RU2790820C1 - Means for protecting the skin from photoaging - Google Patents

Abstract

FIELD: pharmacology; dermatology; anti-aging medicine.

SUBSTANCE: disclosed is the use of echinochrome A as an agent with inhibitory activity against matrix metalloproteinases 1 and 2 (MMP 1 and MMP 2), tryptase and chymase.

EFFECT: protection of skin against photoaging.

1 cl, 8 dwg, 7 ex

Description

The invention relates to the field of pharmacology, dermatology and anti-aging medicine, namely to the use of echinochrome A as an agent that protects the skin from photoaging.

Human skin is the largest and most complex organ of the human body, and acts as a barrier to protect the body from the harmful effects of both external and internal factors that lead to skin aging. Genetic factors and hormonal changes cause so-called chronological aging or natural aging. Along with chronological aging, solar UV exposure is recognized as an essential component of skin aging, which is defined as photoaging [Rittie, L.; Fisher, G.J. UV-light-induced signal cascades and skin aging. Aging. Res. Rev. 2002, 1, 705-720]. Skin exposed to UV radiation produces large amounts of reactive oxygen species (ROS), which trigger a complex array of cellular responses that result in oxidative damage. Thus, ROS-induced oxidative stress, especially when exposed to UV radiation, is an important factor modulating skin changes. The human body has several endogenous systems that eliminate oxidative stress, which are depleted with age and with constant exposure to UV radiation [Fisher G.J. et al. Pathophysiology of premature skin aging induced by ultraviolet light. NEngl. J. Med. 1997, 337, 1419-1428].

In order to find candidates for anti-aging skin, studies have been conducted of natural substances with antioxidant properties, including flavonoids, polyphenols, carotenoids, and vitamins C and E [Petruk G. et al. Antioxidants from Plants Protect against Skin Photoaging. Oxidative Med. cell. Longev. 2018, 2018, 1-11]. Known attempts of topical and oral application of plant extracts, plants such as soybeans, sea buckthorn, turmeric, green tea, grapes, soybeans, milk thistle, pomegranate, horsetail, high gastrodia, water lily.

However, the disadvantage of extracts of biologically active substances is the low content of target products, the presence of concomitant unidentified components, therefore, there is a danger of harmful substances entering the body. In addition, the bioavailability of active substances from extracts is not always satisfactory.

Vietnamese researchers, together with us, described extracts of quinoid pigments from Vietnamese sea urchins D. setosum and Tripneustes gratilla containing echinochrome A, which have antioxidant and antimicrobial activity, do not have irritating and allergic effects, which can be used in cosmeceuticals [Vo M. N. N. et al. Polyhydroxynaphthoquinone pigment from Vietnam sea urchins as a potential bioactive ingredient in cosmeceuticals. Natural Product Communications. 2020. Vol.15, N11. P. 1-8]. A moisturizing cream for protecting the skin from UV radiation, containing 0.5% of the crude pigment extract of sea urchins, is proposed.

The ability of these extracts to significantly inhibit the growth of the tyrosinase enzyme, which catalyzes the multistage reaction of the conversion of tyrosine into melanin, which plays an important biological role in protecting the body from harmful ultraviolet rays, has been shown. Melanin has antioxidant activity, scavenging free radicals generated by UV damage before skin cell DNA damage occurs. Therefore, the proposed skin care products with quinoid pigment components from sea urchins are more likely to be effective in whitening or correcting hyperpigmentation, which is of great importance, in particular, for the population of Southeast Asia. The article describes a test with a free radical diphenylpicrylhydrazine (DPPH), the results of which were used to evaluate the ability of the studied extracts to quench model free radicals. This reaction has nothing to do with UV-induced oxidative stress.

The test showed the antiradical activity of the studied extracts of sea urchins, but not antioxidant. This is because all test systems using stable free radicals (e.g., DPPH, ABTS, etc.) provide information only on the potential ability of compounds to interact with free radicals, and in many cases this ability does not necessarily reflect their antioxidant activity. In order to obtain information about the antioxidant activity of compounds, it is necessary to conduct studies on experimental models in relation to specific lipid, protein, high-molecular substrates, cells or organs in the presence of specific radicals, such as ROS, which are photoaging factors. In addition, the authors suggest using extracts of quinoid pigments topically, that is, applied to the skin, and it is known that natural antioxidants under certain conditions can exhibit prooxidant properties [Vardanyan R. et al. Antioxidant and prooxidant effects of ascorbic acid. Chemistry of plant raw materials, 2015. No. 1. pp. 113-119].

Based on the foregoing, it can be assumed that the described extracts of Vietnamese sea urchins and skin care products based on these extracts will not protect the skin from oxidative stress during prolonged UV exposure, that is, protect against photoaging.

Expanding the arsenal of effective agents that reduce the effects of oxidative stress caused by long-term UV exposure is an urgent task.

The problem was solved by using echinochrome A as a means to protect the skin from photoaging.

Many studies have shown that echinochrome A, the most common quinoid pigment in sea urchins, has a wide spectrum of biological activity, and all of them are accompanied by its experimentally confirmed antioxidant effect. So the antiviral properties of echinochrome A are associated with the ability to inactivate the surface proteins of the virus, but also to reduce the level of ROS in the host cell induced by the virus [Mishchenko N. P. et al. Antiviral potential of sea urchin aminated spinochromes against herpes simplex virus type 1. Marine Drugs. 2020. Vol. 18, N 11. 550]. In other studies, it was shown that echinochrome A reduces the level of mitochondrial ROS in cardiomyocytes and contributes to the restoration of its own antioxidant defense system [Jeong S.H. et al. Echinochrome A protects mitochondrial function in cardiomyocytes against cardiotoxic drugs. Mar. drugs. 2014, 12, 2922-2936], reduces oxidative stress and secretion of pro-inflammatory cytokines in a model of acute uveitis [Lennikov A. et al. Amelioration of endotoxin-induced uveitis treated with the sea urchin pigment echinochrome in rats. Mol. Vis. 2014, 20, 171-177].

Known pharmaceutical composition for stimulating the differentiation of cardiomyocytes derived from embryonic stem cells containing echinochrome A as an active ingredient [KR 20170104223 A, 09/15/2017].

Known pharmaceutical composition containing echinochrome as an active ingredient used for the prevention or treatment of neurodegenerative diseases caused by acetylcholine deficiency [KR 20150114096 A, 10/12/2015].

Echinorochrome A is the active substance in Histochrome®, which is registered in the Russian Federation. In the dosage form, Histochrom® solution for intravenous administration is used in cardiology in acute myocardial infarction in combination with thrombolytic drugs to eliminate the reperfusion complications caused by them, to reduce the size of myocardial infarction and to prevent reperfusion myocardial injury (state registration number R N002363 / 01-2003) [RU 2137472 C1, 04/23/1999]. Histochrom® in the dosage form, injection solution 0.2 mg/ml is used as a treatment for degenerative diseases of the retina and cornea, diabetic retinopathy of the retina, is indicated for hemorrhages in the vitreous body, retina, anterior chamber, and for dyscirculatory disorders in the central artery and vein retina (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]. Histochrome is known to be used as a diuretic [RU2408367 C1, 01/10/2011], as well as an antiviral agent against tick-borne encephalitis and herpes simplex type I [RU 2697887 C1, 08/21/2019].

However, studies on the effectiveness of echinochrome A against photoaging of the skin have not been conducted. No description of such studies has been found in the available patent and other scientific and technical literature.

This new function of echinochrome A does not follow clearly from its known properties.

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

It is known that oxidative stress caused by long-term UV exposure initiates a signaling cascade that leads to increased production of matrix metalloproteinases (MMPs) capable of degrading the collagen extracellular matrix that forms the basis of the dermal connective tissue (dermis) [Masaki, H. Role of antioxidants in the skin: Anti-aging effects. J. Dermatol. sci. 2010, 58, 85-90]. Destruction of the dermal extracellular matrix, which is mainly composed of type I collagen, weakens the structural integrity of the skin and manifests itself in the morphological features of photoaged skin, such as deep wrinkles, laxity and uneven pigmentation, as well as the loss of the functional protective properties of the skin, which leads to an increase in transepidermal water loss and reduced hydration of the stratum corneum [Lavker R.M. et al. Cumulative effects from repeated exposures to suberythemal doses of UVB and UVA in human skin. J. Am. Acad. Dermatol. 1995, 32, 53-62].

The authors of the claimed invention first discovered the ability of echinochrome A to inhibit the activity of MMP 1, MPP 2, tryptase and chymase.

Being an inhibitor of MMP 1 and MPP 2, echinochrome A prevents the destruction of extracellular collagen, helps to maintain the structural integrity of the skin and its functionality during long-term UV irradiation.

As an inhibitor of tryptase and chymase, the main proteins of mast cells, echinochrome A helps prevent the development of inflammatory and allergic reactions.

In the present study, the therapeutic potential of echinochrome A (EchA) against UV-induced photoaging was confirmed in a mouse model, and its effectiveness was evaluated in terms of aspects of the general visual condition of the skin, its barrier function using immunohistochemical studies and histological analysis.

Echinochrome A has shown a protective effect against UVB-induced photoaging, both functionally and structurally. This is thought to be due to its antioxidant and anti-inflammatory properties affecting mitochondrial metabolism. Echinochrome A injections attenuate inflammatory changes and degradation of extracellular collagen caused by UV irradiation, significantly reduce transepidermal water loss, increase the level of hydration of the stratum corneum, help maintain the structural integrity of the skin and its functional protective properties during prolonged UV irradiation.

The invention is illustrated by the following figures:

- figure 1 shows the analysis of the content of proteins MMP 1, 2, 9, tryptase and chymase relative to tubulin in skin biopsies of mice of the experimental groups, carried out by Western blotting;

- figure 2 shows a 100 times enlarged photo of biopsy specimens of the skin of the back of mice stained with monoclonal antibodies to collagens 1, 3 and antibodies to MMP 1,2,9;

- figure 3 shows the result of a histological analysis of a section of the skin of the back of a mouse, stained with toluidine blue, a mast cell reagent, and a statistical analysis of the number of mast cells;

- figure 4 shows a histological analysis of the skin of the back of mice of the experimental groups with staining with hematoxylin and eosin (H&E), trichrome staining according to Masson (MT) and toluidine blue (TV);

- figure 5 presents the results of histometry and statistical analysis of the thickness of the epidermis, the width and depth of papillomatosis in the skin of the back of mice;

- Fig.6 presents the results of macroscopic observation of the skin of the back in mice of the experimental groups;

- Fig.7 presents data on changes in the degree of hydration of the stratum corneum (SCH) in the skin of control mice irradiated with UV-B, and mice treated with Ex A, for 8 weeks;

- Fig.8 presents data on changes in the degree of transepidermal water loss of the skin of the back in mice that were not irradiated, irradiated with UV-B and treated with EchA during the 8 weeks of the experiment.

In the experiments, we used echinochrome A, obtained at the Pacific Institute of Bioorganic Chemistry. G.B. Elyakova FEB RAS.

Six-week-old SKH-1 hairless mice purchased from Orient Bio Laboratory Animals (Daejeon, Korea) were used in the experiment. All mice were housed on a 12-hour light/dark cycle at 22±2° C. and 45±5% humidity, and were given ad libitum access to a standard diet and water. The study was conducted in compliance with the scientific and ethical standards established by the Animal Care and Use Committee of Inye University (Korea).

Mice (n=20) were divided into three groups: control (animals not exposed to any treatment, n=4) and two groups of animals exposed to UV irradiation according to the scheme: one group of mice (UV-B group, n=8) were injected intraperitoneally 200 μl of phosphate-buffered saline (PBS), and the second group (UV-B + EhA group, n=8) was injected intraperitoneally with echinochrome A 0.1 mg/kg in 200 μl of PBS.

A rectangular box shaped UV device 60 cm wide x 40 cm long x 40 cm high was manufactured by NSC, Inc. (Hwasung, Korea). The device was equipped with a TL20W/01RS lamp (Phillips™, Eindhoven, The Netherlands) which emitted UV-B at a narrow peak wavelength of 311 nm. The distance between the UV lamp and the mouse was maintained at 30 cm, and the amount of UV-B radiation emitted was measured using an ILT-1700 research radiometer (International Light Technology™, USA).

The eight-week exposure schedule included exposure every two days, with the mice resting for two days after every third exposure each week.

UV-B was administered at 1 minimum erythemal dose (1 MED) during the first week, gradually increasing to 2 MED, 3 MED, and 4 MED over the next 3 weeks, and then plateauing at 4 MED until the end of week 8. The MED is commonly used as a practical indicator to determine the extent of UV exposure. To establish MED level 1, a tissue with a 1 cm hole was used to cover the dorsal skin of a mouse, and six different areas were irradiated at 60, 80, 100, 120, 140, and 160 mJ. The amount of UV-B at the mouse level was calculated as about 1 mJ per second, so installing the MED took about 11 minutes of exposure. After 24 hours, it was observed whether erythema occurred at any of the six points.

Photographs of skin changes on the back of hairless mice were taken with a camera and a dermatoscope (DermLite™ DLCAM, Dermlite, USA) before each first day of exposure of each week. Mice were photographed under anesthesia, which was administered by short-term inhalation of isoflurane.

The invention is illustrated by the following examples.

Example 1

Effect of EchA on the expression of matrix metalloproteinases, tryptase and chymase from mouse skin biopsy

Western blotting, an analytical method used to detect specific proteins in a sample, is used to determine the effect of EchA on the expression of matrix metalloproteinases, tryptase, and chymase in mouse dorsal skin biopsies. Epidermal dorsal skin samples are 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 the addition of a protease and phosphatase inhibitor 200 mM phenylmethylsulfonylfluoride (PMSF). Protein concentration is determined using a Bio-Rad DC protein assay kit (Bio-Rad Laboratories, Inc., USA). The lysates were separated by electrophoresis in 8-15% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) and transferred to a nitrocellulose membrane (GE Healthcare, USA) for 100 min at 110 V and 4°C. The membranes are blocked with 5% skim 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 are incubated overnight with primary antibodies diluted to 1:1000-1:5000 in 3% bovine serum albumin (BSA) in TBST. After washing three times for 15 minutes, the membranes are incubated for 1 hour with secondary antibodies labeled with horseradish peroxidase, diluted to 1:1000-1:5000 in 3% BSA. Protein bands are visualized with a chemiluminescence enhancement kit (Santa Cruz Biotechnology, USA) and observed with an AI 600 imager (GE Healthcare, USA).

It was shown that the relative expressions of MMP 1 and MMP 2, tryptase and chymase were significantly increased in the UV-B group compared to the control and the UV-B+ExA group (FIG. 1). However, there was no significant difference in the expression of these proteins between the control group and the UV-B+EchA group, which means that EchA can be considered an inhibitor of the expression of these proteins. Although the expression of MMP 9 was slightly higher in the UV-B group, there was insufficient confidence in the expression of this protein in all groups.

Example 2

Effect of EchA on the expression of collagen and matrix metalloproteinases according to immunohistochemical data

Collagen 1 and 3 are important components of tissue tensile strength, they are abundantly distributed in the dermis and polymerized into elongated fibrils. Therefore, it is widely accepted that the density and distribution of dermal collagen is responsible for the formation of wrinkles in photoaging skin.

Immunohistochemical staining was performed using anti-collagen 1 antibody and anti-MMP 9 antibody purchased from Abeam® (Cambridge, UK) and anti-collagen 3 polyclonal antibody, anti-MMP 1 polyclonal antibody and anti-MMP 2 monoclonal antibody purchased from Invitrogen™ (Carlsbad, USA). Stained sections are observed and photographed using optical microscopy Olympus™ BH2 (Tokyo, Japan) and Tucsen™ Digiretina 16 (Fuzhou, China).

The results showed that the UV-B+ExA group had higher amounts of collagen 1 and 3 than the UV-B group, while the MMP metalloproteinases 1, 2, and 9 were more densely stained in the UV-B group compared to the UV-B group. -B+EhA, which once again confirms that EhA is an inhibitor of metalloproteinases (figure 2).

Example 3

Histological examination of mast cells in sections of the skin of the back of a mouse As shown by Western blotting under conditions of oxidative stress caused by UV radiation, the expression of tryptase and chymase, the main protein components of the secretory granules of mast cells (figure 1), increases. Mast cells are involved in the development of inflammation, type 1 hypersensitivity reactions, and other processes. Mast cells underlie the development of allergies and anaphylaxis.

Figure 3 shows the results of a histological examination of a section of the skin of the back of a mouse, stained with toluidine blue, at a magnification of 100 times (A) and a statistical analysis of the number of mast cells (B). The number of mast cells stained with toluidine blue (FIG. 3A) was significantly higher in the UV-B group than in the UV-B+ExA group (FIG. 3B).

Example 4

Histological examination of skin sections of the back of a mouse for the presence of inflammatory cells, the number and distribution of collagen fibers and mast cells

The results of a histological examination of sections of the skin of the back of a mouse for the presence of inflammatory cells staining with hematoxylin and eosin (H&E), the number and distribution of collagen fibers (determined by staining with Masson's trichrome stain (MT)) and mast cells (toluidine blue (TB) with an increase in 100 times, shown in Fig.4.

In the dermis, UV irradiation induces an infiltration of perivascular interstitial inflammatory cells, mainly composed of lymphocytes. When examining H&E sections, the UVB group showed significantly denser inflammatory cell infiltration and epidermotropism characterized by lymphocyte exocytosis, spongiosis, and epidermal proliferation compared to the UVB+ExA group. In addition, the distribution and number of dermal collagen fibers were diffuse and dense in the UV-B+ExA group, while the UV-B group showed a marked decrease in the number of collagen fibers.

Epidermal and dermal changes were more evident in UVB than in the EchA group. When stained with Masson's trichrome (MT, xIOO), there is a significant decrease in the amount of dermal collagen fibers in the UV-B group compared to the UV-B+ExA group. Toluidine blue is often used to identify mast cells due to the heparin in their cytoplasmic granules. Toluidine blue staining (TB, x100) shows denser mast cell infiltration in the UV-B group than in the EchA-treated group.

Example 5

Histological studies of the effect of EchA on the skin of mice

Shown are stained with hematoxylin and eosin (H&E) skin tissue sections of mice in the control group, the UV-B irradiated group, and the UV-B+ExA group at a magnification of 200 times (figure 5). Mouse epidermal thickness (yellow line) and papillomatosis width (blue line) and depth (green line) of mice are calculated as averages from five different sites in H&E skin tissue sections of each mouse using the Tucsen™ TCapture computer program (Fuzhou, China). The histopathological effect of UV irradiation is well understood, and hairless mice are known to exhibit various epidermal and dermal changes similar to those of human skin, including hyperkeratosis, acanthosis, dermal perivascular inflammatory cell infiltration, excessive deposition of abnormal elastin complex, and most importantly, destruction of collagen fibers.

On histological examination of dorsal skin tissue, it can be seen that the UV-B group shows marked changes in both the epidermis and dermis. In the epidermis of the UV-B group, more severe hyperkeratosis, acanthosis, spongiosis, and papillomatosis were found than in the UV-B+ExA group. In particular, mean epidermal thickness, papillomatosis width and depth were significantly increased in the UV-B group compared to the UV-B+ExA group, measured as the mean of five randomly selected areas of each H&E tissue section.

Example 6

Effect of EchoA on the macroscopic appearance of mouse skin

Photographs of the skin of the back of mice taken the next day after the completion of the experiment are shown (Fig. 6). On close examination, it can be seen that the skin of the back in the UV-exposed groups had fine superficial wrinkles, hypopigmented patches, or a yellowish discoloration compared to the control group. Similar features are observed on dermoscopic examination: more pronounced intersecting wrinkles, irregular pigmentation with hypopigmentation and yellowish dots, and small telangiectasias. However, there was a significant difference in skin condition between the UV-B and UV-B+ExA groups. With EchA treatment, wrinkles and pigmentation were less pronounced.

Example 7

Effect of EchoA on the physiological function of the skin

The next day after the completion of each two-week UV irradiation, transepidermal water loss and the degree of stratum corneum hydration are measured with a taumeter and corneometer using a Cutometer ™ MPA 580 (Courage and Khazaka Electronics ™, Germany). The measuring probe is placed on the mid-dorsal skin of mice that are anesthetized by inhalation of low concentration isoflurane. The transepidermal water loss values are read at the plateau of the evaluation plot, while the stratum corneum hydration values are taken as the average of the three measurements.

It has been shown that starting from the 4th week of the experiment in mice irradiated with UV-B, there is a decrease in the hydration of the stratum corneum. However, there is no significant difference in the degree of stratum corneum hydration between the UV-B and UV-B+ExA groups at the end of the experiment (FIGS. 7A-E).

It was shown that at the 2nd week of the experiment there was no significant difference between any of the groups in the amount of transepidermal water loss. A significant increase in transepidermal water loss was observed in UVB-irradiated mice starting at 4 weeks. The UV-B group showed a faster increase in transepidermal water loss compared to the UV-B+ExA group, which was maintained until the end of the experiment (p<0.05) (Fig. 8B, C). At the end of the experiment at week 8, the degree of transepidermal water loss was significantly higher in the UV-B group compared to the UV-B+ExA group (Fig. 8D, E).

Claims (1)

The use of echinochrome A as an agent with inhibitory activity against matrix metalloproteinases 1 and 2 (MMP 1 and MMP 2), tryptase and chymase.

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