JP2010241712A - Tie2-activating agent and lymphatic vessel-stabilizing agent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a novel Tie2-activating agent and a lymphatic vessel-stabilizing agent comprising the activating agent. <P>SOLUTION: The Tie2-activating agent comprises diphenhydramine or diphenhydramine hydrochloride. The lymphatic vessel-stabilizing agent comprises the activating agent. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention provides a novel Tie2 activator comprising diphenhydramine or diphenhydramine hydrochloride and a lymphatic stabilizer comprising the activator.

  Blood is fed from the heart and returns to the heart via capillaries and veins. A lymphatic vessel forms a drainage path for tissue fluid separately from the vascular system. The lymphatic vessels maintain a closed circulatory system by keeping blood volume constant by circulating interstitial fluid, proteins, fats, cells, and the like leaked from the blood vessels in peripheral tissues to the vascular system. In the capillaries existing in the skin, the basement membrane surrounds the outside of the endothelial cells, and pericytes are further attached. On the other hand, in capillary lymphatic vessels, there is almost no basement membrane outside the endothelial cells, and pericytes are not attached. This structure is useful for efficiently taking in body fluids and cells from the interstitium (Non-patent Document 1). To date, it has been shown that the tyrosinase receptor VEGFR (vascular endothelial growth factor receptor) -3 is specifically expressed in lymphatic endothelial cells, and its ligands VEGF-C and VEGF-D are the neovascularization of lymphatic vessels. It was shown to induce Moreover, it has been clarified that VEGF-A induces lymphangiogenesis through VEGFR2 expressed in lymphatic endothelial cells (Non-patent Document 2). Furthermore, regarding the function of lymphatic vessels, there are the following reports. In mouse ears infected with adenovirus expressing VEGF-A, remarkable lymphangiogenesis was observed, but along with structural abnormalities, the recovery function of lymphatic vessels was also remarkable from experiments in which colloidal carbon was injected into the ear (Non-patent Document 3). That is, it is considered that lymphatic endothelial cells must be appropriately arranged and lined for the function of lymphatic vessels. We define this as “lymphatic vessel stabilization”.

  Physical or chemical stimulation to the skin induces angiogenesis and vascular permeability such as by VEGF-A, resulting in fluid retention and edema. On the other hand, these stimuli are also known to directly induce lymphangiogenesis and expansion. So far, lymphatic dilation has been observed due to ultraviolet inflammation, and experiments with dye injection have revealed that lymphatic function is impaired. It is considered that lymphatic vessels are expanding and collecting interstitial fluid as water leaks into the dermis due to vasodilation. However, it was thought that excessive lymphatic vessel dilatation reduced the recovery function and delayed edema (Non-patent Document 4). That is, it is considered that “stabilization of lymphatic vessels” that does not induce excessive expansion of lymphatic vessels is necessary for rapid recovery of interstitial fluid.

  So far, pathological conditions involving dysfunction of lymphatic vessels are known to include congenital lymphedema as well as filaria, surgery, malignant tumors, and secondary lymphedema associated with inflammation. Congenital lymphedema includes Milroy disease, Meige disease, and lymphedema-distichiasis syndrome. Milroy's disease has been reported to be aplastic or hypoplastic lymphatics, while lymphedema-distichiasis syndrome has been reported to have lymphatic hyperplasia. Also from these, it is considered necessary to maintain the recovery function not only by lymphatic neovascularization but also by stabilizing the lymphatic vessels (Non-patent Document 5).

Experimental Medicine Vol. 24, No. 18 (2006), pp. 133-138 Jussila L and Alitalo K, (2006) Vascular growth factors and lymphangiogenesis. Phisiol Rev 82: 673-700 Nagy et al., (2002) Vascular permeability factor / vascular endothelial growth factor induces lymphangiogenesis as well as angiogenesis.J Exp Med 196: 1497-1506 Kajiya K., Hirakawa S., and Detmar M., (2006) VEGF-A mediates UVB-induced impairment of lymphatic vessel function. Am J Pathol 169: 1496-1503 Experimental Medicine Vol. 24, No. 18 (2006), pp. 139-143 Kajiya K., et al., (2005) Hepatocyte growth factor promotes lymphatic vessel formation and function.EMBO J 24: 2885-95 Experimental Medicine Vol. 20, No.8 (2002), pp. 52-57

  An object of the present invention is to provide a novel Tie2 activator, and further to provide a drug effective for stabilizing and improving the recovery function of lymphatic vessels by stabilizing the lymphatic vessels.

  Beginning with molecular cloning of VEGF, molecules of the VEGF family and angiopoietin (Angopoietin; Ang) family have been identified one after another as factors that act specifically on angiogenesis. VEGF and its receptors are involved in a very wide range of angiogenesis, from the initial development of blood vessels called angiogenesis to subsequent angiogenesis. On the other hand, Ang functions in luminal formation accompanied by cellular phenomena such as germination, branching, insertion, and retraction by vascular endothelial cells after angiogenesis. Ang is a receptor-type tyrosine kinase expressed in vascular endothelial cells, Tie (tyrosine kinase with Ig and EGF homology domain) -2, and vascular endothelial cells and vascular walls such as pericytes and vascular smooth muscle cells. Although it is understood that it controls adhesion to cells and functions for structural stabilization of blood vessels (Non-patent Document 7), the relationship between Tie2 and lymphatic vessels has not been fully elucidated. It was.

As a result of the present inventors investigating the function of Ang-1 focusing on the relationship between Ang-1 and Tie2 expressed in lymphatic endothelial cells, Ang-1 recovered lymphatic vessels through the activation of Tie2. It became clear that the function was promoted (Japanese Patent Application No. 2008-159623). Furthermore, when the present inventor screened for a compound having Tie2 activation ability, it was found that diphenhydramine and its hydrochloride, which are H ₁ receptor antagonists, have Tie2 activity in the same manner as Ang-1. Has led to the completion of the invention:
(1) A Tie2 activator comprising diphenhydramine or diphenhydramine hydrochloride.
(2) A lymphatic vessel stabilizer comprising the Tie2 activator.
(3) A cosmetic method for improving or preventing swelling, which comprises applying the lymphatic vessel stabilizer of (2).

  By using the lymphatic vessel stabilizer according to the present invention, swelling and the like can be improved / prevented.

FIG. 1 is a Western blot diagram showing changes in the amount of Tie2 and phosphorylated Tie2 in normal human umbilical vein endothelial cells (HUVEC) and graphs showing changes in the amount of phosphorylated Tie2 when diphenhydramine is added. FIG. 2 is a Western blot diagram showing changes in the amount of Tie2 and phosphorylated Tie2 in HUVEC and graphs showing changes in the amount of phosphorylated Tie2 when diphenhydramine hydrochloride is added.

Tie2 Activator In one aspect, the present invention provides a Tie2 activator comprising diphenhydramine or a hydrochloride salt thereof.

  Activation of Tie2 refers to the ability to convert Tie2 into its active form (phosphorylated Tie2) by phosphorylation. As activators of Tie2, there are those well known to activate Tie2, such as Angiopoietin 1.

Diphenhydramine (2-benzhydryloxy-N, N-dimethylethanamine) is a compound known as an H ₁ antagonist and has the following chemical structure:

  In addition, diphenhydramine hydrochloride is usually used as an antihistamine, and recently, it is also used as a sleep-improving drug based on its hypnotic action. However, none of diphenhydramine and its hydrochloride is known to have Tie2 activity or to stabilize lymphatic vessels. This time, when this inventor investigated Tie2 activity about various chemical substances, it discovered that diphenhydramine and diphenhydramine hydrochloride increased the amount of phosphorylated Tie2 of a vascular endothelial cell.

  As shown in FIGS. 1 and 2, diphenhydramine and its hydrochloride show a Tie2 activation action in a concentration-dependent manner. Therefore, from such a viewpoint, the blending amount of diphenhydramine in the Tie2 activator of the present invention is 0.0001 to 10% by mass, preferably 0.001 to 0.01% by mass, more preferably 0.001% by mass in the total amount of the agent. . In the case of hydrochloride, it is 0.001-10 mass%, Preferably it is 0.001-0.01 mass%, More preferably, it is 0.01 mass%.

  Diphenhydramine hydrochloride is preferred as the drug compounded in the Tie2 activator. However, other inorganic or organic salts such as citrate, salicylate, lauryl sulfate, etc. are also considered to exhibit Tie2 activity.

Lymphatic vessel stabilizer In another aspect, the present invention provides a lymphatic vessel stabilizer comprising the Tie2 activator.

  The lymphatic vessel stabilizer according to the present invention can be used as a pharmaceutical or cosmetic effective for the treatment / prevention of various skin diseases such as edema (swelling) caused by leakage of lymph due to instability of the structure of lymphatic vessels. Examples of edema include secondary lymphedema associated with ultraviolet radiation, filaria, surgery, malignant tumors, inflammation, and congenital lymphedema such as Milroy disease, Meige disease, and lymphedema-distichiasis syndrome.

  The dose, usage, and dosage form of the lymphatic vessel stabilizer according to the present invention can be appropriately determined according to the purpose of use. For example, the dosage form of the lymphatic vessel stabilizer of the present invention is not particularly limited, and may be oral, parenteral, external use and the like. Preferably it is an external preparation. Examples of the dosage form include external preparations such as ointments, creams, emulsions, lotions, packs, bath preparations, parenteral preparations such as injections, drops, or suppositories, or tablets, powders, capsules, granules, Oral administration agents such as extract and syrup can be mentioned.

  The compounding amount of the Tie2 activator in the lymphatic vessel stabilizer of the present invention can be appropriately determined depending on the use, but is generally 0.0001 to 20.0 mol%, preferably 0.0001 to 10.0 mol% in the total amount of the agent.

  In addition to the Tie2 activator, the lymphatic vessel stabilizer of the present invention includes, for example, excipients, moisture-proofing agents, preservatives, strengthening agents, thickeners, emulsifiers, and the like used in ordinary foods and pharmaceuticals. Antioxidants, sweeteners, acidulants, seasonings, colorants, fragrances, whitening agents, moisturizers, oily ingredients, UV absorbers, surfactants, thickeners, alcohols, powders commonly used in cosmetics, etc. Components, colorants, aqueous components, water, various skin nutrients, and the like can be appropriately blended as necessary.

  Furthermore, when the lymphatic vessel stabilizer of the present invention is used as an external preparation for skin, auxiliary agents commonly used for external preparations for skin, such as disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate Sequestering agents such as gluconic acid, caffeine, tannin, verapamil, tranexamic acid and its derivatives, licorice extract, grabrizine, hot water extract of karin fruit, various herbal medicines, tocopherol acetate, glycyrrhizic acid and its derivatives or their derivatives Drugs such as salts, whitening agents such as vitamin C, magnesium ascorbate phosphate, glucoside ascorbate, arbutin, kojic acid, sugars such as glucose, fructose, mannose, sucrose, trehalose, retinoic acid, retinol, retinol acetate, palmiticin Acid retinoic Such as vitamin A like may also be appropriately blended.

Cosmetic method In still another aspect, the present invention provides a cosmetic method for improving or preventing swelling, comprising applying a lymphatic vessel stabilizer.

  The cosmetic method according to the present invention is intended to reduce or prevent swelling and eye bags. This cosmetic method is applied to, for example, a site with swelling of the lymphatic vessel stabilizer according to the present invention and left as it is, or subjected to, for example, massage according to the direction of the flow of the lymphatic vessel. For example, the liquid flow can be promoted. This part can be applied to all parts of the body such as the face, neck, limbs, and the like.

  Next, the present invention will be described in more detail with reference to examples. In addition, this invention is not limited by this.

Western blotting of vascular endothelial cells
Normal human umbilical vein endothelial cells (HUVEC) expressing Tie2 were purchased from Sanko Junyaku. After culturing HUVEC in EBM-2 (Cambrex; Verviers, Belgium) to which additional factors such as growth factors were added, proteins in HUVEC in the presence of diphenhydramine and diphenhydramine hydrochloride at various concentrations (0.01% to 0.0001% by mass) Extracted with Phosphosafe Extraction Reagent (Novagen, Madison, WI). As a control, HUVEC to which solvent ethanol was added was also prepared.

  The total protein amount was quantified with RC DC Protein Assay Kit (BIO-RAD, Hercules, CA) and detected by Western blotting as follows. SDS-PAGE was performed on 7.5% acrylamide gel (NPU-7.5L, ATTO, Japan) using an equal amount of total protein. The color was developed with ECL Kit. The results are shown in FIGS.

  From the results of FIG. 1 and FIG. 2, it can be seen that diphenhydramine and its hydrochloride show a remarkable Tie2 activation action compared to control ethanol, and that the action is concentration-dependent. Since Ang-1 having the ability to activate Tie2 promotes the recovery function of lymphatic vessels, it is considered that these drugs also have the effect of stabilizing lymphatic vessels in the same manner as Ang-1.

Claims (3)

  A Tie2 activator comprising diphenhydramine or diphenhydramine hydrochloride.   A stabilizer for lymphatic vessels comprising the Tie2 activator according to claim 1.   A cosmetic method for improving or preventing swelling, which comprises applying the lymphatic vessel stabilizer according to claim 2.