RU2221547C1 - Cosmetic product - Google Patents

Abstract

FIELD: cosmetology. SUBSTANCE: the suggested innovation deals with cosmetic products of immunomodeling activity. The present cosmetic product contains an active substance and an acceptable carrier; moreover, as an active substance it contains dolargin and could be manufactured in the form of cream, mask or lotion. This products protects skin against unfavorable impacts of harmful factors, it, also, increases body capacity to fight with alien antigens, heals wounds, burns and skin chaps. EFFECT: higher efficiency. 1 cl, 2 ex, 1 tbl

Description

 The invention relates to the field of cosmetology and relates to cosmetics with immunomodulatory activity.

 The main function of skin care cosmetics is to maintain the normal physiological state of skin tissue cells, including normalizing metabolic processes, including water-salt balance, protecting the skin from the adverse effects of environmental factors, and eliminating defects in damaged skin tissue by activating regenerative processes.

 Known hundreds of formulations of cosmetics for skin care, with one or another combination of favorable skin tissue characteristics. Typically, cosmetics are complex and include natural compounds in the form of extracts from materials of plant or animal origin. Such agents have a complex effect on skin tissue, and the effect of individual compounds on the condition of the skin is not always visible.

 Known, for example, is a cosmetic cream for skin care that prevents its aging, containing the collase enzyme, inactivated semen of the cock and / or gonad of sea urchins, and / or the bacterial cell detritus of Bifidobacterium bifidum, and / or the hyaluronidase enzyme isolated from the testes of animals [Patent RF 2.139.039, A 61 K 7/48, 1999].

 A liquid cosmetic skin care product having a tonic effect is described, which contains castor oil, alcohol and active substances of the rootstock and angelica root, or the rhizome of the cinquefoil, or the grass of the string, or the root of the incense [RF Patent 2.134.102, A 61 K 7 / 48, 1999].

 A cream for the skin of the face is proposed that promotes the normalization of fluid balance in the skin, containing a biologically active additive-complex of biologically active substances isolated from medical leeches [RF Patent 2.134.569, A 61 K 7/48, 1996].

 It is proposed to use a natural complex of collagenolytic enzymes isolated from the digestive organs of aquatic organisms as active components of cosmetics that increase skin elasticity and smooth wrinkles [RF Patent 2.141.31, A 61 K 7/48, 1997].

 A cosmetic product is known that has a tonic, anti-inflammatory, antibacterial, immunomodulating and anti-aging effect, which contains therapeutic actives, sapropel, blue clay and kaolin as active principles [RF Patent 2.180.213, A 61 K 7/48, 2002].

 Known cream for face and body skin, normalizing metabolic processes in the skin, having anti-inflammatory effects, stimulating local immunity, which includes a complex of polypeptides isolated from koumiss [RF Patent 2.137.467, A 61 K 7/48, 1999].

 Closest to the proposed can be considered a cosmetic product with stimulating and wound healing activity, containing peat extract as the main active principle, to which immunomodulating activity is attributed [RF Patent 2.126.682, A 61 K 35/10, 1999].

 The aim of the present invention is the creation of cosmetics that reliably have an immunomodulatory effect on skin tissue and exhibit, for this reason, a set of favorable properties, including the ability to optimize the function of skin tissue cells by normalizing metabolic processes in skin tissue, protecting it from adverse environmental factors, her regenerative processes.

 The goal is achieved as a result of the use of dalargin as the main active principle in cosmetics.

 The technical result achieved by using the invention is to expand the arsenal of immunomodulating agents suitable for effective use in the field of cosmetology.

 The invention is as follows.

 To impart immunomodulatory activity to cosmetics, dalargin is introduced into their composition.

 Dalargin is a synthetic derivative of the natural opioid peptide leienkephalin, is a synthetic diacetylated hexapeptide and has the following primary structure: Tur-d-Ala-Gly-Phe-Leu-Arg.

 Dalargin exhibits antiulcer and hypotensive effects, inhibits gastric secretion, promotes the activation of reparative processes and accelerates the healing of defects in the mucous membrane of the gastrointestinal tract. It is allowed in the prescribed manner for use in medical practice and is indicated for peptic ulcer of the stomach and duodenum, obliterating endarteritis, obliterating atherosclerosis of the vessels of the lower extremities, alcoholism [Russian Drug Register. Encyclopedia of drugs. M, "Radar-2001", 2001, p. 253].

 The prerequisites for the creation of the present invention were two scientific facts.

 On the one hand, it was found that dalargin exhibits an affinity for opioid receptors [Yu.A. Zolotarev et.al. The solid-state catalitic isotope exchange of hydrogen in Dalargin./Russian journal of Bioorganic Chemistry, 2000, v. 26, N 7, p.457-460] and is able to modulate the processes associated with the functional, proliferative and differentiating activity of target cells of the immune system [Zozulya A.A. et al. Importance of opiate receptor ligands in the regulation of proliferative activity of lymphocytes. [Pharmacology and Toxicology, 1987, v. 5b N3, p. 58-59].

 On the other hand, it was found that Langengars cells, the main antigen-presenting skin cells [J. Banchereau, R. M. Steinman. Dendric cells and the control of immunity. // Nature, 1988, v.392, p.245-252; V. Makarenkova et al. The system of dendritic cells: a role in the induction of immunity and in the pathogenesis of infectious, autoimmune and oncological diseases. // Immunology, 2002, v. 23, N 2, p. 68-76], as well as a number of other types of immunocompetent cells, express opioid receptors on their surface [A.A. Zozulya, S.F. Pshenichkin. Opioids and immunity. // In the book. "Neuroimmunology: molecular and clinical aspects", Results of science and technology, a series of "Immunology", t.25, M., 1990, p. 48-120; J. Hosoi et al. Beta-endorphin binding and regulation of cytokine expression in Langerhans cells. // Ann.N.Y. Acad. Sci., 1999, v. 885, p. 405-413].

 Despite the a priori evidence of the interaction of dalargin as an agonist of opioid receptors with opioid receptors of cells of the immune system of the skin, information on the severity of this interaction and on the concentrations of dalargin necessary for the manifestation of the effect of the peptide during cutaneous use was not found in the scientific literature.

 The mentioned facts gave reason to assume the ability of dalargin to cause immunomodulating effects when applied topically, in particular for cutaneous application.

 Some results of an experimental study of the immunomodulatory effects of dalargin are given below.

 Dalargin normalizes the proliferative activity of human peripheral blood lymphocytes. Moreover, the direction of the normalizing effect depends on the initial state of the cells.

The proliferation of lymphocytes induced by polyclonal mitogens is one of the main characteristics of the functional state of immunocompetent cells, reflecting their ability to respond to foreign antigens. In this regard, the effect of dalargin on the ability of lymphocytes to include ³ H-thymidine under stimulation with phytohemagglutinin was studied.

 Lymphocytes were isolated in the ficoll-verographin system from the peripheral blood of healthy donors (34 people) and donors with secondary immunodeficiencies (9 people). The proliferative activity of lymphocytes was evaluated in a known manner.

 As a result of studying the effect of dalargin (0.1 nM) on the proliferative activity of lymphocytes, a multidirectional, depending on the initial state, but invariably normalizing effect of the drug was demonstrated.

 In healthy donors, dalargin by 29% (p <0.01, Student's t-test) reduces the proliferative activity of lymphocytes stimulated by the optimal dose of mitogen. This effect cannot be considered as a manifestation of immunosuppression, since in vitro proliferation stimulated by polyclonal mitogens is an artificial situation simulating in vitro hyperactivation of the immune response.

 However, when studying the effects of dalargin on the lymphocytes of donors with immunodeficiency states, a completely opposite picture was found. In the absence of a peptide, the proliferative activity of the lymphocytes of these donors was 54% of that of healthy donors, in the presence of 93% (the effect of dalargin is significant - p <0.01, Student's t-test).

 Thus, the peptide stimulates the mitogen-activated proliferative activity of lymphocytes when proliferation is below normal and inhibits under conditions of increased proliferative activity. That is, the effect of the drug is aimed at maintaining homeostasis.

 Dalargin stimulates immediate and delayed hypersensitivity with intradermal administration.

 During the study, male Wistar rats weighing 200-230 g were immunized subcutaneously with bovine serum albumin (BSA) in the back left foot pad in complete Freund's adjuvant. The animals of the experimental group (10 rats) were given dalargin injections (1 μg / kg of weight in 50 μl of physiological saline), and the control group (11 animals) of physiological saline. Injections were performed for 14 days (once a day in the morning) intradermally, at points located in the immediate vicinity of the immunization site.

 14 days after subcutaneous immunization, 30 μg of BSA in 0.1 ml of physiological saline and BCG 1:10 in the same volume of physiological saline were injected intradermally (subcutaneously) into animals. The intensity of the Arthus reaction and delayed hypersensitivity was evaluated using a skin test 4 and 24 hours after resolving immunization with BSA and tubercle bacille, which are part of Freund's complete adjuvant. In this case, the diameter of the hyperemia zone (with an accuracy of 0.1 mm) and the “degree of inflammation” (the presence of edema and infiltration), which were conditionally expressed on a scale from 0 to ++++, were determined.

 As a result of the experiment, it was shown that the dalargin injection course stimulates the development of an immediate type of hypersensitivity (Arthus reaction) to BSA and tubercle bacille in Wistar rats. This result was obtained by determining the average diameter of the hyperemia zone in animals re-immunized with both BSA (10.0 ± 0.7 mm in the control and 13.2 ± 1.4 mm in the experiment, p <0.05) and tubercle bacille (4.9 ± 0.4 mm in the control and 6.7 ± 0.7 mm in the experiment, p <0.01). In addition, the stimulating effect of the peptide on the intensity of the Arthus reaction was also confirmed in assessing the degree of inflammation. Dalargin injections increase the density of the hyperemic area by 35% (p <0.05) with resolving BSA immunization and 2.1 times (p <0.01) with tubercle bacille immunization.

 An insignificant but significant (p <0.05) stimulating effect was also noted when studying the effect of dalargin on delayed-type hypersensitivity to BSA and tubercle bacille.

 Thus, the effect of the drug on skin cells increases the body's ability to fight foreign antigens. It can be assumed that one of the mechanisms of stimulation of the described immunostimulating effects is associated with direct or indirect action on the most important class of antigen-presenting cells - dendritic cells.

 With systemic administration, dalargin stimulates antibody production (as measured by local hemolysis in gel using the Erne test).

Dalargin (1ng / kg weight), dissolved in 0.5 ml of physiological saline, was injected into the tail vein of mice (CBA / C57Bl ₆ ) F1 30 minutes after immunization with sheep erythrocytes. In the control series of experiments, instead of dalargin, 0.5 ml of physiological saline was administered to immunized mice. Red blood cells (10 ⁸ / 0.5 ml of physiological saline), previously washed three times with physiological saline (300 g, 10 min at 4 ^° C), were also taken into the tail vein. At the peak of the immune response (on the fifth day after immunization), the animals were killed. The extracted spleen was homogenized in a glass-glass homogenizer with a slightly ground pestle. Cell suspensions were prepared at the rate of 10-50 mg wet weight of the spleen / ml of medium and introduced 0.1 ml into tubes thermostatted at 43 ^o C. The tubes contained 2.5 ml of a 0.5% agarose solution prepared on a Hanks solution ( pH 7.2) and 7.5-10.0 • 10 ⁷ red blood cells washed three times with physiological saline (300 g, 10 min at 4 ^o C). After that, the samples were transferred to a Petri dish and incubated for 1 h at 37 ^° C. At the end of the incubation, 2.5 ml of complement of guinea pig diluted with physiological saline 1: 5 was added to each cup. Then the samples were again incubated for 30 min at 37 ^o C and counted antibody-forming spleen cells.

 Similarly, experiments were conducted to study the effect of dalargin on anti-lobe production using a peptide in doses of 5 ng, 250 ng, 500 ng, 3.75 μg, 5.00 μg, 2.50 mg per 1 kg of experimental animal weight. The data obtained are presented in the table at the end of the description.

Radioreceptor study of delta opioid dendritic cell receptors
Radioreceptor analysis allows you to determine the affinity and the number of binding sites on target cells, which in the context of the study is necessary for the science-based choice of the range of concentrations of dalargin in a cosmetic product. In this regard, using the tritium-labeled synthetic analogue of dalargin - ³ H-DADLE (d-Ala ² -Leu-enkephalin), the characteristics of ligand receptor binding to opioid delta-type receptors, the most effective antigen-presenting cells, and mouse dendritic cells, were studied.

As a result of Scatchard's analysis of the saturation curve of ³ H-DADLE binding to mature dendritic cell receptors, an equilibrium constant was determined - receptor binding dissociation of 4.8 ± 1.0 nM and a maximum receptor density of 30 ± 3 fmol / mg of membrane protein fraction of dendrint cells.

 The current concentration of ligands that cause a biological effect mediated by receptors of target cells is comparable to the equilibrium dissociation constant. However, the complexity of the penetration of peptides through skin tissue [A.A. Margolin and others. New cosmetology. M., Cosmetics and medicine, 2000, 204 s; A.A. Zozulya et al. Cell therapy in cosmetology: protein-peptide complexes of fetal tissues as an active link in anti-age therapy in cosmetics. // Cosmetics and medicine, 2001, N 4 (23), p. 32-39], preceding the interaction with dendritic cells, determines the need to increase the effective concentration by orders of magnitude.

 The presented results make it possible to determine the concentration range of dalargin, at which stimulation of immunocompetent skin cells is observed, and even immunomodulating effects are observed that optimize the immune status of the whole organism.

 The results obtained made it possible to recommend the use of dalargin in cosmetology to increase the resistance of skin tissues to adverse external factors and effects, to heal wounds, burns and cracked skin, and to suppress allergic reactions.

 Cosmetics containing dalargin are especially suitable for dry and sensitive skin, including for patients prone to allergic skin reactions.

 Dalargin can be included in a wide variety of cosmetics - creams, cosmetic masks, lotions, cosmetic milk and others, and can also be used as part of oral care products, for example, as part of toothpastes.

 An effective concentration of dalargin in the composition of cosmetics may be 0.001-0.0150 wt.%.

 The following examples only illustrate the substance of the proposal and should not be limiting.

 EXAMPLE 1. To prepare 200 kg of cosmetic cream for aging skin, 0.45 kg of Ultrapres 10 carbopol is poured onto a surface of 50 kg of water and left to obtain a homogeneous mass containing no swollen lumps of carbopol.

In parallel, an emulsion is prepared by mixing 6 kg of isopropyl myristate, 6 kg of vaseline oil, 0.1 kg of vitamin E, 2 kg of cocoa butter, 1.6 kg of DC200 dimethicone, 3 kg of lanolin, 3 kg of stearic acid, 4 kg of polavax, 3 kg of glyceryl monostearate, 2 kg of almond oil, 2 kg of PEG-8 stearate, 0.25 kg of arnica oil, after which the mass is heated in a melting pot to 65 ^o C and stirred until a homogeneous product is formed.

 10 kg of water are poured into the reactor, the prepared emulsion and another 97.34 kg of water are added and the mixture is homogenized. Then add swollen carbopol, 6 kg of glycerol, 2 kg of hermaben II, 0.2 kg of panthenol and 0.45 kg of triethanolamine, after which they are thoroughly mixed.

The resulting homogeneous mass is cooled to 40 ^o C, add 0.2 kg of fragrance Fleur de Matin, 0.05 kg of sodium ascorbyl phosphate and a mixture of 2 kg of aquaftam and 0.002 kg of dalargin. The finished product is stirred and adjusted to pH 6.8 with triethanolamine.

EXAMPLE 2. For the preparation of 200 kg of soothing cream for sensitive skin, 0.4 kg of Ultrez 10 carbopol is poured onto a surface of 50 kg of water and left to swell until a homogeneous mass is obtained. At the same time, an emulsion is prepared, for which 6 kg of isopropyl myristate, 2 kg of petroleum jelly, 2 kg of cetyl alcohol, 2 kg of dimethicone, 2 kg of cetearyl alkanoate, 4 kg of Polavax GP 200.4 kg of almond oil, 4 kg of glycerol stearate and PEG-100 stearate are mixed, 0.2 kg of BHT and heated in a swimming boiler to a temperature of 70 ^o C and stirred until a homogeneous mass. 10 kg of heated water are poured into the reactor, the resulting emulsion is added, 97.56 kg of heated water are added, homogenized. Swollen carbopol, 4 kg of glycerol, 4 kg of propylene glycol, a solution of 0.4 kg of proxanol in 4 kg of water, 2 kg of germaben II, 0.4 kg of panthenol, 0.4 kg of triethlamine are added and a stirrer is added. After obtaining a homogeneous mass, it is cooled to 45 ^o C, add 0.35 kg of fragrance Fleur de Matin, 0.2 kg of aloe gel, a solution of 0.03 kg of dalargin in 2 kg of aquaftem, 0.09 kg of a blue dye solution (0.18 g of dye per 90 g of inputs) and mixed again until a homogeneous mass having a pH of 6.9 is obtained.

 The use of dalargin in cosmetics to impart immunomodulatory activity to these agents has the advantage over the use of other immunomodulatory drugs used in cosmetology that dalargin is a synthetic product with a specific structural formula, which greatly facilitates the standardization of finished products according to their biological activity. Since dalargin can be assigned to a relatively new group of immunomodulators (opioid immunomodulators), the capabilities of cosmetics containing dallargin are expanding due to the achievement of an immunomodulating effect by a different mechanism than was provided by previously known drugs of a similar orientation.

 According to the applicant, dalargin was not used in cosmetology for this purpose and it was not possible a priori to judge its applicability and effectiveness in this area.

 It is known, however, that dalargin was included in the composition of a skin care cream recommended to reduce the function of sebum secretion and exhibit anti-inflammatory effects on skin prone to acne [RF Patent 2.045.949, A 61K 7/48, 1995].

 In the known cosmetic composition, dalargin was used on the basis of its other functional properties, its mechanism of action was not disclosed, and the possibility of using dalargin as an immunomolulating agent in cosmetology was not anticipated.

 Does not refute the novelty of the proposal and the popularity of the parenteral use of dalargin for the treatment of psoriasis [RF Patent 2.017.488, A 61 K 31/485, 1994].

 The applicant believes that the claimed proposal meets all the requirements for the invention.

Claims (2)

1. An agent that protects the skin from the adverse effects of damaging factors, containing an acceptable carrier and an active principle, characterized in that as an active principle contains dalargin in a concentration of from 0.001 to 0.0150 wt.%. 2. The tool according to claim 1, characterized in that it is prepared in the form of a cream, mask, lotion.

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