Urokinase inhibitors

Abstract

Inhibition of urokinase suppresses plasmin production and contributes to the improvement of rough skin and / or blemishes. For this reason, ingredients for improving and / or preventing rough skin and / or blemishes have been developed in the fields of cosmetic and dermatology. However, there is still a need to search for extracts with urokinase inhibitory activity that can be incorporated into cosmetics. [Solution] A urokinase inhibitor is provided, comprising one extract selected from the group consisting of witch hazel extract and saxifrage extract, or a combination thereof.

Description

Technical Field

[0001] The present invention relates to the technical field of urokinase activity inhibition.

Background Art

[0002] Urokinase is a type of serine protease called urokinase-type plasminogen activator (uPA). Urokinase is a substance discovered in urine and has been isolated and used as a thrombolytic agent in the treatment of coronary artery thrombosis and cerebral thrombosis. Urokinase is present not only in urine but also in blood and the extracellular matrix, and acts on the substrate plasminogen to produce plasmin. Plasmin is a serine protease having various physiological activities and is involved in thrombolysis, activation of matrix metalloproteases, etc. Urokinase contained in blood or tissue fluid interacts with the urokinase receptor (uPAR) present on the cell membrane surface. The urokinase receptor is expressed particularly in cells such as fibroblasts, vascular smooth muscle cells, leukocytes, and bone marrow cells. The urokinase receptor has three highly homologous regions (D1, D2, D3) and a glycosylphosphatidylinositol (GPI) anchor, and is tethered to the cell membrane via the GPI anchor. It is considered that when the urokinase receptor and urokinase interact, the plasmin produced by urokinase acts locally around the cells expressing the urokinase receptor.

[0003] In the normal keratinization process of skin, proteases within epidermal cells are thought to play a crucial role. Changes in the activity of serine proteases, particularly those involved in the fibrinolytic system such as plasmin and plasminogen activator, are deeply involved in the development of various skin diseases. In human skin, excessive plasmin, produced by the action of urokinase, damages the epidermis and causes rough skin. Furthermore, it is known that urokinase activity increases in areas with age spots. Therefore, research is underway on cosmetics that inhibit urokinase activity to improve and / or prevent rough skin and / or age spots. While tranexamic acid and tranexamic acid methylamide are known to inhibit urokinase, there is still a need to search for novel ingredients that inhibit urokinase activity. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] Japanese Patent Publication No. 2003-2821 [Patent Document 2] Japanese Patent Publication No. 2006-8550 [Non-patent literature]

[0005] [Non-Patent Document 1] Journal of Translational Medicine volume 20, Article number: 135 (2022) [Non-Patent Document 2] Drug Discovery Today Volume 26, Issue 4, April 2021, Pages 1076-1085 [Non-Patent Document 3] Current Pharmaceutical Design, Volume 17, Number 19, 2011, pp. 1874-1889 (16) [Overview of the project] [Problems that the invention aims to solve]

[0006] Inhibition of urokinase suppresses plasmin production and contributes to the improvement of rough skin and / or blemishes. For this reason, ingredients for improving and / or preventing rough skin and / or blemishes have been developed in the fields of cosmetic and dermatology. However, there is still a need to search for extracts with urokinase inhibitory activity that can be incorporated into cosmetics. [Means for solving the problem]

[0007] As a result of diligent research, the inventors discovered witch hazel extract and saxifrage extract as novel components that inhibit urokinase. Therefore, the present invention relates to the following: [1] A urokinase inhibitor comprising one extract selected from the group consisting of witch hazel extract and saxifrage extract, or a combination thereof. [2] A urokinase inhibitor as described in item 1, containing witch hazel extract. [3] A urokinase inhibitor as described in item 1, containing Saxifraga stolonifera extract. [4] A urokinase inhibitor as described in item 2, containing 0.001% to 5% by weight of witch hazel extract. [5] A urokinase inhibitor as described in item 3, containing 0.001% to 5% by weight of Saxifraga stolonifera extract. [6] A urokinase inhibitor as described in item 1, comprising 0.001% to 5% by weight of witch hazel extract and 0.001% to 5% by weight of saxifrage extract. [7] A topical skin composition comprising a urokinase inhibitor as described in any one of items 1 to 6. [8] The composition according to item 7, wherein the aforementioned external skin composition is a cosmetic composition. [9] A method for inhibiting urokinase activity, comprising applying one extract selected from the group consisting of witch hazel extract and saxifrage extract, or a combination thereof, to a subject in which inhibition of urokinase activity is desired.

[10] The use of one extract selected from the group consisting of witch hazel extract and saxifrage extract, or a combination thereof, in the manufacture of urokinase inhibitors.

[11] A beauty method comprising applying one extract selected from the group consisting of witch hazel extract and saxifrage extract, or a combination thereof.

[12] One extract or a combination thereof, selected from the group consisting of witch hazel extract and saxifrage extract, for use in inhibiting urokinase activity. [Effects of the Invention]

[0008] One extract selected from the group consisting of witch hazel extract and saxifrage extract, or a combination thereof, can inhibit urokinase activity. [Brief explanation of the drawing]

[0009] [Figure 1] Figure 1 shows the urokinase activity inhibition rates for a sample with no extract added (none), a sample with 0.5% by weight tranexamic acid added, a sample with 0.01% and 0.02% by weight witch hazel extract added, a sample with 0.01% and 0.02% by weight saxifrage extract added, a sample with 0.01% and 0.1% by weight lotus germ extract added, and a sample with 0.01% and 0.1% by weight aloe vera extract added. [Modes for carrying out the invention]

[0010] The present disclosure relates to a urokinase inhibitor containing one extract selected from the group consisting of Hamamelis extract and Saxifraga extract or a combination thereof. The urokinase inhibitor of the present disclosure can improve and / or prevent rough skin and / or spots by inhibiting urokinase activity.

[0011] Urokinase is a serine protease, which is one of the thrombolytic enzymes, and has the function of converting plasminogen, a precursor enzyme of fibrinolytic enzyme, into active plasmin. Urokinase in the skin is known to be secreted from vascular endothelial cells by ultraviolet damage. In vivo, urokinase exists as its inactive precursor, prourokinase, and is activated by being cleaved by the action of proteases such as plasmin and kallikrein, and expresses its activity. In the skin with reduced skin barrier function, it is known that the activity of urokinase is enhanced in the skin before the appearance of the skin changes. When plasmin is activated by the action of urokinase and the fibrinolytic system is activated, the proteins constituting the skin barrier are decomposed, damage occurs to the epidermis, and rough skin is caused. In addition, plasmin activates melanocytes and causes the appearance of spots through the production of melanin. In addition, it is known that the activity of urokinase is increased at the spot site. By inhibiting urokinase activity with the urokinase inhibitor according to the present disclosure, the activation of melanocytes can be inhibited, and rough skin and / or spots can be improved and / or prevented.

[0012] Furthermore, plasmin generated by the action of urokinase also acts in the dermis, degrading the extracellular matrix and proteoglycan, and activating proteases downstream of the plasmin action. Physiological actions downstream of the action of active plasmin include matrix metalloprotease (MMP) and collagenase activation, thrombolysis, promotion and activation of cytokine and chemokine expression (Non-Patent Document 1: Journal of Translational Medicine volume 20, Article number: 135 (2022), Non-Patent Document 2: Drug Discovery Today Volume 26, Issue 4, April 2021, Pages 1076-1085, Non-Patent Document 3: Current Pharmaceutical Design, Volume 17, Number 19, 2011, pp. 1874-1889 (16)). Therefore, the urokinase inhibitor of the present disclosure brings about inhibition of MMP activity, inhibition of thrombolysis, and / or inhibition and inactivation of cytokine and chemokine expression by suppressing plasmin formation.

[0013] Matrix metalloproteinases (MMPs) are a type of metalloproteinase in which a metal ion is coordinated to the active site. Currently, MMP1 to MMP28 are known as the MMP family. Zinc ions and calcium ions are coordinated to matrix metalloproteinases. Matrix metalloproteinases degrade extracellular matrix components such as collagen, proteoglycans, elastin, fibronectin, and laminin, and also degrade precursors of physiologically active proteins such as cytokines, contributing to their activation. Through this activity, they are involved in wound healing, bone remodeling, inflammation, cancer metastasis, invasion, and proliferation (Non-Patent Literature 1). Matrix metalloproteinases are produced as propeptides, and their activity is exerted when a propeptide site containing an amino acid (PRCGVP) called the cysteine ​​switch is removed. Matrix metalloproteinases have a plasmin-centered mutual activation pathway. In particular, MMP9 and MMP3 are activated by plasmin action, sequentially activating downstream matrix metalloproteinases, and also activating plasminogen to produce plasmin. In addition to MMP9 and MMP3, other matrix metalloproteinases that are activated by plasmin include MMP8, MMP7, MMP14, MMP13, and MMP1.

[0014] In the skin, dermal fibroblasts are known to secrete MMP2, while keratinocytes secrete both MMP9 and MMP2. MMPs produced by dermal fibroblasts can be activated by plasmin and other proteases. By degrading collagen (especially type I / III collagen), elastin, and proteoglycans that make up the extracellular matrix of the dermis, they lead to thinning of the dermis, resulting in decreased skin elasticity, sagging, and wrinkles. MMPs secreted by keratinocytes act on the basement membrane, degrading collagen anchored to the basement membrane, such as type IV and type VII collagen, and impairing the stability of the basement membrane. When the stability of the basement membrane is lost, the migration and proliferation of epidermal stem cells and the proliferation of keratinocytes on the basement membrane are suppressed, causing a disruption in the turnover of the stratum corneum. Therefore, the urokinase inhibitors of this disclosure inhibit the activation of MMPs by suppressing plasmin formation, and can be described as inhibitors of dermal thinning, elasticity enhancers, and wrinkle and / or sagging improvement agents for the dermis, and as agents for maintaining and / or improving turnover and health for the epidermis, improving skin barrier function, and preventing and / or improving skin blemishes, melasma, dullness, and fine wrinkles.

[0015] In the circulatory system and tissues, matrix metalloproteinases are involved in angiogenesis by acting on the basement membrane of blood vessels. Angiogenesis is necessary for cancer growth, and matrix metalloproteinases also act when cancer invades tissues from blood vessels and metastasizes. Therefore, the urokinase inhibitors of this disclosure can inhibit the activation of MMPs by suppressing plasmin formation and can act as angiogenesis inhibitors and / or cancer inhibitors (Non-Patent Documents 1 and 2). Matrix metalloproteinases are also involved in bone destruction and regeneration. The urokinase inhibitors of this disclosure can act on bone and can be used as prophylactic or therapeutic agents for osteoporosis (Non-Patent Document 2). Since matrix metalloproteinases are involved in the activation of inflammatory cytokines, the urokinase inhibitors of this disclosure can also be used as prophylactic or therapeutic agents for inflammatory diseases (Non-Patent Document 2).

[0016] Plasmin breaks down fibrin contained in blood clots and is involved in thrombolysis. By dissolving fibrin, it contributes to vascular regeneration after hemostasis. It also contributes to the dissolution of blood clots in thrombosis such as myocardial infarction. On the other hand, it is also known that plasmin action can cause bleeding. Therefore, the urokinase inhibitors of this disclosure can be used as bleeding control agents by suppressing plasmin formation. Furthermore, plasmin is involved in promoting and / or activating cytokine and chemokine expression, either directly or indirectly through the action of metalloproteinases, and is also involved in the degradation of cell membrane targets (Non-Patent Literature 3). Therefore, the urokinase inhibitors of this disclosure can also be used as anti-inflammatory agents.

[0017] Hamamelis extract and / or Saxifraga extract inhibit urokinase activity. Therefore, an agent comprising one extract selected from the group consisting of Hamamelis extract and Saxifraga extract, or a combination thereof, can inhibit urokinase activity.

[0018] The plant extracts described herein can be obtained by conventional methods, for example, by immersing or refluxing the source plant with an extraction solvent at room temperature or under heating, followed by filtration and concentration. Any solvent commonly used for extraction can be used as the extraction solvent, such as aqueous solvents, e.g., water, physiological saline, phosphate buffer, borate buffer, or organic solvents, e.g., alcohols such as ethanol, propylene glycol, 1,3-butylene glycol, glycerin, aqueous alcohols, chloroform, dichloroethane, carbon tetrachloride, acetone, ethyl acetate, hexane, etc., each can be used alone or in combination. Preferably, a mixed solvent of water and alcohol, such as 1,3-butylene glycol, is used as the solvent. The extract obtained by extraction with the above solvent can be used as is, or concentrated by methods such as freeze-drying. If necessary, an extract obtained by adsorption, for example, by removing impurities using an ion exchange resin, or by adsorption using a porous polymer (e.g., Amberlite XAD-2) column, followed by elution with a desired solvent and further concentration can also be used. The plant extracts used can be commercially available extracts used as cosmetic ingredients, and these commercially available extracts can be incorporated at a predetermined concentration.

[0019] Hamamelis extract is an extract obtained from plants belonging to the genus Hamamelis in the family Hamamelidaceae. Examples of plants belonging to the genus Hamamelis include Hamamelis japonica and Hamamelis Virginiana. Hamamelis is native to the eastern United States, Canada, and Mexico. Furthermore, according to the Japan Cosmetic Industry Association's standard for labeling cosmetic ingredients and the INCI (International Nomenclature for Cosmetic Ingredients) international labeling system, it is represented as Hamamelis Virginiana (Witch Hazel) Leaf Extract. The plant part may be the bark, leaves, stems, flowers, roots, or fruit, but leaves and / or bark are particularly used. It can be prepared by extracting the plant, especially the leaves and / or bark, with water, alcohol, or a mixture thereof. Suitable alcohols include ethanol, glycerol, propylene glycol, and / or butylene glycol. More preferably, the product can be extracted with a mixture of water and alcohol, such as 1,3-butylene glycol, in any proportion, for example, a mixture of 10:90 to 90:10, preferably 30:70 to 70:30, and even more preferably 50:50.

[0020] Saxifraga sarmentosa extract is an extract obtained from plants belonging to the genus Saxifraga in the family Saxifragaceae. Examples of plants belonging to the genus Saxifraga include Saxifraga stolonifera. Saxifraga sarmentosa is native to Japan and China. In addition, the display name used in the full ingredient list of cosmetics as defined by the Japan Cosmetic Industry Association and the international display name according to the INCI (International Nomenclature for Cosmetic Ingredients) are expressed as: Display name / INCI name: Saxifraga sarmentosa extract / Saxifraga Sarmentosa Extract. The plant part may be the bark, leaves, stems, flowers, roots, or fruits, but the whole plant is particularly used. It can be prepared by extracting the plant, especially the whole plant, with water, alcohol, or a mixture of these solutions. As alcohol, ethanol, glycerol, propylene glycol, and / or butylene glycol can be used. More preferably, the product can be extracted with a mixture of water and alcohol, such as 1,3-butylene glycol, in any proportion, for example, a mixture of 10:90 to 90:10, preferably 30:70 to 70:30, and even more preferably 50:50.

[0021] In one aspect of this disclosure, witch hazel extract is contained in the present preparation at a concentration of 0.001% to 5% by weight, with 0.005% to 0.5% by weight being preferred from the viewpoint of the strength of inhibition. Saxifraga stolonifera extract is contained in the present preparation at a concentration of 0.001% to 5% by weight, with 0.005% to 0.5% by weight being preferred from the viewpoint of the strength of inhibition.

[0022] The urokinase inhibitors of this disclosure may be incorporated into topical skin compositions. The topical skin compositions are not particularly limited as long as they are applicable to the skin, and any dosage form can be used, such as solution, emulsion, solid, semi-solid, powder, powder dispersion, water-oil two-layer separation, water-oil-powder three-layer separation, ointment, gel, aerosol, mousse, stick, etc. The transdermal compositions may contain bases and excipients commonly used in transdermal compositions, such as preservatives, emulsifiers, and pH adjusters.

[0023] The skin-applied composition may also be a cosmetic composition. When incorporated into a cosmetic composition, it can be used in facial or body cosmetics such as lotions, emulsions, serums, creams, packs, essences, and gels, as well as makeup cosmetics such as foundations, makeup bases, and concealers, and even bath additives.

[0024] The topical skin compositions containing the urokinase inhibitors disclosed herein can be used in cosmetic methods to improve rough skin and / or blemishes by being applied to the skin. The methods disclosed herein do not include medical procedures, i.e., surgeries, treatments, or diagnoses performed on humans by a physician or a person under their direction, and may therefore be considered non-medical procedures. The use and dosage of the topical skin compositions disclosed herein in such cosmetic methods are not particularly limited. Typically, an appropriate amount can be rubbed directly onto the entire face, or an appropriate amount can be soaked into gauze or similar material and then applied to the skin, once or multiple times per day.

[0025] Unless otherwise specified in this specification, % means weight percent.

[0026] All references made herein are incorporated herein by reference in their entirety.

[0027] The embodiments of the Disclosure described below are for illustrative purposes only and do not limit the technical scope of the Disclosure. The technical scope of the Disclosure is limited solely by the claims. Modifications to the Disclosure, such as adding, deleting, or replacing constituent elements of the Disclosure, may be made without departing from the spirit of the Disclosure. [Examples]

[0028] Evaluation of urokinase inhibitory activity The buffer solution used was 50 mM Tris-HCl buffer (pH 7.5) containing 0.1 M NaCl. Urokinase (manufacturer: BIOMEDICA) solution was prepared to 1000 IU / ml using the above buffer. In addition, pGlu-Gly-Arg-pNA (manufacturer: DIAPHARMA) was prepared to 10 mM as a substrate using the same buffer. 85 μl of the above buffer, 5 μl of urokinase solution, 5 μl of 10 mM substrate solution, and 5 μl of witch hazel extract (manufacturer: Gatephosé), saxifrage extract (manufacturer: Ichimaru Falcos), lotus germ extract (manufacturer: Maruzen Pharmaceutical), aloe vera extract (manufacturer: Ichimaru Falcos), or tranexamic acid solution were added to 96 wells. The results were: none (sample with no extract added), 0.5% tranexamic acid (0.5 wt% tranexamic acid sample), 0.01% witch hazel extract (0.01 wt% witch hazel extract sample), and 0.02% tranexamic acid. Witch hazel extract (0.02 wt% witch hazel extract sample), 0.01% saxifrage extract (0.01 wt% saxifrage extract sample), 0.02% saxifrage extract (0.02 wt% saxifrage extract sample), 0.01% lotus germ extract (0.01 wt% lotus germ extract sample), 0.1% lotus germ extract (0.1 wt% lotus germ extract sample), 0.01% aloe vera extract (0.01 wt% aloe vera extract sample), and 0.1% aloe vera extract (0.1 wt% aloe vera extract sample) were prepared. The absorbance at 405 nm was immediately measured using a microplate reader (Powerscan HT, BioTek). After incubation at 37°C for 30 minutes, the absorbance was measured again (n=4 for each). The urokinase inhibition rate (%) compared to the control without extract was calculated using ΔA405(30 min - 0 min), which was obtained by subtracting the absorbance at the start (0 min) from the absorbance at 405 nm after 30 minutes of incubation for the control without added extract, and ΔA405(30 min - 0 min), which was obtained by subtracting the absorbance at the start (0 min) from the absorbance at 405 nm after 30 minutes of incubation for each added extract. The results are shown in Figure 1 and Table 1. Tranexamic acid, a known urokinase inhibitor, was used as a positive control. The samples supplemented with witch hazel extract and saxifrage extract significantly inhibited urokinase activity (Figure 1). Table 1

Claims

1. A urokinase inhibitor comprising one extract selected from the group consisting of witch hazel extract and saxifrage extract, or a combination thereof.

2. A urokinase inhibitor according to claim 1, comprising witch hazel extract.

3. A urokinase inhibitor according to claim 1, comprising Saxifraga stolonifera extract.

4. The urokinase inhibitor according to claim 2, comprising 0.001% to 5% by weight of witch hazel extract.

5. The urokinase inhibitor according to claim 3, comprising 0.001% to 5% by weight of Saxifraga stolonifera extract.

6. The urokinase inhibitor according to claim 1, comprising 0.001% to 5% by weight of witch hazel extract and 0.001% to 5% by weight of saxifrage extract.

7. A topical skin composition comprising the urokinase inhibitor described in claim 1 or 6.

8. The composition according to claim 7, wherein the external skin composition is a cosmetic composition.

Images