RU2794841C1 - Means for the treatment of atopic dermatitis - Google Patents

Abstract

FIELD: medicine; dermatology.

SUBSTANCE: invention can be used to treat skin papillomas. Histochrome is used as a treatment for atopic dermatitis, where histochrome is applied topically or administered intraperitoneally.

EFFECT: use of the invention makes it possible to eliminate epidermal skin lesions, reduce transdermal water loss and improve the hydration of the stratum corneum, thereby alleviating the clinical symptoms of atopic dermatitis, such as edema, erythema and dry skin by reducing the expression of pro-inflammatory cytokines interferon-γ (INF-γ), interleukin-4 (IL-4) and interleukin-13 (IL-13), which play a major role in maintaining allergic inflammation in the skin, and inhibition of inflammatory response-induced infiltration of mast cells in the affected skin under the action of histochrome.

1 cl, 7 dwg, 1 tbl, 6 ex

Description

The invention relates to medicine and the pharmaceutical industry and concerns the use of histochrome for the prevention and/or treatment of atopic dermatitis.

Atopic dermatitis (AD) is a skin disease characterized by chronic skin inflammation that leads to pruritus, erythema, skin barrier dysfunction, increased transepidermal water loss, and immune-redox disorders [Cork M.J. et al. J. Investig. Dermatol. 2009, 129, 1892-1908; Leung, D.Y.M. Atopic dermatitis: More than a rash. Ann. Allergy Asthma Immunol. 2018, 120, 555-556; Souto E.B. et al. Int. J. Mol. sci. 2019, 20, 5659]. AD is detected in all countries of the world, in representatives of both sexes and different age categories. The prevalence of AD has more than doubled over the past two decades. According to various authors, atopic dermatitis occupies from 20 to 40% in the structure of skin diseases. The incidence of AD among children in the world varies up to 10-20%, among adults - up to 3%.

The pathogenesis of atopic dermatitis remains unclear, but it is thought to be related to both genetic factors such as immune system dysfunction in which type 2 T-helper cells secrete excessive amounts of interleukin-4 (IL-4) and interleukin-13 (IL-13). 13), interferon-gamma (IFN-γ), and with environmental factors, for example, house dust mites [Yoon, H.J.; et al. Protective effect of diet supplemented with rice prolamin extract against DNCB-induced atopic dermatitis in BALB/c mice. BMC Complement. Altern. Med. 2015, 15, 353]. In addition, AD lesions are usually characterized by excessive infiltration of granular mast cells, which are activated by antigen-specific immunoglobulin E (IgE), and which are critical for the development of inflammatory skin diseases [Ku J. M. et al. The prevention of 2,4-dinitrochlorobenzene-induced inflammation in atopic dermatitis-like skin lesions in BALB/c mice by Jawoongo. BMC Complement. Altern. Med. 2018, 18, 215], and promote skin hypersensitivity reactions by releasing histamine [Klubal R. et al. The high-affinity receptor for IgE is the predominant IgE-binding structure in lesional skin of atopic dermatitis patients. J. Investig. Dermatol. 1997, 108, 336-342., Galli S. J., Tsai M. IgE and mast cells in allergic disease. Nat. Med. 2012, 18, 693-704.; Park S.J. et al. Effect of eriodictyol on the development of atopic dermatitis-like lesions in ICR mice. Biol. Pharm. Bull. 2013, 36, 1375-1379].

Typically, therapeutic strategies for AD include topical (topical) use of corticosteroids, calcineurin inhibitors, and T-cell inhibitors [Carr W.W. Topical calcineurin inhibitors for atopic dermatitis: Review and treatment recommendations. paediatr. Drugs 2013, 15, 303-310.; Papp, K. A.; Werfel, T.; Folster-Holst R. et al. Long-term control of atopic dermatitis with pimecrolimus cream 1% in infants and young children: A two-year study. J. Am. Acad. Dermatol. 2005, 52, 240-246.; Kang L.J. et al. 3'-Sialyllactose prebiotics prevents skin inflammation via regulatory T cell differentiation in atopic dermatitis mouse models. sci. Rep.2020, 10, 5603.]. However, long-term treatment with these drugs is known to cause side effects such as immunosuppression and epidermal barrier dysfunction. The use of systemic (injectable) corticosteroids is indicated only in severe cases of the disease, but the time of their use is limited due to possible side effects, since corticosteroids can induce T-cell lysis and block the transcription of anti-inflammatory cytokine genes IL-1, IL-6, TNFα.

The objective of the invention is to expand the arsenal of effective means for the prevention and treatment of atopic dermatitis.

The problem was solved by using histochrome as a means for the prevention and/or treatment of atopic dermatitis. In this case, histochrome is used topically and / or parenterally.

The technical result consists in the implementation of the specified purpose.

Histochrome® is a dosage form of an individual substance - a natural quinoid pigment of sea urchins echinochrome A (2,3,5,6,8-pentahydroxy-7-ethyl-1,4-naphthoquinone, state registration number P N002362 / 01-2003) . In the dosage form, a solution for intravenous administration of 10 mg / ml Histochrome® is used in cardiology for acute myocardial infarction (state registration number P N002363 / 01-2003) [RU 2137472 C1, 04/23/1999] and Histochrome® in the dosage form injection solution 0 ,2 mg/ml is used as an agent for the treatment of eye diseases (state registration number R N002363/02-2003) [RU 2134107 C1, 10.08.1999].

Known is the use of histochrome for the treatment of hemorrhagic stroke [RU 2266737 C1, 12/27/2005], as well as the use as a treatment for cerebral ischemia in acute cerebrovascular accidents [RU 2625740 C1, 07/18/2017].

It is known to use histochrome as a diuretic [RU 2408367 C1, 01/10/2011], as an antiviral agent against tick-borne encephalitis and herpes simplex type I [RU 2697887 C1, 08/21/2019], as a neuroprotective agent [RU 2765956 C1, 07.02 .2022].

An indication of the use of histochrome for the prevention and/or treatment of atopic dermatitis was not found in the available patent and other scientific and technical literature.

This new function of histochrome does not follow with obviousness from its known properties and composition.

The authors first discovered the ability of histochrome to reduce the expression of pro-inflammatory cytokines interferon-γ (IFN-γ), interleukin-4 (IL-4) and interleukin-13 (IL-13), which play a major role in maintaining allergic inflammation in the skin. It has been shown that histochrome has an anti-inflammatory effect on 2,4-dinitrochlorobenzene-induced atopic dermatitis-like lesions in the NC/Nga mouse model and has the ability to inhibit inflammatory response-induced infiltration of mast cells in the affected skin. The use of Histochrome, topically and/or parenterally, effectively eliminates epidermal skin lesions, reduces transdermal water loss and improves stratum corneum hydration, thereby alleviating the clinical symptoms of atopic dermatitis, such as edema, erythema, and dry skin.

A study of the ability of histochrome to influence the main pathophysiological processes of atopic dermatitis in the skin of mice was carried out on mice of the inbred Nc/Nga line, bred in Japan at the University of Nagoya in 1957 and used as a model of human atopic dermatitis. Six week old male NC/Nga mice were purchased from Orient Bio (Daejeon, Korea). Mice were acclimatized at a temperature of 22±2°C and a humidity of 45±5% with alternating dark/light 12/12 hours for 1 week. The animal study was approved by the Inge University Animal Care and Use Committee.

Skin lesions in mice similar to atopic dermatitis were induced by topical application of 100 µl of 1% 2,4-dinitrochlorobenzene a (DNCB) dissolved in a mixture of olive oil and acetone (1:3) according to the scheme: three times a week for 2 weeks, after sensitization continued to apply 0.4% solution of DNCB three times a week for 5 weeks.

Histochrome was applied topically, applied to the damaged area of the skin of the mouse's back, or administered intraperitoneally, in which case the drug entered the systemic circulation.

Table 1 shows the designations of the experimental groups of animals and a description of the experiments.

Direct exposure of the etiologically significant allergen DNCB to the skin of mice activates antigen-specific cells that infiltrate the epidermis and carry IgE antibodies on their surface. The latter migrate to the lymph nodes, where they activate Th2 lymphocytes that secrete pro-inflammatory cytokines IL-4, IL-13, and IFN-γ, which play a major role in maintaining allergic inflammation in the skin.

Description of experimental research methods

Measurement of transepidermal water loss (TEWL) and stratum corneum hydration (HRH) levels

After sensitization of mice with 1% and 0.4% DNCB according to the above scheme, TWT and HRS were measured using Tewameter® sensors and a Cutometer® MP A 580 corneometer (Courage + Khazaka Electronic GmbH, Cologne, Germany) every 2 weeks. The measuring probe was placed on the middle area of the back skin of mice that were anesthetized by inhalation of isoflurane at a low concentration. TEWL levels were analyzed at the plateau of the estimated curve and HRM levels were taken as the average of the three measurements

Skin monitoring

The skin condition of the area of the back, which caused atopic dermatitis, was observed every 2 weeks, for this, photographs were taken using a dermatoscopy camera (DermLite™ DLCAM, DermLite, San Juan Capistrano, CA, USA). Mice were photographed under anesthesia, which was administered by short-term inhalation of isoflurane.

Histological analysis

To obtain dorsal skin samples, mice were euthanized using CO2 gas. Parts of dorsal skin biopsies were fixed in 4% paraformaldehyde and then transferred to 70% ethanol for 24 h at room temperature. The samples were embedded in paraffin and cut into 4 µm sections. Skin sections were stained with hematoxylin and eosin (H&E) and toluidine blue. Stained slides were examined using the NanoZoomer Digital Pathology slide scanner and HCImage software (Hamamatsu Photonics, Milan, Italy).

Western blotting

Epidermal dorsal skin samples were lysed with radioimmunoprecipitation assay buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% sodium deoxycholate, 1% Triton X-100, 2 mM EDTA, and 0.1% sodium lauryl sulfate with by addition of 200 mM phenylmethylsulfonylfluoride (PMSF) Protein concentration was determined by Bio-Rad DC protein assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA) Lysates were separated by electrophoresis in 8-15% sodium dodecyl sulfate (SDS) polyacrylamide gel -PAGE) and transferred to a nitrocellulose membrane (GE Healthcare, Chicago, IL, USA) for 100 min at 110 W and 4° C. The membranes were blocked with 5% skimmed milk in TBST buffer (20 mmol/L Tris-HCl, pH 7, 5, 50 mmol/l NaCl and 0.1% Tween 20) After blocking, the membranes were incubated overnight with primary antibodies diluted to 1:1000-1:5000 in 3% bovine serum albumin (BSA) in TBST. washings for 15 min membranes were incubated for 1 h with secondary antibodies labeled with horseradish peroxidase, diluted to 1:1000-1:5000 in 3% BSA. Protein bands were visualized with a chemiluminescence enhancement kit (Santa Cruz Biotechnology, Dallas, TX, USA) and observed with an AI 600 imager (GE Healthcare, Chicago, IL, USA).

Statistical analysis

All data are presented as the mean of at least three independent experiments. Statistical comparisons were analyzed using one-way analysis of variance (ANOVA) followed by Tukey's test of fair significance for multiple comparisons (GraphPad Prism 8.0, GraphPad Software Inc., San Diego, CA, USA). Statistical significance was set at p<0.05.

The invention is illustrated by the following figures:

- figure 1 presents the results of dermatoscopic observation of lesions of the skin of the dorsal surface of mice in six groups of animals during the entire experiment with a frequency of 2 weeks;

- figure 2 shows the level of immunoglobulin IgE in the serum of mice of the experimental groups;

- figure 3 shows the result of Western blot analysis of the anti-inflammatory effect of histochrome in skin lesions caused by DNCB; mean ± SEM of three independent experiments, *p<0.05;

- figure 4 shows the result of a histological analysis of a section of the skin of the back of a mouse, stained with toluidine blue - a reagent for mast cells, and a statistical analysis of the number of mast cells; mean ± SEM, *** p<0.001, scale bar = 100 μm;

- figure 5 presents the results of histometry and statistical analysis of the thickness of the epidermis in the skin of the back of mice; mean ± standard error of the mean, ***p<0.001, *p<0.05, scale bar = 100 µm;

- figure 6 presents the measurement data of the degree of transepidermal water loss and hydration of the stratum corneum in the skin of experimental mice; mean ± SEM (n=6), ***p<0.001, *p<0.05.

- figure 7 presents a diagram of the stages of development of atopic dermatitis initiated by 2,4-dinitrochlorobenzene in mice.

The invention is illustrated by the following examples.

Example 1

Changes in AD-like symptoms induced by DNCB in dorsal skin of a mouse model

The Nc/Nga mice were divided into six groups, which are described in Table 1. Every 2 weeks of the experiment, AD-like skin lesions were observed dermoscopically on the dorsal surface of the animals. 4 weeks after the creation of experimental blood pressure on the dorsal skin of mice of groups D, FB and IFB, mild edema, irritation, dryness and erythema were observed. After 6 weeks, more noticeable swelling, irritation, dryness and erythema appeared. In the G and IG groups and after 4 and 6 weeks, the skin condition was significantly improved compared to the FB and IFB groups. The results are presented on slides (figure 1).

Example 2

The effect of histochrome on the level of IgE immunoglobulin in the serum of experimental animals

Enzyme immunoassay (ELISA, ELISA) showed that groups G and IG had lower levels of IgE immunoglobulin in serum compared with group D (figure 2). These results indicate that histochrome, both topically and systemically (parenterally), reduces the pro-inflammatory response in atopic dermatitis.

Example 3

Anti-inflammatory effect of histochrome in skin lesions caused by DNCB

To test whether histochrome suppresses the expression of pro-inflammatory cytokines, immunoblot analysis of the expression of pro-inflammatory-associated proteins IFN-γ, IL-4, and IL-13 was used. Histochrome treatment showed an anti-inflammatory effect in the AD-induced mouse model (FIG. 3A). The relative intensities of the bands of the immune proteins IFN-γ, IL-4 and IL-13, estimated using the ImageJ image processing program, are presented in Figs. 3B-D. The levels of expression of IFN-γ, IL-4 and IL-13, normalized by the expressed reference protein β-actin, in group E were significantly increased compared to the control (group H), and in animals of groups D and IG, these indicators were significantly below the norm. Moreover, the effect of histochrome on the expression of IFN-γ was greater when the drug was administered intraperitoneally, compared with its local application. Thus, it was shown that histochrome exhibits an anti-inflammatory effect in mouse models with induced atopic dermatitis by reducing the level of expression of the pro-inflammatory cytokines IFN-γ, IL-4, and IL-13. It is known that a high level of IFN-γ expression is associated with the development of autoimmune diseases.

Example 4

Investigation of the effect of histochrome on skin infiltration by mast cells in AD-induced mice

Histological observation of sections stained with toluidine blue showed a decrease in skin infiltration by mast cells in groups G and IG compared with groups D, FB, IFB (Fig.4A). The number of infiltrated mast cells in stained skin sections of experimental animals was counted in five typical high magnification fields using a NanoZoomer digital scanner (n=6) and HCImage software (Hamamatsu Photonics, Milan, Italy) (FIG. 4B).

Example 5

Investigation of the effect of histochrome on epidermal damage When atopy occurs, the epidermis hardens and increases in thickness, which damages the skin and reduces its protective properties. Figure 5 presents the result of a comparative analysis of the thickness of the epidermis, measured using a digital scanner NanoZoomer. Histological studies of skin sections stained with hematoxylin and eosin (H&E, scale bar = 100 μm) showed that histochrome, both locally and intraperitoneally, reduces the thickness of the skin epidermis (Fig. 5A). The average values of the thickness of the epidermis of all groups of experimental animals are shown in Fig.5B. The presented data allow us to conclude that in atopic dermatitis, histochrome retains the protective functions of the skin, especially when administered intraperitoneally, and facilitates the treatment of symptoms of this disease.

Example 6

Study of the effect of histochrome on transepidermal water loss and stratum corneum hydration in mice with induced atopic dermatitis

The epidermis is a complex structure, its main component is the stratum corneum, which prevents the penetration of exogenous substances through the skin. Important for the integral assessment of the state of the epidermal barrier is the quantitative measurement of skin parameters, such as the loss of transepidermal water and the level of hydration of the stratum corneum. For this, non-invasive methods for studying the skin barrier in vivo were used, which are given in the description of experimental research methods.

Figure 6 presents data on the effect of histochrome on indicators of transepidermal water loss (TEWL, Fig.6A) and stratum corneum hydration (SCH, Fig.6B) in mice with induced atopic dermatitis. TEWL levels in the skin epidermis were assessed every 2 weeks using Tewameter® measurements. The SCH score was determined using Corneometer® measurements every 2 weeks of the experiment.

As can be seen from the graphs, transepidermal water loss and stratum corneum hydration were improved in the groups of mice treated with histochrome both locally and intraperitoneally. Although these indicators did not reach the baseline values, but throughout the experiment (6 weeks), the indicators of transepidermal water loss in groups D and IG were lower than in groups D, FB, IFB. Conversely, the indicators of stratum corneum hydration were higher in the D and IG groups than in the D, FB, and IFB groups. Thus, these results indicate the effective effect of histochrome on the protective functions of the skin in experimental atopic dermatitis.

Claims (1)

The use of histochrome as a treatment for atopic dermatitis, where histochrome is applied topically or administered intraperitoneally.

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