Composition for hair growth and wound regeneration

Abstract

The invention relates to the field of dermatology and cosmetology and can be used for regenerating wounds and preventing and correcting age-related skin changes, as well as for stimulating hair growth. The proposed composition contains as active ingredient the secretome of mesenchymal stromal cells (MSCs) derived from mammalian adipose tissue, the secretome being provided in an amount of 50-70 ‎μg / ml and being characterized by the presence of the following secretion products: FGF-2, KGF, TGF-β, PDGF, TGF-α, IGF-1, EGF, HGF, VEGF, G-CSF, GM-CSF, PGE2, ANG-1, ANG-2, MCP-1, MMPs, cysteine, tyrosine, arginine, glycine, histidine, isoleucine, lysine hydrochloride, methionine, phenylalanine, serine, threonine, tryptophan and valine. Also proposed is a method for producing the claimed composition, which includes techniques and conditions for harvesting and cultivating MSCs, making it possible to obtain a secretome having constituents effective for the treatment and regeneration of skin and hair.

Description

[0001] Composition for hair growth and wound regeneration

[0002] Field of technology

[0003] The invention relates to the field of dermatology and cosmetology and can be used for wound regeneration, prevention and correction of age-related skin changes, as well as for stimulating hair growth.

[0004] Prior art

[0005] Regenerative medicine is a field that is growing in popularity due to high hopes for stimulating tissue repair in situ. Stem (stromal) cell therapy remains the focus of regenerative medicine due to early reports of their multi-patent differentiation capacity. However, more recent studies have established that the therapeutic effect of stem cells is due to their paracrine activity rather than direct differentiation (Gnecchi M. et al. Paracrine action accounts for marked protection of ischemic heart by Akt-modified mesenchymal stem cells / / Nat Med 2005; 11:367-8). Harnessing the paracrine activity of stem cells is possible without directly introducing the cells themselves into the body, through the use of a medium conditioned by stem cells (secretome).In recent years, the regenerative effects of factors secreted by mesenchymal stem cells (MSCs) have been studied for the treatment of various organs and systems, including the regeneration of the heart, kidneys, central nervous system, and muscle tissue. Numerous studies are also devoted to the treatment of skin and hair (see, for example, RU2716159, RU2280459, etc.).

[0006] As numerous studies have shown, cell isolation and culture methods significantly impact cell quality and the composition of the resulting secretome. Therefore, both the composition and efficacy of the resulting secretome depend significantly on the method of production. Furthermore, changes in cell culture conditions can lead to either improvement or deterioration in cell function and even cell death. The search for new solutions for creating in vitro MSC culture conditions that enable the development of new qualitative and quantitative secretome compositions for the development of compositions for the treatment of various diseases and the correction of age-related changes in skin and hair remains relevant.

[0007] Disclosure of invention

[0008] The objective of the invention is to create new medicinal and cosmetic products with a complex effect on the main cellular processes, promoting the treatment and regeneration of skin and hair, the prevention and correction of age-related skin changes, stimulation of hair growth and wound regeneration.The solution to the set problem is achieved by developing a composition for topical application for the treatment and / or regeneration of skin and / or hair, containing as an active component the secretion of mesenchymal stromal (stem) cells (MSC) of mammalian adipose tissue in an amount of 50-70 μg / ml and at least one pharmaceutically and / or cosmetically acceptable auxiliary component, wherein the MSC secretion is characterized by the presence of the following secretion products: FGF-2, KGF, TGF-P, PDGF, TGF-a, IGF-1, EGF, HGF, VEGF, G-CSF, GM-CSF, PGE2, ANG-1, ANG-2, MCP-1, MMPs cysteine, tyrosine, arginine, glycine, histidine, isoleucine, lysine hydrochloride, methionine, phenylalanine, serine, threonine, tryptophan, valine.

[0009] In some embodiments of the invention, the secretion products are contained in the MSC secretome in the following amount: FGF-2 - 940 + 160 pg / ml, KGF - 970 ± 160 pg / ml, TGF-β - 550 ± 90 pg / ml, PDGF - 62 ± 20 pg / ml, IGF-1 - 56 ± 19 pg / ml, EGF - 510 ± 90 pg / ml, HGF - 520 ± 90 pg / ml, VEGF - 550 ± 190 pg / ml, ANG-1 - 6890 ± 500 pg / ml, MMPs - 976, ± 40 ng / ml.

[0010] In some embodiments of the invention, the pharmaceutically and / or cosmetically acceptable excipient is a carrier, diluent and / or filler.

[0011] In some embodiments of the invention, the carrier, diluent and / or filler are selected from emulsifiers, structure formers, phospholipid base, preservatives, antioxidants, colorants, excipients, flavorings, antibacterial agents, fungicides.

[0012] In specific embodiments of the invention, the MSC secretome is incorporated into nanostructured lipid carriers. In some specific embodiments, the nanostructured lipid carriers are liposomes, niosomes, or ectosomes.

[0013] In some embodiments of the invention, the composition is presented in the form of a gel, cream, serum, lotion, tonic, shampoo, balm, mask, scrub or mousse for the face, hair, eyebrows and / or eyelashes.

[0014] The stated problem is also solved by developing a method for obtaining the secretome of mammalian adipose tissue MSCs for inclusion in a composition for the treatment and / or regeneration of skin and / or hair, comprising the following steps:

[0015] - obtaining mammalian adipose tissue MSCs from primarily isolated adipose tissue cells using type II collagenase for enzymatic processing and subsequent filtration by step filtration using filters with a 70 μm and 40 μm filter membrane;

[0016] - culturing the obtained mammalian adipose tissue MSCs in Dulbecco's modified Eagle's growth medium (DMEM) with phenol red, with the addition of 10% fetal bovine serum (FBS), antibiotics and antimycotics, until a confluence of at least 80% is achieved; - conditioning the obtained cells in Dulbecco's modified Eagle's growth medium (DMEM) with the addition of glucose - 4.5 g / l and L-glutamine - 2-4 mM / l, but without the addition of phenol red, antibiotics, antimycotics and FBS;

[0017] - collection of the conditioned medium and its filtration using filters with a 0.45 μm filter membrane, followed by concentration using 10 kDa and then 5 kDa centrifugal concentrators and subsequent sterilizing filtration of the resulting secretome of mesenchymal stromal cells using filters with a 0.22 μm filter membrane.

[0018] In some embodiments, the mammal is a human, a horse, a rabbit, a guinea pig, a cow, a bull, a cat, a dog, or a mini league.

[0019] The stated problem is also solved by developing a method for producing a composition for topical application for the treatment and / or regeneration of skin and / or hair, according to which, at the first stage, a secretome of mammalian adipose tissue MSCs is obtained, after which the obtained secretome is combined with at least one pharmaceutically and / or cosmetically acceptable auxiliary component, to produce a composition containing 50-70 μg / ml of a secretome of mammalian adipose tissue MSCs in the total volume of the composition.

[0020] The stated problem is also solved by using the developed composition for the treatment and / or regeneration of skin and / or hair.

[0021] In some embodiments of the invention, the composition is used for conditions selected from: acne, rosacea, rosacea, keratosis, dermatitis, psoriasis, acne, urticaria, age wrinkles, pigmentation.

[0022] In some particular embodiments of the invention, the composition is used for wound regeneration.

[0023] In some other embodiments of the invention, the composition is used to stimulate hair growth and / or prevent hair loss (in particular embodiments, the hair is the hair of the scalp, eyelashes, eyebrows, beard and / or moustache), in particular, the composition is used for conditions selected from alopecia areata, diffuse alopecia, alopecia totalis, androgenetic alopecia, seborrhea, gray hair (insufficient pigmentation).

[0024] The stated objective is also achieved by implementing a method for the treatment and / or regeneration of skin and / or hair, including the use of the developed composition. Additional means or procedures, such as mesoroller or micropeeling, can also be used to implement the proposed method. The proposed method can be implemented as monotherapy or in combination with other procedures and / or means aimed at the treatment and / or regeneration of skin and / or hair. The invention achieves the following technical results:

[0025] - a method has been developed for obtaining the secretome of mammalian adipose tissue MSCs, effective for the treatment and regeneration of skin and hair due to the regenerative action of a complex of growth factors, biologically active molecules and amino acids (protein-amino acid complex) included in the secretome, which effectively stimulate the restoration of cellular processes and signaling pathways, namely, FGF-2, KGF, TGF-|3, PDGF, TGF-a, IGF-1, EGF, HGF, VEGF, G-CSF, GM-CSF, PGE2, ANG-1, ANG-2, MCP-1, MMPs cysteine, tyrosine, arginine, glycine, histidine, isoleucine, lysine hydrochloride, methionine, phenylalanine, serine, threonine, tryptophan, valine;

[0026] • - obtained from the secretion of mammalian adipose tissue MSCs, which can be effectively used as a means for stimulating the regeneration of skin and hair, as part of various compositions containing the obtained secretion as an active component and promoting the treatment and regeneration of skin and hair, the prevention and correction of age-related skin changes, stimulation of hair growth and regeneration (healing) of wounds.

[0027] Brief description of the drawings

[0028] Fig. 1. Results of quantitative determination of growth factors in the secretome obtained from mesenchymal stromal cells using ELISA or Luminex multiplexing technology.

[0029] Fig. 2. Proliferative index of dermal papilla (DP) cells when cultured with the addition of MSC secretome in various concentrations. Data are presented as mean ± SEM.

[0030] Fig. 3. Proliferative index of HEK cells using MSC secretome at different concentrations. Data are presented as mean ± SEM.

[0031] Fig. 4. Evaluation of collagen synthesis by HEK cells during their cultivation with the addition of MSC secretome in various concentrations. Data are presented as mean ± SEM.

[0032] Fig. 5. Proliferative index of dermal papilla (DP) cells cultured with the addition of MSC secretome or Minoxidil at various concentrations. Data are presented as mean ± SEM.

[0033] Terms and definitions

[0034] The following terms and definitions are used in this document unless otherwise explicitly stated. References to techniques used in describing this invention refer to well-known techniques, including variations of these techniques and their replacement with equivalent techniques known to those skilled in the art. In the documents of this invention, the terms "includes," "including," and the like, as well as "contains," "comprising," and the like, are interpreted to mean "includes, among other things" (or "contains, among other things"). These terms are not intended to be construed as "consists only of."

[0035] The term "and / or" means one, more than one, or all of the listed elements.

[0036] The term "optional" or "optional" or "optionally" as used herein means that the subsequently described event or circumstance may, but does not necessarily, occur, and that the description includes instances in which the event or circumstance occurs and instances in which it does not occur.

[0037] The terms "subject" and "patient" imply a human being.

[0038] The terms "treatment" and "therapy" mean curing, slowing, halting, or reversing the progression of a disease or disorder (alleviating the severity of the disease). In this document, "treatment" also means alleviating the symptoms associated with the disease or disorder.

[0039] The term "prophylaxis" encompasses the elimination of risk factors, as well as prophylactic treatment of subclinical stages of the disease in humans, aimed at reducing the likelihood of developing the clinical stage of the disease. Primary prophylaxis is defined as prophylactic administration of the composition to patients who have not yet reached the clinical stage of the disease. Secondary prophylaxis is the prevention of recurrence of the same or a similar clinical condition.

[0040] The term "composition" refers to a composition comprising a mixture of active substances with established pharmacological activity (biologically active substances) and, optionally, at least one excipient (pharmaceutically acceptable additive). The term "excipient" ("excipient component") is generally known to mean a carrier, diluent, and / or filler used in formulating compositions that are effective for the desired application and that are inert (i.e., safe, non-toxic, and neither biologically nor otherwise undesirable). Within the framework of the present invention, excipients that are pharmaceutically (dermatologically) and / or cosmetically acceptable are used. Such excipients refer to carriers, diluents, and / or fillers that can be applied to the skin or hair and that will allow the composition to diffuse to the site of its action.When selecting excipients, consideration is given to their compatibility with the biologically active substances included in the composition, the route of administration, and any additional functions possessed by the excipients that are required to create a specific composition. For example, excipients include solid or liquid diluents, hydrotropes, surfactants, structure-forming agents, phospholipid bases, and encapsulating substances. In particular, excipients may include nanostructured lipid carriers (liposomes, micelles, niosomes, ectosomes) and polymeric nanoparticles; excipients may also include, but are not limited to, preservatives, antioxidants, emulsifiers, colorants, excipients, thickeners, fragrances, flavorings, antibacterial agents, and fungicides.

[0041] The choice of excipient depends on the method by which the active ingredients will be used and the composition form. The composition may be presented in various forms suitable for topical administration (application) to the skin and hair, including eyebrows and eyelashes, for example, as a gel, cream, serum, lotion, tonic, shampoo, balm, mask, scrub, or mousse for the face and hair. For example, a mixture of surfactants, solvents, thickeners, conditioning agents, and other functional additives, which may also include plant extracts or oils, can be used as a cosmetically acceptable additive in shampoos. Excipients of the proposed composition may also include, for example, acidity regulators, such as citric acid and / or sodium citrate, chelating agents, etc. The compositions according to the invention are used in effective doses.An "effective dose" refers to the amount of composition administered (applied) to the patient's skin and / or hair that is most likely to achieve the desired effect. The exact required amount may vary from subject to subject depending on numerous factors, such as the degree of disturbances (deviations from the norm) in the body requiring normalization, age, body weight, general health, the combined use of other medications, etc. Application of the composition according to the invention may be performed either once or multiple times, for example, several times a week, several times a month, or more often as a course of application over a period of time sufficient to achieve the desired effect (from several days to a week, several weeks, or even months), with courses of application being repeatable.In particular, for moderately severe disorders, the single dose, frequency, and / or duration of administration of the composition(s) according to the invention may be increased. The dosage regimen may be adjusted based on observed results, using standard efficacy criteria known in the art.

[0042] Unless otherwise defined, technical and scientific terms in this application have the standard meanings generally accepted in the scientific and technical literature. Detailed description of the invention and its implementation

[0043] According to the results of numerous studies, the culture medium conditioned by adipose-derived MSCs (conditioned medium, secretome) possesses significant regenerative potential, promoting accelerated healing of skin lesions, and the treatment and regeneration of skin and hair. It is also known that the composition and, consequently, the regenerative properties of the MSC secretome are significantly influenced by the method of its production, including both the method of MSC isolation and the conditions of their cultivation (Skalnikova H. et al. Mapping the secretome of primary isolates of mammalian cells, stem cells, and derived cell lines / / Proteomics, 2011 Feb; 11 (4): 691-708).

[0044] Therefore, the aim of the present invention was to develop conditions for the isolation and cultivation of MSCs in vitro, enabling the production of a new qualitative and quantitative composition of secretome components for the creation of a composition effective for the treatment and / or regeneration of skin and / or hair. To obtain the secretome of adipose-derived MSCs, the inventors used pinni liga adipose tissue cells. However, it is known that the key role in obtaining a secretome of a specific composition is not the origin of the fat cells, but the methods and conditions used during secretome production. Thus, to obtain the secretome of adipose-derived MSCs for inclusion in the compositions of the invention, fat cells from mammals such as mini-pig, horse, rabbit, guinea pig, cattle, cat, and dog can be used. Human adipose-derived MSCs can also be used. The quality criterion for the resulting secretome composition is its composition.

[0045] The mammalian adipose tissue MSC secretome according to the invention is a protein-amino acid complex also containing prostaglandin E2, which includes the following biologically active molecules: FGF-2, KGF, TGF-|3, PDGF, TGF-a, IGF-1, EGF, HGF, VEGF, G-CSF, GM-CSF, ANG-1, ANG-2, MCP-1, MMPs, PGE2, cysteine, tyrosine, arginine, glycine, histidine, isoleucine, lysine hydrochloride, methionine, phenylalanine, serine, threonine, tryptophan, valine. The biological properties of the molecules that affect the treatment and regeneration of skin and hair are shown in Table 1.

[0046] Table 1.

[0047] Thus, the MSC secretome according to the invention contains a wide range of biologically active compounds: signaling proteins, growth factors, lipid mediators, cytokines, and amino acids, which possess high regenerative potential and are capable of not only temporarily stabilizing the disease but also leading to its complete remission by restoring key signaling pathways and cellular mechanisms. Furthermore, the MSC secretome according to the invention has a positive effect on cell proliferation and migration and possesses immunomodulatory properties.Due to the unique composition of the obtained MSC secretome, cosmetic products based on it have pronounced regenerative and geroprophylactic effects, promote vascularization of skin tissues and hair follicles, improving their trophism, which allows them to be used as means for skin and hair regeneration, prevention and correction of age-related skin changes, stimulation of hair growth and regeneration (healing) of wounds, as well as for the treatment of such diseases and skin conditions as acne, rosacea, rosacea, keratosis, dermatitis, psoriasis, acne, urticaria, age wrinkles, pigmentation, and in such conditions as alopecia (focal, diffuse, total, androgenetic), seborrhea, gray hair (insufficient pigmentation).

[0048] The preparation of the mammalian adipose tissue MSC secretome according to the invention includes the following steps:

[0049] - obtaining mammalian adipose tissue MSCs from primarily isolated adipose tissue cells using type II collagenase for enzymatic processing and subsequent filtration by step filtration using filters with a 70 μm and 40 μm filter membrane;

[0050] - cultivation (passaging) of the obtained mammalian adipose tissue MSCs in Dulbecco's modified Eagle's medium (DMEM) with phenol red, with the addition of 10% fetal bovine serum (FBS), antibiotics and antimycotics, 2-5 passages until a confluence of at least 80% is achieved (usually up to 80-90%);

[0051] - conditioning of the obtained cells (optimal conditions are 37°C, 5% CO2, 70% humidity) in Dulbecco's modified Eagle's growth medium (DMEM) with the addition of glucose - 4.5 g / l and L-glutamine - 2-4 mM / l, but without the addition of phenol red, antibiotics, antimycotics and FBS); conditioning implies the synthesis and secretion of biologically active compounds (cytokines, proteins and extracellular matrix components) by the cells into a fresh medium during contact, incubation, exchange and interaction between the cells and the medium. In accordance with aspects of the present invention, the conditioning period can vary, preferably from 36 to 60 hours. At this stage, it is important to exclude the introduction of phenol red, serum (FBS), antibiotics and antimycotics into the medium;

[0052] - collection of the conditioned medium and its filtration using filters with a 0.45 μm filter membrane to remove cellular debris (Note: At this stage, the secretome can be frozen to -20°C for subsequent storage) with further concentration using 10 kDa and then 5 kDa centrifugal concentrators and subsequent sterilizing filtration of the obtained secretome of mesenchymal stromal cells using filters with a 0.22 μm filter membrane (Note: The obtained secretome can be lyophilized and subsequently restored using methods known to a person skilled in the art).

[0053] The resulting secretome is used as an active ingredient in compositions for the treatment and / or regeneration of skin and / or hair at a concentration of 50-70 μg / ml, in combination with excipients. Depending on the intended use, such compositions may contain various excipients, the choice of which depends on the method of application of the active ingredients and the composition form. The composition may be presented in various forms suitable for topical administration (application) to the skin and hair, including eyebrows and eyelashes, such as gel, cream, serum, lotion, tonic, shampoo, balm, mask, scrub, or mousse for the face and hair.For example, in one embodiment of the invention, the composition is a water-based composition that also contains hydroxyethylcellulose, glycerin, propylene glycol, pentylene glycol, polysorbate-20, polysorbate-60, sorbitan stearate, cholesterol, dicetyl phosphate, sodium chloride, calcium chloride, magnesium sulfate, calcium chloride, and sodium phosphate. In another embodiment, the composition may be presented, for example, in the form of a gel containing a suspension of niosomes containing the MSC secretome.

[0054] To obtain the compositions according to the invention, the obtained secretion of mammalian adipose tissue MSCs in an amount of 50-70 μg / ml is mixed with selected auxiliary components using technologies well known in this field.

[0055] In addition to excipients, the composition according to the invention may also contain additional biologically active substances that have a beneficial effect in the treatment and / or regeneration of skin and / or hair, including the prevention or correction of age-related skin changes, stimulation of hair growth, and wound regeneration. Such biologically active substances may include, but are not limited to, vitamins, plant extracts, or oils. Specifically, in the above-mentioned example of the composition, such additional biologically active substances may include nicotinamide, pyridoxine chloride, riboflavin, thiamine hydrochloride, folic acid, elastin, and collagen.

[0056] The present invention is illustrated, but not limited, by the following examples, given to illustrate methods, compositions and results in accordance with the disclosed essence of the invention.

[0057] Example 1. Obtaining the secretome of mammalian adipose tissue MSCs

[0058] Mammalian adipose tissue (mini pigs) was used to obtain MSCs. A mini pig adipose tissue sample weighing 3.5-6.3 g was minced and washed with phosphate-buffered saline (PBS) supplemented with 10-27 μg / ml penicillin-streptomycin, 15 μg / ml antibiotic-antimycotic, and 1 μg / ml gentamicin. The mixture was then incubated with a type II collagenase solution at 37°C for 45 minutes. Collagenase was inactivated by adding an equal volume of DMEM culture medium containing 10% fetal bovine serum (FBS). The resulting cell suspension was centrifuged for 10 minutes at 700 g (the acceptable range is 600-800 g) and then filtered by stepwise filtration using filters with a 70 μm and 40 μm filter membrane. The viability of the obtained cells was assessed with a vital dye (trypan blue).

[0059] The resulting cells were placed in a T175 culture flask (SPL Life Sciences 70175) and cultured in Dulbecco's modified Eagle's medium (DMEM) with phenol red, supplemented with 10% FBS, antibiotics (penicillin 100 U / ml and streptomycin 100 μg / ml) and an antifungal agent (amphotericin B 25 ng / ml) for 3 passages until 80% confluence was achieved.

[0060] Upon reaching passage 3 and 80% confluency, the cells were washed twice with PBS and then incubated for 45 hours in Dulbecco's modified Eagle's medium (DMEM) without phenol red, containing 4.5 g / L glucose, supplemented with 3 mM / L L-Glutamine, and without antibiotics, antifungals, or FBS. The conditioned medium was then collected and centrifuged twice at 600 g (the acceptable range is 500–800 g) for 10 minutes at room temperature, followed by filtration of the non-caging liquid with a 0.45 μm syringe filter (Cytiva 6780-1304) to remove cellular debris.

[0061] The conditioned medium was concentrated using centrifugal concentrators at 10 kDa and then at 5 kDa for 15 minutes at 3200 g (the acceptable range is 3200–4500 g) and a temperature of 4°C. Subsequent sterilizing filtration of the resulting mesenchymal stromal cell secretome was performed using filters with a 0.22 μm filter membrane.

[0062] Example 2. Evaluation of the composition of the obtained MSC secretome

[0063] The resulting mesenchymal stromal cell secretome is a cell-free culture medium conditioned by the metabolic products and secretions of mammalian mesenchymal stromal cells (mini-pig). To assess the composition of the resulting secretome, enzyme-linked immunosorbent assay (ELISA) kits were used according to the manufacturer's instructions, or multiplexing was performed using Millipore's proprietary Luminex® technology, which utilizes internally color-coded microspheres with two fluorescent dyes mixed in varying ratios so that each molecule has its own unique identity. Each of these molecules is labeled with specific capture antibodies. Analytes contained in the test samples and standards are captured by the labeled molecules, after which a secondary biotinylated detection antibody is introduced.The reaction mixture was then incubated with a streptavidin-BPE conjugate, which served as a reporter. The microspheres were passed through two lasers, one of which excited the molecule and the other the fluorophore. The readings were recorded on a Luminex 200TM instrument, and values ​​were generated based on their relative positions and intensities. The resulting quantitative values ​​(pg / mL) were interpolated from a standard graph generated by the system's built-in software.

[0064] As a result of the evaluation of the obtained secretome of mesenchymal stromal cells, the presence of a wide range of growth factors (GF) and amino acids in its composition was revealed, namely: cysteine, tyrosine, arginine, glycine, histidine, isoleucine, lysine hydrochloride, methionine, phenylalanine, serine, threonine, tryptophan, valine, FGF-2, KGF, TGF-β, PDGF, TGF-α, IGF-1, EGF, HGF, VEGF, G-CSF, GM-CSF, PGE2, ANG-1, ANG-2, MCP-1, MMPs. The results of the quantitative analysis of the secretome composition are shown in Fig. 1. Analysis of growth factors showed that the composition of the obtained secretome of mesenchymal stromal cells (MSCs) is very diverse in relation to various growth factors associated with the proliferation and migration of cells of various lines, angiogenesis, epithelialization and wound healing, hematopoiesis and neurostimulation.

[0065] Among the growth factors involved in cell proliferation, several proteins of the fibroblast growth factor family were identified, such as FGF-2 and KGF, which are known to induce fibroblast and keratinocyte proliferation. EGF, TGF, and PDGF, which promote epithelial cell proliferation and migration, were also detected. Furthermore, angiogenic factors such as VEGF, IGF-1, and HGF were detected. A number of growth factors, such as G-CSF and GM-CSF, which are known to play a role in hematopoiesis, were also detected. The resulting secretome also contains numerous amino acids, which play an important role in biological processes, improve the skin's water and protein balance, and nourish and strengthen the hair.

[0066] Example 3. Treatment with MSC secretome promotes proliferation of dermal papilla cells

[0067] Human dermal papilla (DP) cells were cultured in DMEM medium supplemented with 20% FBS with the addition of an antibiotic-antimycotic, and then (1.5*10 4cells per well) were transferred to 96-well plates. The cells were treated with the secretome of mesenchymal stromal cells at various concentrations of 0.01, 0.1, 1, 0, 10 μM daily for 3 days; medium without the secretome of MSCs was used as a control.

[0068] To study the role of the mesenchymal stromal cell secretome in the proliferative effect on DP cells, the cells were pretreated with the ERK inhibitor PD98059 for 1 hour. Since signaling via the ERK pathway leads to cell survival, proliferation, and increased motility, adding an inhibitor of this signaling pathway allows us to assess the effect of the secretome on proliferation. Absorption was measured at 570 nm using an ELISA reader. The study demonstrated that even low concentrations of the secretome according to the invention exert a proliferative and anti-apoptotic effect on DP cells (see Fig. 2), demonstrating the high potential for using the resulting secretome to stimulate hair growth.

[0069] Example 4. Treatment with MSC secretome promotes proliferation and migration of keratinocytes and increases collagen synthesis

[0070] Proliferation and migration of dermal fibroblasts are important steps in the process of skin regeneration and repair. A human epidermal keratinocyte (HEK) cell line was used as an in vitro system to evaluate the proliferative effect and migratory potential of the mesenchymal stromal cell secretome. As an initial proof of concept, the secretome was tested on HEK cells at various dilutions. It was found that the HEK secretome promoted HEK proliferation starting from a dilution of 5%, although significant differences in the proliferative potential between the secretome and the control medium (medium without the HEK secretome) were observed at dilutions of 20% and 50%. The proliferative effect of the secretome on HEK cells was maximal at a concentration of 100% (Fig. 3).

[0071] In addition to human epidermal keratinocyte proliferation, collagen synthesis in HEK cells pretreated with the MSC secretome was also assessed. Treatment of HEK cells with 1x secretome for 72 hours resulted in increased collagen levels. This increase was 103% and 376% for 1x secretome and 10x secretome, respectively, compared to the corresponding 1x or 10x control media (Fig. 4).

[0072] Example 5. Comparison of the effectiveness of the MSC secretome and Minoxidol

[0073] To evaluate the effectiveness of the secretome according to the invention, a comparative study was conducted, assessing the proliferative index of dermal papilla (DP) cells with the addition of the secretome or Minoxidil. Minoxidil is a drug currently widely used to stimulate hair growth. The mechanism for enhancing hair growth with Minoxidil is associated with vasodilation and improved microcirculation in the skin and hair follicle nutrition, stimulation of the transition of hair follicles from the resting phase (telogen phase) to the growth phase (anagen phase), and stimulation of new follicle growth.

[0074] In the study, DP cells were cultured in DMEM medium supplemented with 20% FBS with an antibiotic-antimycotic, and then treated with the secretome of mesenchymal stromal cells or Minoxidil at concentrations of 0.001, 0.01, 0.1, 1.0, and 10 μM for 3 days. As shown by the studies, the proliferative index of DP cells with the addition of the secretome according to the invention is significantly higher (see Fig. 5), indicating its high effectiveness in stimulating hair growth.

[0075] Thus, as the conducted studies have shown, the obtained MSC secretion effectively stimulates the proliferation and functional activity of skin cells and hair follicles and can be effectively used as a means for stimulating the regeneration of skin and hair, as part of various compositions containing the obtained secretion as an active component and promoting the treatment and regeneration of skin and hair, the prevention and correction of age-related skin changes, stimulation of growth and prevention of hair loss and regeneration (healing) of wounds.

[0076] Although the invention has been described with reference to the disclosed embodiments, it will be apparent to those skilled in the art that the specific experiments described in detail are provided merely for the purpose of illustrating the present invention and should not be construed as limiting the scope of the invention in any way. It should be understood that various modifications are possible without departing from the spirit of the present invention.

Claims

Invention formula 1. A composition for topical application for the treatment and / or regeneration of skin and / or hair, comprising as an active component the secretome of mesenchymal stromal cells (MSCs) of mammalian adipose tissue in an amount of 50-70 μg / ml and at least one pharmaceutically and / or cosmetically acceptable auxiliary component, wherein the MSC secretome is characterized by the presence of the following secretion products: FGF-2, KGF, TGF-[3, PDGF, TGF-a, IGF-1, EGF, HGF, VEGF, G-CSF, GM-CSF, PGE2, ANG-1, ANG-2, MCP-1, MMPs, cysteine, tyrosine, arginine, glycine, histidine, isoleucine, lysine hydrochloride, methionine, phenylalanine, serine, threonine, tryptophan, valine.

2. The composition according to claim 1, wherein the secretion products are contained in the MSC secretome in the following amount: FGF-2 - 940 ± 160 pg / ml, KGF - 9701 160 pg / ml, TGF-β - 550 ± 90 pg / ml, PDGF - 62 ± 20 pg / ml, IGF-1 - 56 ± 19 pg / ml, EGF - 510 ± 90 pg / ml, HGF - 520 ± 90 pg / ml, VEGF - 550 ± 190 pg / ml, ANG-1 - 6890 ± 500 pg / ml, MMPs - 976 ± 40 ng / ml.

3. A composition according to any one of paragraphs 1-2, in which the pharmaceutically and / or cosmetically acceptable auxiliary component is a carrier, diluent and / or filler.

4. The composition according to paragraph 3, in which the carrier, diluent and / or filler are selected from the group including emulsifiers, structure formers, phospholipid base, preservatives, antioxidants, dyes, excipients, flavorings, antibacterial agents, fungicides.

5. A composition according to any one of paragraphs 1-4, in which the MSC secretome is included in nanostructured lipid carriers.

6. The composition according to claim 5, wherein the nanostructured lipid carriers are liposomes, niosomes or ectosomes.

7. A composition according to any one of paragraphs 1-6, presented in the form of a gel, cream, serum, lotion, tonic, shampoo, balm, mask, scrub or mousse for the face, hair, eyebrows and / or eyelashes.

8. A method for obtaining a composition according to any of paragraphs 1-7, characterized in that at the first stage, the secretion of MSCs from adipose tissue of a mammal is obtained, which includes the following stages: - obtaining mammalian adipose tissue MSCs from primarily isolated adipose tissue cells using collagenase type II for enzymatic processing and subsequent filtration by step filtration using filters with a 70 µm and 40 µm filter membrane; - cultivation of the obtained mammalian adipose tissue MSCs in Dulbecco's modified Eagle's medium (DMEM) with phenol red, with the addition of 10% fetal bovine serum (FBS), antibiotics and antimycotics, 2-5 passages until a confluence of at least 80% is achieved; - conditioning the obtained cells in Dulbecco's modified Eagle's medium (DMEM) with the addition of glucose - 4.5 g / l and L-glutamine - 2-4 mM / l, but without the addition of phenol red, antibiotics, antimycotics and FBS; - collecting the conditioned medium and filtering it using filters with a 0.45 μm filter membrane with further concentration using 10 kDa centrifugal concentrators, and then 5 kDa and subsequent sterilizing filtration of the obtained secretome of mammalian adipose tissue MSCs using filters with a 0.22 μm filter membrane; the obtained secretome is combined with at least one pharmaceutically and / or cosmetically acceptable auxiliary component, to obtain a composition containing 50-70 μg / ml of the secretome of mammalian adipose tissue MSCs in the total volume of the composition.

9. The method of claim 8, wherein the mammal is a human, a horse, a rabbit, a guinea pig, cattle, a cat, a dog, or a mini pig.

10. Use of a composition according to any of paragraphs 1-7 for the treatment and / or regeneration of skin and / or hair.

11. The use according to claim 10 for stimulating hair growth and / or preventing hair loss.

12. The use according to claim 11, characterized in that the hair is hair from the scalp, eyelashes, eyebrows, beard and / or moustache.

13. Use according to item 10 for conditions selected from: acne, rosacea, rosacea, keratosis, dermatitis, psoriasis, acne, urticaria, age wrinkles, pigmentation.

14. Use according to claim 10 for wound regeneration.

15. Use according to claim 10 for conditions selected from focal alopecia, diffuse alopecia, total alopecia, androgenetic alopecia, seborrhea, gray hair (insufficient pigmentation).

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