Extracts of bacteria of the genus Sphingomonas

Abstract

Bacteria of the genus Sphingomonas and their extracts possess several anti-inflammatory activities combined with enhanced skin barrier function, enabling the prevention and / or treatment of skin reactions caused by allergic reactions. [Solution] The present invention provides extracts of bacteria of the genus Sphingomonas, and compositions comprising extracts of bacteria of the genus Sphingomonas or at least one bacterium of the genus Sphingomonas, for use in the prevention and / or treatment of skin reactions caused by allergic reactions, and for use in regulating immune responses and optionally in enhancing the skin barrier function.

Description

[Technical Field]

[0001] The present invention relates to extracts of bacteria of the genus Sphingomonas, and compositions comprising extracts of bacteria of the genus Sphingomonas or at least one bacterium of the genus Sphingomonas, for use in the prevention and / or treatment of skin reactions caused by allergic reactions, and for use in the modulation of immune responses and optionally in the enhancement of skin barrier function. [Background technology]

[0002] Allergies are complex phenomena in which an excessive inflammatory response occurs after an organism is exposed to an allergen. In particular, allergies depend on two factors: the porosity of the barrier between the organism and the external medium, and the regulation of the immune system.

[0003] Therefore, allergies depend on skin permeability, which is not only a barrier against external elements, particularly particles, chemicals, or chemical attacks, but also to limit the leakage of water and other interstitial compounds. This property of the skin is called barrier function. In particular, barrier function is ensured by tight junctions and associated proteins. By stimulating the expression of these proteins and the formation of tight junctions, activation of Toll 2 (TLR2) receptors allows for the enhancement of barrier function.

[0004] Allergies also depend on the regulation of the immune system, particularly through type E antibodies (IgE), prostaglandin E2 (PGE2), and various interleukins (ILs).

[0005] IgE is involved in immediate-type allergic reactions that can cause urticaria or angioedema.

[0006] PGE2 and IL-6 / IL-8 also play a role in attracting neutrophils. They have complementary mechanisms of action: on the one hand, PGE2 due to increased vascular permeability, and on the other hand, IL-6 or IL-8 through a neutrophil-attracting chemokine gradient. Thus, these two different, combined mechanisms of action improve the immune response. [Overview of the Initiative] [Means for solving the problem]

[0007] The inventors discovered that an extract of Sphingomonas activates the hTLR2 receptor in vitro and promotes the expression of genes associated with enhancing the skin barrier function. The inventors also showed that this extract can inhibit the secretion of the pro-inflammatory mediator PGE2 and modulate the expression of interleukins that promote IL-10 (an anti-inflammatory cytokine) without significantly affecting other pro-inflammatory interleukins, such as IL-12, IL-6, and IL-8. Finally, the inventors showed that the extract can limit IgE production by human B lymphocytes.

[0008] Therefore, bacteria of the genus Sphingomonas and their extracts possess several anti-inflammatory activities combined with enhanced skin barrier function, enabling the prevention and / or treatment of skin reactions caused by allergic reactions.

[0009] Therefore, the present invention relates to an extract of bacteria of the genus Sphingomonas for use in the prevention and / or treatment of skin reactions caused by allergic reactions.

[0010] The present invention also relates to a composition comprising an extract of a bacterium of the genus Sphingomonas or at least one bacterium of the genus Sphingomonas, for use in the prevention and / or treatment of skin reactions caused by allergic reactions.

[0011] The present invention also relates to extracts of bacteria of the genus Sphingomonas for use in modulating the immune response and, optionally, in enhancing the skin barrier function.

[0012] Furthermore, the present invention relates to a composition comprising an extract of a bacterium of the genus Sphingomonas or at least one bacterium of the genus Sphingomonas for use in regulating the immune response and, optionally, enhancing the skin barrier function. [Modes for carrying out the invention]

[0013] general definition The term "bacteria" refers to living, semi-active, inactive, or dead forms of prokaryotic microorganisms, preferably proteobacteria.

[0014] The bacteria used in this invention or the bacteria in the extract used in this invention are bacteria of the genus Sphingomonas. In one embodiment, the bacteria include Sphingomonas abaci, Sphingomonas adhaesiva, Sphingomonas aerolata, Sphingomonas aquatilis, Sphingomonas asaccharolytica, Sphingomonas aurantiaca, Sphingomonas azotifigens, Sphingomonas dokdonensis, Sphingomonas echinoides, and Sphingomonas phaeni. Sphingomonas faeni), Sphingomonas fennica, Sphingomonas haloaromaticamans, Sphingomonas jaspsi, Sphingomonas koreensis, Sphingomonas mali, Sphingomonas melonis, Sphingomonas natatoria, Sphingomonas oligophenolica, Sphingomonas panni, Sphingomonas parapausimovilis (Sphingomonas Sphingomonas paucimobilis, Sphingomonas phyllosphaerae, Sphingomonas pituitosaSphingomonas pituitosa, Sphingomonas pruni, Sphingomonas roseiflava, Sphingomonas sanguinis, Sphingomonas soli, Sphingomonas sp, Sphingomonas suberifaciens, Sphingomonas trueperi, Sphingomonas wittichii, Sphingomonas xenophaga, Sphingomonas yabuuchiae The bacterium is a species selected from among Sphingomonas yabuuchiae, Sphingomonas yunnanensis, and Sphingomonas yanoikuyae. In one embodiment of the present invention, the bacterium is Sphingomonas xenophagum. In a preferred embodiment, the bacterium is Sphingomonas xenophagum submitted in accordance with the Budapest Convention on November 21, 2019, to the National Collection of Microorganism Culture (CNCM), Paris, France, under the number CNCM I-5455 by L'Oreal, 101 Avenue Gustave Eiffel, 37390 Notre Dame d'Oe.

[0015] As used herein, the terms “prevent” or “prevention” refer to any action aimed at preventing the appearance of a disorder or one or more symptoms associated with that disorder. Thus, the term also includes slowing, interrupting, or limiting the symptoms.

[0016] As used herein, the terms “to treat” or “treatment” refer to any action aimed at slowing, alleviating, or preventing the progression of a disorder or one or more symptoms associated with that disorder. Thus, the terms encompass the reduction, mitigation, or suppression of symptoms.

[0017] "Prevention and / or treatment of skin reactions caused by allergic reactions" should be understood as slowing, interrupting, limiting, reducing, alleviating, or suppressing skin symptoms caused by allergic reactions in the subject. Generally, these symptoms include the appearance of urticaria, skin rashes, and / or redness.

[0018] In this specification, "subject" should be understood to mean a human being, preferably a human being suffering from an allergy. In one embodiment of the present invention, the subject already has an allergic reaction that is causing a skin reaction.

[0019] The term "of the skin" should be understood to mean at the level of the skin, particularly at the level of areas of skin that have been or may be exposed to the allergen. According to one embodiment of the present invention, skin reactions caused by an allergic reaction include the appearance of urticaria, skin rashes, and / or redness.

[0020] In preferred embodiments, the extracts and compositions used in the context of the present invention are used topically. In preferred embodiments, the extracts and compositions used in the context of the present invention are intended for application to the skin.

[0021] In the context of this invention, the term “skin” means any skin surface of the body, preferably the skin of the face, the skin of the neck, the skin of clefts, and especially the skin of the face.

[0022] By "allergy reaction", it should be understood the meaning commonly used by those skilled in the art, i.e., an excessive inflammatory reaction that occurs after the exposure of an organism to an allergen. This reaction is generally harmless and is a sign of the organism's hypersensitivity to substances present in the environment, which are allergens. These substances can be found in the atmosphere, cosmetics, etc.

[0023] The so-called immediate allergic reaction is characterized by the appearance of symptoms most frequently within a few minutes (less than 2 hours) after contact with an allergen. In this case, it consists of urticaria, which spreads and may be accompanied by IgE. The so-called delayed allergic reaction is generally characterized by the appearance of symptoms 48 hours after contact with an allergen. In this case, it consists of local eczema at the site of contact and is accompanied by chemical molecules.

[0024] In a preferred embodiment, the extracts and compositions implemented in the context of the present invention are used for the prevention and / or treatment of skin reactions caused by immediate allergic reactions.

[0025] By "modulation of the immune response", a deceleration, interruption or attenuation of the immune response should be understood herein. In particular, the modulation of the immune response according to the present invention is a decrease in the activity of pro-inflammatory cytokines and / or an increase in the activity of anti-inflammatory cytokines.

[0026] In a preferred embodiment of the present invention, the modulation of the immune response is achieved through an increase in the activity of IL-10 without significantly affecting pro-inflammatory interleukins IL-12, IL-6 and / or IL-8.

[0027] By "enhancement of the skin barrier function", it should be understood herein to improve the skin barrier function, particularly through an increase in the number of tight junctions and / or through an increase in related proteins at the skin level, such as claudin-1, corneferin and / or occludin-1.

[0028] Extracts of bacteria of the genus Sphingomonas This invention relates to an extract of bacteria of the genus Sphingomonas for use in the prevention and / or treatment of skin reactions caused by allergic reactions.

[0029] In the context of the present invention, the term “bacterial extract” refers to both a set of compounds produced and / or secreted by bacteria and thus present in the culture medium (also called the extracellular medium) of a bacterial culture, and / or a set of compounds contained in bacteria and thus present in the bacterial pellet of a bacterial culture after centrifugation, such as the intracellular medium and / or components of the cell wall and / or membrane.

[0030] The extracts according to the present invention can be prepared by any conventional method well known to those skilled in the art. Typically, these methods include steps of fermentation, separation, and purification.

[0031] Typically, bacteria of the genus Sphingomonas are cultured in a suitable medium at a desired density. Preferably, the bacteria themselves are concentrated by any known process, such as centrifugation. The concentrated bacteria may then be used directly as a crude preparation, or they may undergo additional processing steps well known to those skilled in the art, such as freeze-drying, dehydration, filtration, purification, freezing and optionally subsequent thawing, sterilization, column chromatography, grinding, lysis, etc.

[0032] Preferably, the bacterial extract is a bacterial lysate. Such a lysate contains compounds that are present in the bacteria and therefore in the bacterial pellet of the bacterial culture after centrifugation. Preferably, the bacterial extract is a bacterial lysate containing intracellular media and / or components of the cell wall and / or membrane.

[0033] Lysates, also known as cell lysis, typically refer to the substances obtained upon completion of the destruction or lysis of a biological cell, which is a phenomenon that induces the release of intracellular biological components naturally present in the cells of the assumed microorganism.

[0034] In the context of the present invention, the term "lysate" is often used to refer to either the whole or part of the lysate obtained by the lysis of the assumed microorganism.

[0035] Therefore, the lysates produced are formed entirely or partially by intracellular biological components, as well as by components of the cell wall and membrane.

[0036] Therefore, preferably, the lysate to be carried out is formed entirely or partially by intracellular biological components, as well as by cell wall and membrane components.

[0037] In a preferred embodiment, the lysate contains less than 1% by dry mass of extracellular medium; more preferably, the lysate contains between 0.5% by dry mass and 1% by dry mass of extracellular medium; and even more preferably, the lysate contains between 0.7% by dry mass and 0.8% by dry mass of extracellular medium.

[0038] This cell lysis can be completed using various techniques, such as osmotic shock, thermal shock, freezing and subsequent thawing, ultrasound, high-pressure processing, or under centrifugal mechanical load.

[0039] Preferably, the solution carried out in the context of the present invention is obtained by a method comprising the following steps: i) Centrifugal separation process, ii) The process of recovering the pellets, iii) The process of freezing the pellets, iv) Thawing the pellets, v) Optionally, a sterilization step, particularly by autoclaving at 121°C.

[0040] In certain embodiments of the present invention, the bacterial extract comprises inactivated bacteria.

[0041] The term "inactivated" should be understood in the sense commonly used by those skilled in the art, namely, the suppression of bacterial activity under the influence of various causes (heat, chemical, mechanical, enzymatic). In particular, bacteria are inactivated by heat, especially by autoclaving.

[0042] Preferably, the extract according to the present invention is active in activating the hTLR2 receptor and / or promoting the expression of genes related to the enhancement of the skin barrier function.

[0043] Preferably, the extract is active in inhibiting the secretion of the pro-inflammatory mediator PGE2.

[0044] Preferably, the extract is active in stimulating the expression of IL-10 without significantly affecting the expression of IL-12, IL-6, and IL-8.

[0045] Preferably, the extract is active in inhibiting IgE production by B lymphocytes.

[0046] All of these activities can be detected and / or analyzed by methods known to those skilled in the art, including the specific examples described herein.

[0047] Therefore, bacteria of the genus Sphingomonas and their extracts possess several anti-inflammatory activities combined with enhanced skin barrier function, enabling the prevention and / or treatment of skin reactions caused by allergic reactions.

[0048] The present invention also relates to bacterial extracts according to the present invention for use in modulating the immune response and, optionally, in enhancing the skin barrier function.

[0049] composition The present invention also relates to compositions, particularly intended for topical application, comprising an extract of a bacterium of the genus Sphingomonas or at least one bacterium of the genus Sphingomonas, for use in the prevention and / or treatment of skin reactions caused by allergic reactions.

[0050] The present invention also relates to a composition comprising an extract of a bacterium of the genus Sphingomonas or at least one bacterium of the genus Sphingomonas for use in modulating the immune response and, optionally, enhancing the skin barrier function.

[0051] In one embodiment, the composition comprises the extract described above herein.

[0052] In one embodiment, the composition comprises at least one bacterium of the genus Sphingomonas as described in the General Definitions section.

[0053] The amount of bacteria or bacterial extracts in the composition according to the present invention varies depending on the intended type of composition. Preferably, the amount of microorganisms or extracts is between 0.0001 and 30% by dry mass, between 0.001 and 15% by dry mass, between 0.01 and 10% by dry mass, preferably between 0.01 and 5% by dry mass, or between 0.01 and 3% by dry mass, relative to the total mass of the composition.

[0054] When bacteria are incorporated into the composition in a living form, the amount of living bacteria is 10 per gram of composition. 3 from 10 15 Up to ufc / g, especially 10 5 from 10 15 Up to ufc / g, especially 10 7 from 10 12 Even the bacteria in the UFC / G can change.

[0055] Preferably, the composition according to the present invention comprises an aqueous phase.

[0056] Preferably, the composition according to the present invention has a water content in the range of 20% to 95% by mass, preferably 30% to 70% by mass, based on the total mass of the composition.

[0057] The composition according to the present invention may further contain one or more water-miscible organic solvents in a concentration ranging from 0.5% to 25% by mass, preferably 5% to 20% by mass, and more preferably 10% to 15% by mass, based on the total mass of the composition.

[0058] As previously shown, the compositions used in the context of the present invention are preferably used topically.

[0059] As is well known to those skilled in the art, compositions for topical application may also contain common additives, such as hydrophilic or lipophilic gelling agents, hydrophilic or lipophilic active substances different from the extracts according to the present invention, preservatives, antioxidants, solvents that are not miscible with water, fragrances, odor absorbers, and colorants. The amounts of these various additives are those commonly used in the intended field, for example, 0.01 to 20% of the total mass of the composition.

[0060] Naturally, a person skilled in the art would carefully select these optional additional components and / or active substances, and / or their amounts, so as to ensure that the advantageous properties of the extract according to the present invention are not altered, or substantially altered, by the intended additions.

[0061] In one embodiment, a bacterium of the genus Sphingomonas or an extract of a bacterium of the genus Sphingomonas is the sole active substance of the composition.

[0062] In another embodiment, the composition of the present invention may further contain other active substances different from the bacteria or extracts according to the present invention. These active substances may have effects on the skin, particularly sedative effects.

[0063] In certain embodiments of the present invention, the composition according to the present invention further comprises at least one active substance selected from acetyl dipeptide-1 cetyl ester, an extract of tangin's (Salvia miltiorrhiza) root, thermal water, such as La Roche-Posay water, and mixtures thereof.

[0064] The compositions according to the present invention may be in any Galenic form commonly used for topical application, particularly in the form of aqueous, aqueous-alcoholic solutions, oil-in-water (O / W) or water-in-oil (O / W) or multiple (triple: W / O / W or O / W / O) emulsions, aqueous gels, or dispersions of a lipid phase in an aqueous phase using spheres, which may consist of ionic and / or nonionic lipid vesicles (liposomes, niosomes, oleosomes). These compositions are prepared using a prescribed procedure.

[0065] Advantageously, the compositions according to the present invention are in the form of a gel, emulsion, or paste. Furthermore, the compositions according to the present invention may be fluid to varying degrees and may have the appearance of a white or colored cream, ointment, emulsion, lotion, serum, paste, foaming or non-foaming gel, exfoliant, mask, treatment, tonic, or foamy substance. It may be applied to the skin in the form of a spray. It may also be in the form of a solid, such as a stick or soap.

[0066] The composition according to the present invention may include a fatty phase, particularly an oily phase.

[0067] Examples of oils suitable for use in the composition according to the present invention include: - Hydrocarbon oils, - In particular, oils of fatty acids, synthetic esters and ethers, for example, oils of formulas R'COOR2 and R'COR2 (wherein R' represents a fatty acid residue containing 8 to 29 carbon atoms, and R2 represents a branched or unbranched hydrocarbon chain containing 3 to 30 carbon atoms), - Linear or branched hydrocarbons of inorganic or synthetic origin, - Fatty alcohols having 8 to 26 carbon atoms, - Partially hydrocarbonized and / or siliconeized fluorinated oils, - Silicone oil, - A mixture of those.

[0068] In the list of oils enumerated above, hydrocarbon oils should be understood as any oil that primarily contains carbon atoms and hydrogen atoms.

[0069] The oily phase may include other fatty bodies that may be present in the oily phase, such as fatty acids containing 8 to 30 carbon atoms, waxes, silicone resins, and silicone elastomers. These fats may be selected in various ways by those skilled in the art to prepare compositions having desired properties, such as consistency or texture.

[0070] According to a particular embodiment of the present invention, the composition according to the present invention is a water-in-oil (W / O) or oil-in-water (O / W) emulsion. The proportion of the oily phase of the emulsion may be in the range of 5 to 80% by mass, preferably 30 to 70% by mass, based on the total mass of the composition. The emulsion generally contains at least one emulsifier selected from amphoteric, anionic, cationic, or nonionic emulsifiers, and optionally a co-emulsifier, used alone or in a mixture. The emulsifier is appropriately selected depending on the emulsion to be obtained (W / O or O / W). Generally, the emulsifier and co-emulsifier are present in the composition in a proportion of 0.3% to 30% by mass, preferably 0.5% to 20% by mass, based on the total mass of the composition.

[0071] For W / O emulsions, examples of emulsifiers include dimethicone copolyols and alkyl-dimethicone copolyols. It is also possible to use a cross-linked elastomer solid organopolysiloxane containing at least one oxyalkylene group as a surfactant in the W / O emulsion.

[0072] For O / W emulsions, a nonionic emulsifier may be used as an emulsifier, for example.

[0073] The composition may or may not be rinsed off after application to the skin.

[0074] method The present invention also relates to a method for preventing or treating skin reactions caused by allergic reactions, comprising the step of administering, preferably topically, an extract of a bacterium of the genus Sphingomonas, or a composition comprising an extract of a bacterium of the genus Sphingomonas or at least one bacterium of the genus Sphingomonas, to the surface of the skin.

[0075] The use of extracts of Sphingomonas bacteria, or compositions containing extracts of Sphingomonas bacteria or at least one species of Sphingomonas bacteria, in the manufacture of pharmaceuticals for the prevention or treatment of skin reactions caused by allergic reactions is also proposed.

[0076] The present invention also relates to a method for modulating an immune response and optionally enhancing the skin barrier function, comprising the step of administering, preferably topically, an extract of a bacterium of the genus Sphingomonas, or a composition comprising an extract of a bacterium of the genus Sphingomonas or at least one bacterium of the genus Sphingomonas, preferably on the surface of the skin.

[0077] The use of extracts of Sphingomonas bacteria, or compositions containing extracts of Sphingomonas bacteria or at least one species of Sphingomonas bacteria, in the manufacture of pharmaceuticals for modulating immune responses and, optionally, for enhancing the skin barrier function is also proposed.

[0078] In certain embodiments, compositions comprising an extract of a bacterium of the genus Sphingomonas or at least one bacterium of the genus Sphingomonas are as described in the above section of the Specified Compositions.

[0079] In certain embodiments, the extract of bacteria of the genus Sphingomonas is as described in the following section on extracts of bacteria of the genus Sphingomonas.

[0080] Preferably, the application is carried out with an effective amount of an extract of at least one bacterium of the genus Sphingomonas, or at least one composition containing an extract of a bacterium of the genus Sphingomonas or at least one bacterium of the genus Sphingomonas, to the target that requires it.

[0081] As used herein, the term “effective amount” means, as a whole, the amount of extract of Sphingomonas bacteria that enables the prevention and / or reduction of skin reactions caused by allergic reactions, or as a whole, the amount of Sphingomonas bacteria that enables the prevention and / or reduction of skin reactions caused by allergic reactions.

[0082] The effective dose naturally depends on the intended active substance, mode of administration, therapeutic indication, patient's age, and patient's condition.

[0083] Advantageously, the composition or extract may be used particularly in the form of a dermatological composition further comprising physiologically acceptable excipients as an option.

[0084] "Physiologically acceptable" in this specification means that compositions and molecular entities that, when administered to a subject, do not cause undesirable secondary reactions, allergies, etc. Therefore, physiologically acceptable excipients or vehicles are encapsulating materials, diluents, supports, or any other non-toxic compounding aids of liquid, semi-solid, or solid.

[0085] Dermatological compositions used in the context of the present invention are typically prepared to suit a mode of administration. Physiologically acceptable excipients are typically determined not only in part by the composition being administered, but also by the specific method used to administer the composition.

[0086] The dosage of a compound to be administered depends on the individual case and, as is well known to those skilled in the art, should be adapted to the specific circumstances to obtain an effective dose and optimal effect. The effective dose level is specific to each target and depends in particular on various factors, such as the disorder being treated and its severity, the activity of the specific compound used, and the specific composition used. For example, it is well known to those skilled in the art to start with a dose of the compound at a level lower than the level required to achieve the desired effect and gradually increase the dose until the desired effect is obtained.

[0087] Preferably, the method according to the present invention includes the step of topically applying at least one bacterium of the genus Sphingomonas, or an extract of at least one Sphingomonas, or a composition containing the same.

[0088] In a preferred embodiment, topical application in the context of the present invention refers to application to the skin.

[0089] The method of the present invention may consist of just a single dose. In another embodiment, the dose may be repeated, for example, two to three times a day over one or more days, or applied daily, whether for several consecutive days or not, and may be repeated for a long period of at least four weeks or four to fifteen weeks, or for as long as necessary, with interruptions as may occur.

[0090] In another embodiment, the method of the present invention may include the step of administering to a skin area before and / or after exposure of that area to an allergen.

[0091] In this description and the following examples, unless otherwise indicated, percentages are mass percentages, and ranges expressed as "between ~ and ~" include specified upper and lower limits. The components are mixed in an order and under conditions readily determined by those skilled in the art before packaging.

[0092] The present invention is further illustrated by the following examples herein. [Brief explanation of the drawing]

[0093] [Figure 1] This graph shows the effect of Sphingomonas extract on cytokine secretion by monocytes. [Examples]

[0094] (Example 1) Preparation of extract according to the present invention Sphingomonas xenophagum strain CNCM-I 5455 is cultured in batch mode in a 3000 effective liter bioreactor using complete culture medium. During this process, the pH is not adjusted, the temperature is maintained at 26°C, and 30% oxygen is dissolved.

[0095] The composition of the initial culture medium is described in Table 1 below.

[0096] [Table 1]

[0097] Once a plateau level is reached, cell extraction and separation are performed by centrifugation (continuously at 10,000 g). The pellet containing the cells, also called biomass, is then collected, frozen at -20°C, and then thawed, thereby allowing the cells to burst and thus enabling the acquisition of lysates. The lysates are then placed in small bags and finally stabilized by sterilization at 121°C for 30 minutes.

[0098] The solution obtained upon completion of the method described in Example 1 contains 4.5% by mass of dry material relative to the total mass of the solution.

[0099] (Example 2) In vitro trial - Dose-response analysis of TLR (Invivogen). HEK293-TLR cell line: Extracts and controls are tested in pairs against recombinant HEK-293 cell lines. These cell lines functionally overexpress not only human TLR proteins but also a reporter gene for secreted alkaline phosphatase (SEAP). The generation of this reporter gene is controlled by an NFκB-inducible promoter. The results of TLR reporter activation are given in the form of optical density (OD) values.

[0100] Positive control: CU-T12-9 and FSL-1 were used on cells expressing hTLR2, and TLR1 / TLR2 and TLR2 / TLR6 activity were evaluated, respectively.

[0101] FSL-1 will be tested at 10 pg / ml and 30 pg / ml. CU-T12-9 will be tested at 200 nM and 300 nM.

[0102] Negative control: A HEK-293 cell line, recombinant only in the reporter gene, was used as a negative control for cell lines expressing hTLRs. To confirm the efficacy of the inducible reporter gene NFkB(SEAP), this HEK-293 TLR cell line was stimulated with TNFα at the following concentrations: 100 ng / ml, 30 ng / ml, 10 ng / ml, 3 ng / ml, 1 ng / ml, 0.3 ng / ml, and 0.1 ng / ml.

[0103] These negative control cell lines were also stimulated with various concentrations of the extracts being tested.

[0104] Testing of the extract: Reporter hTLR cell lines in a 200 μl reaction volume were stimulated with 20 μl of extract. For dose-response testing of the HEK-Blue-hTLR5 cell line, liquid extracts obtained according to Example 1 were tested in two series at the following concentrations: 1000 μg / ml, 500 μg / ml, 250 μg / ml, 125 μg / ml, 62.5 μg / ml, and 31.5 μg / ml.

[0105] For TLR1 / 2 and / or TLR2 / 6 specificity, the liquid extracts obtained according to Example 1 were tested in two series at the following concentrations: 100 μg / ml, 30 μg / ml, and 10 μg / ml.

[0106] result:

[0107] [Table 2]

[0108] [Table 3]

[0109] [Table 4]

[0110] Conclusion: The extract does not activate negative control cell lines, but strongly activates the reporter cell line hTLR2(1 / 6), even at 10 μg / ml. It activates hTLR1 / 2 and hTLR2 / 6 in a nonspecific manner.

[0111] (Example 3) In vitro study - Regulation of gene expression in normal human keratinocytes Materials and methods Cytotoxicity: Normal human epidermal keratinocytes were seeded in 96-well culture plates and cultured in culture medium at 37°C and 5% CO2 for 24 hours. The medium was then replaced with a culture medium containing or not containing the compound of interest (8 concentrations tested), and the cells were incubated for another 24 hours. All conditions were repeated in two cycles. At the end of incubation, cell viability was measured by a standard test for mitochondrial activity.

[0112] Analysis of gene expression related to barrier function / hydration using RT-qPCT: Normal human epidermal keratinocytes (NHEKs) were seeded in 48-well culture plates and then cultured in culture medium at 37°C and 5% CO2 for 3 days, with the culture medium replaced after the first 24 hours of incubation. At the end of incubation, the culture medium was replaced with either a control medium without the compound to be tested or a test medium containing the compound (supplemented with 1.5 mM CaCl2), and the cells were incubated for 24 hours. All conditions were repeated in two cycles.

[0113] At the end of the process, the culture medium was removed and the cells were rinsed twice with PBS (without CaCl2 and MgCl2). Total RNA was then isolated using an extraction kit. RNA quantification and quality control were performed using Labchip GX (Perkin Elmer).

[0114] The expression of selected transcripts was analyzed by quantitative PCR in two steps. First, cDNA was transcribed from RNA in the reverse direction. Subsequently, quantitative PCR experiments were performed using a real-time PCR system.

[0115] Experimental protocol Normal human epidermal keratinocytes are cultured and grown to produce enough cells to evaluate up to 72 samples per campaign. Cells (50,000 cells per well) are seeded into 48-well microplates and incubated for 48 hours in a controlled environment of temperature, humidity, and CO2. When changing the culture medium, the samples to be tested are added to the cells, and then the cells are incubated for 24 hours.

[0116] The cells are washed and freeze-dried at -80°C to preserve the RNA. The RNA is extracted, quantified, and its quality is checked before reverse transcription to cDNA. Finally, RT-qPCR is performed for each experimental condition to quantify the expression of the selected set of genes.

[0117] [Table 5]

[0118] In this example, the tested Sphingomonas batch corresponds to the lyophilized (100% MS) product of the liquid extract (4.5% MS) obtained according to Example 1.

[0119] In conclusion, considering the fact that 1 g / L corresponds to a concentration of 0.1%, the liquid extract obtained according to Example 1 allows for significant regulation of the expression of the gene claudin-1, corniferin, and closure zone 1 at concentrations of 0.0009 (calculation: 0.2 × 0.1 × 0.045) and 0.0045 (calculation: 1 × 0.1 × 0.045) in NHEK.

[0120] (Example 4) In vitro study: "Evaluation of regulation of PGE2 release by human bone marrow cells" Anti-inflammatory efficacy study of the U937 human bone marrow cell line (provided by Dr. Kenneth Nilsson, Professor of Cellular Pathology, Department of Immunology, Genetics and Pathology, Uppsala University, Rapphonsvagen 11, 75653 Uppsala, Sweden).

[0121] Principles of the test This cell line is used in this protocol to model the monocyte / macrophage immune response in vitro. Treatment with PMA and LPS induces the secretion of pro-inflammatory mediators and cytokines. Co-treatment with anti-inflammatory agents allows for the identification of intrinsic regulatory activity of the pro-inflammatory response in vitro.

[0122] Cell processing Human bone marrow cell lines were treated with Sphingomonas xenophagum extract in RPMI 1640 culture medium supplemented with L-glutamine, penicillin, streptomycin, 10% heat-treated fetal bovine serum, 10 μg / mL phorbol 12-myristate 13-acetate (PMA), and 60 μg / mL lipopolysaccharide (LPS) for Escherichia coli (E. coli). Seven concentrations of Sphingomonas xenophagum extract were tested: 1, 3, 10, 30, 100, 300, and 1000 μg / mL.

[0123] Cell viability was measured for each condition 24 hours after treatment in a humid atmosphere of 5% CO2 at 37°C.

[0124] Subsequently, the supernatant was collected for the PGE2 assay.

[0125] Cytokine assay PGE2 levels are determined by ELISA testing.

[0126] Calculating Survival Rate - CV75 CV75=C1+((V1-75) / (V1-V2))×(C2~C1) (In the formula, C1 = Lowest concentration with a survival rate > 75% C2 = Highest concentration with a survival rate of <75% V1=minimum survival percentage >75% V2=maximum survival percentage<75%)

[0127] Calculation of the Reactivity Index - RI RI = Amount of marker in the treated supernatant (pg / ml) × 100 / Amount of analyte in the untreated supernatant (pg / ml).

[0128] Calculation of inhibitory concentration at 50% - IC50 I C 50 =C1+((50-RI1) / (RI2-RI1))×(C2~C1) (In the formula, The highest concentration with C1=RI<50 Lowest concentration with C2=RI>50 RI1=maximum RI<50 RI2=minimum RI>50)

[0129] [Table 6]

[0130] Sphingomonas extract is equivalent to IC199 μg / ml. 50 This inhibits the secretion of pro-inflammatory mediators.

[0131] (Example 5) An in vitro study of the effects of extracts on the secretion of cytokines (IL-6, IL-8, IL-10, and IL-12p40) by monocytes. The purpose of the experiment is to study the effect of the extract on cytokine secretion by monocytes 36 hours later.

[0132] Materials and methods: Cell isolation and culture: Mononuclear cells were obtained from the leukocyte layer of healthy blood donors using a standard Ficol-Hypak gradient method. Monocytes were isolated from mononuclear cells by adhering them to plastic in serum-free medium (M-SFM) optimized for macrophage culture at 37°C in a humid atmosphere containing 5% CO2 for 2 hours. Adhering cells highly enriched with monocytes were incubated with the compound under test for 36 hours.

[0133] Cytotoxicity test: During the last six hours, Alamar Blue was introduced into the cell culture. Resorphin, a fluorescent molecule obtained by the conversion of one of the components of Alamar Blue, was measured as an indicator of cell death. The measurement was performed using a Tecan Infinite F500 fluorescence spectrophotometer.

[0134] Cytokine assays: After 36 hours, the supernatant was collected and frozen at -80 °C until assay. Measurements were performed using a milliplex assay kit (IL-6, IL-8, IL-10, and IL-12p40) on a Luminex LX100 instrument.

[0135] Calculation method for result evaluation: Since the value ranges are different for each cytokine, multiplication factors are different for each cytokine compared to IL-10 to obtain values that are easy to compare between compounds.

[0136] Comparison between IL-10 and IL-6: The ratio is IL-10 × 100 / IL-6 Comparison between IL-10 and IL-12p40: The ratio is IL-10 / IL-12p40 Comparison between IL-10 and IL-8: The ratio is IL-10 × 100000 / IL-8

[0137] The results are shown in Figure 1. Product 2 corresponds to the liquid extract obtained according to Example 1.

[0138] The extract has an immunomodulatory effect on monocytes: IL10 > IL-6 / IL-8 / IL-12(p40).

[0139] The extract induces a high production of IL-10 (an anti-inflammatory cytokine) with respect to the dose response compared to IL-6 and IL-8 (inflammatory cytokines).

[0140] (Example 6) In vitro test of the effect of the extract on the production of IgE by human B lymphocytes stimulated with a combination of anti-CD40 and IL-4 In this study, purified human B lymphocytes (CD19 from peripheral blood mononuclear cells) +The effect of extracts on IgE production (induced by lymphocyte isolation) was evaluated. More specifically, IgE production was induced by stimulating B lymphocytes with a combination of anti-CD40 antibody and IL-4. After 14 days of incubation, the amount of IgE released into the culture supernatant was quantified using a specific ELISA kit. Three other molecules were evaluated simultaneously with the extract being tested. Reference standards CpG-ODN 2006 (TLR9 ​​ligand), as well as IFN-γ and PGE2, were tested as reference compounds.

[0141] [Table 7]

[0142] Culture and processing B lymphocytes CD19 + The cells were isolated, seeded in 24-well plates, and incubated overnight in culture medium. Then, the cells were pre-incubated for 2 hours with or without the addition of the compound to be tested, either alone or in combination with the reference compound (control). Subsequently, the treatment with the test or reference compound was repeated, either with or without the addition of the inducing factor (CD40 + IL-4 combination). Finally, the cells were raised to a final density of 1.7 × 10⁶. 6 The cells were incubated at a concentration of [number] cells / ml for 14 days.

[0143] All experimental conditions were performed with n=3.

[0144] Enzyme-linked immunosorbent assay (ELISA) The amount of IgE released into the culture supernatant was measured using a specific ELISA kit according to the supplier's instructions.

[0145] statistics Group comparisons were performed using an independent Student's t-test. Statistical analysis can be interpreted when n ≥ 5, but when n < 5, the statistical values ​​are given for reference only.

[0146] Formulas used in this report: Mean standard error: MSE = Sd / √n

[0147] The mean standard error (MSE) is a measure of the deviation between the mean of a sample and the actual mean of the population. The MSE is calculated as the sample standard deviation (Sd), divided by the square root of the sample size (n).

[0148] Results and Conclusions Under non-stimulation conditions, baseline IgE release by B lymphocytes was very low (<199 pg / ml, near the limit of detection). 14 days of stimulation with the anti-CD40+IL-4 combination resulted in high levels of IgE production (3657 pg / ml). The TLR9 CpG-ODN2006 agonist, tested at 3 μM as a reference compound, completely inhibited IgE production induced by anti-CD40+IL-4 association (<3% of stimulated controls). These results were expected and confirmed the validity of the study.

[0149] Two other potential reference compounds also showed inhibitory effects on IgE production, but to a lesser degree compared to CpG-ODN2006. IFN-γ tested at 10 ng / ml also showed a very strong and significant inhibitory effect (20% of stimulated controls). However, the effect of PGE2 tested at 1 μM was far more limited and not statistically significant (58% of stimulated controls).

[0150] The extracts from Example 1, tested at 0.2% and 0.4%, showed almost complete inhibition of IgE production by B lymphocytes stimulated by combined anti-CD40+IL-4 (<3% and <11%, respectively, compared to the stimulated control).

[0151] Under the experimental conditions of this study, extracts from Example 1 at various concentrations showed a very potent inhibitory effect on IgE production by cell B stimulated by the anti-CD40 + IL-4 combination.

[0152] [Table 8]

[0153] (Example 7) A gel with the following composition was prepared:

[0154] [Table 9]

[0155] The resulting composition was applied to the face of a person with skin prone to allergies.

[0156] [Table 10]

Claims

1. An extract of bacteria of the genus Sphingomonas, for use in the prevention and / or treatment of skin reactions caused by allergic reactions.

2. A composition comprising an extract of a bacterium of the genus Sphingomonas or at least one bacterium of the genus Sphingomonas, for use in the prevention and / or treatment of skin reactions caused by allergic reactions.

3. The extract for use according to claim 1, or the composition for use according to claim 2, wherein the bacterium of the genus Sphingomonas is a bacterium of the species Sphingomonas xenophaga.

4. The extract for use or composition for use according to claim 3, wherein the bacterium of the species Sphingomonas xenophagum is a strain of the bacterium submitted under CNCM accession number I-5455.

5. Preferably, an extract for use according to any one of claims 1, 3, and 4, or a composition for use according to any one of claims 2 to 4, for topical use on the surface of the skin.

6. The composition for use according to any one of claims 2 to 5, wherein the concentration of the extract is between 0.0001% by mass and 30% by mass relative to the total mass of the composition.

7. The composition according to any one of claims 2 to 6, further comprising at least one active substance selected from the group consisting of acetyl dipeptide-1 cetyl ester, an extract of tangin's (Salvia miltiorrhiza) root, thermal water, for example, La Roche-Posay water, and mixtures thereof.

8. An extract for use according to any one of claims 1, 3 to 5, or a composition for use according to any one of claims 2 to 7, wherein the allergic reaction is an immediate-type allergic reaction.

9. An extract for use according to any one of claims 1, 3 to 5 and 8, or a composition for use according to any one of claims 2 to 8, wherein the reaction comprises the appearance of urticaria, skin rash, and / or redness.

10. Extracts of Sphingomonas bacteria for use in regulating immune responses and, optionally, enhancing skin barrier function.

11. A composition comprising an extract of a bacterium of the genus Sphingomonas or at least one bacterium of the genus Sphingomonas, for use in modulating the immune response and, optionally, enhancing the skin barrier function.

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