RU2641041C1 - Composition for atopic dermatitis therapy based on fulleren - Google Patents

Abstract

FIELD: medicine.SUBSTANCE: composition includes an aqueous solution of fullerene C, vaseline, sucrose palmitate, as well as parabens - nipasol and nipagin in a weight ratio of 4:3.78:2.2:0.001:0.001. The final content of fullerene in the composition is 0.005 to 0.012 wt %.EFFECT: invention allows reduction of inflammatory leukocyte infiltration of the epidermis.1 cl

Description

The invention relates to pharmacology and medicine, mainly to medicines in the form of an ointment, and can find application in the treatment of atopic dermatitis and other inflammatory diseases associated with an allergic reaction.

Atopic dermatitis (AD) is very widespread, including acute and chronic forms, in the whole world it affects up to 30% of children and up to 5% of adults. The disease is a socially significant problem, it significantly affects the quality of life of patients, limits their performance, up to disability. The basis of pathogenesis is the dominance of an Independent immune response with the production of IgE-class antibodies, enhanced by mast cell degranulation, deficiency of filagrin, which plays a key role in the barrier function of the skin [Peng W., Novak N. Pathogenesis of atopic dermatitis. Clin. Exp. Allergy, 2015, 45 (3). DOI: 10.1111 / cea.12495; Leung D.Y.M., Boguniewicz M., Howell M.D., Hamid Q. New insights into atopic dermatitis. J. Clin. Invest., 2004, 113: 651-657; Novak N., Bieber T., Leung D.Y.M. Immune mechanisms leading to atopic dermatitis. J. Allergy Clin. Immunol., 2003, 112: 128-138]. A typical clinical picture of AD is characterized by itching of the skin, persistent hyperemia or transient erythema, papular vesicular eruptions, exudation, dry skin, peeling, excoriation, lichenification, which are widespread or limited. The immune response is characterized by the production of Th2 cells of cytokines such as IL-4, IL-5, IL-13, as well as TNF-α, which leads to increased levels of IgE and leukocyte infiltration of the dermis [Sinke JD, Rutten VPMG, Willemse T. Immune dysregulation in atopic dermatitis. Vet. Immunol. Immunopathol., 2002, 87: 351-356]. Mast cells, blood basophils secreting allergamators make a significant contribution to the development of inflammation: histamine, heparin, leukotrienes, prostaglandins, bradykinin, serotonin [Ryan JJ, Kashyap M, Bailey D, Kennedy S, Speiran K, Brenzovich J, Barnstein B, Oskeritz , Gomez G. Mast cell homeostasis: a fundamental aspect of allergic disease. Crit. Rev. Immunol., 2007, 27: 15-32]. It is believed that a prerequisite for the development of AD is a genetic predisposition to the disease. In particular, it is known that in 15-20% of patients with AD, a gene is encoded that encodes the most important skin barrier protein, filagrin. This mutation indirectly confirms the genetic predisposition to blood pressure, which can be inherited [Oyoshi MK, Murphy G.F., Geha R.S. Filaggrin deficient mice exhibit Th17-dominanted skin inflammation and permissiveness to epicutaneous sensitization with protein antigen. J. Allergy Clin. Immunol., 2009, 124: 485-493]. In the acute phase of blood pressure, its indicators are cytokines, IL-4, IL-5, and IL-13 secreted by Th2 skin cells. In the chronic phase, Th1 and Th17 cells become dominant along with topically released interferon-gamma. With regard to the causes of blood pressure, various hypotheses are expressed, the cause may be endogenous, genetically determined factors, as well as exogenous, associated with the external environment. One of the possible mechanisms is considered to be a violation of the balance of redox homeostasis of the skin. An inflammatory reaction, including during blood pressure, can be induced as a result of oxidative stress, accompanied by the generation of reactive oxygen species (ROS), free radicals and nitric oxide, as damaging factors. Inflammation is a complex biological response of tissues to such factors. In patients with AD, the profile of urinary markers of oxidative stress changes significantly, including an increase in the levels of 8-hydroxydeoxyganidine nitrite / nitrate, selenium, suggesting a worsening of redox system homeostasis. With exacerbations of blood pressure, there is a noticeable increase in the level of lipid peroxides, the marker of which is an increase in the concentration of malondialdehyde (MDA), a decrease in the level of antioxidant vitamins A, C and E, an increase in the oxidation products of bilirubin [Amin M.N., Liza K.F., Sarwar M .S. et al. Effect of lipid peroxidation, antioxidants, macro minerals and trace elements on eczema. Arch. Derm. Res., 2015, 307 (7): 617-623; Tsukahara H., Shibata R., Ohta N. et al. High levels of urinary pentosidine, an advanced glycation end product, in children with acute exacerbation of atopic dermatitis: relationship with oxidative stress. Metabolism: Clinical and Experimental, 2003, 52 (12): 1601-1605]. It has been shown that the antioxidant enzyme, hemoxygenase, slows the development of blood pressure in humans, including experimental blood pressure in mice [Kirino M., Kirino Y., Takeno M. et al. Heme oxygenase 1 attenuates the development of atopic dermatitis-like lesions in mice: implications for human disease. J. Allergy Clin. Immunol., 2008, 122 (2): 290. e8-297. E8].

Treatment of blood pressure is often a big problem and this forces us to look for new therapeutic approaches. The traditional basis for the treatment of blood pressure, especially with exacerbations, are topical glucocorticosteroids (GCS) and, more rarely, topical calcineurin inhibitors, non-steroidal drugs with anti-inflammatory and immunosuppressive effects. However, prolonged use of topical corticosteroids may suppress adrenal cortex function and develop skin atrophy due to suppression of collagen synthesis and delayed fibroblast proliferation.

Therapy with systemic corticosteroids is indicated only in severe cases (psoriasis), but in this case, their use should be limited in time due to possible side effects. Corticosteroids induce lysis of certain subtypes of T cells and also block the transcription of IL-1, IL-6 TNFα genes in macrophages. The most well-known systemic drugs for suppressing inflammation: prednisone, methylprednisolone, dexamethasone, methotrexate. Other drugs in this class, especially immunosuppressants (more than 30 drugs are known), have even greater restrictions on regular use. These include cyclosporine (Sandimun), interferon-gamma, Cellsept, tazathioprine (Imuran) and several others [J. Am. Acad. Dermatol., 2005; 52: 156; J Am Acad Dermatol. 2004; 50: 391-404]. Cyclosporin blocks the transcription of the cytokine genes IL-2, IL-4, IFN-α and IL-2R, thereby inhibiting the production of cytokines themselves, which cause the induction, activation and differentiation of T cells, elements of the inflammatory response. The use of these drugs causes prolonged use of drug resistance, and a variety of side effects with complications.

It has been found that in patients with AD, the profile of urinary markers of oxidative stress significantly changes, including an increase in the levels of 8-hydroxydeoxyguanidine, nitrite / nitrate, selenium, suggesting a deterioration in homeostasis of the redox system. The reason for its occurrence, in addition to genetic factors, can be xenobiotics: nitrogen oxides, formaldehyde, dust nanoparticles, car exhausts, cigarette smoke, ultraviolet radiation. If the protective threshold of the redox system of the skin is exceeded, its homeostasis may be disrupted with the start of the inflammatory process such as blood pressure. Oxidative stress induces a complex set of signaling events (Xu Y., Shao Y., Voorhees JJ, Fisher GJ Oxidative inhibition of receptor-type protein-tyrosine phosphatase k by ultraviolet irradiation activates epidermal growth factor receptor in human keratinocytes. J. Biol. Chem. , 2006, 281: 27389-27397), which, in particular, lead to increased production of matrix metalloproteinases that destroy the collagen extracellular matrix of the dermis. In this regard, the role of antioxidants may be crucial in controlling the development of blood pressure. It has been shown that antioxidant therapy with markedly improves the skin condition in patients with mild to moderate symptoms of AD in double-blind randomized and placebo-controlled trials [Jaffray F., Faghihi G., Mokhtarian A., Hosseini S.M. Effects of oral vitamin E on treatment of atopic dermatitis: A randomized controlled trial. J. Res. Med. Sci., 2015, 20 (11): 1053-1057]. [Kirino M., Kirino Y., Takeno M. et al. Heme oxygenase 1 attenuates the development of atopic dermatitis-like lesions in mice: implications for human disease. J. Allergy Clin. Immunol., 2008, 122 (2): 290. e8-297. E8].

As the strongest electron acceptor, fullerene C ₆₀ exhibits high antioxidant activity and is able to effectively inactivate all ROS forms and free radicals in vivo [Krustic PJ, Wasserman E., Keizer PN Radical reactions of C ₆₀ . Science, 1991, 254: 1183-1185; Gharbi N., Pressac M., Hadchouel M., Szwarc H., Wilson SR, Moussa F. [60] Fullerene is a powerful antioxidant in vivo with no acute or subacute toxicity. Nano Letters, 2005, 5 (12): 2578-2585]. Such activity suggests that fullerene is a promising therapeutic agent for the treatment of diseases associated with oxidative stress. Significant anti-inflammatory activity of various forms of C ₆₀ (fullerenol, cationic amino acid derivatives of C ₆₀ on a model of type 4 allergy (delayed-type hypersensitivity, HRT) has been shown [Dragojevic-Simic V., Jacevic V., Dobric S. et al. Anti-inflammatory activity of fullerenol C60 (OH) 24 nanoparticles in a model of acute inflammation in rats. Digest J. Nanomater. Biostr., 2011, 6 (2): 819-827; Bashkatova EH, Andreev S.M., Shershakova N.N. ., Babakhin AA Shilovsky IP, KHaitov Study MR modulating activity of fullerene derivatives C ₆₀ on delayed type hypersensitivity reaction. The physiology and pathology of immune System 2012, №2, 17-27]. Poligidroksifulleren (C ₆₀ (OH) _n and N-etilpoliaminofulleren (C ₆₀ (NHCH ₂ CH _{3) n} inhibited in vitro IgE-dependent mast cell degranulation, cytokine secretion, histamine and prostaglandins in response to allergen stimulation [Magoulas G.E., Garnelis T., Athanassopoulos C.M et al. Synthesis and anti-oxidative / anti-inflammatory activity of novel fullerene-polyamine conjugates. Tetrahedron, 2012, 68 (35): 7041-7049].

The invention is known (US 7947262 B2), where water-soluble C ₄₀ , C ₅₀ , C ₆₀ or C ₇₀ fullerene, modifications by covalent attachment of one or more fragments selected from the group consisting of pyridine, hydroxyl, cyclodextrin, polyvinylpyrrolidone, bis- (monosuccinimide- , p, p'-bis (2-amino-ethyl) diphenyl, polyamines and N-ethylpolyamines, are supposed to be used to treat diseases mediated by the reaction of mast cells and peripheral blood basophils in response to an allergen. A very wide range of diseases from allergy to cardiac istyh. unmodified fullerene C ₆₀ was not used for these purposes.

The invention is known (WO 2009145982), where compounds of various classes with antioxidant properties are claimed for treating damaged skin, including lipophilic cations aimed at transport to mitochondria, keratinocytes and skin fibroblasts. Vitamin E, ascorbic acid, lipoic acids, acetyl-cysteine and other classes of substances, including chemical derivatives of fullerene, are also used as antioxidants.

The invention is known (US 20110251158), where fullerene compounds are claimed as anti-inflammatory drugs for the treatment of vascular atherosclerosis. The patent claims a wide range of C ₆₀ fullerene derivatives with the formula Zm-F-Yn, where F is fullerene. However, unmodified fullerene C _{60 is} not declared for these purposes.

Describes compositions designed to smooth wrinkles and improve skin condition (WO 200913426 A1), which is a solution of fullerenes C ₃₂ , C ₄₄ , C ₆₀ , C ₇₀ , C ₇₆ , C ₉₀ , C ₉₆ and others in oil.

Describes agents for the treatment of inflammatory diseases (US 8680125 B2), which are chemically modified fullerenes, consisting of C ₇₀ fullerene, to which are polyadditioned: glycolic acid, inositol, phenylpropionic acid, triazole, malonic acid amide. However, unmodified C ₆₀ fullerene is not declared for these purposes.

A method for treating itchy skin is described (patent WO 2009114087), where the active ingredients are fullerene derivatives of the formula C _p or X @ C _P , where the fullerene has substituents; C _p is fullerene with the number p of carbon atoms (from 60 to 200), and X @ C _P is endofullerene with group X (metal) inside the fullerene cavity. Unmodified fullerene C _{60 is} not declared for these purposes.

A composition is described comprising solubilized fullerene as a component of cosmetics (JP 2005060380). Fullerene C ₆₀ or C _{70 is} solubilized in water or aqueous media with acrylic acid, esters of acrylic acid, homopolymers.

Known antioxidant composition intended for cosmetic products for external use only, containing oxygen derivatives of fullerene C ₆₀ and C ₇₀ and fullerenes in clathrate form (with cyclodextrin, crown ether, organic polymer)) (US 20060134095 A1), as well as included components with antioxidant activity (e.g., SOD, tryptophan, quercetin, polyphenol, etc.).

A known method (patent US 20110021630 A1) for the treatment of wound lesions using the compositions described in patent WO 2009114087.

Known invention (WO 2006053839 Al), intended for use in the field of perfumes, cosmetics and / or dermatological agents, where the active ingredient is fullerene derivatives of the general formula F (X) m (-YZ) n and their salts (where F is fullerene; X = —OH, —NH ₂ , COO, —SO ₃ , —PO ₃ , —O — CH ₂ , —CH ₂ , —NH +, NH ₃ ⁺ , H ⁺ , etc .; Y = AB, where A: CH ₂ -, -O-, -S-, -NHCONH-, or -NHCO-, and B: RO [Si (Me) 2-O-, -alkyl (divalent radical), C _6-40 aryl (divalent radical) , C _7-60 aryl or alkyl alkylaryl (divalent radical), etc. Z = CD, where C = R, R-Ar, Ar or Ar-R). The purpose of the invention is to increase storage stability, increase oxidation stability and reduce the allergic effects of perfumes.

A method for producing pharmaceutical compositions based on fullerene derivatives with the formula C ₆₀ (H) ₃ {NH (CH ₂ ) _n COOH} ₃ ⋅ xH ₂ O, where C ₆₀ is fullerene, n = 5, 6, 7, x = 8- 10 (patent WO 2012105873 A1) having activity against herpes virus, influenza viruses of various nature, HIV, as well as antitumor and antiporiatic activity.

Compositions for use as medicaments for the treatment of skin diseases or tumors under radiofrequency or microwave irradiation are described (CN 103191427 A). The active ingredient is the polymer derivatives of fullerene: fullerene-polyethyleneimine, fullerene-polyethylene glycol fullerene-hyaluronic acid. Unmodified fullerene C _{60 is} not declared for these purposes.

The closest set of essential features to the claimed one is a cosmetic composition comprising fullerene bonded to porphyrin or water-soluble polymers, polyvinylpyrrolidone, PEG, polypropylene glycol, vinylpyrrolidone copolymers (WO 2006001784 A1 - Cosmetic composition containing fullerene clusters). The composition is proposed to prevent or reduce the processes of oxidation and damage to healthy skin in the skin under the influence of free radicals.

However, in the invention, fullerene, for conversion to a soluble form, is first dissolved in toxic solvents, benzene or toluene, and then complexed with the polymer. The authors indicate that in an alternative form, fullerene crystals are simply crushed and introduced into the composition, however, in this form, fullerene is biologically inaccessible due to its complete insolubility in the aqueous (biological) medium.

The task of the invention claimed by us is to create an ointment composition containing a non-steroidal active substance, fullerene in a water-soluble form, having a therapeutic effect against atopic dermatitis. The following is a detailed description of the preparation and use of the drug.

The technical result of the claimed invention is to reduce the inflammatory leukocyte infiltration of the epidermis, associated with the therapeutic effect of the ointment.

To obtain the composition, a freshly prepared aqueous solution of fullerene (VRF) is used, obtained by the method described in patent RU 2548971. Non-toxic components - petrolatum and sucrose palmitate (emulsifier and stabilizer of the composition) are used as the basis for the ointment, and parabens are used as preservatives. Using the optimal ratios of these components, the ointment produced has a homogeneous, odorless, milky color structure and is easily applied to the skin (Fig. 1). The pH of the ointment is in the range of 6-6.5. The ratio of components has a strong influence on its rheological properties, dynamic viscosity, stability and application ability.

The therapeutic effect of the ointment was evaluated by measuring serum IgE antibody concentrations, cytokine concentrations, expression of filagrin (FLG), IL-13, Foxp3 and TNFα genes, as well as histological analysis of skin samples. The level of specific IgE in serum was one of the main indicators of the intensity of development in mice of an allergenic response. Its decrease, as can be seen in Fig. 3, indicates the presence of an antiallergic effect in the preparation of ointment. It was found that the level of IL-4 in the supernatants of stimulated splenocytes of mice receiving fullerene was significantly lower relative to the blood pressure model. A similar pattern was observed when determining the level of IL-5 and IL-13 in the supernatants of OA-stimulated mouse splenocytes. A clear decrease in these levels was observed only in the C60 20 group (20 μg / mouse). An upward trend in IFN-γ and IL-12 was observed in mice treated with C ₆₀ fullerene at a dose of 20 μg / mouse, indicating a change in the polarization of the immune response from Th2 (allergenic) to Th1. Analysis of a regulatory cell marker, FoxP3, aimed at reducing the allergic response through the expression of IL-10, showed an increase in this production. An increase in Foxp3 may indicate an increase in the number of regulatory cells, which may lead to the development of remission of an allergic disease. The ability of fullerene to influence the expression of this marker can be considered as a key point in the mechanism of its therapeutic action. The same can be attributed to the stimulation of expression of filagrin (> 2 times), an important protective component of the skin, when exposed to fullerene (Fig. 4). Thus, the application of ointment with fullerene at a dose of 20 μg C ₆₀ / mouse leads to a noticeable decrease in the allergic immune response.

A histological microscopic examination of skin sections (Fig. 5-7) confirmed the findings of immunochemical analysis. The regeneration process is clearly observed when comparing figures 5 (blood pressure model) and 6 (after treating the inflamed skin with ointment with fullerene). Visually, the image of the skin section in Fig. 6 is very similar to the image of the skin section in Figure 7 taken from the intact region. A pathomorphological examination of the skin also revealed a striking contrast in detail between the sample “blood pressure model” (Fig. 5) and the sample treated with ointment (Fig. 6). In the first case, pronounced inflammatory infiltration of the epidermis and dermis with leukocytes, including eosinophils, with necrotic areas and scabs from keratinous masses was detected. In the second case (Fig. 6), there was a clear decrease in necrotization and a decrease in inflammatory leukocyte infiltration, obviously associated with the therapeutic effect of the ointment. It should be noted that the area of the inflamed area of the skin was about 6 cm ² and the optimal dose for the active substance, C ₆₀ fullerene, was 20 μg. Other doses used gave the worst result. Based on these figures, the optimal dose per 1 cm ^{2 of} inflamed skin is 3-4 μg, or 66 mg of ointment / cm ^{2 of} skin with a content of 50 μg C ₆₀ / g of ointment.

Brief Description of the Drawings

FIG. 1. Sample composition (ointment) according to example 1.

FIG. 2. A photo of an ointment sample deposited on a glass plate under a microscope (backlight up). Magnification x140.

FIG. 3. Serum anti-OA IgE antibody levels.

FIG. 4. Evaluation of immunological changes in mice with experimental blood pressure after treatment with an ointment containing fullerene (20 μg / mouse).

FIG. 5. Histological examination: sample "model of blood pressure."

FIG. 6. Histological examination: skin sample after ointment therapy

7. Histological examination: sample of intact skin.

Example 1. Obtaining a composition

An aqueous solution of fullerene, VRF, was mixed with petrolatum as a base, and then sucrose palmitate, nipazole and nipagin were added in a weight ratio of 4: 3.78: 2.2: 0.001: 0.001, respectively, sucrose palmitate (emulsifier and stabilizer composition) in the ratio 4: 3: 2 (weight), respectively. The resulting mixture was vigorously mixed on a ULTRA-TURRAX T25 digital homogenizer (up to 25,000 rpm, IKA) without heating for 20 minutes, controlling the temperature (not higher than 40 ° C). After homogenization, samples were taken for analysis for foreign inclusions by microscopy (a biological microscope equipped with an MOB-1 ocular micrometer with an eyepiece magnification of 15x and an 8x objective), and the appearance of the samples was evaluated visually in daylight, color was measured in reflected light against a white background. The pH value was measured at room temperature using a SevenEasy pH meter (Mettler Toledo). For analysis of dynamic viscosity, a Brookfield RVDV2T rotational viscometer was used. The analysis of the content of fullerene C ₆₀ was carried out spectrophotometrically, pre-extracting fullerene, at 340 nm (mol. Extinction 50200). The obtained values of the ointment: pH 6.1, viscosity 11.3 pz, fullerene content 52 μg / g, odorless, milky color with a cream tint (Fig. 1). The homogeneity of ointments is determined microscopically (Fig. 2).

In a similar way, samples of ointments with different fullerene concentrations were obtained by changing the introduced fraction of a solution of fullerene, VRF. Thus, samples of ointment with a content of C ₆₀ were obtained: from 20 to 120 μg / g. In a similar way, samples of ointments with different fullerene concentrations were obtained by changing the concentration of fullerene in the VRF solution added to the composition (more concentrated VRF solutions are obtained by evaporation in vacuum). Thus, samples of ointment with a content of C ₆₀ were obtained: from 50 to 120 μg / g (0.005-0.012%).

Example 2. Ointment

The composition of the ointment,% (wt.): Fullerene C ₆₀ 0.005 Water 40.0 Sucrose palmitate 21.795 Petroleum jelly 37.75 Nipazole 0.1 Nipagin 0.1

Example 3. Modeling blood pressure and method of therapeutic effect

The sensitization of mice was performed with the model allergen ovalbumin (Sigma). A 1 × 1 cm sterile gauze impregnated with a 0.1% OA solution was applied to the pre-shaved area of the back of mice, securing it with a special material (Bioclusive, Johnson and Johnson Medical Limited), which prevents drying. The area of the skin subjected to OA sensitization was ~ 6 cm ² . The allergen was applied for 7 days, after which the dressings were removed. After two weeks, the 7-day application of 0.1% OA was repeated two more times at a two-week interval (AD model group). Analysis was carried out at the end of the period of the third application. The negative controls were mice that received epidermal phosphate buffered saline (PBS) instead of OA.

The ointment was applied to damaged areas of the skin with a concentration of fullerene of 20 μg / mouse ("C60 20 ointment") was carried out 5 times at 2-week intervals.

Analysis of the level of specific antibodies and cytokines

The levels of specific IgE antibodies in individual sera of mice were tested according to the manufacturer's instructions ("BD", USA). For sorption, OA (10 μg / ml) in PBS was used. Quantitative determination of cytokines in supernatants of stimulated splenocytes was performed by ELISA using kits from BD OptEIA ™ (USA) in accordance with the manufacturer's guidelines. The concentration of cytokines was determined by the calibration curve obtained by titration of the standard samples of mouse interleukins included in the sets. The results are presented in fig. 3 (analysis of IgE antibodies) and in qualitative terms in Fig. four.

Analysis of the expression of cytokines IL-13, Foxp3, FLG and TNFα

The polymerase chain reaction was carried out using a Syntol reagent kit (Russia). Accounting and analysis of the results was carried out using the software for the IQ5 Bio-Rad device according to the manufacturer's instructions. CDNA samples were normalized to the mHPRT gene. The results are shown in FIG. four.

Histological analysis

Samples of excised skin were fixed in a 10% buffered formalin solution (pH 6.8–7.2). Dehydration, sample transfer and paraffin impregnation were carried out in an automatic type processor SLEE MAINZ (Germany), paraffin was filled using a SLEE MAINZ filling station (Germany). Microtomination of paraffin blocks to obtain sections with a thickness of 4 μm was carried out using an automated rotational microtome Finesse E + (Finland), histological sections stained using hematoxylin and eosin according to the generally accepted method were enclosed in a mounting medium under coverslips to obtain constant micropreparations. Microscopic analysis of histological preparations of the skin of mice of different groups and photographing was performed using a Leica DM2000 microscope with a DFC295 camera (Leica, Germany).

Claims (1)

Composition for the treatment of atopic dermatitis in the form of an ointment, comprising: an aqueous solution of fullerene C ₆₀ , petrolatum, sucrose palmitate, as well as parabens - nipazole and nipagin in a weight ratio of 4: 3.78: 2.2: 0.001: 0.001, with the final content fullerene in the composition is from 0.005 to 0.012 wt. %

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