CN116801861A - Extracts of Sphingomonas bacteria - Google Patents
Extracts of Sphingomonas bacteria Download PDFInfo
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- CN116801861A CN116801861A CN202180085393.9A CN202180085393A CN116801861A CN 116801861 A CN116801861 A CN 116801861A CN 202180085393 A CN202180085393 A CN 202180085393A CN 116801861 A CN116801861 A CN 116801861A
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Abstract
The present invention relates to an extract of a bacterium of the genus Sphingomonas, and a composition for preventing and/or treating skin reactions caused by allergic reactions and for modulating immune responses and optionally for enhancing the barrier function of the skin, comprising an extract of a bacterium of the genus Sphingomonas or at least one bacterium of the genus Sphingomonas.
Description
Technical Field
The present invention relates to an extract of a bacterium of the genus Sphingomonas and 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 and for regulating immune responses and optionally for enhancing the barrier function of the skin.
Background
Allergy is a complex phenomenon in which an excessive inflammatory reaction occurs after an organism is exposed to an allergen. Specifically, allergy depends on two titers: the porosity of the barrier between the organism and the external medium and the modulation of the immune system.
Thus, allergy depends on the permeability of the skin, which is a barrier against external, in particular particulate, chemical or chemical attack, but also to limit leakage of water and other interstitial compounds. This property of the skin is called barrier function. In particular, the barrier function is ensured by tight junctions and related proteins. Activation of Toll 2 (TLR 2) type receptors allows to enhance the barrier function by stimulating the expression of these proteins and the formation of tight junctions.
Allergy is also dependent on modulation of the immune system, in particular by antibodies of the E-type (IgE), prostaglandin E2 (PGE 2 ) And various Interleukins (IL).
IgE is involved in immediate allergic reactions that can cause urticaria or angioedema.
PGE 2 And IL-6/IL-8 also play a role in neutrophil attraction. They have complementary modes of action, on the one hand, by PGE 2 On the other hand, by attracting a gradient of neutrophils by chemokines due to IL-6 or IL-8. Thus, the two different and combined modes of action improve the immune response.
Disclosure of Invention
The inventors have found that extracts of Sphingomonas allow activation of hTLR2 receptors in vitro and promote expression of genes associated with enhancing the barrier function of the skin. They also demonstrated that this extract allows the inhibition of the pro-inflammatory mediator PGE 2 Promote the expression of interleukin IL-10 (anti-inflammatory cytokine) without significantly affecting other pro-inflammatory interleukins such as IL-12, IL-6 and IL-8. Finally, the inventors have demonstrated that this extract allows to limit IgE production by human B lymphocytes.
Thus, sphingomonas bacteria and extracts thereof have various anti-inflammatory activities in combination with enhancing the barrier function of skin, thereby enabling prevention and/or treatment of skin reactions caused by allergic reactions (cutaneous reaction).
The present invention therefore 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.
The invention also relates to a composition for preventing and/or treating skin reactions caused by allergic reactions, comprising an extract of Sphingomonas bacteria or at least one Sphingomonas bacteria.
The invention also relates to an extract of a bacterium of the genus Sphingomonas for modulating the immune response and optionally for enhancing the barrier function of the skin.
Furthermore, the present invention relates to a composition for modulating an immune response and optionally for enhancing the barrier function of skin, the composition comprising an extract of a Sphingomonas bacterium or at least one Sphingomonas bacterium.
General definition
The term "bacteria" refers to a prokaryotic microorganism, preferably of the order Proteus (Proteus), in viable, semi-active, inactivated or dead form.
The bacteria used according to the invention or the bacteria of the extracts used according to the invention belong to the genus Sphingomonas. In one embodiment, the bacterium is a species selected from the group consisting of Sphingomonas abaci, sphingomonas adhaesiva, sphingomonas aerolata, sphingomonas aquatilis, sphingomonas asaccharolytica, sphingomonas aurantiaca, sphingomonas azotifigens, sphingomonas dokdonensis, sphingomonas echinoides, sphingomonas faeni, sphingomonas fennica, sphingomonas haloaromaticamans, sphingomonas jaspsi, sphingomonas koreensis, sphingomonas mali, sphingomonas melonis, sphingomonas natatoria, sphingomonas oligophenolica, sphingomonas panni, sphingomonas parapaucimobilis, sphingomonas paucimobilis, sphingomonas phyllosphaerae, sphingomonas pituitosa, sphingomonas pruni, sphingomonas roseiflava, sphingomonas sanguinis, sphingomonas soli, sphingomonas sp (Sphingomonas sp), sphingomonas suberifaciens, sphingomonas trueperi, sphingomonas wittichii, sphingomonas xenophaga, sphingomonas yabuuchiae Sphingomonas yunnanensis, and Sphingomonas yanoikuyae. In one embodiment of the invention, the bacteria is Sphingomonas xenophaga. In a preferred embodiment, the bacterium is Sphingomonas xenophaga submitted by L 'Oreal, 101Avenue Gustave Eiffel,37390Notre Dame d'O e under the national collection of microorganisms ((CNCM), paris, france) under CNCM I-5455 according to Budapest strip at about 11.21.2019.
As used herein, the term "prevent" or "prevention" refers to any effect intended to prevent the occurrence of or one or more symptoms associated with a disorder. Thus, this term also relates to alleviation, cessation, or limitation of symptoms.
As used herein, the term "treatment" or "treatment" refers to any action intended to inhibit, reduce or prevent the progression of a disorder (one or more symptoms associated with the disorder). Thus, this term covers alleviation, alleviation or inhibition of symptoms.
By "preventing and/or treating a skin response caused by an allergic reaction" is understood a slowing, stopping, limiting, alleviating or inhibiting of the skin symptoms of a subject caused by an allergic reaction. Generally, these symptoms include urticaria, rash, and/or redness.
"subject" is to be understood here as a person, preferably a person suffering from allergy. In an embodiment of the invention, the subject has already an allergic reaction which has caused a skin reaction.
"skin" should be understood at the level of skin, and more specifically at the level of areas of skin that are or are likely to be exposed to allergens. According to embodiments of the invention, skin reactions caused by allergic reactions include urticaria, rash, and/or redness.
In a preferred embodiment, the extracts and compositions used in the context of the present invention are used locally. In a preferred embodiment, the extracts and compositions used in the context of the present invention are intended for application to the skin.
In the context of the present invention, the term "skin" refers to any skin surface of the body, preferably the skin of the face, the skin of the neck, the skin of the milk folds, more particularly the skin of the face.
"allergic reaction" is understood to mean the meaning generally used by those skilled in the art, i.e. an excessive inflammatory reaction which occurs after exposure of an organism to an allergen. This reaction is an indication that the organism is allergic to substances (allergens) that are normally harmless and present in the environment. These substances may be present in air, cosmetics, etc.
The so-called immediate allergic reaction is characterized by symptoms that most often occur a few minutes (less than two hours) after exposure to the allergen. In this case, this consists of urticaria which can spread and involve IgE. The so-called delayed allergic reaction is characterized by symptoms that usually occur 48 hours after exposure to the allergen. In this case, it consists of local eczema of the contact site involving chemical molecules.
In a preferred embodiment, the extracts and compositions applied in the context of the present invention are used for the prevention and/or treatment of skin reactions caused by immediate allergic reactions.
By "modulating an immune response" is herein understood slowing, stopping or alleviating the immune response. In particular, modulating an immune response according to the invention is reducing the activity of a pro-inflammatory cytokine and/or increasing the activity of an anti-inflammatory cytokine.
In a preferred embodiment of the invention, modulation of the immune response is achieved by increasing the activity of IL-10 without significantly affecting the pro-inflammatory interleukins IL-12, IL-6 and/or IL-8.
By "enhancing the barrier function of the skin" is herein understood improving the barrier function of the skin, in particular at the skin level by increasing the number of tight junctions and/or by increasing related proteins such as Claudin-1, keratin and/or zonules 1 obliquus 1.
Extracts of Sphingomonas bacteria
The present invention relates to an extract of Sphingomonas bacteria for preventing and/or treating skin reactions caused by allergic reactions.
In the context of the present invention, the term "bacterial (extracts)" refers to: a series of compounds produced and/or secreted by bacteria and thus present in the medium of the bacterial culture (also called extracellular medium); and/or a series of compounds, such as intracellular matrix and/or cell wall and/or membrane components, contained in the bacteria and thus present in the bacterial sediment of the bacterial culture after centrifugation.
The extracts of the present invention may be prepared by any conventional method known to those skilled in the art. Typically, these methods include fermentation, isolation, and purification steps.
Typically, bacteria of the genus Sphingomonas are cultured in a suitable medium and at a desired density. Preferably, the bacteria themselves are concentrated by any known method, such as centrifugation. The concentrated bacteria may then be used directly as the original formulation, or may undergo other processing steps known to those skilled in the art such as lyophilization, dehydration, filtration, purification, freezing (possibly followed by thawing), sterilization, column chromatography, crushing, lysis, etc.
Preferably, the bacterial extract is a bacterial lysate. Such lysates include in particular compounds which are contained in bacteria and which are therefore present in bacterial precipitates of the centrifuged bacterial culture. Preferably, the bacterial extract is a bacterial lysate comprising components of the intracellular matrix and/or cell wall and/or membrane.
Lysates generally represent substances obtained after the destruction or disintegration of biological cells by a phenomenon known as cell lysis, resulting in the release of intracellular biological components naturally contained in the cells of the microorganism under consideration.
In the context of the present invention, the term "lysate" is used indiscriminately to refer to the whole lysate or only a portion thereof obtained by lysing the microorganism in question.
Thus, the lysate implemented is formed in whole or in part by intracellular biological components as well as by components of the cell wall and membrane.
Thus, preferably, the lysate implemented is formed substantially entirely or partly by intracellular biological components and by components of the cell wall and membrane.
In a preferred embodiment, the lysate comprises less than 1% by dry weight of extracellular medium, more preferably the lysate comprises from 0.5% to 1% by dry weight of extracellular medium, even more preferably the lysate comprises from 0.7% to 0.8% by dry weight of extracellular medium.
This cell lysis can be accomplished by the following different techniques: such as osmotic shock, thermal shock such as thawing after freezing, sonication, high pressure treatment or under centrifugal mechanical stress.
Preferably, the lysate implemented in the context of the present invention is obtained by a method comprising the following steps:
i) A centrifugation step;
ii) a step of recovering the precipitate;
iii) Freezing the precipitate;
iv) a step of thawing the precipitate;
v) optionally by autoclaving, in particular a sterilization step at 121 ℃.
In a specific embodiment of the invention, the bacterial extract comprises inactivated bacteria.
"inactivated" is understood to mean that the person skilled in the art generally uses, i.e. inhibits the activity of bacteria under the influence of various factors (thermal, chemical, mechanical, enzymatic). In particular, the bacteria are inactivated by heating, more particularly by autoclaving.
Preferably, the extract according to the invention has the following activity: activating hTLR2 receptors and/or promoting expression of genes associated with enhancing skin barrier function.
Preferably, the extract has the effect of inhibiting the pro-inflammatory mediator PGE 2 Secreted activity.
Preferably, the extract has activity to stimulate IL-10 expression without significantly affecting the expression of IL-12, IL-6 and IL-8.
Preferably, the extract has an activity of inhibiting the production of IgE by B-lymphocytes.
All of these activities can be detected and/or analyzed by techniques known to those skilled in the art, including the specific examples described in the examples below.
Thus, sphingomonas bacteria and extracts thereof have various anti-inflammatory activities in combination with enhancing the barrier function of skin, thereby enabling prevention and/or treatment of skin reactions caused by allergic reactions.
The invention also relates to a bacterial extract according to the invention for modulating an immune response and optionally for enhancing the barrier function of the skin.
Composition and method for producing the same
The invention also relates to a composition for preventing and/or treating skin reactions caused by allergic reactions, which is intended in particular for topical application, comprising an extract of Sphingomonas bacteria or at least one Sphingomonas bacteria.
The invention also relates to a composition for modulating an immune response and optionally for enhancing the barrier function of skin, the composition comprising an extract of a Sphingomonas bacterium or at least one Sphingomonas bacterium.
In one embodiment, the composition comprises an extract as described above.
In one embodiment, the composition comprises at least one Sphingomonas bacterium as described in the general definition section.
The amount of bacteria or bacterial extracts in the composition according to the invention varies according to the type of composition considered. Preferably, the amount of microorganisms or extracts is between 0.0001 and 30% by dry weight relative to the total weight of the composition, between 0.001 and 15% by dry weight relative to the total weight of the composition, between 0.01 and 10% by dry weight relative to the total weight of the composition, preferably between 0.01 and 5% by dry weight relative to the total weight of the composition, or between Ji Jie.01 and 3% by dry weight relative to the total weight of the composition.
In the case where the bacteria are formulated in the composition in a viable form, the amount of viable bacteria may be from 10 per gram of composition 3 To 10 15 Ufc/g, especially from 10 5 To 10 15 ufc/g, more particularly from 10 7 To 10 12 Ufc/g changes.
Preferably, the composition according to the invention comprises an aqueous phase.
Preferably, the composition according to the invention has a water content ranging from 20% to 95% by weight, preferably from 30% to 70% by weight, relative to the total weight of the composition.
The composition according to the invention may further comprise one or several water-miscible organic solvents in the following concentrations: a concentration in the range of 0.5 to 25% by weight, preferably 5 to 20% by weight, more preferably 10 to 15% by weight, relative to the total weight of the composition.
As previously mentioned, the compositions used in the context of the present invention are preferably applied topically.
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 other than the extracts according to the invention, preservatives, antioxidants, water-immiscible solvents, fragrances, odor absorbers, colorants. The amounts of these various additives are those generally used in the field under consideration, for example from 0.01 to 20% by weight relative to the total weight of the composition.
Of course, the person skilled in the art will choose these optional additional ingredients and/or active substances, and/or their amounts, e.g. the advantageous properties of the extract according to the invention are not or substantially not altered by the additives considered.
In one embodiment, the extract of Sphingomonas bacteria or Sphingomonas bacteria is the only active material of the composition.
In another embodiment, the composition of the present invention may further contain other active substances than the bacteria or extract of the present invention. These actives may have an effect on the skin, in particular a soothing effect.
In a specific embodiment of the invention, the composition according to the invention further comprises at least one active substance selected from the group consisting of: acetyl dipeptide-1 cetyl ester; extract of root of red-rooted salvia; spring water (thermal water), such as skin water (La Roche Posay water); and mixtures thereof.
The composition according to the invention may be in the form of any galenic formulation generally used for topical application, in particular in the form of a water, a water-alcohol solution, an oil-in-water (O/W) or water-in-oil (W/O) emulsion or multiple (triple: W/O/W or O/W/O) emulsions, hydrogels or dispersions of fatty phases in aqueous phase using globules, which may consist of ionic and/or non-ionic lipid vesicles (liposomes, vesicles, oleosomes). These compositions are prepared by conventional methods.
Advantageously, the composition according to the invention is in the form of a gel, emulsion (emulgation) or paste. Furthermore, the composition according to the invention may be more or less fluid and have the appearance of a white or coloured cream, ointment, emulsion (mill), lotion, serum, paste, foaming or non-foaming gel, exfoliating agent, mask, care agent, toner (tonic) or foam. It may be applied to the skin in the form of a spray. It may also be in solid form, for example in the form of a bar or soap.
The composition according to the invention may comprise a fatty phase, in particular an oil phase.
As oils suitable for the composition according to the invention, mention may be made of, for example:
-a hydrocarbon oil which is present in the mixture,
-synthetic esters and ethers, in particular esters and ethers of fatty acids, such as oils of the formula R ' COOR2 and R ' COR2, wherein R ' represents a fatty acid residue comprising from 8 to 29 carbon atoms, R2 represents a branched or unbranched hydrocarbon chain comprising from 3 to 30 carbon atoms;
-linear or branched hydrocarbons of mineral or synthetic origin;
-fatty alcohols having 8 to 26 carbon atoms;
-partially hydrocarbonated and/or siliconized fluorinated oil;
-silicone oil;
-mixtures thereof.
The hydrocarbon oils in the list of oils cited above should be understood to be any oil comprising mainly carbon and hydrogen atoms.
The oil phase may include: other fatty bodies that may be present in the oil phase, for example fatty acids comprising 8 to 30 carbon atoms; wax, silicone; and a silicon elastomer. These fats can be selected by one of skill in the art in a variety of ways to prepare compositions having the properties sought (e.g., the viscosity properties or texture properties).
According to a specific embodiment of the present invention, the composition according to the present invention is a water-in-oil (W/O) or an oil-in-water (O/W) emulsion. The proportion of the oil phase in the emulsion may be in the range 5 to 80% by weight, preferably 30 to 70% by weight, relative to the total weight of the composition. Emulsions generally contain: at least one emulsifier selected from amphoteric, anionic, cationic or nonionic emulsifiers used alone or in combination; and optionally a co-emulsifier. The emulsifier is appropriately selected according to the emulsion (W/O or O/W) to be obtained. In general, the emulsifier and co-emulsifier are present in the composition in a proportion ranging from 0.3% to 30% by weight, preferably from 0.5% to 20% by weight, relative to the total weight of the composition.
As emulsifiers, mention may be made, for example, of disilane copolyols (dimethicone copolyols) and alkyldisilane copolyols (alkyl-dimethicone copolyols) for W/O emulsions. As surfactants for the W/O emulsion, crosslinked elastomeric solid organopolysiloxanes comprising at least one alkylene oxide group may also be used.
As emulsifiers, mention may be made, for example, of nonionic emulsifiers for O/W emulsions.
The composition may or may not be rinsed after it has been applied to the skin.
Method
The invention also relates to a method for preventing or treating skin reactions caused by allergic reactions, comprising the application, preferably topically on the surface of the skin: an extract of a bacterium of the genus Sphingomonas; or a composition comprising an extract of a Sphingomonas bacterium or at least one Sphingomonas bacterium.
The use of the following extracts or compositions in the manufacture of a medicament for the prevention or treatment of skin reactions caused by allergic reactions is also proposed: an extract of a Sphingomonas bacterium, the composition comprising an extract of a Sphingomonas bacterium or at least one Sphingomonas bacterium.
The invention also relates to a method of modulating an immune response and optionally enhancing skin barrier function, the method comprising the administration, preferably topically on the surface of the skin: an extract of a bacterium of the genus Sphingomonas; or a composition comprising an extract of a Sphingomonas bacterium or at least one Sphingomonas bacterium.
The use of the following extracts or compositions in the manufacture of a medicament for modulating an immune response and optionally enhancing the skin barrier function is also proposed: an extract of a Sphingomonas bacterium, the composition comprising an extract of a Sphingomonas bacterium or at least one Sphingomonas bacterium.
In specific embodiments, a composition comprising an extract of a sphingomonas bacterium or at least one sphingomonas bacterium is described in the composition section above.
In specific embodiments, the extract of Sphingomonas bacteria is described in the following Wen Qiao extract section of Sphingomonas bacteria.
Preferably, the extract of at least one sphingomonas bacterium or the at least one composition comprising the extract of a sphingomonas bacterium or the at least one sphingomonas bacterium is applied in an effective amount on a subject in need thereof.
As used herein, the term "effective amount" refers to the amount of an extract of a bacterium of the genus sphingomonas: which as a whole allows preventing and/or reducing skin reactions caused by allergic reactions; or the amount of Sphingomonas bacteria, which as a whole allows the prevention and/or reduction of skin reactions caused by allergic reactions.
The effective amount naturally depends on the active substance under consideration, the mode of administration, the therapeutic indication, the age of the patient and his/her condition.
Advantageously, the composition or the extract is used in particular in the form of a dermatological composition, possibly further comprising physiologically acceptable excipients.
By "physiologically acceptable" is herein understood compositions and molecular entities that do not produce an undesired secondary, allergic or other reaction when administered to a subject. Thus, a physiologically acceptable excipient or carrier is an encapsulating material, diluent, support, or any other liquid, semi-solid, or solid non-toxic formulation aid.
Dermatological compositions for use in the context of the present invention are generally prepared to suit the mode of administration. Physiologically acceptable excipients are generally determined in part by the composition being administered and the particular technique used to administer the composition.
The dose of the compound to be administered (dosage) depends on the individual situation and should be adapted to the individual situation in order to obtain an effective amount and optimal effect, as is well known to the person skilled in the art. The effective dosage level is specific to each subject and depends on a variety of factors including the disease being treated and the severity of the disease, the activity of the particular compound being used; the specific combination used. For example, it is well known to those skilled in the art to start with a dose (dose) of the compound below the level required to achieve the desired effect and gradually increase the dose until the desired effect is achieved.
Preferably, the method according to the invention comprises topical application of at least one Sphingomonas bacterium or at least one Sphingomonas extract, or a composition comprising the same.
In a preferred embodiment, a topical application in the context of the present invention is an application on the skin.
The methods of the invention may comprise a single administration. In another embodiment, the administration is repeated, for example, 2 to 3 times per day for one day or more, or daily application for several consecutive days or for several discontinuous days, and typically for an extended duration of at least 4 weeks or 4 to 15 weeks, or as long as desired, with possible interruption if desired.
In another embodiment, the methods of the invention may comprise applying to an area of skin before and/or after exposure to an allergen.
In the description and in the examples below, percentages are by weight unless otherwise indicated, and ranges of values are expressed as "between … … and … …" including the specified upper and lower limits. The ingredients are mixed in sequence prior to packaging under conditions readily ascertainable by those skilled in the art.
The invention will be further illustrated by the following examples.
Drawings
FIG. 1 shows the effect of Sphingomonas extract on monocyte secretion of cytokines.
Detailed Description
Examples:
example 1: preparation of the extract according to the invention
Cultures of strain Sphingomonas xenophaga CNCM-I5455 were prepared in batch mode in complete medium in a 3000-liter bioreactor. In this step, the pH was not adjusted, the temperature was kept at 26℃and the oxygen solubility was 30%.
The composition of the initial medium is described in table 1 below.
TABLE 1
Once the plateau level is reached, the extraction and isolation of the cells is carried out by centrifugation (10000 g, continuously). The cell-containing pellet (also known as biomass) is then recovered for subsequent freezing at-20 ℃ and then thawed, enabling the cells to burst, thus obtaining a lysate. The lysate is then packaged in a pouch and finally stabilized by sterilization at 121 ℃ for 30 minutes.
The lysate obtained after completion of the process according to example 1 contains 4.5% by weight of dry matter with respect to the total weight of the lysate.
Example 2: in vitro test-dose response analysis of TLR (invitrogen).
HEK293-TLR cell line:
extracts and controls were tested in duplicate against recombinant HEK-293 cell lines. These cell lines functionally overexpress human TLR proteins and are reporter genes for secreted alkaline phosphatase (SEAP). The production of this reporter gene is controlled by the NFkB inducible promoter. The activation results of TLR reporters are given in the form of Optical Density (OD) values.
Positive control:
activities of TLR1/TLR2 and TLR2/TLR6 were assessed on hTLR2 expressing cells using CU-T12-9 and FSL-1, respectively.
FSL-1 was tested at 10pg/ml and 30 pg/ml. CU-T12-9 was tested at 200nM and 300 nM.
Negative control:
HEK-293 cell line recombinants directed against only this reporter gene served as negative controls for cell lines expressing hTLR. To confirm the effectiveness of the inducible reporter gene NFkB (SEAP), this HEK-293TLR cell line was stimulated with tnfα at the following concentrations: 100ng/ml, 30ng/ml, 10ng/ml, 3ng/ml, 1ng/ml, 0.3ng/ml, 0.1ng/ml, etc.
The negative control cell line was also stimulated with varying concentrations of the test extract.
Testing of the extracts:
the reporter hTLR cell line was stimulated with 20 μl of extract at a reaction volume of 200 μl. For dose response of HEK-Blue-hTLR5 cell line, the liquid extracts obtained according to example 1 were tested in duplicate at the following concentrations: 1000. Mu.g/ml, 500. Mu.g/ml, 250. Mu.g/ml, 125. Mu.g/ml, 62.5. Mu.g/ml, 31.5. Mu.g/ml.
For TLR1/2 and/or TLR2/6 specificity, the liquid extracts obtained according to example 1 were tested in duplicate at the following concentrations: 100. Mu.g/ml, 30. Mu.g/ml and 10. Mu.g/ml.
Results:
TABLE 2 results for cell lines with hTLR2
TABLE 3 results for cell lines with hTLR2-1
TABLE 4 results for cell lines with hTLR2-6
Conclusion:
the extract did not activate the negative control cell line, but strongly activated the reporter cell line hTLR2 (1/6), even at 10. Mu.g/ml. It activates hTLR1/2 and hTLR2/6 in a non-specific manner.
Example 3: in vitro test-modulation of Gene expression in Normal human keratinocytes
Materials and methods
Cytotoxicity:
normal human epidermal keratinocytes were inoculated in 96-well plates and then incubated in medium at 37℃and 5% CO 2 Incubate for 24 hours. Thereafter, the medium was replaced with medium with or without (control) the compound to be tested (8 test concentrations), after which the cells were incubated for 24 hours. All conditions were performed in duplicate. At the end of incubation, cell viability was measured by standard tests for mitochondrial activity measurements.
Expression of genes related to barrier function/hydration was analyzed by RT-qPCT:
normal Human Epidermal Keratinocytes (NHEK) were seeded in 48-well plates and then incubated at 37 ℃ and 5% co in medium 2 Incubate for 3 days, wherein the medium is refreshed after the first 24 hours of incubation. At the end of incubation, the incubation was performed with test medium (supplemented with 1.5mM CaCl 2 ) The medium was replaced and the cells were then incubated for 24 hours. All conditions were performed in duplicate.
At the end of the treatment, the medium was removed and used with PBS (CaCl-free) 2 Does not contain MgCl 2 ) The cells were washed twice. The total RNA was then isolated using an extraction kit. Quantification of RNA and quality control thereof were analyzed by means of Labchip GX (Perkin Elmer).
The expression of the selected transcripts was analyzed in two steps by quantitative PCR. First, cDNA is transcribed in the opposite direction to RNA. Then, a quantitative PCR experiment was performed using a real-time PCR system.
Experimental protocol
Normal human epidermal keratinocytes are cultured and expanded to produce enough cells to evaluate up to 72 samples per activity. Inoculating cells (50000 cells per well) in a 48-well microplate and incubating under controlled temperature, humidity and CO 2 Is incubated for 48 hours in the environment. The sample to be tested is added to the cells while the medium is being refreshed, and the cells are then incubated for 24 hours.
The cells were washed and frozen dry at-80 ℃ to preserve RNA. RNA was extracted, quantified and checked for quality prior to reverse transcription of RNA into cDNA. Finally, RT-qPCR was performed for each experimental condition to quantify the expression of the selected series of genes.
TABLE 5 results
In this example, the Sphingomonas batch tested corresponds to a lyophilisate (100% MS) of the liquid extract (4.5% MS) obtained according to example 1.
In summary, the liquid extract obtained according to example 1 allows significant regulation of the expression of the genes of claudin-1, keratins and zonula occludens 1 in NHEK at a concentration of 0.0009 (calculated: 0.2x0.1x0.045) and at a concentration of 0.0045% (calculated: 1 x 0.1x0.045), taking into account the fact that 1g/L corresponds to a concentration of 0.1%.
Example 4: evaluation of in vitro test for modulation of PGE2 released by human bone marrow cells "
Anti-inflammatory efficacy test of U937 human bone marrow cell lines (cytopathology professor ken nisin nalson doctor, sweden, uppsala 75653, lappa Feng Siwei, 11, from the university of uppsala, sweden).
Principle of testing
Cell lines were used in this protocol to mimic the immune response of monocytes/macrophages in vitro. Treatment with PMA and LPS induced secretion of pro-inflammatory mediators and cytokines. The combination treatment with anti-inflammatory agents enables the identification of intrinsic regulatory activity of the pro-inflammatory response in vitro.
Cell treatment
Human bone marrow cell lines were treated with Sphingomonas xenophaga extract in RPMI 1640 medium supplemented with L-glutamine, penicillin, streptomycin, 10% heat treated fetal bovine serum, 10 μg/mL phorbol 12-tetradecanoate 13-acetate (PMA) and 60 μg/mL E.coli Lipopolysaccharide (LPS). Seven concentrations of Sphingomonas xenophaga extracts were tested: 1. 3, 10, 30, 100, 300 and 1000. Mu.g/mL.
At 37℃and 5% CO 2 After 24 hours of treatment in a humid atmosphere, the cell viability was measured under each condition.
Thereafter, the supernatant was collected for PGE 2 And (5) analyzing.
Cytokine assay
PGE2 levels were determined by ELISA test.
Vitality calculation-CV 75
CV75＝C1+((V1-75)/(V1-V2))×(C2-C1)
Wherein the method comprises the steps of
C1 Minimum concentration of =vitality >75%
C2 Highest concentration of =activity <75%
V1=minimum percent viability >75%
V2=highest percent viability <75%
Calculation of reactivity index-RI
RI = amount of label of treated supernatant (pg/ml) ×100/amount of analyte in untreated supernatant (pg/ml).
Calculation of 50% inhibition concentration-IC 50
IC 50 ＝C1+((50-RI1)/(RI2-RI1))×(C2-C1)
Wherein the method comprises the steps of
C1 Highest concentration of =ri <50
C2 Minimum concentration at =ri >50
rI1=highest RI <50
rI2=lowest RI >50
TABLE 6 results
Sphingomonas extract inhibits the pro-inflammatory mediator portion, wherein the IC50 is equal to 199 μg/ml.
Example 5: in vitro testing of the effect of extracts on monocyte secretion cytokines (IL-6, IL-8, IL-10 and IL-12p 40).
The purpose of this experiment was to investigate the effect of the extract on monocyte secretion of cytokines after 36 h.
Materials and methods:
cell isolation and culture:
monocytes were obtained from the leukocyte layer of healthy donors by standard Ficoll-Hypaque gradient method. At 37℃with 5% CO 2 Monocytes were isolated from monocytes by adhesion to plastic in serum-free medium (M-SFM) optimized for macrophage culture for 2 hours. Adherent cells highly enriched for monocytes were incubated with the tested compounds for 36 hours.
Cytotoxicity test:
within the last 6 hours, alamar Blue (Alamar Blue) was introduced into the cell culture. The resorufin, a fluorescent molecule obtained by converting a component of alamar blue, was measured as an indicator of cell death. Measurements were made with a Tecan Infinite F500 fluorescence spectrophotometer.
Analysis of cytokines:
after 36 hours, the supernatant was collected and frozen at-80 ℃ until testing. Measurements were performed on a Luminex LX100 instrument with the aid of a multifactorial (IL-6; IL-8; IL-10 and IL-12p 40) detection kit.
The calculation method for the evaluation result comprises the following steps:
since the range of values for different cytokines is different, and in order to obtain values that are easy to compare between compounds, the multiplication factor varies from cytokine to cytokine compared to IL-10.
Comparison of IL-10 with IL-6: the ratio is IL-10X 100/IL-6
Comparison of IL-10 with IL-12p 40: the ratio is IL-10/IL-12p40
Comparison of IL-10 with IL-8: the ratio is IL-10X 100 000/IL-8
The results are shown in FIG. 1. Product 2 corresponds to the liquid extract obtained according to example 1.
The extract has immunoregulatory effect on monocytes: IL10> IL-6/IL-8/IL-12 (p 40).
The extract induces high yields of IL-10 (anti-inflammatory cytokine) in terms of dose response compared to IL-6 and IL-8 (pro-inflammatory cytokine).
Example 6: in vitro test of the effect of extracts on IgE production by human B lymphocytes stimulated by a combination of anti-CD 40 and IL-4
In this study, the extract was evaluated for purified human B lymphocytes (CD 19 isolated from peripheral blood mononuclear cells + Lymphocytes) IgE production. More specifically, igE production is induced by stimulating B lymphocytes with a combination of anti-CD 40 antibodies and IL-4. After 14 days of incubation, the amount of IgE released in the culture supernatant was quantified using a specific ELISA kit. At the same time as the extract was tested, 3 other molecules were also evaluated in this test. The reference standard CpG-ODN2006 (a TLR9 ligand) was tested as a reference compound for IFN-gamma and PGE 2.
TABLE 7 Compounds investigated
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Cultivation and treatment
Isolation of B lymphocyte CD19 + Inoculated in 24-well plates and incubated overnight in medium. Thereafter, the test compound (alone or in combination) and the reference compound, or no compound (control) are added and the cells are pre-incubated for 2 hours. After that, the inducer (combination of CD40+ IL-4) was added, or no (unstimulated control), and the treatment with test compound or reference compound was repeated. Thereafter, the cells were grown at 1.7X10 6 Final density of cells/ml for 14 days).
All experimental conditions were performed with n=3.
Enzyme-linked immunosorbent assay (ELISA)
The amount of IgE released in the culture supernatant was measured using a specific ELISA kit according to the instructions of the supplier.
Statistics
Group comparisons were made by t-test of unpaired students. If n.gtoreq.5, statistical analysis can be interpreted, but for n <5, statistical values are given for reference.
The formula used in this report:
average standard error:
MSE＝Sd/√n
the Mean Standard Error (MSE) is a measure of the deviation between the sample mean and the overall actual mean. The MSE is calculated as the standard deviation of the sample (Sd) divided by the square root of the sample size (n).
Results and conclusions
In the unstimulated condition, the baseline of IgE release by B lymphocytes was very low (< 199pg/ml, approaching the limit of detection). Stimulation by a combination of anti-d40+il-4 for 14 days resulted in high levels (3657 pg/ml) of IgE production. TLR9 CpG-ODN2006 agonist as a reference compound was tested at a concentration of 3 μm, completely inhibiting IgE production induced by the anti-cd40+ IL-4 complex (< 3% of stimulated control). These results were expected and validated for testing.
The other two potential reference compounds also showed inhibitory effects on IgE production, but to a lesser extent compared to CpG-ODN 2006. IFN-gamma tested at 10ng/ml also showed a fairly strong and significant inhibition (20% of stimulated control). However, PGE tested at 1. Mu.M 2 The effect of (2) is much more limited and not statistically significant (58% of stimulated control).
The extracts of example 1 tested at 0.2% and 0.4% showed almost complete inhibition of IgE production by B lymphocytes stimulated by the combination of anti-cd40+ IL-4 (< 3% and <11% of stimulated controls, respectively).
Under the experimental conditions of this study, the extracts of example 1 at different concentrations showed very strong inhibition of IgE production by cell B stimulated by the anti-cd40+il-4 combination.
TABLE 8 summary of results
ns: 0.05 is not significant
* :0.01 to 0.05 is remarkable
* *:0.001 to 0.01 are very remarkable
< or >: below or above the detection limit
Undiluted sample <123.457 or >90000 pg/ml
In the case where the value is below or above the detection limit, the extrapolation proceeds with the following calculation.
Example 7
A gel having the following composition was prepared:
TABLE 9 composition of gel
The obtained composition is applied to the face with hypersensitive skin.
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Claims (11)
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 for use in the prevention and/or treatment of skin reactions caused by allergic reactions, the composition comprising an extract of a sphingomonas bacterium or at least one sphingomonas bacterium.
3. The extract for the use according to claim 1 or the composition for the use according to claim 2, wherein the Sphingomonas bacteria are Sphingomonas xenophaga.
4. An extract for the use or a composition for the use according to claim 3, wherein the Sphingomonas xenophaga bacteria are the strain submitted under deposit number CNCM I-5455.
5. The extract for the use according to any one of claims 1, 3 and 4 or the composition for the use according to any one of claims 2 to 4, for topical use, preferably on the skin surface.
6. Composition for the use according to any one of claims 2 to 5, wherein the concentration of the extract is between 0.0001% and 30% by weight relative to the total weight 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; extract of root of red-rooted salvia; active spring water such as skin care spring water; and mixtures thereof.
8. The extract for the use according to any one of claims 1, 3 to 5 or the composition for the use according to any one of claims 2 to 7, wherein the allergic reaction is an immediate type allergic reaction.
9. The extract for the use according to any one of claims 1, 3 to 5 and 8, or the composition for the use according to any one of claims 2 to 8, wherein the reaction comprises urticaria, rash and/or the appearance of redness.
10. An extract of a bacterium of the genus Sphingomonas for modulating the immune response and optionally for enhancing the barrier function of the skin.
11. A composition for modulating an immune response and optionally for enhancing the barrier function of skin, the composition comprising an extract of a sphingomonas bacterium or at least one sphingomonas bacterium.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FRFR2013724 | 2020-12-18 | ||
| FR2109095 | 2021-08-31 | ||
| FR2109095A FR3117777A1 (en) | 2020-12-18 | 2021-08-31 | Extract of bacteria of the genus Sphingomonas |
| PCT/EP2021/086534 WO2022129549A1 (en) | 2020-12-18 | 2021-12-17 | Extract of bacteria of the genus sphingomonas |
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| CN116801861A true CN116801861A (en) | 2023-09-22 |
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| CN202180085393.9A Pending CN116801861A (en) | 2020-12-18 | 2021-12-17 | Extracts of Sphingomonas bacteria |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117821362A (en) * | 2024-03-05 | 2024-04-05 | 广州蕊特生物科技有限公司 | Embryo bovine serum freeze-dried powder and preparation method thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117821362A (en) * | 2024-03-05 | 2024-04-05 | 广州蕊特生物科技有限公司 | Embryo bovine serum freeze-dried powder and preparation method thereof |
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