RU2641091C1 - Application of fullerene c60 aqueous solution as therapeutic agent at atopic dermatitis disease - Google Patents

Abstract

FIELD: pharmacology.SUBSTANCE: invention is application of a therapeutic agent for parenteral administration in atopic dermatitis, which is a water-salt solution consisting of fullerene C60, pluronic F-127 and physiological saline, where the components in the agent are in a certain ratio per ml of solution.EFFECT: reduction of allergic inflammation, skin regeneration improvement.1 tbl, 5 dwg

Description

The invention relates to pharmacology and medicine, mainly to allergology, dermatology, and can be used to treat allergic dermatoses and other diseases associated with an allergic reaction.

The treatment of blood pressure often has great difficulties and urgently needs to develop new approaches. The traditional basis of drug treatment of atopic dermatitis, especially with exacerbations, is 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. The most well-known calcineurin inhibitors are tacrolimus and pimecrolimus, which block indirectly, via signaling pathways, the production of pro-inflammatory cytokines [Sidbury R, Hanifin JM. Systemic therapy of atopic dermatitis. Clin Exp Dermatol. 2000, 25: 559-66]. However, despite their high activity, comprehensive data on their long-term clinical safety have not yet been submitted [Kutasevich Y.F., Umanets TR The efficacy and safety of topical calcineurin inhibitors in pediatrics. http://www.umj.com.ua/wp/wp-content/uploads/2014/06/4255.pdf]. 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.

In recent years, monoclonal antibodies against the cytokine IL-4 and its receptors, immunoglobulin E (IgE), activated T and B cells have also become available as drugs [Yalcin A.D. An overview of the effects of anti-IgE therapies, "Medical Science Monitor, 2014, 20: 1691-1699; Ganzetti G., Campanati A., Molinelli E., Offidani A. Biologic therapy in inflammatory and immunomediated skin diseases: safety profile Current Drug Safety, 2015, 10 (999): 1-10]. However, the use of such drugs for many groups of patients is limited due to their very high cost. In addition, none of these drugs is not yet recommended for the treatment of blood pressure, in view of insufficient data on their effectiveness and safety.

Patients with proven sensitization to certain groups of allergen may carry out allergen-specific immunotherapy (ASIT). The effectiveness of existing treatment methods has been proven by clinical studies, however, at present, there is a tendency to develop severe, continuously recurring forms of blood pressure that are resistant to standard therapy.

On the other hand, fullerene C ₆₀ is a promising substance for high-tech materials and new drugs. After the discovery of a simple method for its preparation, multiple fullerene derivatives were synthesized, many of which have obvious biological activity [Kratschmer W., Lamb LD, Fostiropoulos K., Huffman DR Solid C60: a new form of carbon. Nature; 1990, 347: 354-356; Bosi, S., T. Da Da Ros, G. Spalluto, et al. Fullerene derivatives: an attractive tool for biological applications. Eur. J. Med. Chem., 2003, 38: 913-923; Piotrovsky L.B. Fullerenes in Biology / LB Piotrovsky, O.I. Kiselev. SPb .: Rostok Publishing House LLC, 2006. 336 p.]. C ₆₀ is the only molecular form of pure carbon having the form of an ideal spheroid with 60 atoms. Three important properties of the unique action of fullerene are its hydrophobicity, electron-acceptor Activity polarizability and light particles, is defined as its intramolecular interaction and facilitated the transfer of electrons from donor molecules. As a strong electron acceptor, C ₆₀ exhibits high antioxidant activity and capable of in vivo and in vitro and effectively activate all forms of ROS and free radicals [Krustic PJ, Wasserman EM, 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 may be a promising therapeutic agent for the treatment of diseases associated with oxidative stress. An important property of fullerene is its low toxicity and the ability to use different routes of administration. So, long-term (6 years) experiments on rats showed that fullerene, dissolved in olive oil, does not exhibit chronic toxicity, moreover, it significantly extends the life time of animals. The data showed that its effect is apparently associated with the leveling of oxidative stress [Baati T., Bourasset F., Gharbi N., Njim L. et al. The prolongation of the lifespan of rats by repeated oral administration of [60] fullerene. Biomaterials, 2012, 33: 4936-4946]. Significant anti-inflammatory activity of various forms of C ₆₀ (fullerenol C ₆₀ (OH) ₂₄ , cationic amino acid derivatives C ₆₀ ) was also shown in the model of foot and mouse edema (delayed-type hypersensitivity reaction model, HRT) caused by macromolecular agents [Dragojevic-Simic V. , Jacevic V., Dobric S. et al. Anti-inflammatory activity of fullerenol C ₆₀ (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 A.A., Shilovsky I.P., Khaitov M.R. Study of the modulating activity of C ₆₀ fullerene derivatives on a delayed-type hypersensitivity reaction. Physiology and pathology of the immune system, 2012, No. 2, 17-27].

With regard to the antiallergic effects of fullerene, it has been shown that polyhydroxyfullerene (C ₆₀ (OH) _n and N-ethylpolyaminofullerene (C ₆₀ (NHCH ₂ CH ₃ ) _n ) inhibited in vitro IgE-dependent mast cell degranulation, secretion of cytokines and prostaglandins in response to allergen stimulation. The authors suggested that this effect is associated with a decrease in the ROS level in cells, i.e., blocking of oxidative stress during an allergic reaction due to the antioxidant activity of fullerene [Magoulas G.E., Garnelis T., Athanassopoulos C.M et al. Synthesis and anti -oxidative / anti-inflammatory activity of novel fullerene-polyamine conjug ates. Tetrahedron, 2012, 68 (35): 7041-7049]. When they were incubated with mast cells and human blood basophils, they suppressed their reaction to the allergen, and in the anaphylaxis model they slowed down the release of histamine. It was also noted that such derivatives inhibit the production of pro-inflammatory cytokines whose secretion is mediated by tumor necrosis factor (TNF-α) in synovial fibroblasts, lymphocytes and macrophages, and it is obviously a promising drug for the treatment of arthritis [Park E. - J., Kim N., Kim Y., Yi J ., Choi K., and Park K. Carbon fullerenes (C60s) can induce inflammatory responses in the lung of mice. Toxicology and Applied Pharmacology, 2010, 244 (2): 226-233].

Known invention (US 7947262 B2), where a water-soluble C ₄₀ , C ₅₀ , C ₆₀ or C ₇₀ fullerene modified by covalent addition 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 basophils of peripheral blood in response to an allergen. A very wide range of diseases from allergy to cardiac -so udistyh. However, 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 C60 fullerene derivatives with the formula Zm-F-Yn, where F is fullerene. However, unmodified fullerene C _{60 is} not declared for these purposes.

A composition is described for smoothing wrinkles and improving the condition of the skin (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. However, the compositions presented are non-aqueous and cannot be administered parenterally.

Thus, experimental data indicate clear antiallergic properties of fullerene. However, fullerene is not yet used in clinical practice. One of the reasons is the commercial inaccessibility of affordable and non-toxic water-soluble forms of fullerene, chemically unmodified. Another reason is the lack of results on long-term comprehensive clinical trials of fullerene preparations, the lack of knowledge about the possible ways of its metabolism and excretion. The very low solubility of fullerene C ₆₀ in water (<10 ^-11 g / l) limits the possibility of its direct use for biomedical research. Moreover, chemical modification, the introduction of substituents in the nucleus of fullerene often reduces its antioxidant activity due to a violation of its electronic structure (reducing the level of electron delocalization).

Earlier, we described a simple way to obtain a stable aqueous solution (colloidal solution or dispersion) of C ₆₀ fullerene (VRF), which is toxicologically suitable for biomedical use. The method is based on an exhaustive dialysis of a solution of C ₆₀ in a mixture of water-N-methylpyrrolidone (MP) against water [Andreev S. M., Purgina D. D., Bashkatova E. N., Garshev A. V., Maerle A. V. ., Khaitov M.R. An effective way to obtain aqueous nanodispersions of fullerene. S. 60. Russian Nanotechnology, 2014, No. 7-8: 11-17; Patent RU 2548971 "Method for the production of aqueous nanodispersions of fullerene"]. VRF is an opalescent yellow-brown solution with a concentration of fullerene particles of 130-150 mg / l, has characteristic absorption maxima at 220, 265 and 345 nm, a particle size of 60-80 nm, and a surface charge (z-potential) of ξ = -25-30 mV. In these parameters, the WFD dispersion has similar characteristics with the dispersions obtained by the common toluene-ultrasonic method [Andrievsky GV, Kosevich MV, Vovk OM, Shelkovsky VS, Vashcenko LA Chem. Soc. 1995, 12: 1281-1282], but with one significant difference. HMF nanoparticles contain MP molecules that form a donor – acceptor complex with fullerene, which increases their hydrophilicity and stability in an aqueous medium.

It has been shown that VRF significantly reduces skin inflammation caused by the allergenic protein ovalbumin, provides a decrease in the level of specific IgE and Th2 cytokines (IL-4, IL-5) [Shershakova N, Baraboshkina E, Andreev S, Purgina D, Struchkova I, Kamyshnikov O , Nikonova A, Khaitov M Anti-inflammatory effect of fullerene C ₆₀ in a mice model of atopic dermatitis. Journal of Nanobiotechnology, 2016, 14: 8 doi: 10.1186 / s12951-016-0159-z]. However, the fullerene particles in the WGF are dispersed in deionized / distilled water, i.e. in an environment that is non-isotonic with respect to the biological environment (blood, tissues). It is known from the literature that aqueous solutions (colloidal dispersions) of unmodified fullerene in the presence of salts are unstable. Thus, with the direct introduction of VRF into the bloodstream, due to the presence of salts, fullerene will precipitate. Therefore, it is impossible to use VRF as a systemic remedy, for example, for the treatment of allergic dermatitis (AD). Intramuscular administration of VRF is fraught with serious complications (infiltrate, abscess, phlegmon, etc.).

The essence of the present invention is to use for anti-allergic therapy, including blood pressure, a water-salt solution of fullerene C ₆₀ (HSRF), stable in a biological environment, having low hemolytic activity, and suitable for parenteral administration. The specified method is based on the introduction into VRF of a small additive of the biocompatible nonionic polymer pluronic F-127. In this case, the HRVF can be used in any form: either by skin application, for example, using medical dressings, healing bandages and wipes containing HCVF, or by oral administration, or by intravenous and intramuscular injection.

The technical result of the present invention is to reduce allergic inflammation, improve skin regeneration.

In vivo studies in mice (BALB / c) showed the presence of a marked anti-inflammatory effect in the HSRF. A visual assessment of the residual lesion of the skin showed that the wound healing process with his participation is approximately two times more intense than in the absence of therapy. According to the results of the analysis of the expression of factors such as IgE (Fig. 3), IL-4, IL-5, IL-12 (Fig. 4), IFN-γ (Fig. 5), Foxp3, the filaggrin gene and histology data (Fig. . 2) it was found that HRVF reduces allergic inflammation, stimulates the process of epithelialization of wounds and promotes normal regeneration

The technical result is achieved by using a water-salt solution of fullerene C ₆₀ , containing, in addition to the active substance - fullerene C ₆₀ , pluronic F-127, as a therapeutic agent for parenteral administration in diseases of atopic dermatitis. The ratio of Pluronic C ₆₀ to fullerene of 1: 1 or 1: 2, the solution is filtered through a 0.45 micron filter. The concentration of C ₆₀ in the solution is in the range of 70-120 μg / ml. Content in 1 ml of solution: fullerene C ₆₀ - 120 mcg, F-127 - 120 mcg, the rest is saline.

Below is a detailed description of the preparation and use of a water-salt solution of fullerene C ₆₀ .

A brief description of the drawings.

FIG. 1 UV View of the dispersion spectrum of fullerene C ₆₀ / pluronic F-127 / water. (C60: F-127 = 1: 1) diluted 10 times.

FIG. 2. Histological analysis of the skin: A - model of blood pressure; B - after intravenous administration of HRVF; B - after intramuscular injection of the HRF; G - intact mice.

FIG. 3. Change in the concentration of IgE antibodies against ovalbumin after the introduction of HRVF. 1 - model of blood pressure; 2 - C ₆₀ + lanolin; 3 - C _{60 v} / m; 4 - C60 iv; 5 - C ₆₀ and / g; 6 - PBS. * - significantly different from the group "model of blood pressure", p <0.05. Methods of administration: i / m - intramuscular, i / v - intravenous, and / g - oral. All groups reliably differ from the “intact” (PBS) group.

FIG. 4. Changes in concentrations a - IL-4; b - IL-5 and c - IL-12 after the introduction of the HRF. 1 - model of blood pressure; 2 - C _{60 v} / m; 3 - C60 in / in; 4 - intact. * - significantly different from the group "model of blood pressure", p <0.05. Methods of administration: i / m - intramuscular, i / v - intravenous.

FIG. 5. Changes in the concentration of interferon-gamma (IFNg). 1 - model of blood pressure; 2 - C ₆₀ + lanolin; 3 - C _{60 v} / m; 4 - C60 iv; 5 - C ₆₀ and / g; 6 - PBS. * - significantly different from the group "blood pressure model", p <0.05; ** - significantly different from the PBS group, p <0.05; x - significantly different from the group "C ₆₀ + lanolin", p <0.05.

1. Obtaining HRVF.

100 mg of crystalline fullerene C60 (99.9%) is dissolved in 125 ml of N-methylpyrrolidone (MP), the solution is stirred on a magnetic stirrer until the fullerene is completely dissolved. The resulting solution of C ₆₀ in a dark purple MP is filtered through a 3 μm glass fiber filter. Then, 250 ml of a 0.04% solution of pluronic F-127 in distilled water or in phosphate-buffered saline (PBS) is added to the filtrate with constant stirring. The color of the resulting solutions varies from dark violet to red-orange, and slight opalescence is also observed. The solution was stirred on a magnetic stirrer for about an hour and then subjected to exhaustive dialysis against physiological saline with a pH of 7.4. For dialysis, SpectraPor dialysis membranes with cutoff pores of 10 to 50 kDa are used. The result is a yellow-orange solution. To calculate the concentration of fullerene in the solution, UV-View spectrophotometry based on the molar extinction of fullerene C ₆₀ at 340 nm, ε ³⁴⁰ = 50200 (Fig. 1) is used. The resulting solution was filtered for sterility and poured into 1 ml ampoules of dark clear glass. Content in 1 ml of solution: fullerene C ₆₀ - 120 mcg, F-127 - 120 mcg, the rest is saline. The fullerene content together with F-127 can be changed (down) by diluting the resulting solution with saline for injection.

2. Modeling of blood pressure and the method of therapeutic administration of the drug.

Modeling of blood pressure was carried out by sensitizing mice with the allergen ovalbumin (OA) (Sigma). A 1 × 1 cm sterile gauze impregnated with a 0.1% OA solution was applied to a pre-shaved area of the back of mice, securing it with a special material (Bioclusive, Johnson), which prevents the patch from drying out. The allergen dressings were kept 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 (group “model”). Analysis was carried out at the end of the period of the third application. The negative controls were mice that were epidermally treated with FSB instead of OA (intact group). Intramuscular and intravenous (tail vein) injections were carried out between applications of OA at weekly intervals (5 injections of 5 μg C ₆₀ / mouse, or 50 μl of HRVF solution). At the end of the experiment, material was taken for PCR and histological analysis.

3. 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).

A pathomorphological study of the skin in a group of mice in which atopic dermatitis was modeled without specific treatment revealed the following changes characterizing this pathological process of dermatitis development: severe inflammatory leukocyte infiltration of the epidermis and skin dermis with polynuclear leukocytes, in which a large number of eosinophils, a specific component of allergic inflammation. Infiltration in the dermis was predominantly diffuse or streaky, often involving subcutaneous adipose tissue in the pathological inflammatory process. The skin epidermis in this group of animals over the areas of dermal inflammation was necrotic up to the dermis, and in the adjacent areas it was thickened mainly due to the prickly layer, an increase in the number of mitosis figures was noted in the cells of the basal layer as a sign of the regeneration process. A scab of keratin masses and degeneratively altered white blood cells formed over the epidermis. In addition to leukocyte infiltration of the skin dermis, proliferation of connective tissue fibroblast-like cellular elements occurred as a reaction in response to irritation in order to compensate for damaged connective tissue of the dermis. Also in the dermis, the development of destructive hemorrhages was noted, which looked like clusters of bright red erythrocytes, skin appendages - hair follicles and sebaceous glands in the skin dermis often reduced and disappeared. A micrograph of a histological preparation of the skin of the AD group (blood pressure model) is shown in FIG. 2A. After intramuscular administration of HRVF in mice, a decrease in the severity of polymorphonuclear leukocyte infiltration of the epidermis and dermis was noted in mice, however, the eosinophilic component of the infiltration was preserved. There was also a decrease in the manifestations of necrosis of the epidermis and dermis of the skin, a thickening of the epidermis due to the prickly layer was preserved, an increase in the number of mitosis figures was observed in the cells of the basal layer of the epidermis, which indicates a more pronounced development of regeneration compared to the AD model group. Destructive hemorrhages in the dermis of the skin in this group were detected less frequently. Moderate development of epidermal hyperkeratosis was noted as a manifestation of the compensatory mechanism of protection against damage. The proliferation of fibroblast-like cells of the connective tissue of the dermis was moderate (Fig. 2B). In the group receiving intravenous administration of HRVF, a more pronounced therapeutic effect was observed in comparison with the group of blood pressure, associated with a decrease in the severity of inflammatory leukocyte infiltration with a decrease in the number of eosinophils in the composition of the infiltrate. A decrease in epidermal necrosis and skin dermis was observed, and in the cells of the basal layer of the epidermis an increase in the number of mitosis figures, indicating a more pronounced skin regeneration (Fig. 2B). FIG. 2G reflects the normal structure of the skin of mice in the control group — intact animals. The epidermis is thin, only the basal, spiky and horny layer is clearly distinguishable in it. Subcutaneous fatty tissue is represented by fat cells with vessels and nerve fibers located between them. In this group of animals, there were no obvious signs of inflammation and necrosis; only a few animals met focal lymphocytic infiltrates, which, apparently, are the conditional physiological norm of BALB / c mice.

An important aspect reflecting a decrease in the inflammatory cellular response is a decrease in the concentration of IgE antibodies (Fig. 3) and the level of skin dermis eosinophil infiltration, which was observed in groups of mice treated with HRVF injections, and this correlated with a decrease in IL-5 expression (Fig. 4 ) In addition, analysis of the cytokine profile showed a significant change in the polarization of T-helper cells from the Th2 phenotype (fall of IL-4, IL-5 (Fig. 4)) to Th1 (growth of IFN-gamma (Fig. 5), IL-12 (Fig 4)) and stimulation of expression of filagrin, an important skin barrier factor, and the FoxP3 marker (Table 1), which plays an important role in maintaining immunological tolerance to an allergic attack. The summation of histological analysis indicators (using a point system) revealed that the introduction of HRVF reduces the manifestation of signs of inflammation by about 45% and 27%, in groups with intravenous and intramuscular administration, respectively.

Claims (4)

The use of a therapeutic agent for parenteral administration in atopic dermatitis, which is an aqueous salt solution consisting of fullerene C60, pluronic F-127 and physiological saline, where the components in the agent are in the following ratio per 1 ml of solution: Fullerene C60 120 mcg Pluronic F-127 120 mcg Saline solution

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