Inhibitor of interaction between urokinase and urokinase receptor

Abstract

To provide a drug that inhibits the interaction between urokinase receptor and urokinase, focusing on various physiological actions caused by the inhibition of plasmin action.SOLUTION: Provided is 1-(2-hydroxyethyl)-2-imidazolidinone (HEI) or derivatives thereof, or salts thereof, as a drug that inhibits the interaction between urokinase receptor and urokinase. 1-(2-hydroxyethyl)-2-imidazolidinone (HEI) or derivatives thereof, or salts thereof, inhibits the action of plasmin through inhibition of the interaction between urokinase receptor and urokinase, and exerts various physiological actions such as inhibition of inflammation, prevention of the degradation of extracellular matrix, and inhibition of cancer invasion.SELECTED DRAWING: Figure 2

Description

Technical Field

[0001] The present invention relates to a technology that inhibits the interaction between urokinase (uPA) and urokinase receptor (uPAR), suppresses the actions involving urokinase, and exerts physiological functions.

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 exists not only in urine but also in blood and the extracellular matrix, acts on the substrate plasminogen, and generates plasmin. Plasmin is a serine protease with various physiological activities and is involved in thrombolysis, activation of matrix metalloproteinases, etc.

[0003] 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, white blood cells, 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. When the urokinase receptor and urokinase interact, it is thought that plasmin generated by urokinase acts locally around the cells expressing the urokinase receptor. Cancer cells express the urokinase receptor, and plasmin is generated around cancer cells by the interaction between the urokinase receptor and urokinase. Degradation of the extracellular matrix, which is one of the actions of plasmin, is thought to be involved in cancer invasion, metastasis, and proliferation. Development of a treatment method for suppressing cancer cell invasion by suppressing the interaction between the urokinase receptor and urokinase has been attempted (Non-Patent Document 1: ACS Chem. Biol. 2011. 6. 11. 1232-1243, Non-Patent Document 2: Front. Pharmacol. (2015) 6:154). In addition, analysis of the interaction between the urokinase receptor and urokinase and development of compounds capable of suppressing the interaction by mimicking the shapes between proteins have been carried out (Non-Patent Document 3: Chem Med. Chem Vol.12, Issue 21, P.1794-1809).

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Non-Patent Documents

[0005]

Non-Patent Document 1

Non-Patent Document 2

[0006] Focusing on various physiological effects caused by the inhibition of plasmin action, an object of the present invention is to provide a compound that inhibits the interaction between urokinase receptor and urokinase. [Means for Solving the Problems]

[0007] When the present inventors screened for an agent that inhibits the interaction between urokinase receptor and urokinase, they found that 1-(2-hydroxyethyl)-2-imidazolidinone (HEI) or its derivative, or a salt thereof, inhibits the interaction between urokinase (uPA) and urokinase receptor (uPAR) in a dose-dependent manner, and thus the present invention was achieved. Therefore, the present invention relates to the following: [1-1] An inhibitor of the interaction between urokinase (uPA) and urokinase receptor (uPAR), comprising 1-(2-hydroxyethyl)-2-imidazolidinone (HEI) or its derivative, or a salt thereof. [1-2] Use of 1-(2-hydroxyethyl)-2-imidazolidinone (HEI) or its derivative, or a salt thereof, for the production of an inhibitor of the interaction between urokinase (uPA) and urokinase receptor (uPAR). [1-3] A method for inhibiting the interaction between urokinase (uPA) and urokinase receptor (uPAR), comprising administering 1-(2-hydroxyethyl)-2-imidazolidinone (HEI) or its derivative, or a salt thereof, to a subject in need of inhibiting the interaction between urokinase (uPA) and urokinase receptor (uPAR). [1-4] 1-(2-hydroxyethyl)-2-imidazolidinone (HEI) or its derivative, or a salt thereof, for use in the prevention, treatment or improvement of plasmin-related diseases and conditions through inhibition of the interaction between urokinase (uPA) and urokinase receptor (uPAR). [2-1] The interaction inhibitor according to item 1-1, wherein the interaction inhibitor suppresses plasmin activation. [2-2] The use according to item 1-2, wherein the interaction inhibitor suppresses plasmin activation. [2-3] The method according to item 1-3, wherein plasmin activation is suppressed by administering 1-(2-hydroxyethyl)-2-imidazolidinone (HEI) or its derivative, or a salt thereof. [2-4] 1-(2-hydroxyethyl)-2-imidazolidinone (HEI) or its derivative, or a salt thereof, according to item 1-4, for preventing, treating or improving plasmin-related diseases and conditions through suppressing plasmin activation by inhibiting the interaction between urokinase (uPA) and urokinase receptor (uPAR). [3] The invention according to items 1-1 to 1-4, wherein the suppression of plasmin activation includes activation of matrix metalloproteinase, and 1-(2-hydroxyethyl)-2-imidazolidinone (HEI) or its derivative, or a salt thereof, suppresses the expression of matrix protease. [4] The invention according to item 4, wherein the matrix protease is MMP3 or MMP9. [5-1] An inhibitor of matrix metalloproteinase activation, comprising 1-(2-hydroxyethyl)-2-imidazolidinone (HEI) or a derivative thereof, or a salt thereof. [5-2] Use of 1-(2-hydroxyethyl)-2-imidazolidinone (HEI) or a derivative thereof, or a salt thereof, for the manufacture of an inhibitor of matrix metalloproteinase activation. [5-3] A method for inhibiting matrix metalloproteinase activation, comprising administering 1-(2-hydroxyethyl)-2-imidazolidinone (HEI) or a derivative thereof, or a salt thereof, to a subject in need of inhibiting matrix metalloproteinase activation. [5-4] 1-(2-hydroxyethyl)-2-imidazolidinone (HEI) or a derivative thereof, or a salt thereof, for use in the prevention, treatment or improvement of plasmin-related diseases and conditions through inhibition of matrix metalloproteinase activation. [6] The invention according to items 5-1 to 5-4, wherein the matrix metalloproteinase is MMP3 or MMP9. [7-1] A plasmin generation inhibitor, comprising 1-(2-hydroxyethyl)-2-imidazolidinone (HEI) or a derivative thereof, or a salt thereof, which inhibits plasmin generation by urokinase. [7-2] Use of 1-(2-hydroxyethyl)-2-imidazolidinone (HEI) or a derivative thereof, or a salt thereof, for the manufacture of a plasmin generation inhibitor by inhibiting plasmin generation by urokinase. [7-3] A method for inhibiting plasmin generation, comprising administering 1-(2-hydroxyethyl)-2-imidazolidinone (HEI) or a derivative thereof, or a salt thereof, to a subject in need of inhibiting plasmin generation, and inhibiting plasmin generation by inhibiting plasmin generation by urokinase. [7-4] 1-(2-hydroxyethyl)-2-imidazolidinone (HEI) or a derivative thereof, or a salt thereof, for use in the prevention, treatment or improvement of plasmin-related diseases and conditions through inhibition of plasmin generation by urokinase. [Effect of the Invention]

[0008] It is possible to inhibit the interaction between urokinase (uPA) and urokinase receptor (uPAR). Thereby, physiological actions based on plasmin action can be suppressed. [Brief Description of the Drawings]

[0009]

Figure 1

Figure 2

[0010] One aspect of the present invention relates to an inhibitor of the interaction between urokinase (uPA) and urokinase receptor (uPAR), which contains 1-(2-hydroxyethyl)-2-imidazolidinone (HEI) or its derivative, or a salt thereof. By inhibiting the interaction between urokinase (uPA) and urokinase receptor (uPAR), the activation from plasminogen to plasmin can be suppressed. Thereby, some or all of the physiological actions downstream of plasmin action can be suppressed. Plasmin actions include the following: Matrix metalloproteinase (MMP) activation; Thrombolysis; Promotion and activation of cytokine and chemokine expression; include (Non-Patent Document 4: Journal of Translational Medicine volume 20, Article number: 135 (2022), Non-Patent Document 5: Drug Discovery Today Volume 26, Issue 4, April 2021, Pages 1076-1085, Non-Patent Document 6: Current Pharmaceutical Design, Volume 17, Number 19, 2011, pp. 1874-1889 (16)). 1-(2-Hydroxyethyl)-2-imidazolidinone (HEI) or its derivative, or a salt thereof, can suppress at least one or a combination of these plasmin actions.

[0011] Matrix metalloproteinase (MMP) is a type of metalloproteinase with a metal ion coordinated in the active center, and currently MMP1 to MMP28 are known as the MMP family. Zinc ions and calcium ions are coordinated to matrix metalloproteinase. Matrix metalloproteinase degrades extracellular matrices such as collagen, proteoglycan, elastin, fibronectin, and laminin, and also degrades precursors of bioactive proteins such as cytokines and contributes to their activation. Due to such activities, it is involved in wound healing, bone remodeling, inflammation, cancer metastasis, invasion, and proliferation (Non-Patent Document 4). Matrix metalloproteinase is produced as a propeptide and exerts its activity by removing the propeptide site containing an amino acid (PRCGVP) called the cysteine switch. Matrix metalloproteinase has a mutual activation pathway centered on plasmin. In particular, MMP9 and MMP3 are activated by plasmin action, sequentially activate downstream matrix metalloproteinases, and MMP9 and MMP3 also activate plasminogen to generate plasmin (Figure 1). Matrix metalloproteinases activated by plasmin include, in addition to MMP9 and MMP3, MMP8, MMP7, MMP14, MMP13, and MMP1.

[0012] In the skin area, it is known that in particular, dermal fibroblasts secrete MMP2, and keratinocytes secrete MMP9 and MMP2. MMP produced by dermal fibroblasts can be activated by the action of plasmin and other proteases. By degrading collagen (especially type I / III collagen), elastin, proteoglycan, etc., which constitute the extracellular matrix of the dermis, it causes non-thinning of the dermis, induces a decrease in skin elasticity, sagging associated with decreased elasticity, wrinkles, etc. MMP secreted by keratinocytes acts on the basement membrane and degrades collagen anchored to the basement membrane, such as type IV collagen and type VII collagen, impairing the stability of the basement membrane. When the stabilization 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 disruption of the turnover of the stratum corneum. For the skin, it is possible to act directly transdermally. By transdermally administering an inhibitor of the interaction between urokinase (uPA) and urokinase receptor (uPAR), the action of MMP can be suppressed, maintaining and / or improving the turnover and health of the epidermis, improving the skin barrier function, and thereby exerting a preventive and / or improving effect on skin spots, dullness, fine wrinkles, etc. When acting on the dermal layer, it can exert an effect of suppressing dermal thinning, enhancing elasticity, and thereby improving wrinkles and / or sagging. Therefore, an inhibitor of the interaction between urokinase (uPA) and urokinase receptor (uPAR) can also be referred to as an inhibitor of dermal thinning, an elasticity enhancer, and an agent for improving wrinkles and / or sagging for the dermis, and as an agent for maintaining and / or improving turnover and health, an agent for improving skin barrier function, and an agent for preventing and / or improving skin spots, dullness, fine wrinkles, etc. for the epidermis.

[0013] In the circulatory system and tissues, matrix metalloproteinases are involved in angiogenesis by acting on the basement membrane of blood vessels. Angiogenesis is required for cancer growth, and matrix metalloproteinases also act when cancer invades from blood vessels into tissues and metastasizes. Therefore, an inhibitor of the interaction between urokinase (uPA) and urokinase receptor (uPAR) can also act as an angiogenesis inhibitor and / or a cancer inhibitor (Non-Patent Documents 1 and 2). Matrix metalloproteinases are also involved in bone destruction and regeneration. An inhibitor of the interaction between urokinase (uPA) and urokinase receptor (uPAR) can be used as a preventive or therapeutic agent for osteoporosis by acting on bone (Non-Patent Document 5). Since matrix metalloproteinases are involved in the activation of inflammatory cytokines, an inhibitor of the interaction between urokinase (uPA) and urokinase receptor (uPAR) can also be used as a preventive or therapeutic agent for inflammatory diseases (Non-Patent Document 5).

[0014] Plasmin degrades fibrin contained in thrombi and is involved in thrombolysis. The dissolution of fibrin contributes to blood vessel regeneration after hemostasis. Also, in thrombotic diseases such as myocardial infarction, it contributes to the dissolution of thrombi. On the other hand, it is also known that plasmin action causes bleeding. Therefore, an inhibitor of the interaction between urokinase (uPA) and urokinase receptor (uPAR) can also be used as a hemostatic control agent by suppressing plasmin formation. Also, plasmin is involved in promoting and / or activating the expression of cytokines and chemokines directly or indirectly through the action of metalloproteinases, and is also related to the degradation of cell membrane targets (Non-Patent Document 6). Therefore, an inhibitor of the interaction between urokinase (uPA) and urokinase receptor (uPAR) can also be used as an anti-inflammatory agent.

[0015] 1-(2-Hydroxyethyl)-2-imidazolidinone (HEI) or its derivative has the following formula:

Chemical formula

[0016] 1-(2-hydroxyethyl)-2-imidazolidinone (HEI) or its derivative, or a salt thereof, is a compound having MMP9 inhibitory activity and heparanase inhibitory activity. Based on these uses, it has a physiological effect of stabilizing the epidermal basement membrane and improving the skin barrier function. In this example, at a concentration higher than 0.5%, the interaction between urokinase (uPA) and urokinase receptor (uPAR) can be suppressed. The upper limit is not particularly limited. For example, the interaction between urokinase (uPA) and urokinase receptor (uPAR) can be suppressed at a concentration of 5% or less, 2% or less, or 1.5% or less. On the other hand, the concentration range when formulating into a product can be appropriately selected according to the desired physiological effect and administration route. When formulated into cosmetics or pharmaceuticals, as an example, it can be formulated in the range of 0.00001% to 10%, preferably 0.0001% to 5%, more preferably 0.001% to 3%. As an example, it can also be formulated at 1.5% for cosmetics such as lotion, essence, emulsion, cream, etc.

[0017] The inhibitor of the interaction between urokinase (uPA) and urokinase receptor (uPAR), MMP inhibitor, hemostatic agent, anti-inflammatory agent, angiogenesis inhibitor, and cancer treatment or preventive agent of the present invention can be used compatibly, and each may be formulated in cosmetics, pharmaceuticals, or quasi-drugs. Further, they may be formulated in foods, such as dietary supplements like supplements, or foods with functional claims. These agents may be administered orally or parenterally, for example, transdermally. When administered transdermally, they can be formulated into external skin preparations.

[0018] The external skin preparation is not particularly limited as long as it can be applied to the skin. For example, any dosage form 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. can be applied. When formulated into an external skin preparation, bases and excipients usually used in external skin preparations, such as preservatives, emulsifiers, pH adjusters, etc. may be used.

[0019] When formulated in cosmetics, it can be formulated in facial or body cosmetics such as lotion, emulsion, essence, cream, lotion, pack, essence, gel, etc., makeup cosmetics such as foundation, makeup base, concealer, etc., and further in bath agents. When formulated in foods, it can be formulated in capsules, tablets, beverages, etc. By using cosmetics, foods, pharmaceuticals, and quasi-drugs containing the components of the present invention, various physiological effects are brought about through the suppression of plasmin generation. The inhibitor of the interaction between urokinase (uPA) and urokinase receptor (uPAR) can be administered over a long period as a food or cosmetic of the present invention. From the viewpoint of causing a decrease in cholesterol, it may be administered for several days or more, one week or more, two weeks or more, one month or more, three months or more, or six months or more. The upper limit is not particularly limited, but it may be several years or less, for example, one year or less.

[0020] In the present invention, the subject to which 1-(2-hydroxyethyl)-2-imidazolidinone (HEI) or its derivative, or a salt thereof is administered is a subject in need of suppressing the interaction between urokinase (uPA) and urokinase receptor (uPAR), that is, a subject suffering from symptoms and diseases caused by the action of plasmin. Such subjects include, for example, subjects with thin dermis, problems with epidermal turnover and health, or skin barrier function, subjects suffering from cancer, subjects suffering from inflammation, and subjects suffering from bleeding. In subjects with thin dermis, problems with epidermal turnover and health, or skin barrier function, administration of 1-(2-hydroxyethyl)-2-imidazolidinone (HEI) or its derivative, or a salt thereof can prevent or improve the thinning of the dermis, problems with epidermal turnover and health, or the decline in skin barrier function. In subjects suffering from cancer, cancer invasion, metastasis, or proliferation can be suppressed. In subjects suffering from inflammation, the inflammation can be alleviated. Further, in subjects suffering from bleeding, hemostasis can be achieved.

[0021] All documents mentioned in this specification are hereby incorporated by reference in their entirety.

[0022] The examples of the present invention described below are for illustrative purposes only and do not limit the technical scope of the present invention. The technical scope of the present invention is limited only by the description in the claims. Changes to the present invention, for example, addition, deletion, and substitution of the constituent elements of the present invention, can be made on the condition that the gist of the present invention is not deviated from.

Examples

[0023] Example 1: Measurement of u-PAR-uPA interaction 100 μL of a solution of uPAR dissolved at 2 μg / mL in PBS was dispensed into each well of a uPAR antibody-coated 96-well plate (uPAR ELISA kit, R&D Systems, DUP00), and incubated overnight at 4°C. An aqueous solution of 1-(2-hydroxyethyl)-2-imidazolidinone (HEI) was prepared as the test solution. After washing the uPAR antibody-coated 96-well plate that had been incubated overnight, the test solution was added (final HEI concentrations of 0%, 0.25%, 0.5%, 1%, 1.5%), and incubated at room temperature for 1 hour. Next, 10 μL of a 5 ng / mL uPA-HRP (Molecular Innovations) solution dissolved in PBS was added and incubated for 1 hour. After washing the uPAR antibody-coated 96-well plate that had been incubated for 1 hour, 100 μL of TMB (ThermoFisher) was added and further incubated for 20 minutes. Next, 100 μL of 1N sulfuric acid was added as a stop solution and incubated for 15 minutes to stop the reaction. Signals were detected at 450 nm using a microplate reader. The results are shown in Figure 2. The interaction between uPAR and uPA in the presence of the test solution is shown in Figure 2(A), and the inhibition rate of uPAR-uPA by the test solution is shown in Figure 2(B).

Claims

**Claim 1** 1-(2-Hydroxyethyl)-2-imidazolidinone (HEI) or a derivative thereof, or a salt thereof, comprising the following: The following formula: 【Chemical Formula 1】 (wherein, n is an integer from 1 to 3, R 1 is a hydrogen atom or a hydrocarbon group having 1 to 6 carbon atoms which may be substituted with a hydroxyl group, X is -CH 2 - or a group represented by -N(R 2 )-, and R 2 is a hydrogen atom or a hydrocarbon group having 1 to 6 carbon atoms which may be substituted with a hydroxyl group) An inhibitor of the interaction between urokinase (uPA) and urokinase receptor (uPAR), comprising a compound represented by. **Claim 2** The interaction inhibitor according to claim 1, wherein the interaction inhibitor suppresses plasmin activation. **Claim 3** The interaction inhibitor according to claim 2, wherein the suppression of plasmin activation includes the expression of matrix metalloproteinase, and the interaction inhibitor suppresses the expression of matrix protease. **Claim 4** The interaction inhibitor according to claim 3, wherein the matrix protease is MMP3 or MMP9. **Claim 5** 1-(2-Hydroxyethyl)-2-imidazolidinone (HEI) or a derivative thereof, or a salt thereof, comprising the following formula: 【Chemical Formula 2】 (wherein, n is an integer from 1 to 3, R 1 is a hydrogen atom or a hydrocarbon group having 1 to 6 carbon atoms which may be substituted with a hydroxyl group, X is -CH 2 - or -N(R 2 ) - and is a group represented by R 2 is a hydrogen atom or a hydrocarbon group having 1 to 6 carbon atoms which may be substituted by a hydroxyl group) An inhibitor of matrix protease activation, comprising a compound represented by. **Claim 6** The activation inhibitor according to claim 5, wherein the matrix protease is MMP3 or MMP9. **Claim 7** 1-(2-Hydroxyethyl)-2-imidazolidinone (HEI) or a derivative thereof, or a salt thereof, comprising the following formula: 【Chemical 3】 (wherein, n is an integer from 1 to 3, R 1 is a hydrogen atom or a hydrocarbon group having 1 to 6 carbon atoms which may be substituted with a hydroxyl group, X is -CH 2 - or a group represented by -N(R 2 )-, and R 2 is a hydrogen atom or a hydrocarbon group having 1 to 6 carbon atoms which may be substituted with a hydroxyl group) A plasmin generation inhibitor, comprising a compound represented by, which suppresses plasmin generation by urokinase.

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