Compositions and uses thereof

Abstract

The present invention relates to a composition comprising heterotrophic skin bacteria and / or an extract of heterotrophic skin bacteria for use in modulating cytokine expression in skin cells. The compositions are particularly suitable for use in the prevention and / or treatment of skin lesions caused by ultraviolet rays and for use in the treatment of skin conditions such as solar eruption polymorphic (PLE).

Description

Technical Field

[0001] The present invention relates to a composition for modulating cytokine expression in skin cells, comprising a heterotrophic skin bacterium and / or an extract of a heterotrophic skin bacterium. Specifically, the heterotrophic skin bacterium is Micrococcus luteus (M. luteus) and / or the extract of the heterotrophic skin bacterium is derived from Micrococcus luteus. The composition is highly suitable for mediating cytokine expression in the skin. Specifically, the composition is applicable for preventing and / or treating skin conditions related to cytokine expression, such as those caused by skin exposure to ultraviolet light. Background Art

[0002] The mechanisms by which bacteria exert positive effects on humans are diverse, including inhibiting pathogens, modulating immune responses, and enhancing epithelial barrier function. However, in general, the nature of potential probiotics is not well-defined, and relatively little information is available regarding the molecular mechanisms mediating the observed probiotic effects.

[0003] The topical application of probiotics on the skin has been investigated in a limited number of studies. In general, the mechanisms underlying these effects are not well understood.

[0004] Current theory suggests that immune regulation involves a balance between T helper cell 1 (Th1) and T helper cell 2 (Th2) activities, where Th1 and Th2 helper cells direct different immune response pathways. Overactivation of either pattern can lead to disease, and either pathway can downregulate the other. The Th1 and Th2 pathways are generally associated with the secretion of different cytokines.

[0005] Exposure to ultraviolet radiation (UVR) suppresses the immune response. Data from many laboratories indicate that one consequence of UVR exposure is the inhibition of T helper cell type 1 (Th1) function, while Th2 cell activation is normal, leading to a shift towards a Th2-like phenotype. In addition, exposure to UVR exacerbates or induces many conditions. These are collectively referred to as photodermatoses, including conditions such as urticaria, photoaggravated eczema, and polymorphic light eruption (PLE or PMLE).

[0006] Polymorphic light eruption (PLE or PMLE) is a skin "complaint" caused by sunlight. The name is derived from polymorphic eruption, meaning a rash with many forms. PLE is thought to be caused by an immune response to compounds in the skin that have been altered after exposure to ultraviolet radiation. PLE typically occurs after 2 to 3 days of sunlight exposure, but can also occur with shorter exposures (e.g., direct exposure to incident light through a window, or 15 to 20 minutes in direct sunlight).

[0007] A delayed, blotchy, itchy rash appears on the skin and may take 5 to 10 days to resolve. The rash, typically consisting of small red spots or blisters, may appear anywhere on the body exposed to the sun, but typically avoids the face and backs of the hands. It often heals without scarring. PLE differs from prickly heat, which affects the trunk due to warm weather rather than sun exposure.

[0008] PLE has been observed in 20% to 30% of the population in Northern Europe and the United States (where sun exposure is less common), and in 10% to 20% of the population elsewhere, with women being more affected than men. PLE typically affects patients under 30 years of age, with a median age range of 20 to 40 years. The problem is more common in temperate climates, such as those in Europe and the United States, and typically begins in spring and lasts until summer. PLE is likely related to an immune system response in the skin in some individuals and is more common in those with fair skin. Evidence of PLE has also been found in northern China and Australia. Current data suggest that the development of photodermatoses like PLE may be associated with overexpression of TH1 and suppression of TH2 responses. There are currently no proven therapies to help alleviate skin conditions like polymorphous light eruption (PLE), although calamine lotion, topical steroid creams containing corticosteroids, hydrocortisone, and hydroxychloroquine, and some conventional antipruritic medications have limited efficacy.

[0009] Currently, there is no effective treatment for PLE, and most therapies and compositions are aimed at alleviating symptoms rather than treating or preventing the condition itself. For at least the reasons stated above, there is a need to develop compositions, therapies, and methods to prevent and / or treat and / or ameliorate PLE and skin conditions caused by skin exposure to UV light. Summary of the Invention

[0010] The present invention is defined in the appended claims and also encompasses combinations of the aspects and preferred features described below, unless such combinations are expressly impermissible or explicitly avoided.

[0011] According to one aspect of the present invention, there is provided a composition for modulating cytokine expression in skin cells, comprising a heterotrophic skin bacterium and / or an extract of a heterotrophic skin bacterium. It should be understood that in embodiments where the composition comprises a heterotrophic skin bacterium, the composition may contain whole / intact bacteria. Alternatively, in embodiments, the extract of the heterotrophic skin bacterium is in the form of a lysate. The heterotrophic skin bacterium or the extract of the heterotrophic skin bacterium may be in freeze-dried, lyophilized or lysate form. Preferably, the heterotrophic skin bacterium is of the family Micrococcaceae and / or the extract of the heterotrophic skin bacterium is derived from a bacterium of the family Micrococcaceae. More preferably, the heterotrophic skin bacterium or the extract of the heterotrophic skin bacterium is a saprophytic bacterium or is derived from a saprophytic bacterium. Even more preferably, the heterotrophic skin bacterium is Micrococcus and / or the extract of the heterotrophic skin bacterium is derived from Micrococcus bacteria. Most preferably, the Micrococcus bacteria may be Micrococcus luteus. Those skilled in the art will understand that Micrococcus luteus is a Gram-positive saprophytic coccus and is part of the normal microbiota of mammalian skin. Preferably, the composition comprises Micrococcus luteus and / or a bioactive extract of Micrococcus luteus in lysate form. Micrococcus luteus may be deposited under the accession number 22110401.

[0012] In related embodiments, the composition is formulated for topical application.

[0013] In further related embodiments, the modulation of cytokine expression results in the modulation of one or more of the following cytokines: IL-6, and / or IL-8, PTX-3, VEGF and / or MIP-3α. The modulation of cytokine expression in skin cells may be an increase in the secretion of one or more of the following cytokines: IL-6, and / or IL-8, and / or PTX-3, and / or a decrease in the secretion of one or more of the following cytokines: VEGF and / or MIP-3α.

[0014] In another embodiment, the composition is formulated in the form of a cream, gel, spray, ointment or oil. In another related embodiment, the composition further comprises one or more pharmaceutically acceptable ingredients or excipients. The composition may suitably be in the form of a liquid, solution (e.g., aqueous, non-aqueous), suspension (e.g., aqueous, non-aqueous), emulsion (e.g., oil-in-water, water-in-oil), elixir, syrup, lozenge, mouthwash, douche, drops, granules, powder, ampoule, bolus, suppository, vaginal suppository, tincture, gel, paste, ointment, cream, lotion, oil, foam, spray, mist or aerosol. The composition may suitably be provided as part of a patch, adhesive tape, bandage, dressing, etc., impregnated with one or more active compounds and optionally one or more other pharmaceutically acceptable ingredients, including, for example, penetration, permeation and absorption enhancers. The composition may also suitably be provided in the form of a depot or reservoir. The composition may also be provided in the form of a coating for a medical device, such as an implant, prosthesis, surgical instrument, glove, catheter, valve, pacemaker, etc.

[0015] The composition according to aspects of the present invention may further comprise one or more pharmaceutically or cosmetically acceptable ingredients or excipients. Pharmaceutically acceptable ingredients are well known to those skilled in the art and include, but are not limited to, pharmaceutically acceptable carriers, adjuvants, excipients, diluents, fillers, buffers, preservatives, carriers, excipients, diluents, fillers, buffers, preservatives, antioxidants, lubricants, stabilizers, solubilizers, surfactants (e.g., wetting agents), masking agents, colorants, fragrances and penetration enhancers.

[0016] In some cases, the carrier may include the culture medium that comes into contact with the bacteria during the culturing process. The composition of the culture medium changes during the culturing process, for example due to the secretion of substances by the bacteria. The composition may consist of the culture medium in which the bacteria grow or contain the culture medium in which the bacteria grow.

[0017] Preferably, the composition is formulated for topical administration, particularly for or to be applied to the skin, or to be used on or applied to the skin. The composition may be formulated in the form of a gel, paste, ointment, cream, spray, lotion, oil and patch, adhesive tape, bandage, dressing, depot, adhesive, glue and reservoir for topical administration. Preferably, the composition may be formulated in the form of a cream, gel, spray, ointment or oil for topical administration.

[0018] An ointment is generally prepared from the composition and paraffin or a water-soluble ointment base.

[0019] Creams are usually prepared from extracts and an oil-in-water cream base. If desired, the aqueous phase of the cream base can include, for example, at least about 30% w / w of a polyol, i.e., an alcohol having two or more hydroxyl groups, such as propylene glycol, butane-1,3-diol, mannitol, sorbitol, glycerol, and polyethylene glycol and mixtures thereof. Topical formulations can desirably include compounds that enhance the absorption or penetration of the active compound through the skin or other affected area. Examples of such skin penetration enhancers include dimethyl sulfoxide and related analogs.

[0020] Emulsions are usually prepared from heterotrophic skin bacteria and / or bioactive extracts of heterotrophic skin bacteria and an oil phase, which can optionally contain only an emulsifier (also called an emulgent), or can contain at least one emulsifier and a fat or oil, or a mixture of a fat and an oil. Preferably, a hydrophilic emulsifier and a lipophilic emulsifier as a stabilizer are included. It is also preferred to include both an oil and a fat at the same time. The emulsifier, together with or without the stabilizer, constitutes a so-called emulsifying wax, and the wax, together with the oil and / or fat, constitutes a so-called emulsifying ointment base, forming the oily disperse phase of the cream formulation.

[0021] Suitable emulsions and emulsion stabilizers include Tween 60, Span 80, cetearyl alcohol, myristyl alcohol, glyceryl monostearate, and sodium lauryl sulfate. Suitable oils or fats for the formulation are selected based on achieving the desired cosmetic properties, since the solubility of the active compound in most oils that may be used in pharmaceutical emulsion formulations may be very low. Thus, the cream should preferably be a product that is non-greasy, non-staining, washable, and has a suitable consistency to avoid leakage from a tube or other container. Straight-chain or branched-chain mono- or diesters of alkyls can be used, such as diisoadipate, isocetyl stearate, propylene glycol diester of coconut fatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate, or a blend of branched-chain esters called Crodamol CAP, with the latter three being preferred esters. These can be used alone or in combination depending on the desired properties. Alternatively, high-melting-point lipids such as white soft paraffin and / or liquid paraffin or other mineral oils can be used.

[0022] The composition can be administered alone, or simultaneously or sequentially with other treatments. The composition according to the invention can also contain other active agents, such as antimicrobial agents such as bactericides and fungicides, to prevent the composition from deteriorating during storage.

[0023] In some embodiments, the composition can be provided in the form of a suspension in a pharmaceutically or cosmetically acceptable excipient, diluent, or carrier.

[0024] The composition of the present invention can be formulated as a medicament, that is, formulated as a pharmaceutical product or a medical device. The medicament may include other pharmaceutically acceptable ingredients well known to those skilled in the art, including but not limited to pharmaceutically acceptable carriers, adjuvants, excipients, diluents, fillers, buffers, preservatives, antioxidants, lubricants, stabilizers, solubilizers, surfactants (such as wetting agents), masking agents, colorants, flavoring agents, and sweeteners. The preparation may also contain other active agents, such as other therapeutic agents or prophylactic agents.

[0025] Media suitable for culturing bacteria (such as Micrococcus luteus) are well known to those skilled in the art. As used herein, the term "media" encompasses any liquid containing nutrients in which bacteria can be supported, survive, grow, and / or proliferate. The media may contain the minimum amount of nutrients to support bacterial life, as well as optional other nutrients. Exemplary nutrients contained in broth include sugars, magnesium, phosphates, phosphorus, and sulfur. The media can be made or modified from combinations of nutrients well known in the art, such as tryptic soy agar / broth. The media can be pre-mixed and obtained from commercial sources, or can be made in-house.

[0026] In an embodiment, the composition is cell-free and does not contain any live bacterial cells. In such an embodiment, whole bacterial cells can be removed from the media, for example, by centrifugation and / or filtration (or other suitable methods for removing live bacteria). For example, bacteria can be sedimented from the media by centrifuging in a centrifuge at 15,000 x g for a time sufficient to sediment substantially all of the bacteria from the media, thereby removing the bacteria. A microporous filter with pores of a suitable size can be used to filter the media to remove substantially all of the bacteria from the media. These methods can remove intact bacteria and, if the extract is obtained by cell lysis, can also remove bacterial debris.

[0027] The composition can be sterile. That is, the composition has been subjected to a sterilization process, such as radiation, heating, chemicals, pressure, or filtration, or any combination thereof. For example, the composition can be filter sterilized, which is a term understood by those skilled in the art. Filter sterilization uses a 0.22 micron filter, as understood by those skilled in the art. The sterilization procedure must be adjusted so as not to impair or reduce the efficacy of the heterotrophic skin bacteria and / or the heterotrophic skin bacteria extract. In the case where the media contains an extract, the media may have been sterilized before the introduction and culturing of the heterotrophic skin bacteria and after the removal of the bacteria from the media.

[0028] In some cases, the extract of the composition is substantially free of intact bacteria. The composition may also be substantially free of lysed bacteria or bacterial fragments. The intact bacteria and / or lysed bacteria or bacterial fragments may have been separated from the extract. The separation can be carried out by any suitable method known in the art, such as centrifugation or filtration. "Substantially free" means that the extract is free of or contains a minimal amount of contamination by non-secretory bacterial components, such as whole bacteria, lysed bacteria or bacterial fragments. Thus, the composition may comprise 100% extract, at least 99% extract, at least 95% extract, at least 90% extract, at least 85% extract, at least 80% extract, at least 75% extract or at least 70% extract. The extract may contain additional components of non-bacterial origin, such as a carrier solution, other active agents or preservatives, as described herein.

[0029] The composition described herein can be prepared by culturing heterotrophic skin bacteria in a culture medium, separating the heterotrophic skin bacteria from the culture medium, and preparing the composition from the culture medium. The heterotrophic skin bacteria can be cultured under anaerobic conditions. The heterotrophic skin bacteria can be cultured at a temperature higher than the normal human body temperature. The heterotrophic skin bacteria can be cultured at 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C or 41°C. Preferably, the bacteria are cultured at 37°C. The bacteria can be cultured in the culture medium for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days or 14 days. The bacteria or lysed bacteria or fragments can be separated from the culture medium by centrifugation, for example, at 15000 xg. The culture medium can be separated from the heterotrophic skin bacteria, lysed heterotrophic skin bacteria or fragments of heterotrophic skin bacteria by filtration. The culture medium can be separated by a combination of filtration and centrifugation. The culture medium can be sterilized before or after removing the heterotrophic skin bacteria. For example, the culture medium can be sterilized after separating it from the intact bacteria, lysed bacteria or bacterial fragments. The culture medium can be concentrated such that the proportion of the heterotrophic skin bacteria extract is increased relative to the total volume of the culture medium. The concentration can be carried out by any method known in the art, such as evaporation. The heterotrophic skin bacteria extract can be separated from the culture medium. Any method for separating a substance from a carrier solution can be used. For example, the heterotrophic skin bacteria extract can be separated from the culture medium by chromatography, crystallization, distillation, drying, electrophoresis or precipitation. After separation from the culture medium or concentration in the culture medium, the extract can be dissolved or diluted in a carrier or otherwise formulated into the composition disclosed herein.

[0030] In another embodiment of the present invention, there is provided a composition for preventing and / or treating skin damage caused by ultraviolet rays, comprising heterotrophic skin bacteria and / or an extract of heterotrophic skin bacteria.

[0031] In another embodiment of the present invention, there is provided use of a composition comprising heterotrophic skin bacteria and / or a heterotrophic skin bacteria extract in the preparation of a medicament for preventing, controlling or treating skin damage caused by ultraviolet rays.

[0032] In yet another embodiment of the present invention, a composition comprising heterotrophic skin bacteria and / or a heterotrophic skin bacteria extract is provided for preventing, controlling or treating a skin condition caused by exposure to ultraviolet light. In a related embodiment, the skin condition is PLE.

[0033] In an embodiment, the composition can be used to prevent skin damage caused by UV rays or skin conditions caused by exposure to UV rays. In such an embodiment, the composition can be applied or administered before exposure to UV rays. It will be understood by those skilled in the art that before exposure to UV rays means an amount of UV rays sufficient to cause skin damage or skin conditions (such as PLE).

[0034] In further related embodiments, the composition is formulated for topical application to the skin. In another related embodiment, the skin condition is a skin condition mediated by VEGF and / or MIP-3α. In a further related embodiment, the skin condition is a skin condition mediated by IL-6, and / or IL-8, and / or PTX-3.

[0035] In another embodiment of the present invention, a composition comprising heterotrophic skin bacteria and / or a heterotrophic skin bacterial extract is provided for use in a cosmetic preparation for application to skin damaged by ultraviolet light. Typically, such cosmetic preparations are marketed as "after-sun" and are applied to skin after prolonged exposure to sunlight.

[0036] The compositions and formulations according to the invention may also contain other active agents, for example other antibacterial agents, such as bactericides.

[0037] In some embodiments, the composition may comprise at least about 0.01%, about 0.05%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.5%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6.0%, about 7.0%, about 8.0% by weight. , about 9.0%, about 10.0%, about 11.0%, about 12.0%, about 13.0%, about 14.0%, about 15.0%, about 16.0%, about 17.0%, about 18.0%, about 19.0%, about 20.0%, about 25.0%, about 30.0%, about 35.0%, about 40.0%, about 45.0%, about 50.0% heterotrophic skin bacteria and / or heterotrophic skin bacteria extracts.

[0038] In some embodiments, the composition can comprise at least about 0.01% to about 30%, about 0.01% to about 20%, about 0.01% to about 5%, about 0.1% to about 30%, about 0.1% to about 20%, about 0.1% to about 15%, about 0.1% to about 10%, about 0.1% to about 5%, about 0.2% to about 5%, about 0.3% to about 5%, about 0.4% to about 5%, about 0.5% to about 5%, about 1% to about 5% heterotrophic skin bacteria and / or heterotrophic skin bacteria extract by weight.

[0039] In another embodiment, the present invention provides a method for preventing, controlling or treating a skin condition caused by exposure to ultraviolet light, the method comprising applying a composition as defined in any other embodiment to the skin. The skin condition may be PLE. Also provided is a method for preventing and / or treating skin damage caused by ultraviolet light, the method comprising applying a composition as defined in any other embodiment to the skin. In an embodiment, the composition can be used to prevent skin damage caused by ultraviolet light or a skin condition caused by exposure to ultraviolet light. In such an embodiment, the composition can be applied or applied before exposure to ultraviolet light. It will be understood by those skilled in the art that before exposure to ultraviolet light, it means an amount of ultraviolet light sufficient to cause skin damage or a skin condition such as PLE.

[0040] In a further embodiment, the present invention provides a method of modulating cytokine expression in skin cells, the method comprising administering a composition as defined in any other embodiment.

[0041] The heterotrophic skin bacteria preparation according to the present invention can be formulated into a pharmaceutical composition for clinical use and can contain a pharmaceutically acceptable carrier, diluent or adjuvant. They can be formulated for topical administration.

[0042] Administration is preferably carried out in a prophylactically or therapeutically effective amount, which is an amount sufficient to show benefit to the individual. The actual amount applied, the rate of administration, and the time course of administration will depend on the nature and severity of the disease being treated. Treatment prescriptions (e.g., determination of dosage, etc.) are the responsibility of general practitioners and other physicians, and typically take into account the condition to be treated or prevented, the condition of the individual patient, the delivery site, the method of administration, and other factors known to the physician. Examples of the above techniques and protocols can be found in Remington's Pharmaceutical Sciences, 2000, 20th edition. th

[0014] (Vol. 18, No. 2, 1994, pp. 199-2006), Lippincott, Williams & Wilkins. It will be appreciated by those skilled in the art that the appropriate dosage of the active compound and the composition comprising the active compound may vary from patient to patient.

[0043] The compositions of the present invention can be formulated as medicaments, that is, formulated as pharmaceutical products or medical devices. Such medicaments may include other pharmaceutically acceptable ingredients well known to those skilled in the art, including but not limited to pharmaceutically acceptable carriers, adjuvants, excipients, diluents, fillers, buffers, preservatives, antioxidants, lubricants, stabilizers, solubilizers, surfactants (such as wetting agents), masking agents, colorants, flavoring agents, and sweetening agents. The formulations may also contain other active agents, such as other therapeutic or prophylactic agents.

[0044] The compositions of the present invention can be formulated as cosmetics, that is, formulated as cosmetic products. Cosmetic products may include other cosmetically acceptable ingredients well known to those skilled in the art, including but not limited to cosmetically acceptable carriers, excipients, diluents, fillers, buffers, preservatives, antioxidants, lubricants, stabilizers, solubilizers, surfactants (such as wetting agents), masking agents, colorants, and fragrances.

[0045] Aspects and embodiments of the present invention will now be described by way of example with reference to the accompanying drawings. Other aspects and embodiments will be apparent to those skilled in the art. All documents mentioned herein are incorporated herein by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Embodiments and experiments demonstrating the principles of the present invention will now be discussed with reference to the accompanying drawings, in which:

[0047] Figure 1 FIG. showing the cytotoxic effect of UVB is dose-dependent. NHEK (p3–5) cultured to 80% confluence were irradiated at 5 to 30 mJ / cm 2 Cell viability was assessed 24 h after irradiation by trypan blue (n = 3). Data are presented as mean ± SD. **P < 0.01, *P < 0.05, determined by one-way ANOVA and subsequent Tukey's test.

[0048] Figure 2 FIGS. showing that UVB has no significant effect on IL-10 / IL-12 secretion. Supernatants collected 24 h after irradiation (5 to 30 mJ / cm 2 , p3-5) were assayed for (A) IL-10 (n = 3) and (B) IL-12 (n = 2). Data are presented as mean ± SD. Significance was evaluated by one-way ANOVA and subsequent Dunnett's test.

[0049] Figure 3 FIG. is a collection of array blot images and graphs showing that UVB exerts a dose-dependent effect on inflammatory mediators. Figure 3 A is for 0 mJ / cm2 (i), 10 mJ / cm 2 (ii), 20 mJ / cm 2 (iii) and 30 mJ / cm 2 Array blot images after cytokine release assay in the pooled supernatants collected 24 h after irradiation (n = 3, p3 - 5) at (i), (ii), and (iii). Figure 3 B is a graph showing changes in protein secretion compared to the non - irradiated control. Figure 3 C is a graph showing the quantification of dose - dependent changes of the following by ImageJ: pro - inflammatory mediators (i, ii, and iii): IL - 6 (i), IL - 8 (ii), and MIP - 3α (iii); pro - angiogenic markers (iv and v): VEGF (iv), MMP - 9 (v); and acute - phase protein: PTX3 (vi).

[0050] Figure 4 is a set of graphs showing verification of cytokine array analysis by ELISA. Supernatants collected 24 h after irradiation (5 to 30 mJ / cm 2 , n = 3, p3 - 5) were assayed for (A) IL - 6, (B) IL - 8, (C) MIP - 3α (D), and PTX - 3 (E). Data are presented as mean ± SD. ****P < 0.0001, **P < 0.01, *P < 0.05, determined by one - way ANOVA and subsequent Dunnett's test.

[0051] Figure 5 is a graph showing that the protective effect of Micrococcus luteus is dose - dependent. NHEK (p3–5) were cultured to 80% confluence, treated with medium ± Micrococcus luteus 10 4 / 10 2 CFU / ml for 90 min, and irradiated at 5 to 30 mJ / cm 2 . Cell viability was assessed by trypan blue 24 h after irradiation. Data are presented as mean ± SD. ****P < 0.0001, *P < 0.05, determined by two - way ANOVA and subsequent Tukey's test.

[0052] Figure 6 are two graphs showing that UVB promotes the adhesion of Micrococcus luteus to keratinocytes. NHEK (p3–5) were cultured to 80% confluence, treated with medium ± Micrococcus luteus 10 4 / 10 2 CFU / ml for 90 min, and irradiated at 5 to 30 mJ / cm 2 . 1×10 4 CFU / ml (A, n = 3) and 1×10 2Adhesion of Micrococcus luteus at CFU / ml (n = 4, B). Data are presented as mean ± SD. *P < 0.05, determined by one-way ANOVA and subsequent Dunnett's test.

[0053] Figure 7 These are three graphs showing that Micrococcus luteus exacerbates the production of pro-inflammatory cytokines induced by UVB. Supernatants collected 24 h after irradiation ± application of Micrococcus luteus (5 to 30 mJ / cm 2 , n = 3, p3 - 5) were assayed for (A) IL-6, (B) IL-8, and (C) PTX-3. Data are presented as mean ± SD. ****P < 0.0001, ***P < 0.001, **P < 0.01, and *P < 0.05, determined by two-way ANOVA and subsequent Tukey's test.

[0054] Figure 8 These are two graphs showing that Micrococcus luteus modulates UVB-induced VEGF secretion. Supernatants collected 24 h after irradiation ± application of Micrococcus luteus (5 to 30 mJ / cm 2 , n = 3, p3 - 5) were assayed for (A) VEGF and (B) MIP-3α. Data are presented as mean ± SD. **P < 0.01, *P < 0.05, determined by two-way ANOVA and subsequent Tukey's test.

[0055] Figure 9 This is a graph showing that Micrococcus luteus exerts a protective effect on UVR-treated differentiated keratinocytes. Differentiated NHEK (p2–3) were treated with medium ± Micrococcus luteus 10 4 / 10 2 CFU / mL for 1 h and irradiated at 5 to 30 mJ / cm 2 . Cell viability was evaluated by trypan blue 24 h after irradiation. Data are presented as mean ± SD. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, determined by two-way ANOVA and subsequent Tukey's test.

[0056] Figure 10 These are two graphs showing the adhesion of Micrococcus luteus to keratinocytes in response to UVB exposure. NHEK (p2–3) were treated with medium ± Micrococcus luteus 10 4 / 10 2 CFU / mL for 1 h and irradiated at 5 to 30 mJ / cm 2 . 1×10 4 CFU / ml (A, n = 3) and 1×10 2Adhesion of Micrococcus luteus at CFU / ml (n = 3, B). Data are expressed as mean ± SD. Significance was evaluated by one-way ANOVA and subsequent Dunnett's test. Example

[0057] Example 1: Evaluation of the cytotoxic effect of UVB on epidermal keratinocytes

[0058] Materials and methods

[0059] Keratinocyte culture

[0060] The pooled normal human epidermal keratinocytes (NHEK, n = 3, juvenile foreskin, Promocell C-12005, passages 1-5) were routinely cultured in keratinocyte growth medium (KGM-2, Promocell). The cells were seeded into 12-well plates (2×10 4 cells / well to 5×10 4 cells / well) and incubated (37 °C, 5% CO2) until 80% confluence was reached. The cells were washed with phosphate-buffered saline (PBS, Gibco) and then resuspended in PBS for UV irradiation or seeded with bacteria as described below.

[0061] UV irradiation

[0062] A broadband UVB-emitting TL-12 lamp (Philips) with a maximum power output of 20 W and a wavelength of 270–400 nm (peak: 313 nm) was used. The irradiance output was evaluated for at least five minutes before irradiation using a UVX radiometer (UV Products, California) coupled to a UVX-31 detector until the level stabilized at 0.55 ± 0.01 mW / cm 2 . The irradiation time was then calculated as the radiation exposure (mJ / cm 2 ) / (irradiance × calibration factor), where the calibration factor was 0.63, and radiation exposure doses of 5 to 30 mJ / cm 2 were used for the experiment. The monolayer of cells resuspended in PBS was placed 16 cm from the light source, and half of the plate was covered with foil as an unirradiated control. The cells were exposed to the above doses, the medium was replaced with KGM-2, and the cells were incubated for 24 h (37 °C, 5% CO2).

[0063] Cell viability and bacterial adhesion

[0064] At 24 h post-irradiation, the cells were washed three times with PBS and trypsin (0.04%), and then ethylenediaminetetraacetic acid (EDTA, 0.03%, Promocell) was applied for 7 min (37 °C, 5% CO2). Then an equal volume of trypsin neutralization solution (TNS) was applied to the cells, and the samples were mixed with an equal volume of trypan blue (Gibco). Then cell viability was recorded using a hemocytometer (improved Neubauer). Alternatively, the cell suspension was diluted to 1 mL in PBS and serially diluted to 10 -5 , and then 20 μL aliquots were applied in triplicate to TSB agar to determine the adhesion of microorganisms to NHEK.

[0065] UV regulation of inflammatory cytokine concentration

[0066] Using a human XL cytokine array kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions, the relative expression of 105 inflammatory cytokines in pooled supernatant samples (n = 3, NHEK p3-5) treated with UVB at doses of 10 to 30 mJ / cm 2 was evaluated.

[0067] Human total interleukin-6 (IL-6), IL-8, IL-10, IL-12, pentraxin-3 (PTX-3), macrophage inflammatory protein-3α (MIP-3α), and vascular endothelial growth factor (VEGF) in the supernatant were measured by ELISA according to the manufacturer's protocol (Duoset, R and D systems, Biotechne).

[0068] Statistical analysis

[0069] Data were processed using Excel software (Microsoft) and analyzed using Prism (GraphPad software, CA, USA). Unless otherwise stated, all experiments were performed at least in triplicate, and data were analyzed using Student's t-test (when comparing two groups) or general linear model (GLM), followed by one-way or two-way ANOVA and Tukey / Dunnett tests (when comparing three or more groups).

[0070] Results

[0071] Previous studies have provided evidence that UVB has a dose-dependent cytotoxic effect on epidermal keratinocytes. Our results confirm these findings, showing that UVB doses >20 mJ / cm 2 were sufficient to induce a 34% decrease in cell viability relative to unirradiated controls, as Figure 1As shown. These cytotoxic effects are thought to be attributed to the imbalance between the immunosuppressive cytokine (IL-10) and the pro-inflammatory cytokine (IL-12).

[0072] However, Figure 2 it was shown that UVB had no significant effect on the secretion of IL-10 or IL-12 ( Figure 2 ). Therefore, cytokine arrays were used to characterize UVB-dependent cytokine secretion.

[0073] Example 2: Evaluation of dose-dependent changes in cytokine secretion

[0074] Materials and methods

[0075] The method was carried out according to Example 1 above.

[0076] Results

[0077] Cytokine arrays were used to examine the dose-dependent effects of UVB on the secretion of 107 cytokines. Evaluation of the relative pixel density changes revealed the basal expression of 45 inflammatory proteins. Figure 3 Panel B shows that irradiation at 10 mJ / cm 2 , 20 mJ / cm 2 or 30 mJ / cm 2 led to increased secretion of 15, 27, and 15 proteins, respectively, compared to unirradiated controls. Quantification of the relative pixel density of the blot shown in Figure 3 Panel A revealed dose-dependent changes in a range of inflammatory mediators, as Figure 3 shown in Panel C, and these changes were quantitatively verified by ELISA, as Figure 4 included. As Figure 4 shown in Panels A to Figure 4 C, a dose-dependent increase in pro-inflammatory mediators including IL-6, IL-8, and MIP-3α was observed. At the same time, a dose-dependent increase in the secretion of the innate immune sensor and regulator PTX-3 was also observed, as Figure 4 shown in Panel E. In contrast, Figure 3 the secretion of VEGF in Panel C showed a dose-dependent decrease, which was not verified by ELISA as shown in Figure 4 Panel D, as this trend was attributed to UVB-induced cytotoxicity. Then the mediation of the skin microbiome on UVB-dependent cytotoxicity and inflammation was characterized.

[0078] Example 3: Micrococcus luteus exerts a protective effect against cytotoxic doses of UVB

[0079] Materials and methods

[0080] Bacterial co-culture

[0081] Micrococcus luteus was routinely cultured in tryptic soy broth (TSB, Oxoid) at 37°C for 48 h. The cells were washed twice with PBS and resuspended in KGM to a volume of 1 × 10 4 to 1×10 6 CFU / mL. To confirm the number of viable cells, the inoculum was serially diluted and applied to TSB / WCB agar in triplicate at 20 μL. Medium ± M. luteus was then applied for 90 minutes (37°C, 5% CO2), and non-adherent cells were washed twice with PBS. The cell monolayer was then UV-irradiated as described below.

[0082] The remaining methods are all carried out according to the above embodiment 1.

[0083] result

[0084] Previous studies have documented that Micrococcus luteus can repair UV-induced DNA damage. However, whether it can provide this protection in the skin remains to be determined. The photoprotection induced by Micrococcus luteus is bacterial load-dependent. Figure 5 It shows that with 30mJ / cm 2 Compared with the control without bacterial treatment, 1×10 2 CFU / mL increased cell viability by 53%. 4 CFU / mL inoculation had no significant effect on cell viability. Interestingly, this photoprotective effect was also accompanied by an increase in microbial adhesion to NHEK. Figure 6 As shown, compared with the unirradiated control, at 30 mJ / cm 2 The lower pair was inoculated with 1×10 4 CFU / ml or 1×10 2 UVB application increased the adhesion of NHEKs to CFU / ml of Micrococcus luteus by 97% and 174%, respectively. Without being limited by theory, the inventors believe that this increased adhesion may modulate UVB-associated inflammation, thereby conferring photoprotection. Therefore, the effects of Micrococcus luteus on UVB-regulated inflammatory proteins were evaluated.

[0085] The inflammatory response to M. luteus is thought to be toll-like receptor 4-dependent, leading to increased production of cytokines including IL-8. The inventors observed that this response was M. luteus dose-dependent. Figure 7 B shows that high doses of Micrococcus luteus (1×10 4 CFU / mL) resulted in increased IL-8 secretion, while cytotoxic doses of UVB (30 mJ / cm 2 ) will aggravate this reaction. IL-6 and PTX-3 also reflected this trend, which was similar to that of 30mJ / cm 2±1×10 2 CFU / mL Micrococcus luteus - treated cultures, compared with cultures treated with 30 mJ / cm 2 UVB + 1×10 4 CFU / mL Micrococcus luteus - treated cultures showed a significant increase in cytokine secretion, such as Figure 7 shown. In contrast, inoculation at any microbial density resulted in a significant decrease in the secretion of VEGF (as 2 ) mediated by UVB (30 mJ / cm Figure 8 as shown in a) and MIP - 3α (as Figure 8 shown in b). This indicates that these factors may play an important role in Micrococcus luteus - related photoprotection.

[0086] Example 4: Micrococcus luteus has a photoprotective effect on differentiated keratinocytes

[0087] Materials and methods

[0088] The materials and methods were carried out according to Examples 1 and 3 above.

[0089] Results

[0090] The epidermis is formed by multiple layers of keratinocytes at different differentiation stages. Actively proliferating keratinocytes form the basal layer, while differentiated keratinocytes act on the skin surface and coordinate the initial response to UVR and pathogens. To ensure that the model used in this study is more physiologically relevant, the protective effect of Micrococcus luteus on UVR - treated differentiated keratinocytes was evaluated. Similar to undifferentiated keratinocytes, Figure 9 it was shown that UVR produced a dose - dependent decrease in cell viability, such that compared with the untreated control, it decreased by 52% after irradiation at 30 mJ / cm 2 . Micrococcus luteus effectively alleviated the cytotoxic effect of UVR (30 mJ / cm 2 ), such that compared with the control without bacterial stimulation at 30 mJ / cm 2 , after treatment with high - dose (1×10 4 CFU / mL) and low - dose (1×10 2 CFU / mL) Micrococcus luteus, the cell viability increased by 63% and 49% respectively. In addition, Figure 10 it was shown that this response was accompanied by a trend of UVR dose - dependent increase in the adhesion of Micrococcus luteus to keratinocytes.

[0091] General conclusion

[0092] Data on the immune system's response to UVR have been published previously. Most of these data were generated in humans, and the specific cell types that produce immune mediators were not characterized. This study specifically investigated the response of keratinocytes to UVR. Keratinocytes were selected because they are a major component of the innate immune system in the skin, and their response to UVR has been unclear to date. Second, keratinocytes are likely the major cell type that interacts with the microbiome (as the microbiome is thought to be predominantly present on the skin surface).

[0093] Currently, a wide variety of cytokines produced by keratinocytes have been confirmed. In addition, the data included in this article indicate that the cytokine response can be altered by at least one bacterium, Micrococcus luteus. This can be used to alter the balance between Th1 and Th2 cytokines, demonstrating the effectiveness of this therapy in conditions such as PLE (which currently affects approximately 18% of Europeans).

[0094] Surprisingly, it was found that heterotrophic skin bacteria are able to regulate the response of skin cells to UV exposure. The inventors found that inoculating skin cells with Micrococcus luteus increased the survival rate of the skin cells after exposure to ultraviolet light. Surprisingly, this was due to the ability of Micrococcus luteus to regulate the immune response of skin cells by reducing the expression of certain cytokines and increasing the expression of different sets of cytokines.

[0095] The foregoing embodiments are not intended to limit the scope of protection conferred by the claims, but rather are examples of how the invention may be practiced.

[0096]

Claims

1. A composition for regulating cytokine expression in skin cells, comprising a heterotrophic skin bacterium and / or an extract of a heterotrophic skin bacterium.

2. The composition according to claim 1, wherein, The extract of the heterotrophic skin bacterium is in the form of a lysate.

3. The composition according to any one of the preceding claims, wherein, The heterotrophic skin bacterium is derived from a Micrococcus bacterium.

4. The composition according to claim 3, wherein, The Micrococcus bacterium is Micrococcus luteus.

5. The composition according to any one of the preceding claims, wherein, The composition is formulated for topical application.

6. The composition according to any one of the preceding claims, wherein, Regulation of cytokine expression causes regulation of the secretion of one or more of the following cytokines: IL-6, and / or IL-8, PTX-3, VEGF, and / or MIP-3α in skin cells.

7. The composition according to any one of the preceding claims, wherein, Regulation of cytokine expression causes an increase in the secretion of one or more of the following cytokines: IL-6, and / or IL-8, and / or PTX-3 in skin cells.

8. The composition according to any one of the preceding claims, wherein, Regulation of cytokine expression causes a decrease in the secretion of one or more of the following cytokines: VEGF and / or MIP-3α in skin cells.

9. The composition according to any one of the preceding claims, wherein, The composition further comprises one or more pharmaceutically acceptable ingredients or excipients.

10. The composition according to claim 9, wherein, The composition is formulated in the form of a cream, gel, spray, ointment or oil.

11. The composition according to any one of claims 1 to 10 for preventing and / or treating skin damage caused by ultraviolet light.

12. The composition according to any one of claims 1 to 10 for use in a cosmetic preparation for application to skin damaged by ultraviolet light.

13. The composition according to any one of claims 1 to 10 for preventing, controlling or treating a skin condition caused by exposure to ultraviolet light.

14. The composition according to claim 13, wherein, The skin condition is PLE.

15. The composition according to any one of claims 13 or 14, wherein, The composition is formulated for topical application to the skin.

16. The composition according to any one of claims 11 to 15, wherein, The composition is administered before exposure to ultraviolet light.

17. The composition according to any one of claims 13 to 15, wherein, The skin condition is a VEGF- and / or MIP-3α-mediated skin condition.

18. The composition according to any one of claims 13 to 15, wherein, The skin condition is an IL-6-, and / or IL-8-, and / or PTX-3-mediated skin condition.

19. The composition according to any one of the preceding claims, wherein, The heterotrophic skin bacterium and / or the extract of the heterotrophic skin bacterium comprises Micrococcus luteus (22110401) or is derived from Micrococcus luteus (22110401).

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