EA045003B1 - COMPOSITION BASED ON PROBIOTICS AND ITS APPLICATION - Google Patents

Description

Field of technology to which the invention relates

The present invention relates to a composition containing probiotics, preferably based on bacteria, in particular bacteria of the genus Lactobacillus, for use in the prevention and/or treatment of diseases affecting the skin, in particular inflammatory diseases, in particular autoinflammatory diseases of a dermatological nature, such like atopic dermatitis. In addition, the composition according to the invention is suitable for preventing and/or reducing skin damage caused by UV radiation, in particular skin aging.

Prior Art

Human skin is colonized by a rich microbiome. This aspect was previously thought to represent a potential source of infection, while there has now been an awareness of the fact that the human microbiota in general, and therefore the skin microbiota, performs important and beneficial functions in the host not only due to its ability to counteract adhesion and the development of skin pathogens, but also due to its ability to dialogue and interact with the immune system.

If dysbiosis occurs at the skin level, then probiotics can act as modulators and restore the balance of the skin microbiota.

The use of new technologies in the last decade has greatly facilitated the taxonomic analysis of the skin microbiota, the bacterial population of which can number approximately 1010 species of microorganisms belonging to more than 25 phyla (phyla = taxonomic ranks of microorganisms), the most representative of which are Actinobacteria, Firmicutes ) and proteobacteria (Proteobacteria). Various skin diseases are associated with changes in the skin microbiome.

For example, in the case of acne, P. acnes (Propionibacterium acnes) is considered the bacteria primarily associated with this disease; in fact, with this disease, the optimal environment for the growth of these bacteria is provided by increased production of sebum (sebum).

The traditional approach to these problems is to use antibacterial agents, i.e. in the use of topical (for local use) disinfectants and antibiotics.

On the one hand, antibiotics are undoubtedly effective, but in addition to eliminating beneficial bacteria, they carry with them the risk of developing hypersensitivity and potentially adverse side effects, especially in the case of long-term use of broad-spectrum antibiotics.

For this reason, the applicant identified as a new solution to the problem of restoring the balance of the skin microbiota - the use of probiotic compositions, based, in particular, on the use of bacteria belonging to the genus Lactobacillus. In fact, the applicant has discovered that a composition containing probiotics belonging to the genus Lactobacillus is capable of preventing or, in any case, mitigating the action of specifically those bacteria that are harmful to the skin, such as P. acnes, and is thereby capable of

1) promote the prevention and/or treatment of diseases affecting the skin;

2) stimulate the normal healing process; And

3) maintain and/or improve the normal processes of re-epithelialization and/or scarring.

Brief description of the figures

The present invention is described in detail below and illustrated by way of example with reference to the accompanying figures, among which:

fig. 1 graphically shows P. acnes adhesion as a percentage of live, viable cells attached to keratinocytes after

1A: pre-stimulation by contact of keratinocytes with test probiotics;

1B: co-incubation of keratinocytes with P. acnes and test probiotics;

1C: incubation of keratinocytes with test probiotics after infection of eukaryotic cells with a pathogen.

Hereinafter, in all figures, LP125 is designated the strain Lactobacillus paracasei DG CNCM 1-1572, and LC48 is designated Lactobacillus paracasei LPC-S01 DSM26760.

If P. acnes adhesion in the absence of probiotic stimulation is taken as 100, then the inhibitory ability of the tested strains will be expressed as a % reduction in P. acnes adhesion compared to the positive control. In particular, FIG. 1A shows the ability of both probiotic strains to prevent the adhesion of P. acnes in the indicated % (42% for L. casei DG (LP125) and 35% for L. paracasei LPC-S01 (LC48)).

Fig. Figure 1B shows the ability of L. casei strain DG (LP125) to reduce adhesion by 17%, while L. paracasei strain LPC-S01 (LC48) showed 9%. The mixture of strains gave a statistically significant reduction in P. acnes adhesion, which amounted to 42%, i.e. a clearly higher percentage than that observed in the case of individual strains and their combined effect.

Fig. Figure 1C shows a statistically significant synergistic effect of a mixture of 2 probiotics, which was shown to reduce P. acnes adhesion by 42%. In contrast to this, individual pro

- 1 045003 biotics showed the ability to reduce adhesion, which was less than half the level of reduction in adhesion of their mixture - 18% for L. casei DG (LP125) and 11% for L. paracasei LPC-S01 (LC48).

Fig. 2 graphically shows the results of an assay for the cytokines IL-10, ΓΕ-1β, IL-8, and TSLP in cell supernatants, expressed in picograms (pg)/ml of supernatant, obtained by ELISA assay.

Disclosure of the Invention

One aspect of the present invention relates to a composition containing probiotics, preferably one or more species of probiotic bacteria, for use in the treatment and/or prevention of diseases and/or pathologies, preferably of the inflammatory type, affecting the skin, preferably of the dermatological type.

Said diseases and/or pathologies affecting the skin are selected from dermatitis, preferably associated with irritation and/or excoriation (superficial damage to the integrity of the skin as a result of scratching, etc.); acne, infections, skin inflammation, erythema, ulcers, psoriasis, atopic dermatitis, otitis media, skin cracks, fistulas, hemorrhoids and any other disease or injury with or without a developing inflammatory process and/or combinations and/or complications, and /or the consequences of the above.

Said diseases and/or pathologies affecting the skin are preferably associated with or caused by pathogens. That is, the composition is particularly effective when used not only for preventive/prophylactic purposes, but also for medicinal/therapeutic purposes, i.e. after infection with these pathogens.

According to a preferred aspect of the present invention, the composition may also promote/facilitate/stimulate/accelerate the wound healing process and/or re-epithelialization and/or scarring processes.

Said pathogens are preferably selected from bacteria, fungi, yeast, viruses, and combinations thereof.

Said pathogens are preferably selected from bacteria, and said bacterial pathogens are preferably selected from the genus Propionibacterium, preferably the acnes species; Staphylococcus genus, preferably epidermis, aureus, warneri, pyogenes, mitis; Corynebacterium ssp.; Pseudomonas, preferably aeroginosa; Acinetobacter, preferably johnsonii; Streptococcus, preferably pyogenes; Micrococcus ssp. and Brevibacterium ssp.

In addition, a composition containing probiotics, preferably one or more species of probiotic bacteria, as detailed below, is indicated for use in protecting the skin and/or lips and/or conjunctiva from UV radiation, UVA (UV-A rays). (long wave)) and/or UVB rays (short wave)). In other words, the composition is indicated for use in the prevention and/or mitigation of damage and/or effects caused by/associated with exposure to UV radiation, UVA and/or UVB. The composition is preferably intended to prevent and/or delay skin aging.

From this point of view, a composition containing probiotics, preferably one or more species of probiotic bacteria, as described in detail below, has a cosmetic purpose. The use of the composition is preferably associated with the immunomodulatory effect of the probiotics contained in the composition, preferably probiotics as defined and described below. Said immunomodulation includes modulating, preferably reducing, the expression of at least one cytokine selected from hematopoietic cytokines, preferably hematopoietic growth factors and/or CSF (colony stimulating factor); primary pro-inflammatory cytokines, preferably IL-1 and/or TNF (tumor necrosis factor); anti-inflammatory and/or immunosuppressive cytokines, preferably IL-10 and/or TGF-β (transforming growth factor-beta); secondary proinflammatory cytokines (chemokines); cytokines that regulate a specific immune response, preferably IL-2; and combinations of the above. Said immunomodulation preferably includes modulation, preferably reduction, of the expression of at least one cytokine selected from IL-1e, IL-10, IL-8, TSLP and combinations thereof.

In this regard, it should be noted that cytokines are non-antigen-specific polypeptide mediators that act as communication signals between cells of the immune system and between the latter and different organs and tissues. Cytokines are produced by different types of cells, and once released in the body, they induce specific responses in nearby cells (paracrine effect), in other cells very far away (endocrine effect), or in the cells that created them (autocrine effect). In particular, cytokines produced by cells of the immune system, such as interleukins and chemokines, play a fundamental role in regulating and activating human defense mechanisms and in inflammatory processes. A complex network of cytokines maintains a balance between pro-inflammatory and anti-inflammatory actions. An imbalance between pro-inflammatory and anti-inflammatory cytokines, as well as uncontrolled cytokine production, can lead to inflammatory diseases, allergies or autoimmune pathologies.

- 2 045003 logiam. TSLP (thymic stromal lymphopoietin) is a protein belonging to the cytokine family: it plays an important role in the maturation of T cell populations by activating antigen presenting cells. TSLP is produced primarily by non-hematopoietic cells such as fibroblasts, epithelial cells and various types of stromal cells, and its expression is associated with many pathological conditions, including asthma, inflammatory arthritis, atopic dermatitis, eczema, eosinophilic esophagitis and other allergic conditions.

In particular, the applicant has shown that when keratinocytes are exposed to probiotics, the latter are able to exhibit immunomodulatory effects, as evident from the evaluation of assays for the determination of cytokines in the cell supernatant. In particular, a decrease in the expression of IL1 β, IL10 and IL8 was observed under the influence of certain probiotic strains, in particular the L. paracasei strain LPC-S01 (LC48). The effect of inhibiting cytokine expression became more obvious after stimulation of keratinocytes with lipopolysaccharide (LPS).

As for TSLP (thymic stromal lymphopoietin), contact with probiotics determines a decrease in the expression of this indicator compared to the initial expression (keratinocytes not stimulated by the probiotic). LPS prestimulation of keratinocytes did not result in increased TLSP levels: this would be expected since other molecules are known to be more stimulatory of Toll-like LPS receptors. Probiotic strains - individually and as a mixture - have been shown to be more effective in inducing the expression of this indicator. The indicator is of significant interest because it is overexpressed in several skin pathologies such as atopic dermatitis. In addition, this cytokine is considered a key mediator at the functional interface between keratinocytes and dendritic cells.

According to a preferred aspect of the invention, the composition can be used in wound healing and/or re-epithelialization and/or scarring of damaged and/or diseased skin.

Thus, the data clearly indicate that probiotics, in particular the strains tested, are capable of exerting anti-inflammatory and/or immunomodulatory effects on keratinocytes and thereby on the skin.

In general, given the effects described herein, the probiotic composition disclosed herein can be used to restore the balance of the skin microbiota.

In the context of the present invention, skin means the first line of defense against the external environment; In particular, this protection is realized through the action of keratinocytes scattered in the outer layer of the skin (epidermis), where they can induce the secretion of cytokines and chemokines to transmit an alarm signal to the deeper layers of the skin, thereby generating an inflammatory response. During their development, keratinocytes migrate from deeper layers to layers closer to the surface with increasing deposition of keratin, which is responsible for the protective effect towards the underlying cells.

In the context of the present invention, skin aging means a completely natural and inevitable physiological process that occurs in all individuals. Over time, the skin undergoes structural changes caused by a number of factors of various origins, which cause loss of moisture in the skin, the appearance of fine wrinkles, loss of elasticity, hyperkeratosis and the formation of hyperpigmented spots called age spots.

Said aging may preferably be intrinsic - or chronological - aging, which is largely dependent on genetic (or intrinsic) factors. Alternatively, said skin aging may be extrinsic aging - or aging caused by environmental factors, i.e. caused by external factors.

Generally speaking, intrinsic aging usually begins after age 25. It usually involves a number of changes, which in most cases result in thinning and/or changes in the structure of the skin.

Extrinsic aging is preferably caused by exposure to external agents and/or environmental factors, preferably selected from UV radiation (responsible for photoaging), cigarette smoking, alcohol abuse, pollution, chronic exposure to irritants, and combinations thereof.

Skin damage and/or effects associated with UV exposure are preferably selected from erythema, pigmentation, keratosis, hyperkeratosis, skin redness, tanning, burns, actinic photoaging or solar elastosis, cortical cataract, pterygium, reactivation of oral herpes, skin damage of any nature, preferably damage to the lips and/or conjunctiva, skin melanoma, squamous cell carcinoma of the skin, basal cell carcinoma, squamous cell carcinoma of the cornea or conjunctiva, and/or combinations, and/or complications, and/or consequences of the above.

In the context of the present invention, restoring the balance of the skin microbiota means restoring the qualitative and quantitative physiological composition of the skin microbiota, understood as the entire set of microorganisms present on the skin, and thereby restoring the physiological skin microbial ecology.

In the context of the present invention, test infection means any ex

- 3 045003 experimental test or sample or test involving challenge with microorganisms of various species and subsequent assessment of changes in microbial load, usually by plate counting the number of living microorganisms at specified intervals.

In the context of the present invention, probiotics, as defined by FAO/WHO, mean: Live microorganisms which, when taken in adequate quantities, have a beneficial effect on the health of the host. In other words, probiotics are microorganisms that, when taken in suitable quantities, show the ability to exhibit functions that are beneficial to the body.

Said microorganisms are preferably selected from bacteria, fungi, yeasts and combinations thereof.

According to a preferred aspect of the invention, the bacteria belong to at least one genus selected from Lactobacillus, Bifidobacterium, Bacillus, Propionibacterium, Streptococcus, Lactococcus, Aerococcus and Enterococcus. More preferably, the bacteria are of the genus Lactobacillus.

According to another preferred aspect of the invention, bacteria of the genus Lactobacillus are at least one species selected from

Lactobacillus paracasei, Lactobacillus acidophilus. Lactobacillus amylolyticus, Lactobacillus amylovorus, Lactobacillus alimentarius, Lactobacillus aviaries. Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus casei, Lactobacillus cellobiosus, Lactobacillus coryniformis, Lactobacillus crispatus, Lactobacillus curvatus, Lactobacillus delbrueckii, Lactobacillus farciminis, Lactobacillus fermentum, Lactobacillus gallinarum, Lactobacill us gasseri, Lactobacillus helveticus, Lactobacillus hilgardii, Lactobacillus johnsonii, Lactobacillus kefiranofaciens, Lactobacillus kefiri , Lactobacillus mucosae, Lactobacillus panis, Lactobacillus collinoides, Lactobacillus paraplantarum, Lactobacillus pentosus, Lactobacillus plantarum, Lactobacillus pontis, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus sakei, Lactobacillus salivarius, Lactobacill us sanfranciscensis and their combinations.

More preferred are Lactobacillus species Lactobacillus paracasei, preferably Lactobacillus paracasei strain DG CCNCM 1-1572 and/or Lactobacillus paracasei strain LPC-S01.

Both strains were isolated and deposited by SOFAR S.p.A., in particular, the bacterial strain Lactobacillus paracasei DG was deposited in the National Collection of Cultures of Microorganisms (CNCM) of the Pasteur Institute (Paris) on 05/05/1995 with the deposit number CNCM 1-1572. The strain was originally named Lactobacillus casei DG ssp. casei.

The bacterial strain Lactobacillus paracasei LPC-S01 has been deposited in the DSMZ with accession number DSM26760.

These strains are particularly effective in the applications described herein if they are used in an association and/or combination where they actually show an additive or synergistic effect.

According to another preferred aspect of the invention, bacteria of the genus

Bifidobacterium are at least one species selected from B. animalis, B. bifidum, B. breve, B. infantis, B. longum, B. adolescentis, B. catenulatum, B. angulatum, B. asteroides, B. bourn , B. choerinum, B. coryneforme, B. cuniculi, B. denticolens, B. dentium, B. gallicum, B. gallinarum, B. indicum, B. inopinatum, B. lactis, B. magnum, B. merycicum, B minimum, B. pseudocatenulatum, B. pseudoIongum, B. pullorum, B. ruminantium, B. saeculare, B. subtile, B. thermacidophilum, B. thermophilum and B. tsurumiense', more preferably selected from Bacillus clausii, Bacillus subtilis, Bacillus coagulans, Bacillus megaterium, Bacillus halodurans, Bacillus thuringiensis, Bacillus insolitus and Bacillus marinus.

- 4 045003

According to another preferred aspect of the invention, bacteria of the genus Propionibacterium are at least one species selected from

R. shermanii,

P. acnes, P. australiense, P. avidum, P. cydohexanicum, P. freudenreichii, P. granu/osum, P. jensenii, P. microaerophilum, P. propionicum and P. thoenii.

According to another preferred aspect of the invention, bacteria of the genus Streptococcus are at least one species selected from Streptococcus thermophilus, Streptococcus salivarius, Streptococcus agalactiae, Streptococcus anginosus,

Streptococcus bovis, Streptococcus canis, Streptococcus constellatus, Streptococcus downei, Streptococcus dysgalactiae, Streptococcus equinus. Streptococcus ferus, Streptococcus infantarius, Streptococcus iniae, Streptococcus intermedius, Streptococcus milleri, Streptococcus mitis, Streptococcus mutans, Streptococcus oralis, Streptococcus orisratti, Streptococcus parasanguinis, Streptococcus peroris, Streptococcus pneumoniae, Strept ococcus pseudopneumoniae, Streptococcus pyogenes, Streptococcus ratti, Streptococcus tigurinus, Streptococcus sanguinis , Streptococcus sobrinus, Streptococcus suis, Streptococcus uberis, Streptococcus vestibularis, Streptococcus viridans and Streptococcus zooepidemicus.

According to another preferred aspect of the invention, bacteria of the genus Lactococcus are at least one species selected from L. chungangensis, L. formosensis, L. fujiensis, L. garvieae, L. lactis, L. piscium, L. plantarum, L. raffinolactis and L. taiwanensis.

According to another preferred aspect of the invention, bacteria of the genus Aerococcus are at least one species selected from A. urinae, A. sanguinicola, A. christensenii, A. suis, A. urinaeequi and A. urinaehominis.

According to a further preferred aspect of the invention, the bacteria of the genus Enterococcus are at least one species selected from

Enterococcus avium, Enterococcus durans, Enterococcus faecalis, Enterococcus faecium, Enterococcus gallinarum, Enterococcus haemoperoxidus, Enterococcus hirae, Enterococcus malodoratus, Enterococcus moraviensis, Enterococcus mundtii, Enterococcus pseudoavium, Enterococcus raffinosus and Enterococcus solitarius.

According to another preferred aspect of the invention, the yeast is of the genus Saccharomyces, more preferably the species Saccharomyces cerevisiae and/or Saccharomyces boulardii.

The microorganisms - preferably the bacterial strain L. casei DG® and/or the bacterial strain Lactobacillus paracasei LPC-S01 - are preferably used alive, i.e. used as probiotics.

Alternatively, the microorganisms, preferably the bacterial strain L. casei DG® and/or the bacterial strain Lactobacillus paracasei LPC-S01, are used dead or tindalized (ie, subjected to fractional sterilization with flowing steam).

In a further embodiment of the invention, microorganisms - preferably the bacterial strain L. casei DG® and/or the bacterial strain Lactobacillus paracasei LPC-S01 - are used in the form of a lysate and/or extract, i.e. they are used as a paraprobiotic. Alternatively, microorganisms - preferably the bacterial strain L. casei DG® and/or the bacterial strain Lactobacillus paracasei LPC-S01 - are used in the form of bacterial products, supernatants; derivatives, preferably bacterial derivatives, preferably metabolites; metabolic bioproducts, postbiotics, cell walls and components of the above, exopolysaccharides or compounds containing immunogenic components, preferably selected from ribosomes and glycoproteins, immunostimulant derivatives such as glucans and other polysaccharides, lipopolysaccharides and any supernatant component.

In most cases, these microorganisms are used individually or in combinations of microorganisms (or consortia) of any species listed on the QPS (Qualified Presumption of Safety) list of the European Food Safety Authority (EFSA).

In any case, the composition according to the invention may contain any type of microorganisms, including

- 5 045003 ness, bacteria showing probiotic effect/action/function, and/or microorganisms as defined above, preferably bacteria that are capable of stably colonizing the skin, intestines and/or other parts of the body, taking away space from pathogenic microorganisms/bacteria and/ or directly fighting them. The cosmetic composition and/or composition for medical purposes as described above preferably contains a combination of the strains described above with other microorganisms as described above, preferably selected from bacteria, fungi, yeast and combinations thereof.

Microorganisms, preferably bacteria, are present in minimal quantities sufficient to provide temporary colonization of the skin, intestines and/or other parts of the body. Said amount preferably ranges from 10 ⁶ to 10 ¹¹ units of microorganisms, more preferably from 10 ⁸ to 10 ⁹ units of microorganisms, and said amount is preferably an amount/dose/day (daily).

The composition additionally contains auxiliary substances (excipients) and/or additional pharmaceutically acceptable substances and/or carriers.

The composition of the present invention preferably also contains a substance selected from plasma, PRP (platelet rich plasma), healing agents, re-epithelializing agents, wetting agents, humectants, emollients, adsorbents, analgesic and phlebotonic agents, anti-inflammatory agents, muscle relaxants, peptide and/or protein substances and/or proteins such as collagen; substances related to connective tissue, such as glycosaminoglycans, preferably chondroitin sulfate, and/or combinations of the above.

In one embodiment of the present invention, the formulated composition is preferably for topical use, preferably in the form of a cream, gel, oil, emulsions, sprays, gauze pads, patches, dressings, lotions, mousses, ointments, pastes or liquid formulations into which the composition preferably added immediately before application to the skin.

In one embodiment of the present invention, the composition is used for skin microbiota transplantation. In the context of the present invention, skin microbiota transplantation means the introduction, preferably locally, of the skin microbiota (and/or part thereof) of healthy subjects not suffering from skin diseases, in order to restore the correct balance and thereby treat dysbiosis that may be present. For these purposes, the composition according to the invention preferably contains bacteria, preferably the bacterial strain L. casei DG® and/or the bacterial strain Lactobacillus paracasei LPC-S01, and optionally also fungi, viruses, peptides, protein and/or peptide substances, carbohydrates, vitamins, microelements or any other substance characterizing the microbiota of the skin and/or other parts of the body. For such purposes, the said composition is preferably used in combination with amino acids, additives, vitamins, microelements such as zinc and selenium; macro- and micronutrients, enzymes and/or prebiotic substances such as fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS), inulin, guar gum, and/or combinations of the above. In one embodiment of the present invention, the composition is formulated for oral administration, preferably in the form of a solid formulation, preferably in the form of pills, capsules, tablets, granular powder, hard shell capsules, orodissolving granules, sachets or lozenges.

Alternatively, the composition is formulated as a liquid, preferably for immediate administration.

Alternatively, the composition is in a form capable of exerting a topical effect, such as an enema, cream, spray, gel, patch, lotions, mousses, ointments, pastes or liquid formulations, to which the composition is preferably added immediately before application to the skin.

According to a preferred embodiment of the invention, the composition of the invention is used in combination with amino acids, additives, vitamins, microelements such as zinc and selenium; macro- and micronutrients, enzymes and/or prebiotic substances such as fructooligosaccharides (FOS), galactooligosaccharides (GOS), inulin, guar gum or combinations of these.

Example

Bacterial strains and cells.

The invention is demonstrated by way of example using the following bacterial strains:

L. casei DG (Lactobacillus paracasei CNCM 1-1572);

L. paracasei LPC-S01;

L. casei DG + L. paracasei LPC-S01 (1:1 mixture).

Bacterial strains were cultivated in a selective nutrient medium consisting of MRS (agar medium developed by de Man, Rogosa, Sharpe) for 16 hours at 37°C. Upon reaching the stationary growth phase, bacterial samples were collected and used for tests. They were washed several times with phosphate buffered saline (PBS) and centrifuged to remove

- 6 045003 of waste culture medium. Live cells in the stationary growth phase were used, since it is in this phase that bacteria induce a smaller immune response.

All tests described below were performed in triplicate.

The experiments used normal human skin keratinocytes maintained in culture with suitable additives.

Propionibacterium acnes (P. acnes) CCUG 50865 (purchased from CCUG (Culture Collection, University of Gothenburg) was cultured for 16 h at 37°C in meat nutrient medium (both liquid and solid) prepared according to the supplier's instructions. The concentration was then determined bacteria by spectrophotometry with readings taken at an optical density (O.D.) of 600 nm and used for adhesion tests.

Test for adhesion and pathogen infection of a cell line.

Keratinocytes were seeded at 37°C in a 5% CO2 atmosphere into wells of cell culture plates and allowed to grow until confluent (i.e., a total of approximately 105 cells). The inoculation rate was 2.5x105 cells/0.32 ^cm2 (i.e., cell density 7.8x105 cells/ ^cm2 ). During the experiment, the medium was removed, the cells were washed with antibiotic-free medium, and then bacteria were added for tests that were performed according to various protocols described below.

1) Adhesion of strains to keratinocytes.

After determining the number of keratinocytes per well of the plate, L. paracasei strains were added at an MOI (multiplicity of infection) of 1:10 (105 cells and 106 CFU of bacteria/well). Strains were tested in a 1:1 ratio, both individually and in combination.

The bacteria were left in contact with the cells maintained at 37°C under gentle shaking for 60 minutes. At the end of incubation, the medium was removed and the cells were washed with sterile medium to remove bacteria not attached to the cell monolayer.

To calculate the number of attached bacteria, cells were separated, lysed, and bacteria were counted by supravital staining after plating on MRS agar medium.

2) Adhesion exclusion test.

After determining the number of keratinocytes in each well of the plate, L. paracasei strains were added to the cells in a 1:1 ratio, either individually or in combination.

The bacteria were left in contact with the cells maintained at 37°C under gentle shaking for 60 minutes. At the end of incubation, the medium was removed and the cells were washed with sterile medium to remove bacteria not attached to the cell monolayer.

P. acnes (at an MOI of 1:10) was then brought into contact with the cells, followed by incubation for 60 minutes at 37°C. At the end of the incubation, the culture medium was removed and unattached bacteria were removed by washing with sterile medium.

To calculate the number of P. acnes attached to keratinocytes, cells were separated and lysed, and bacteria were enumerated by supravital staining after plating on 5% sheep blood agar under anaerobiosis for 48–72 h at 37°C. Wells with keratinocytes that were not preincubated with L. paracasei strains, but were inoculated with P. acnes only, were used as a positive control for P. acnes adhesion (P. acnes adhesion control), while wells with cells that were not incubated with no bacteria, was used as a negative control (sterility control).

3) Adhesive competition test.

After determining the number of keratinocytes per well, L. paracasei strains (either individually or in a 1:1 ratio) were added simultaneously with P. acnes. Bacteria were added at a total MOI of 1:10 and a probiotic:pathogen ratio of 1:1.

The bacteria were left in contact with the cells at 37°C under gentle shaking for 60 minutes. At the end of incubation, the medium was removed and the cells were washed with sterile medium to remove bacteria not attached to the cell monolayer.

To calculate the number of P. acnes attached to keratinocytes, cells were separated and lysed, and bacteria were enumerated by supravital staining after plating on 5% sheep blood agar under anaerobiosis for 48–72 h at 37°C. Wells with keratinocytes inoculated with P. acnes only were used as a positive control (P. acnes adhesion control), while wells with cells that were not incubated with any bacteria were used as a negative control (sterility control).

4) Test to remove attached pathogens.

After determining the number of keratinocytes per well, P. acnes was added (at an MOI of 1:10).

The bacteria were left in contact with the cells maintained at 37°C under gentle shaking for 60 minutes. At the end of incubation, the medium was removed and bacteria not attached to the cell monolayer were removed by repeated washes with sterile medium.

Then the L. paracasei strains (both individually and in combination at a 1:1 ratio) leaving

- 7 045003 in contact with cells maintained at 37°C with gentle shaking for 60 minutes. At the end of incubation, the medium was removed and the cells were washed with sterile medium to remove bacteria not attached to the cell monolayer.

To calculate the number of P. acnes attached to keratinocytes, cells were separated and lysed, and bacteria were enumerated by supravital staining after plating on 5% sheep blood agar under anaerobiosis for 48–72 h at 37°C.

Keratinocytes treated with P. acnes were used as a positive control (P. acnes adhesion control), while keratinocytes that were not treated with bacteria were used as a negative control (sterility control).

Tests carried out both on individual strains in pure form and on a 1:1 mixture were repeated twice.

1) Adhesion of strains to keratinocytes - results.

The results of this test showed the ability of the strains (taken separately and also in combination) to adhere to keratinocytes. In particular, L. casei strain DG showed a higher ability to adhere to keratinocytes than L. paracasei strain LPC-S01. Therefore, the adhesive ability observed in the case of the strain combination can be attributed presumably to the contribution of the L. casei DG strain.

Table 1

Adhesion (bacteria no. - CFU/well) L. casei DG 139 x 10 ² - 141 x 10 ² L. paracasei SOI 18.2 x 10 ² - 21.8 x 10 ² Mix 1:1 98 x 10 ² - 122 x 10 ² Propionibacterium acnes 700 x 10 ² - 2000 x 10 ²

Adhesion exclusion test (pre-treatment of eukaryotic cells with probiotics and subsequent incubation with a pathogen).

Results.

As noted above, a monolayer of keratinocytes was first contacted with probiotics and then (after removal of non-adherent bacteria) contacted with P. acnes, which were then removed by washing; after this, the residual viability of P. acnes was quantified.

Eukaryotic cells without stimulation by contact with bacteria served as a negative control, while the ability of P. acnes to adhere to keratinocytes without pretreatment with probiotics was used as a positive control. Fig. 1A shows results regarding P. acnes adhesion as a percentage of live, viable cells attached after pre-stimulating keratinocytes by contact with the various probiotics tested. If P. acnes adhesion in the absence of probiotic stimulation is taken as 100, the inhibitory capacity of the tested strains is expressed as % reduction in P. acnes adhesion compared to the positive control.

Both probiotic strains showed the ability to prevent P. acnes adhesion in the indicated % (42% for L. casei DG (LP125) and 35% for L. paracasei LPC-S01 (LC48)).

Adhesion competition test (co-incubation of eukaryotic cells with probiotics and a pathogen).

Results.

In these tests, the reduction, if any, in P. acnes adhesion to keratinocytes was assessed following co-treatment of the cell line with probiotics and the pathogen to evaluate the possible competitive inhibition effect exerted by the probiotics when co-incubated with P. acnes.

Keratinocytes treated with P. acnes were used as a positive control, while keratinocytes that were not treated with bacteria were used as a negative control (sterility control).

Fig. 1B graphically shows the results regarding P. acnes adhesion as a percentage of live, viable cells attached after co-incubation of keratinocytes with P. acnes and the various probiotics tested. As described previously, if P. acnes adhesion in the absence of probiotic stimulation is taken as 100, the inhibitory capacity of the tested strains is expressed as the % reduction in P. acnes adhesion compared to the positive control.

In this mode of interaction between probiotics and pathogen, the L. casei strain DG (LP125) showed the ability to reduce adhesion by 17%, while the reduction in adhesion by the L. paracasei strain LPC-S01 (LC48) was 9%. The combination of strains gave a statistically significant reduction in P. acnes adhesion, amounting to 42%, i.e. a significantly higher percentage than that observed with the individual strains, and also higher than their combined effect.

Test for removal of attached pathogens (pre-treatment of eukaryotic cells with a pathogen and subsequent incubation with a probiotic).

Results.

In these tests, any reduction in P. acnes adhesion to keratinocytes was assessed after pretreatment of the cell line with the pathogen and subsequent incubation with probiotics after removal.

- 8 045003 detection of a non-attached pathogen.

Keratinocytes treated with P. acnes were used as a positive control, while keratinocytes that were not treated with bacteria were used as a negative control (sterility control).

Fig. 1C graphically shows the results regarding P. acnes adhesion as a percentage of live, viable cells attached following incubation of keratinocytes with the various probiotics tested following infection of the eukaryotic cells with the pathogen. As described above, if P. acnes adhesion in the absence of probiotics is taken as 100, then the inhibitory capacity of the tested strains is expressed as % reduction in P. acnes adhesion compared to the positive control.

In these tests, as with the co-incubation protocol, an interesting statistically significant synergistic effect was observed with the combination of 2 probiotics, which was shown to reduce P. acnes adhesion by 42%. In contrast, individual probiotics showed an ability to reduce adhesion that was less than half that of the probiotic mixture: 18% in casei DG (LP125) and 1% in L. paracasei LPC-S01 (LC48).

Immunomodulation tests.

Immunomodulation tests were performed to test whether probiotic strains had the ability to modulate cytokine release by human keratinocytes negatively stimulated by bacterial lipopolysaccharides (LPS).

Normal human keratinocytes were seeded as described above in cell culture plate wells and, once confluent, were used for experiments. Specifically, cell monolayers were washed and incubated in freshly prepared antibiotic-free medium. L. paracasei, L. casei DG (LP125) and L. paracasei LPC-S01 (LC48) strains were then added individually and in combination in a 1:1 ratio at an MOI (multiplicity of infection) of 1:10.

The bacteria were left in contact with the keratinocytes for 120 minutes at 37°C with gentle shaking. In some cases, cells were prestimulated (infected for approximately 24 h) with lipopolysaccharide (LPS) at a final concentration of 100 ng/ml.

At the end of incubation, the medium was removed, the cells were washed with sterile medium to remove bacteria that were not attached to the cells, and then kept in culture medium for 24 h at 37°C.

At the end of incubation, the culture medium was collected to quantify the cytokines produced by ELISA (IL-10, IL1β, IL-8, thymic stromal lymphopoietin [TSLP]), while cyclooxygenase-2 (COX-2) levels 2) and activated NF-κB induced by LPS or IL-1e were determined by Western blotting on total proteins extracted from lysed keratinocytes.

These markers were chosen because they are involved in both acute phlogosis and allergic dermatitis, as well as chronic associated phlogosis. In particular, IL-10 is considered anti-inflammatory, and its production typically increases in parallel with the production of potent proinflammatory cytokines such as ΓΕ-1β. TSLP is a cytokine that modulates the action of lymphocytes and is involved in the development of chronic keratitis.

Results.

The ability of the tested strains to stimulate the immune response of keratinocytes was assessed in accordance with the protocol described above. The tests were aimed at determining cytokines (TL-8, IL-1e and IL-10) and assessing the activation of 2 markers - COX-2 and NF-kB.

COX-2 (cyclooxygenase-2) is an inducible marker produced by a limited number of cell types in response to a specific inflammatory stimulus. It is overexpressed in several neoplasms (neoplasms), including skin neoplasms. NF-kB (nuclear factor kappa light chain enhancer of activated B cells or nuclear factor kappa bi) is a protein complex with transcription factor function produced by all cell types in response to various stimuli, including inflammatory stimuli.

Cultured keratinocytes were exposed to the probiotics L. paracasei, L. casei DG (LP125) and L. paracasei LPC-S01 (LC48) and a 1:1 combination of the two strains according to the procedures described above. Cell supernatants were then analyzed to determine cytokines, with a particular focus on anti-inflammatory cytokines that were detected in keratinocytes in contact with the probiotic strains. The results of the analysis are summarized in Fig. 2 and graphically show the results of the analysis of interleukins IL-10, IL-1e and IL-8 in cell supernatants, expressed in pg (picograms)/ml of supernatant obtained by ELISA test.

Detailed assessments of the results obtained allowed us to draw the following conclusion.

IL-1e (interleukin-1beta): expression levels of several nanograms/ml of keratin supernatant

- 9 045003 cits. Contact with probiotics did not lead to a statistically significant decrease in the expression of this cytokine compared to the initial level of expression (keratinocytes not stimulated with the probiotic). L. paracasei LPC-S01 (LC48) showed a moderately positive effect compared to L. casei DG (LP125), and alone was comparable in effect to the strain combination. Similar conclusions also apply to pre-stimulation of keratinocytes with lipopolysaccharide (LPS) to maximize the immune response.

IL-10: expression levels of several tens of picograms/ml of keratinocyte supernatant. Contact with probiotics led to a decrease in the expression of this cytokine compared to the initial level of expression (keratinocytes not stimulated by the probiotic). L. paracasei LPC-S01 (LC48) showed a moderate positive effect compared to L. casei DG (LP125) and was comparable in effect to the strain combination. Similar conclusions also apply when keratinocytes are pre-stimulated with LPS to maximize the immune response.

IL-8: expression levels of several nanograms/ml of keratinocyte supernatant. Contact with probiotics led to a decrease in the expression of this cytokine compared to the initial level of expression (keratinocytes not stimulated by the probiotic). L. paracasei LPC-S01 (LC48) showed a positive effect compared to L. casei DG (LP125) and was comparable in effect to the strain combination. When keratinocytes were pre-stimulated with LPS to maximize the immune response, both probiotics showed potential for positive effects on cytokine control, with similar potential for probiotics alone or as a mixture.

TSLP (thymic stromal lymphopoietin): expression levels of several nanograms/ml of keratinocyte supernatant. Contact with probiotics resulted in a decrease in indicator expression compared to baseline expression (keratinocytes not stimulated by the probiotic). Probiotic strains, both individually and in combination, have shown to be effective in establishing the required level of expression of this indicator. The indicator is of significant interest as it is overexpressed in some skin pathologies such as atopic dermatitis. In addition, this cytokine is considered a key mediator at the functional interface between keratinocytes and dendritic cells.

Evaluation of COX-2 expression levels showed that the expression levels of this marker did not appear to undergo significant changes after exposure of keratinocytes to probiotics compared to baseline levels, with the exception of a moderate inhibitory effect upon exposure to L. paracasei LPC-S01 (LC48). Treatment with lipopolysaccharide (LPS) showed an increase in COX-2 expression levels in eukaryotic cells, an increase that was effectively inhibited and limited by probiotics. The presence of L. paracasei LPC-S01 (LC48) abolished marker expression to a greater extent compared to L. casei DG (LP125), while the combination of 2 probiotic strains seemed to have no effect.

Since the expression of IL-8 and COX-2 is regulated by the transcription factor NF-κB, NF-κB release by keratinocytes exposed to probiotics was qualitatively assessed. The results demonstrate that in the absence of the inflammatory stimulus provided by LPS, marker expression appears to be undetectable. However, it is activated by bacterial lipopolysaccharides through phosphorylation of p65. Two probiotic strains were shown to be capable of inhibiting NF-kB expression, with particular emphasis on L. paracasei LPC-S01 (LC48), which, when present either as a single strain or as a mixture with DG, was shown to be particularly effective in inhibiting expression of LPS-induced NF-kB.

Assessing the reduction of UV damage.

The ability of the probiotic strain LPC-S01 to reduce damage caused by UV irradiation was assessed in a complete three-dimensional in vitro 3D model of reconstructed skin, which reproduces the dermis (skin proper) and epidermis compartments, which makes it possible to study modifications of the extracellular matrix of the dermis and differentiation of viable skin layers (model full thickness skin). The study is considered to evaluate the effect of the LPC-S01 strain on the activation of the inflammasome (a multiprotein complex that leads to the launch of an inflammatory response when the cell comes into contact with microorganisms) in response to UV irradiation. Strain LPC-S01 (LC48) was applied directly to the surface of a 3D skin model, incubated overnight and then washed off with saline to remove excess product. The tissue was lightly scratched and then exposed to 1 MED (minimum erythematogenous dose) UV to simulate normal solar exposure. Inflammatory activation was tested 4 and 24 hours after exposure to UV irradiation. Tissue treated with saline and exposed to UV irradiation was used as a positive control. Saline served as a negative control.

Histomorphological analysis with hematoxylin/eosin staining.

At the end of treatment, the tissues were washed with saline and fixed in 10% formaldehyde. For each sample, biological copies (n=3) were embedded in the same paraffin blocks, and 2 5 pm sections were cut and collected from non-consecutive tissue sections. Tissue sections

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Claims (3)

stained with hematoxylin and eosin. Histological samples were analyzed using an optical microscope (magnification 20X and 40X) and morphological changes in the tissue and cytotoxic effect were assessed. NFkB immunostaining. Labeling NFkB makes it possible to assess its translocation from the cell cytoplasm to the nucleus; its translocation can actually activate the inflammatory process. Labeling of NFkB was performed using immunostaining techniques known in the art. Specifically, a primary antibody Anti-NFKB (Abeam) and a secondary antibody stained with Alexa 555 fluorophore were used and counterstained with DAPI to highlight cell nuclei. Images were obtained using a fluorescence microscope at 40X magnification. Quantitative determination of interleukin IL-1 β by ELISA. Antibodies were adsorbed onto the ELISA plate and samples were then incubated. The secondary antibody was added to form a sandwich. Quantification was based on a standard curve. Data were obtained through spectrophotometric measurements. Results in reducing UV damage. After 4 h of UV exposure, untreated tissue (positive control) showed morphology that remained unchanged but with UV-damaged cells in the lower layer of the epidermis. After 4 h, tissue treated with strain LPC-S01 showed no significant change in tissue morphology or overall architecture or burn damage in the lower epidermis. 24 hours after UV exposure, the tissue showed typical epidermal changes associated with burns, with shrunken (pyknosis) nuclei, epidermis, and dermis with changes. The introduction of strain LPC-S01 (LC48) did not lead to improvement in preventing morphological changes in tissue induced by UV radiation. In table Figure 2 summarizes the results of quantitative assessment of NFkB translocation 4 hours after exposure to UV radiation. table 2 Negative control Positive control LPC-S01 (LC48) Number of translocations 25.7 47.2 22.4 4 hours after irradiation, the positive control showed a large number of NFkB translocations, especially in the suprabasal layer of the epidermis. Treatment with the probiotic LPC-S01 (LC48) was able to inhibit NFkB nuclear translocation to a significant extent compared to the positive control. In addition, strain LPC-S01 (LC48) has also been shown to be able to reduce cytoplasmic levels of NFkB. Finally, IL-13 secretion showed an increase at 4, 8, and 24 h after UV damage. Administration of the probiotic LPC-S01 (LC48) allowed the secretion of IL-1e to return to its original state 4, 8 and 24 hours after radiation injury. In particular, a significant decrease was noted 8 and 24 hours after injury. Table 3 Secretion of IL-1β NC 4h PC 4h S01 4h NC 8h PC 8h S01 8h NC 24h PC 24h S01 24h IL-Ιβ (pg/ml) 2.12 2.74 2.10 2.49 3.18 1.92 3.13 4.78 3.44 In summary, the probiotic LPC-S01 (LC48) showed the ability to prevent inflammasome activation by inhibiting the nuclear translocation of NFkB in cells exposed to UV-induced damage. In addition, strain S01 (LC48) directly showed the effect of reducing proinflammatory interleukin 1β levels at 4, 8, and 24 h after radiation-induced injury. CLAIM 1. The use of a composition containing probiotic bacteria strain Lactobacillus paracasei DG CNCM 1-1572 and/or bacteria strain Lactobacillus paracasei LPC-S01 DSM26760 for the prevention or treatment of diseases or pathologies of the inflammatory type affecting the skin, or for the reduction or treatment of symptoms, or consequences associated with these diseases/pathologies. 2. The use of a composition containing probiotic bacteria strain Lactobacillus paracasei DG CNCM 1-1572 and/or bacteria strain Lactobacillus paracasei LPC-S01 DSM26760 to stimulate the process of wound healing, or reepithelialization processes, or scarring of the skin. 3. Use according to claim 1, where said diseases or pathologies affecting the skin are selected from -