L. casei CECT 9104 and B. longum CECT 7347), which retain the ability to reduce and/or improve the development of atopic dermatitis in an individual and therefore are of use in the treatment of atopic dermatitis. An example of a test to test whether an organism has the ability to reduce and/or improve the development of atopic dermatitis in an individual is described in the accompanying examples herein.

In addition, the present invention also provides cellular components, metabolites and molecules secreted by B. lactis, B. longum and L. casei or their respective strains of B. lactis CECT 8145, L. casei CECT 9104 and/or B. longum CECT 7347 , as well as compositions containing these components, whether cells, metabolites or secreted molecules, and their use for the treatment and/or prevention of atopic dermatitis. Cellular components of bacteria can include cell wall components (such as, but not limited to, peptidoglycan), nucleic acids, membrane components, and the like, such as proteins, lipids, and carbohydrates, and combinations thereof (such as lipoproteins, glycolipids, or glycoproteins). Metabolites include any molecule produced or modified by a bacterium as a result of its metabolic activity during growth, its use in manufacturing processes or during product storage (the probiotic composition of the present invention). Examples of these metabolites include, without limitation, organic and inorganic acids, proteins, peptides, amino acids, enzymes, lipids, carbohydrates, lipoproteins, glycolipids, glycoproteins, vitamins, salts, minerals, and nucleic acids. Secreted molecules include any molecule secreted or excreted by a bacterium during growth, its use in technological processes (for example, in food or drug processing), or during product storage (a probiotic composition according to the present invention). Examples of these molecules include, without limitation, organic and inorganic acids, proteins, peptides, amino acids, enzymes, lipids, carbohydrates, lipoproteins, glycolipids, glycoproteins, vitamins, salts, minerals, and nucleic acids.

As will be appreciated by those skilled in the art, the probiotic composition of the present invention may be formulated for pharmaceutical use, i. to form part of the pharmaceutical products to be administered to the subject (eg orally, topically, etc.), and/or to be administered with food, i.e. forming part of the food consumed in the diet of the subject. In the present invention, said composition is intended to reduce, reduce, treat and/or prevent atopic dermatitis. Therefore, in a specific embodiment, the probiotic composition according to the present invention is a pharmaceutical composition and/or food composition.

The pharmaceutical composition is a set of components or compounds, which is formed by at least B. lactis, L. casei and B. longum microorganisms, in particular B. lactis strains CECT 8145, and/or L. casei CECT 9104, and/or B. longum CECT 7347 (or strains derived therefrom) at any concentration, and which may additionally contain one or more components or compounds having any biological, pharmacological and/or veterinary activity which, when administered to a subject, may further increase, enhance and/ or stimulate the activity of the strains included in the probiotic composition according to the present

- 4 040422 of the invention. As will be appreciated by one of skill in the art, the additional components or compounds must be compatible with the strains of the probiotic composition of the present invention. In the context of the present invention, the term pharmaceutical composition also includes veterinary compositions.

Examples of useful ingredients or compounds useful in the treatment of atopic dermatitis that may be part of a pharmaceutical composition include, but are not limited to, conventional HI antihistamines (eg, hydroxyzine, clemastine, and dexchlorpheniramine), newly developed HI antihistamines (eg, dexfenfluramine, loratadine, cetirizine and derivatives) and calcineurin inhibitors (eg tacrolimus and pimecrolimus).

In a specific embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and/or adjuvant.

The term adjuvant refers to a substance which assists in the absorption of any components or compounds of the probiotic composition according to the present invention, namely strains according to the present invention, or stabilizes the components or compounds and/or assists in the preparation of the pharmaceutical composition in the sense of giving it a texture or flavor to make it more enjoyable. Thus, excipients may have the function of, by way of example, without limitation, binding components (e.g. starches, sugars or cellulose), sweetening, coloring, protecting the active ingredient (e.g. isolating it from air and/or moisture), filling the tablet, capsule or any other form of release, or a disintegrating function that promotes the dissolution of the components, without excluding other excipients not listed in this paragraph. As a consequence, the term excipient is defined as the material included in galenic forms that is added to the active ingredients or their associations to enable their preparation and stability, modify their organoleptic properties and/or determine the physico-chemical properties of the pharmaceutical composition and its bioavailability. The pharmaceutically acceptable adjuvant must provide the activity of the components or compounds of the pharmaceutical composition, i.e. must be compatible with strains according to the present invention.

A galenic form or pharmaceutical form is a configuration to which active ingredients and excipients are adapted to provide a pharmaceutical composition or drug product. It is determined by the combination of the form in which the pharmaceutical composition is presented by the manufacturer and the form in which it is administered.

The carrier medium or carrier is preferably an inert substance. The function of the carrier is to facilitate incorporation of other components or compounds, to provide better dosage and/or administration, and/or to impart consistency and form to the pharmaceutical composition. Therefore, a carrier is a substance used in a medicinal product to dilute any of the components or compounds of the pharmaceutical composition according to the present invention to a given volume or weight; or even without diluting these components or compounds, it is capable of providing better dosage and administration and/or imparting consistency and shape to the drug product. When the release form is liquid, the pharmaceutically acceptable carrier is the diluent. The carrier may be of natural or non-natural origin. Examples of pharmaceutically acceptable carriers include, without limitation, water, saline solutions, alcohol, vegetable oils, polyethylene glycols, gelatin, lactose, starch, amylose, magnesium stearate, talc, surfactants, silicic acid, viscous paraffin, perfume oil, fatty monoglycerides and diglycerides. acids, petroetrals fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidone, and the like.

In addition, the adjuvant and the carrier must be pharmacologically acceptable, ie. the adjuvant and the carrier permit and determine that it does not cause harm to the subject to whom it is administered. In addition, the carrier and/or carrier medium may be natural, ie. natural origin, or non-natural, i.e. they may or may not occur naturally, but if they are natural, they do not occur naturally in combination with the strains of the present invention.

In each case, the dosage form of the pharmaceutical composition will be adapted to the type of administration used. Thus, the composition may be presented in the form of solutions or any other form of clinically acceptable administration and in a therapeutically effective amount. The pharmaceutical composition may be formulated as solid, soft or liquid preparations such as tablets, capsules, powders, granules, solutions, suppositories, gels or microspheres. In a particular embodiment, the pharmaceutical composition is formulated for administration in liquid form or solid form.

In another specific embodiment, the solid dosage form is selected from the group consisting of tablets, lozenges, candies, chewable tablets, chewing gums, capsules, sachets, powders, granules, coated particles or coated tablets, tablets, pills, troches, tablets and enteric-coated capsules; and dispersible strips and/or films.

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In another specific embodiment, the liquid dosage form is selected from the group consisting of oral solutions, suspensions, emulsions and syrups.

Similarly, various systems are known that can be used to administer the sustained release probiotic composition of the present invention, including, for example, encapsulating it in liposomes, microvesicles, microparticles or microcapsules, and the like. Suitable sustained release formulations, as well as materials and methods for their preparation, are well known in the art. Thus, the orally administered form of the probiotic composition of the present invention is in a sustained release form further comprising at least one coating or matrix. The sustained release coating or matrix includes, without limitation, natural semi-synthetic or synthetic polymers, water-insoluble or modified, waxes, fats, fatty alcohols, fatty acids, natural, semi-synthetic or synthetic plasticizers, or a combination of two or more of all of the foregoing. Enteric coatings can be applied using conventional methods known to those skilled in the art.

In addition to the above, the present invention also includes the possibility that the probiotic composition of the present invention may be administered to a subject along with other components or compounds, although they are not part of the probiotic composition. Examples of such components or compounds have been mentioned in the previous paragraphs.

When the probiotic composition of the present invention is formulated as a nutritional composition, said nutritional composition may be in the form of a food product or may be included in a food product or food product intended for both human and animal consumption. Thus, in a particular embodiment, the nutritional composition is selected from a food (which may be a food for specific nutritional purposes or a health food) and a food supplement.

In the present invention, the term food composition refers to that food product, which, regardless of the nutrients provided to the subject who consumes it, beneficially affects one or more bodily functions in order to provide said subject with better health and well-being. In the present invention, said nutritional composition is intended to alleviate, reduce, treat and/or prevent atopic dermatitis.

The term supplement, synonymous with any of the terms dietary supplement, nutritional supplement, nutritional supplement, nutritional supplement, or nutritional complex, refers to products or preparations whose purpose is to supplement the normal diet and which consist of sources of concentrated nutrients or other substances that provide nutritional benefits. or physiological effect on the subject. In the present invention, the substance that has a nutritional or physiological effect on an individual when taking a nutritional supplement is B. lactis, L. casei and B. longum microorganisms, in particular B. lactis strains CECT 8145, and/or L. casei CECT 9104 , and/or B. longum CECT 7347, which are part of the probiotic composition according to the present invention. The nutritional supplement may be in single or combined form and may be sold as a dosage form, i. e. in capsules, pills, tablets and other similar forms, powder sachets, liquid ampoules and drop spray bottles and other similar forms such as liquids and powders intended to be taken in a single dose.

There is a wide range of nutrients and other elements that may be present in dietary supplements, including, without limitation, vitamins, minerals, amino acids, essential fatty acids, fiber, enzymes, plants, and plant extracts. Since their role is to supplement the provision of nutrients in the diet, they should not be used as a substitute for a balanced diet, and consumption should not exceed the daily dose specifically/specially recommended by a physician or nutritionist. The probiotic composition can also be part of a so-called food for special groups, ie. foods that meet specific nutritional needs.

Examples of food products that may contain a probiotic composition according to the present invention (microorganisms B. lactis, L. casei and B. longum, in particular strains of B. lactis CECT 8145, and/or L. casei CECT 9104, and/or B. longum SEST 7347 (or strains derived therefrom)), include, without limitation, food, dairy, vegetable, meat, snack foods, chocolate, beverages, baby food, cereals, fried foods, industrial baked goods, and biscuits. Examples of dairy products include, but are not limited to, products derived from fermented milk (eg, but not limited to yogurt or cheese) or unfermented milk (eg, but not limited to ice cream, butter, margarine, or whey). The herbal product is, for example, without limitation, cereals in any form, fermented (eg, but not limited to soy yogurt or oat yogurt) or unfermented, and snacks. The beverage may be, without limitation, unfermented milk. In a particular embodiment, the food product or food is selected from the group consisting of fruit or vegetable juices, ice cream, infant formula, milk, yogurt, cheese, fermented milk, milk powder, cereals,

- 6 040422 bakery products, milk-based products, meat products and drinks.

In addition, the probiotic composition according to the present invention may contain other microorganisms in addition to B. lactis, L. casei and B. longum, in particular B. lactis strains CECT 8145 and/or L. casei CECT 9104 and/or B. longum CECT 7347. Thus, in a specific embodiment, the probiotic composition of the present invention further comprises a microorganism selected from the group consisting of Lactobacillus sp., Streptococcus sp., Bifidobacterium sp., Saccharomyces sp., Kluyveromyces sp. and their combinations.

In another even more specific embodiment, Lactobacillus sp. is L. rhamnosus, L. delbrueckii subsp. bulgaricus, L. kefir, L. parakefir, L. brevis, L. casei, L. plantarum, L. fermentum, L. paracasei, L. acidophilus, L. paraplantarum or L. reuteri; Streptococcus sp. represents St. thermophilus; Bifidobacterium sp. is B. longum, B. breve, B. bifidum, B. catenulatum, B. adolescentis or B. pseudocatenulatum; Saccharomyces is S. cerevisiae or S. boulardii; or Kluyveromyces sp. is K. lactis or K. marxianus.

In another specific embodiment, the probiotic composition of the present invention is administered to a subject via diet.

As will be appreciated by one of skill in the art, B. lactis, L. casei and B. longum microorganisms, in particular B. lactis CECT 8145, L. casei CECT 9104 and/or B. longum CECT 7347 strains, must be present in the probiotic composition. according to the present invention in a therapeutically effective amount, so that they can have their effect of alleviating, reducing, treating and/or preventing atopic dermatitis.

In the present invention, a therapeutically effective amount is that amount of a component or compound of a pharmaceutical composition that, when administered to a subject, is sufficient to produce the desired effect. Said component or compound of the pharmaceutical composition refers to B. lactis, L. casei and B. longum microorganisms, in particular B. lactis CECT 8145, L. casei CECT 9104 and/or B. longum CECT 7347 strains. As is known to those skilled in the art , the therapeutically effective amount may vary depending on, for example, the subject's age, body weight, general health, sex, and diet, as well as the route and time of administration, rate of elimination, or combination of drugs (among other factors).

Thus, in a specific embodiment, the total concentration of B. lactis, L. casei and B. longum microorganisms, in particular strains of B. lactis CECT 8145 and/or L. casei CECT 9104 and/or B. longum CECT 7347, in the composition is between 103 and 10 ¹² cfu, preferably 109 cfu. In another specific embodiment, the administered dose of microorganisms B. lactis, L. casei and B. longum, in particular B. lactis CECT 8145, L. casei CECT 9104 and/or B. longum CECT 7347, in the composition is between 10 ⁶ and 10 ¹² cfu/day, preferably 109 cfu/day, and in another even more specific embodiment, the administration schedule is at least once a day, specifically twice a day, and more specifically three times a day, one with each meal (breakfast, dinner and supper).

In another specific embodiment, the concentration of B. longum, specifically B. longum strain CECT 7347, relative to the total concentration of microorganisms present in the composition, is selected from any of the following values: 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or 80%. In another specific embodiment, the concentration of B. lactis, specifically B. lactis strain CECT 8145, relative to the total concentration of microorganisms present in the composition, is selected from any of the following values: 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or 80%. In another specific embodiment, the concentration of L. casei, in particular strain L. casei CECT 9104, relative to the total concentration of microorganisms present in the composition, is selected from any of the following values: 10, 15, 20, 25, 30, 35, 40, 45 , 50, 55, 60, 65, 70, 75 or 80%. As should be clear to the expert, the composition according to the present invention contains any combination of concentrations for each microorganism described above.

In another specific embodiment, the concentration of B. longum relative to the total concentration of microorganisms present in the composition is at least between 25 and 45%, preferably at least 35%; the concentration of B. lactis relative to the total concentration of microorganisms is at least between 25 and 45%, preferably at least 35%; and/or the concentration of L. casei relative to the total concentration of microorganisms is between 20 and 40%, preferably 30%.

The probiotic composition according to the present invention is useful in the treatment and/or prevention of atopic dermatitis in an individual, as well as improving the development of atopic dermatitis after the suspension of the probiotic composition described herein.

Thus, another purpose described herein relates to the probiotic composition described herein for use as a drug.

The term drug, as used herein, refers to any substance used to prevent, diagnose, alleviate, treat, or cure diseases in humans and animals. In the context of the present invention, the disease is AD.

Another purpose described herein relates to a probiotic composition such as

- 7 040422 described above, in the treatment and/or prevention of AD in an individual suffering from this disease.

In the present invention, the term subject is equivalent to the term individual; so that both terms can be used interchangeably in this document.

Subject means, in addition to any individual, any animal belonging to any species. Examples of subjects include, without limitation, animals of commercial interest such as birds (chickens, ostriches, chickens, geese, partridges, etc.); rabbits, hares, domestic animals (dogs, cats, etc.); sheep, goat-type cattle (goats, etc.); pigs (boars, piglets, etc.); livestock such as horses (horses, ponies, etc.); cattle (bulls, cows, oxen, etc.); animals of hunting interest such as deer, fallow deer, reindeer, etc.; and people. However, in a particular embodiment, the subject is a mammal, preferably a human of any race, sex, or age.

In the present invention, the term prevention means preventing the occurrence of a disease or condition in an individual, especially when the individual has a predisposition to the condition but has not yet been diagnosed. In the present invention, the disease or pathological condition is an outbreak of atopic dermatitis.

In the present invention, the term treat or treatment includes the suppression of a disease or pathological condition, i.e. cessation of its development;

alleviation of a disease or pathological condition, i. e. initiating regression of the disease or pathological condition; and/or stabilization of a disease or pathological condition in an individual.

In the present invention, the disease or pathological condition is atopic dermatitis.

In the present invention, the term atopic dermatitis (AD) refers to inflammatory dermatoses, pruritus, chronic course, characterized by the development of eczematous lesions with a characteristic distribution pattern that affects individuals with skin hyperreactivity to various environmental factors that are harmless individuals without atopy. As a chronic inflammatory process, outbreaks of an inflammatory nature occur due to significant hyperactivity of the skin, causing lesions both in the area of \u200b\u200bthe folds of the skin and in areas of stretching like acute or subacute eczema. Atopic dermatosis can manifest in many forms and places other than the skin.

A method for the treatment and/or prevention of inflammatory skin diseases.

Another aspect of the invention relates to a method of treating atopic dermatitis in a subject, further to a method of treatment according to the present invention, comprising the introduction of a probiotic composition described in the present invention, an individual who has atopic dermatitis.

All specific terms, definitions and embodiments of the previous aspects of the invention are applicable to the method of treatment according to the present invention.

Throughout the description and claims, the word comprise and variations thereof are not intended to exclude other technical features, additives, components, or steps. For those skilled in the art, other objects, advantages and characteristics of the invention will become apparent in part from the description and in part by practice of the invention. The following examples and figures are provided by way of illustration and are not intended to limit the scope of the present invention.

Brief description of the figures

In FIG. 1 is a graph showing the analysis of the principal components of the microbiological profiles of fecal samples taken from patients with atopic dermatitis (square symbol and black) compared with healthy individuals belonging to enterotype 1 (round symbol and dark gray), to enterotype 2 (triangular symbol). and grey) and enterotype 3 (diamond symbol and light grey).

In FIG. 2 is a graph showing the analysis of variability in stool samples from patients with dermatitis compared to the healthy population in each of the enterotypes. The figure shows statistical significance after applying the Wilcoxon test.

In FIG. 3 is a graph showing principal component analysis of patients with dermatitis or classified as enterotype 1 (square symbol and black) compared to samples from the healthy enterotype 1 population (round symbol and gray). Categories that are most represented in the samples located in the upper right part of the figure are shown in black, while categories that are underrepresented in this arrangement are shown in gray. As for the samples located in the lower left corner, the trend is opposite: less numerous are marked in black, and numerous are marked in gray.

In FIG. 4 is a graph showing principal component analysis of patients with dermatitis or classified as enterotype 2 (square symbol and black) compared to samples from the healthy enterotype 2 population (round symbol and gray). Categories that are most represented in the samples located in the upper right part of the figure are shown in black, while categories that are underrepresented in this arrangement are shown in gray.

As for the samples located in the lower left corner, the trend is opposite: less numerous are marked in black, and numerous are marked in gray.

In FIG. 5 is a graph showing principal component analysis of patients with dermatitis or classified as enterotype 3 (square symbol and black) compared to samples from the healthy enterotype 3 population (round symbol and gray). Categories that are most represented in the samples located in the upper right part of the figure are shown in black, and categories that are underrepresented in this arrangement are shown in gray. As for the samples located in the lower left corner, the trend is opposite: less numerous are marked in black, and numerous are marked in gray.

In FIG. 6 shows the results of the SCORAD index one month after the start of treatment with a probiotic composition according to the present invention or placebo. The results show the absolute value of SCORAD for each treatment group (FIG. 6A) and the percentage (%) reduction in the SCORAD index over the course of treatment in both groups (FIG. 6B). V3, examination after 1 month.

Group A: placebo group;

group B: group receiving the probiotic composition.

In FIG. 7 shows the results of the SCORAD index two months after the start of treatment with a probiotic composition according to the present invention or placebo. The results show the absolute value of SCORAD for each treatment group (FIG. 7A) and the percentage (%) reduction in the SCORAD index over the course of treatment in both groups (FIG. 7B). V5, examination after 2 months.

Group A: placebo group;

group B: group receiving the probiotic composition.

In FIG. 8 shows the results of the SCORAD index three months (end of study) after initiation of treatment with a probiotic composition of the present invention or placebo. The results show the absolute value of SCORAD for each treatment group (FIG. 8A) and the percentage (%) reduction in the SCORAD index over the course of treatment in both groups (FIG. 8B). V7, examination after 3 months.

Group A: placebo group;

group B: group receiving the probiotic composition.

In FIG. 9 shows the percentage (%) improvement in the SCORAD score during the trial period measured at one month, two months and three months (end of study) compared to baseline. The differences between the two treatment groups were statistically significant from the first month, and these differences increased after two and three months of the trial. The dotted line shows the group receiving the probiotic composition; continuous line - placebo group.

In FIG. 10 shows trends in the SCORAD index over time measured one month, two months, and three months (end of study) after baseline. The differences between the two treatment groups are statistically significant. The dotted line shows the group receiving the probiotic composition; the continuous line shows the placebo group.

In FIG. 11 shows the trend in the number of patients with new AD flare (FIG. 11A), the total number of patients with new AD flare (FIG. 11B), and the percentage of patients without new AD flare (FIG. 11C) during the three months of follow-up after discontinuation. experimental treatment with a placebo or probiotic composition according to the present invention. Differences between the two treatment groups are statistically significant at 3 months post-monitoring for the three analyzed variables. Black line - placebo patients; gray line - patients who received the probiotic composition.

Examples

The invention will be illustrated by tests carried out by the inventors, which show the properties of the probiotic composition according to the present invention.

Example 1 Determination of bacterial microbiota in patients with AD.

I. Materials and methods.

Based on stool samples from 49 patients with dermatitis, extraction of genetic material (DNA) was performed using a combination of mechanical and enzymatic disruption of cell walls and membranes in order to increase the extraction yield rather than alter the presence of bacteria with a cell wall (Gram+). The genetic material obtained by extraction was analyzed for quality and quantity using Nanodrop 2000 ThermoScientific to check ratios of 260/280 and 260/230 indicating the quality of the extraction (presence of PCR inhibitors, pigment, etc.). Later, after checking their quality, large-scale sequencing of the libraries was performed, capturing the hypervariable region V3-V4 of the 16S rRNA gene of bacteria (based on Klindworth A. et al. (2013), Nucleic Acids Res., 41: e1) according to the protocol described Illumina for microbial composition analysis based on 16s rRNA capture. Each library was quantified with Invitrogen's QuantiT PicoGreen and equimolarly mixed for subsequent sequencing.

- 9 040422

Samples were sequenced on the MiSeq platform in a combination of 300 cycles of paired ends. The resulting FASTQ files were processed to provide high quality sequence analysis. For this, a quality control was carried out, consisting of

1) end joins to recreate unique sequences using 'pear' v0.9.6. (Zhang J. et al. (2014), Bioinformatics 30(5):614-20);

2) elimination of sequencing adapters and capture primers from hypervariable regions V3 and V4 using cutadapt version 1.9.1. (Martin M. (2010), EMBnet.joumal [S.L.], 17(1):10-12, ISSN 2226-6089);

3) elimination of low-quality sequences using FASTX-ToolKit version 0.91;

4) elimination of chimeras obtained by PCR using the UCHIME program (December 2015) (Edgar R.C. et al. (2011) Aug 15, 27 (16):2194-200) and the latest database of chimeras.

The resulting samples were compared to the 16S rRNA sequence database (NCBI) using local BLAST alignment (Altschul S. F. et al. (1990), J. Mol. Biol., 215:403-410).

Each of the sequences, for which a score of ninety-five percent (95%) identity was obtained, was tested at different taxonomic levels: type, family, genus and species.

The R statistical package was used for statistics and for plotting for Principal Component Analysis (PCA).

It is interesting to note that there are significant differences in the microbial populations of the digestive tract of each individual; epidemiological studies show that the microbiota of almost all of them can be classified as belonging to three different categories known as enterotypes (Arumugam et al. (2011), Nature, 473:174-180). This classification is based on the predominance in each of these three enterotypes of members of the genera Bacteroides, Prevotella, or Ruminococcus. Enterotype 1 (ent1) is mentioned when the genus Bacteroides predominates, enterotype 2 (ent2) if the genus Prevotella predominates, and enterotype 3 (ent3) when the genus Ruminococcus predominates (Dave et al. (2012), Trans. Res., 160, 246-257).

II. Results.

As mentioned above, an analysis of the microbial composition in stool samples from individuals suffering from atopic dermatitis was performed in comparison with stool samples from healthy individuals.

The results show that, as seen in FIG. 1, the microbiological profiles of samples from patients with AD are closer to healthy individuals with enterotype 3 than to other healthy individuals. As shown in the upper left corner of the above FIG. 1, patients with AD (square and black symbol) are grouped together with healthy individuals with enterotype 3 (diamond and light gray symbol), but with more variance in the case of patients with AD than in healthy individuals with enterotype 3. In contrast, individuals in the healthy population with enterotype 1 (round dark gray symbol) or enterotype 2 (triangular gray symbol) are grouped at the top from left to right and on the left side from top to bottom, respectively. It is also notable that the accessions grouped on the right side of the graph have a higher content of the genus Bacteroides and less Prevotella and Faecalibacterium, while the samples grouped on the top of the graph in FIG. 1 represent the apparent absence of Prevotella; therefore, samples from healthy patients with enterotype 2 (more Prevotella species) are located in the lower left corner and show an increase in Faecalibacterium and Ruminococcus. These data allow the inventors to state that the microbiota of patients with AD is closer to that of healthy individuals with enterotype 3, but with a different composition and variability of organisms.

This different microbial variability and composition among healthy enterotype 3 individuals and AD patients appears in greater detail when bacterial population variability is analyzed in healthy population samples (with each of three separate enterotypes) for AD patient samples using the Shannon index (Fig. 2 ). As shown in FIG. 2, in general, patients with AD have greater variability compared to healthy individuals, having a mean and interquartile range of 2.97 (2.84 to 3.06) for patients with atopic dermatitis and 1.99 (1.37 to 2 .52); 2.14 (from 1.50 to 2.62) and 2.23 (from 1.91 to 2.57) for healthy people with enterotypes 1, 2 and 3, respectively. In particular, there is a significantly higher bacterial variability in AD patients than in any of the enterotypes of healthy individuals, because when comparing the bacterial variability of AD patients with that of a healthy population with different enterotypes, all p<0.0001.

In order to identify the bacteria that distinguish the healthy population from the atopic dermatitis population, the inventors proceeded to classify patients with atopic dermatitis based on the predominance of Bacteroides (ent1), Prevotella (ent2) or Ruminococcus (ent3). Further, the inventors investigated whether there were differences between each of the different enterotypes of the healthy population in relation to the different enterotypes in people with AD. Initially, out of 49 AD patients, 25 were classified as ent1, two as ent2, and 22 as ent3. Similarly, out of 324 healthy subjects, 298

- 10 040422 was classified as entl, 21 as ent2 and 5 as ent3. Bacterial variability was then analyzed within each enterotype, 1, 2, or 3, among AD patients compared to healthy subjects.

In FIG. 3 shows the bacterial variability among healthy subjects compared to AD patients, all with enterotype 1. As shown in FIG. 3, healthy individuals have higher levels of Bacteroides than patients with AD, while patients with AD show more genera Faecalibacterium, Bifidobacterium and especially low levels of species of the genus Alistipes.

In FIG. 4 shows the bacterial variability among healthy subjects compared to AD patients, all with enterotype 2. With respect to bacterial genera, it is not possible to differentiate AD patients with enterotype 2 from healthy individuals with the same enterotype in this case. On the other hand, this figure demonstrates the low rate of AD patients who were classified as enterotype 2.

As with healthy individuals and AD patients with enterotype 1, in AD patients classified as enterotype 3 (FIG. 5), there are marked differences between AD patients compared with healthy individuals. As shown in FIG. 5, AD patients have fewer species belonging to the genus Ruminococcus than healthy patients, as well as fewer species belonging to the genus Faecalibacterium.

Considering all the results shown above, it is clear that the microbiota of AD patients differs in composition and variability from the microbiota of the healthy population. It has also been demonstrated that fewer AD patients belong to enterotype 2 and, in addition, that AD patients also show increased variability in microorganisms, but with fewer bacteria belonging to species of important genera in each enterotype, in particular the genera Bacteroides and Ruminococcus for enterotypes 1 and 3, respectively.

Example 2 Analysis of the effectiveness of a probiotic composition according to the present invention in the prevention and/or treatment of atopic dermatitis (AD).

I. Materials and methods.

In order to evaluate the effectiveness of the probiotic composition of the present invention in reducing symptoms together with the use of topical corticosteroids in the treatment of atopic dermatitis, a randomized pilot clinical, double-blind, placebo-controlled study was conducted in a group of 20 patients suffering from atopic dermatitis, aged between 4 and 17 years.

To this end, patients included in the study were diagnosed with atopic dermatitis according to the criteria of Hanifin and Reik (Hanifin J.M. et al., Acta Derm Venereol., 1980, 92:44-7), meeting all inclusion criteria and none of the criteria exceptions identified in the study. For the treatment of atopic dermatitis flares, study participants also used or may have used topical corticosteroids along with other active ingredients used as conventional treatment such as emollients, systemic corticosteroids, antihistamines, etc.

Each patient was assigned to either treatment or placebo using a 1:1 stratified randomization with blocks based on the variables sex, age, family history of first-degree atopic or atopic dermatitis consanguinity, and onset of atopic dermatitis before the age of four years.

The probiotic composition according to the present invention contains Bifidobacterium lactis CECT 8145, Bifidobacterium longum CECT 7347 and Lactobacillus casei CECT 9104 based on tapioca with sugar and maltodextrin with a content of 5x10 ¹⁰ CFU/g.

The form and appearance of the placebo administered was the same as the composition of the present invention, but contained only tapioca, maltodextrin and sugar.

The dosage regimen for the probiotic composition of the present invention or placebo was one capsule per day.

Methylprednisolone aceponate was used as a topical corticosteroid for the treatment of flare-ups of atopic dermatitis, which may have occurred in patients included in the study, and the duration of such treatment did not exceed two weeks. Deflazacort was prescribed if treatment with systemic corticosteroids was required. Desloratadine has been used to treat pruritus and, in cases of infected atopic dermatitis, a commonly used antibiotic such as fusidic acid has been used.

The treatment period lasted twelve weeks from the time of recruitment of patients to the last examination. Following study entry examination (baseline) and treatment assignment (treatment group or placebo), seven examinations were programmed at 4, 8 and 12 weeks from the start of treatment with the composition of the present invention or placebo. During these surveys, the SCORAD index was assessed and the data collection log (DCL) of each patient was updated at 2, 6 and 10 weeks.

All medications taken by the patient during the trial were recorded in the patient's medical records and in the DCL, indicating doses, route of administration and duration of treatment (start and end dates) and inclusion of the medication. In addition, each examination was recorded in the DCL, as well as the number of capsules returned. In table. 1 shows baseline demographic and clinical characteristics of patients in each treatment group. In table. 2 shows the test schedule.

Table 1

Baseline demographic and clinical characteristics of patients in each treatment group

Treatment Average SD SEM Age placebo 8.96 3,940 0.804 Composition 9.35 3.577 0.702 SCORAD placebo 31.6417 5.05129 1.03109 Composition 33.3077 3.51351 ,68906 Baseline IgE placebo 292.08 636.537 129.933 Composition 428.44 723.861 144.772 Baseline eosin. placebo 4.73 4.274 0.872 Composition 4.96 5.799 1.160 Total corticosteroids V2* placebo 3.68 4.224 0.901 Composition 3.00 3.240 0.648 Total antihistamines V2 drugs* placebo 0.32 0.780 0.166 Composition 1.28 2.072 0.414

* Days of treatment after 2 weeks;

SD: standard deviation;

SEM: standard error of the mean.

table 2

Clinical trial protocol.

Surveys 0/1 2 3 4 5 6 7 weeks 0 2 4 6 8 10 12 Inclusion/exclusion criteria X Signed informed consent X Medical card X Physical examination X X X X Pregnancy test X X Additional Research X X SCORAD X X X X Treatment (probiotic composition or placebo) X X X Data collection log X X X X Carrying out treatment X X X Refund and calculation of unspent poepaoat X X X Assessment of adverse events X X X X X X

All statistical analyzes performed to obtain the final results shown below were performed using SPSS 20.0 software.

In addition, the test was carried out in accordance with the Declaration of Helsinki, as amended by subsequent world assemblies. A medical record and a letter of informed consent were obtained for all participants (signed by the patient or his/her guardian or legal

- 12 040422 representative) in accordance with the Declaration of Helsinki and approved by the Ethics and Institutional Research Committee.

II. results

First, the inventors analyzed whether the samples included in each study group, ie. in the placebo group and the group receiving the probiotic composition according to the present invention, homogeneous. Tab. 3 demonstrates that this requirement was met as no differences were found between any of the variables analyzed for each study group.

Table 3

Homogeneity of the two samples. research statistics ^a

Age SCORAD Total corticosteroids V2* Total antihistamines V2* U-criterion Manna-Uitney 286.500 260,500 271,000 223.500 Criterion Wilcoxon 586.500 560,500 596,000 476.500 Z-test -0.497 -1,000 -0.088 -1.435 Asymptotic Significance (two-tailed) 0.619 0.317 0.930 0.151

^a Grouping of variables: no differences in major variables at baseline.

* Days of treatment after 2 weeks.

As mentioned above, means (mean and median) and ± standard deviation (SD) were calculated for global data at baseline, one month, and two and three months of treatment. The values reported by the patient at each of these time points were compared with the values taken at the start of the trial using the Wilcoxon test.

The effectiveness of the composition according to the present invention one month after the start of treatment.

After one month of treatment, the SCORAD index was analyzed, comparing values between groups as well as baseline values. Tab. 4 shows the changes in absolute numbers and percentages.

Table 4

SCORAD index one month after initiation of treatment with a probiotic composition of the present invention or placebo

Treatment SCORAD in 1 month % change in SCORAD after 1 month placebo Average value 25.8500 16.0015 SO 8.02355 23.17611 Median 24.8500 18.0150 Composition Average value 19.6182 41.7586 SO 7.33580 19.27088 Median 19.5000 40.8150 Mann-Whitney U test 124,500 79,000 Wilcoxon test 377.500 289,000 Z-test -2.406 -3.551 Asymptotic Significance (two-sided) 0.016 0.000

As the results in Table. 4 and in FIG. 6, after a month of treatment, there were significant differences in the SCORAD index for the placebo group compared to the group receiving the probiotic composition of the present invention. In FIG. 7 and 8 show changes in the SCORAD index at examinations corresponding to two months (Fig. 7) and three months (Fig. 8) of treatment compared to baseline between the group receiving the probiotic composition according to the present

-13040422 of the invention, and a placebo group.

In FIG. 9 shows that the composition treatment group according to the present invention has a lower SCORAD index measured at one month, two months and three months from baseline compared to the placebo group. As seen in FIG. 9, the differences between the two treatment groups are statistically significant as early as the first month of treatment, with these differences increasing after 2 (V5) and 3 (V7) months from baseline.

In FIG. 10 shows trends in the SCORAD index measured one (V3), two (V5), and three (V7) months after the start of the trial. The differences between the group treated with the composition of the present invention and the placebo group were statistically significant, demonstrating the benefits of using the composition in the treatment of patients with AD.

Regarding the use of topical corticosteroids in each study group, after one month of treatment, there were no statistically significant differences between the two treatment groups for different number of days of topical corticosteroid use (Table 5) when comparing both treatment groups using the Wilcoxon test with a statistical significance level of five percent (5%). On the contrary, there was a trend when topical corticosteroids were not used, which was more in the group receiving the probiotic composition according to the present invention, compared with the placebo group, especially at 2 and 3 months of the trial. Notably, it did not reach statistical significance, mainly due to the sample size.

Table 5

Analysis of topical corticosteroids used in each treatment group one month after trial initiation

Statistical analysis General quantity days treatment corticosteroids V3 Mann-Whitney U test 210,500 Wilcoxon test 441.500 Z-test -0.512 Asymptotic Significance (two-sided) 0.108

The grouping variable used to obtain the data shown is the type of treatment, ie. probiotic composition according to the present invention or placebo.

In the analysis of the variable days of antihistamine use in the first month of significant differences between the two treatment groups, i.e. probiotic composition according to the present invention compared with placebo was not observed (table. 6). This variable was analyzed using the Wilcoxon test with a five percent (5%) level of statistical significance for each treatment group.

Table 6

Analysis of antihistamines used in each treatment group one month after baseline

Statistical analysis Total number of days of treatment antihistamines V3 Mann-Whitney U test 220,500 Wilcoxon test 473.500 Z-test -0.578 Asymptotic Significance (two-sided) 0.563

The grouping variable used to derive the data shown is the type of treatment: probiotic composition of the present invention versus placebo.

In table. 7 summarizes the efficacy of the probiotic composition of the present invention in the treatment of AD after one month of administration, especially with respect to the analyzed variables: SCORAD score, topical corticosteroids and antihistamines compared with the placebo group.

- 14 040422

Table 7

SCORAD index, total days of corticosteroid use and total days of antihistamine use in the group treated with the probiotic composition of the present invention,

compared to placebo group Treatment Medium range Sum of ranges Statistical Significance SCORAD in 1 month placebo 26.28 525.50 0.016 Composition 17.16 377.50 % improvement SCORAD in 1 month placebo 14.45 289.00 0.000 Composition 27.91 614.00 Total corticosteroids V3 placebo 21.02 441.50 0.608 Composition 22.93 504.50 Total antihistamines V3 placebo 22.50 472.50 0.563 Composition 21.52 473.50

A multiple linear regression analysis was performed to evaluate the independent effect of treatment with a probiotic composition of the present invention compared to other variables that may affect SCORAD. In this analysis, the SCORAD variable is included as the dependent variable. In addition to the variable, treatment type included the following variables: total days of antihistamine treatment, total days of corticosteroid treatment, and SCORAD at baseline in the trial.

One month after treatment, the only variable significantly associated with the SCORAD score is the type of treatment. Accordingly, the group treated with the probiotic composition of the present invention is associated with an absolute reduction in SCORAD of 9.07 points. Those. of the total reduction in SCORAD observed in the group receiving the probiotic composition of the present invention during the first month, the composition of the present invention is responsible for sixty six percent (66%) of this change (9.07 out of 13.69 SCORAD scores). Neither the total duration of antihistamine treatment variable nor the total number of days of corticosteroid treatment was associated with the SCORAD score.

In addition, systemic corticosteroid consumption was assessed in the same way as described for topical corticosteroid use, as a predefined secondary variable. The results obtained for systemic consumption of corticosteroids showed no significant differences associated with the use of these compounds between the two treatment groups: the composition according to the present invention and placebo.

Patients included in the study indicated that they experienced minor adverse effects such as flatulence in the first five days of treatment: thirty percent (30%) of patients treated with the composition of the present invention compared to fourteen percent (14%) in the group placebo.

In conclusion, in this example, the results show that the administration of the probiotic composition of the present invention for the treatment of AD provides advantages in terms of efficacy and tolerability compared to the results of patients receiving placebo. Thus, the analyzed parameters show a significant improvement in the SCORAD index in patients with AD treated with a probiotic composition according to the present invention, compared with a group of patients treated with placebo. This improvement could be observed from the first month of taking the probiotic composition according to the present invention, and this improvement was maintained or increased in further analyzes after 2 and 3 months of treatment.

In addition, with regard to the total number of days that patients in each group were required to use topical corticosteroids, although the mean is slightly higher in the group receiving the probiotic composition of the present invention compared to the placebo group (3.3% vs. 6%), this difference is insignificant. Conversely, when analyzing corticoid consumption at the within-subject or within-group level (comparing baseline and end-of-study corticosteroid use in the same group), there is a trend of decreasing steroid consumption over time in the group taking the probiotic composition of the present invention (steroid consumption decreases fifty-five percent (55%) compared to the placebo group (eight percent (8%) reduction during the observation period), reaching statistical significance in this case.

Example 3 Analysis of the effect of using a probiotic composition according to the present invention on the prevention of AD flare-ups.

Of the twenty (20) patients with AD included in the study, an interim analysis of patients with a SCORAD index of 6 was performed. This group of patients was subjected to special monitoring until the completion of the full three-month treatment period. Ten (10) patients received a placebo (Fig. 11, black line) and nine (9) patients received a probiotic (Fig. 11, gray line).

In FIG. 11 shows the results of monitoring the number of AD flares in all these patients from the end of 3 months of probiotic treatment to 12 weeks after the end of treatment. Thus, in FIG. 11 shows the trend in the number of patients suffering from a new AD flare (FIG. 11A), the total number of patients with new AD flares (FIG. 11B), and the percentage of patients without new flares (FIG. 11C). Considering all previously treated patients, in FIG. 11 shows that the number of flare-ups, the number of patients, and the percentage of patients experiencing new flare-ups of AD in a 12-week no-treatment period are higher among those who previously received a placebo than among those who previously received a probiotic.

Thus, in this subgroup, patients treated with the probiotic composition of the present invention had a lower rate of relapse and/or worsening than the placebo group (1 out of 10 (10%) versus 5 out of 9 (55%)), respectively. (Fig. 11B).

Differences between the two treatment groups are statistically significant over the three-month evaluation period after the start of monitoring for the three analyzed variables.

Data supporting the effectiveness of the probiotic in preventing the occurrence of new AD flares is as follows: Three months after discontinuation of treatment with the above probiotic composition, individuals treated with the above composition had fewer flare-ups and therefore a longer disease-free period than in those patients who did not receive treatment.

These results demonstrate that the use of the composition of the present invention is effective in preventing new outbreaks of AD. Thus, three months after cessation of treatment with a probiotic composition of the present invention, patients treated with said composition had fewer disease flares and therefore a longer disease-free period than patients who received placebo.

Claims (14)

1. Probiotic composition for the treatment and/or prevention of atopic dermatitis, containing Bifidobacterium animalis subsp. lactis (B. lactis), Bifidobacterium longum and Lactobacillus casei, where B. lactis is B. lactis CECT 8145, B. longum is B. longum CECT 7347 and L. casei is L. casei CECT 9104. 2. A probiotic composition according to claim 1, wherein the probiotic composition is a pharmaceutical composition or a food composition. 3. Probiotic composition according to claim 2, wherein the pharmaceutical composition contains a pharmaceutically acceptable carrier and/or excipient. 4. A probiotic composition according to claim 2 or 3, wherein the pharmaceutical composition is formulated to be administered in liquid form or solid form. 5. Probiotic composition according to claim 4, wherein the solid dosage form is selected from the group consisting of tablets, lozenges, candies, chewable tablets, chewing gum, capsules, sachets, powders, granules, coated particles or tablets, tablets and enteric coated tablets and capsules; and dispersible strips and/or films. 6. Probiotic composition according to claim 5, wherein the liquid dosage form is selected from the group consisting of oral solutions, suspensions, emulsions and syrups. 7. Probiotic composition according to claim 2, wherein the food composition is a food product or food supplement. 8. Probiotic composition according to claim 7, wherein the food product is selected from the group consisting of fruit or vegetable juices, ice cream, infant formula, milk, yogurt, cheese, fermented milk, milk powder, cereals, baked goods, milk-based products, meat products and drinks. 9. Probiotic composition according to any one of claims 1 to 8, wherein the composition further comprises a microorganism selected from the group consisting of Lactobacillus sp., Streptococcus sp., Bifidobacterium sp., Saccharomyces sp., Kluyveromyces sp. and their combinations. 10. Probiotic composition according to any one of claims 1 to 9, wherein the total concentration of microorganisms of B. lactis, L. casei and B. longum strains in the composition is between 103 and 10 ¹² cfu, preferably 109 cfu. 11. A probiotic composition according to any one of claims 1 to 10, wherein the concentration of B. longum is at least between 30% and 40% relative to the total concentration of microorganisms present in the composition; and/or the concentration of B. lactis is at least between 30 and 40% relative to the total concentration of microorganisms; and/or the concentration of L. casei is at least between 25 and 35% relative to the total concentration of microorganisms. 12. A probiotic composition according to any one of claims 1 to 11, wherein the concentration of B. lactis is at least between 30 and 40% relative to the total concentration of microorganisms. 13. A probiotic composition according to any one of claims 1 to 12, wherein the concentration of L. casei is at least between 25 and 35% relative to the total concentration of microorganisms. 14. The use of a probiotic composition according to any one of claims 1 to 13 for the treatment and/or prevention of atopic dermatitis. Comparison of patients with dermatitis and healthy