Patent Application
20100292509
The present invention relates to an ameliorant for bloating, lymphedema, wrinkles or obesity by inducing expression of vascular endothelial growth factor C (VEGFC) and activating lymphatic vessel function.
The skin's vascular system is present in the dermis, and is composed of blood vessels and lymphatic vessels. Tissue fluids that have migrated outside blood vessels must again return to veins to maintain homeostasis. Veins of the skin efficiently send blood to the center of the body. However, veins themselves lack the ability to incorporate tissue fluids. Consequently, tissue incorporating tissue fluids, namely lymphatic vessels, have come to be understood to be essential structures in the skin as well.
Lymphatic vessels play an important role in maintaining the microenvironment surrounding cells in a constant state by recovering unnecessary substances present in the skin as well as moisture and proteins that constantly leak from blood vessels. In addition, they are also thought to play a role that resists infectious factors and external factors from the exterior through transport of T lymphocytes.
Known examples of symptoms associated with the pathology of lymphatic vessel dysfunction include bloating and lymphedema (Non-Patent Document 1: Jussila, L. & Alitalo, K. (2002) Physiol. Rev. 82, 673-700). In addition, the functions of lymphatic vessels are also known to play an important role not only with respect to bloating, but also in photoaging (wrinkle formation) of skin accompanying exposure to ultraviolet rays (Non-Patent Document 2: Kajiya, K. & Detmar, M. (2006) J. Invest. Dermatol., 126, 919-21).
On the basis of research conducted thus far, VEGFR-3 has been identified as a transmembrane receptor specifically present in lymphatic vessels, and VEGFC and VEGFD have been found to be ligands thereof. VEGFC activates lymphatic vessel function by promoting growth and migration of lymphatic vascular endothelial cells as well as lumen formation by acting on lymphatic vessels (Non-Patent Document 1: Jussila, L. & Alitalo, K. (2002) Physiol. Rev. 82, 673-700). In addition, studies have been conducted regarding the potential of gene therapy involving introduction of VEGFC for edema as a pathological state of bloating (Non-Patent Document 3: Saaristo, A., Tammela, T., Timonen J., Yla-Herttuala, S., Tukiainen, E., Asko-Seljavaara, S. & Alitalo, K. (2004) Faseb. J., 18, 1707-9).
More recently, genetically mutant mice bred to cause functional abnormalities of lymphatic vessels are known to present with obesity as they mature. Findings have been obtained regarding the mechanism by which abnormalities in lymphatic vessel formation and function cause obesity that indicate that lymph flowing through lymphatic vessels differentiates into fat of preadipocytes (Non-Patent Document 4: Harvey, N. L. et al., Nat. Genet. 2005, 37, 1072-81). In other words, functional abnormalities of lymphatic vessels have been reported to cause leakage of lymph outside lymphatic vessels, which then differentiates into fat and eventually leads to formation of obesity. Thus, a VEGFC promoter is expected to be a therapeutic drug for the prevention of obesity that results in functional lymphangiogenesis.
Members of the VEGF gene family consist of VEGF-A to VEGF-E. Among these, VEGFB and VEGFE have been identified as factors that only act on blood vessels. VEGFA is known to be a factor that is present in skin and but conversely acts to exacerbate the function of lymphatic vessels (Non-Patent Document 5: Nagy, J. A., Vasile, E., Feng, D., Sundberg, C., Brown, L. F., Detmar, M. J., Lawitts, J. A., Benjamin, L., Tan, X., Manseau, E. J., Dvorak, A. M. & Dvorak, H. F. (2002) J. Exp. Med. 196, 1497-506).
In the skin, although VEGFD is reported to only be present in minute amounts in the dermis, since VEGFD knockout mice do not present with any morphological or functional abnormalities in skin lymphatic vessels, they are considered to not be essential factors for the formation of skin lymphatic vessels (Non-Patent Document 6: Baldwin, M. E., Halford, M. M., Roufail, S., Williams, R. A., Hibbs, M. L., Grail, D., Kubo, H., Stacker, S. A. & Achen, M. G. (2005) Mol. Cell. Biol. 25, 2441-9). On the other hand, VEGFC in the skin is strongly expressed in the epidermis. Mice that highly express VEGFC in the epidermis demonstrate increased numbers of lymphatic vessels in the dermis (Non-Patent Document 7: Jeltsch, M., Kaipainen, A., Joukov, V., Meng, X., Lakso, M., Rauvala, H., Swartz, M., Fukumura, D., Jain, R. K. & Alitalo, K. (1997) Science 276, 1423-5), while on the other hand, when the effects of VEGFC in the epidermis were blocked by highly expressing neutralizing antibody of a receptor of VEGFC in the form of VEGFR-3 in the epidermis, the number of lymphatic vessels in the dermis decreased dramatically and findings indicating the formation of edema were obtained (Non-Patent Document 8: Makinen, T., Jussila, L., Veikkola, T., Karpanen, T., Kettunen, M. I., Pulkkanen, K. J., Kauppinen, R., Jackson, D. G., Kubo, H., Nishikawa, S., Yla-Herttula, S. & Alitalo, K. (2001) Nat. Med. 7, 199-205). On the basis of these findings, it has been determined that the function of lymphatic vessels present in the skin dermis is controlled by VEGFC expressed in the skin, and that bloating (edema) occurs when VEGFC no longer functions.
Therefore, compounds that promote expression of VEGFC were screened as a method for eliminating bloating and the like by activating lymphatic vessels.
An object of the present invention is to find a novel drug that is effective for prevention or control of bloating, lymphedema, wrinkle formation or obesity from the viewpoint of activating lymphatic vessel function by inducing expression of VEGFC.
The inventors of the present invention found that a tranexamic acid amide derivative and/or salt thereof induces expression of VEGFC by screening numerous compounds. In other words, a tranexamic acid amide derivative and/or salt thereof that induces expression of VEGFC was found to be extremely effective for preventing or inhibiting bloating, lymphedema, wrinkle formation caused by photoaging of skin accompanying exposure to ultraviolet light or obesity by promoting activation of lymphatic vessel function.
The constitution of the present invention is as described below.
[a] A bloating ameliorant comprising a tranexamic acid amide derivative represented by the following general formula (I):
wherein, R1 and R2 represent hydrogen atoms, linear or branched alkyl groups having 1 to 18 carbon atoms, cycloalkyl groups having 5 to 8 carbon atoms, benzyl groups or groups represented by the following general formula (II):
wherein, X represents a lower alkyl group, lower alkoxy group, hydroxy group, amino group or halogen atom and n=0 to 3, and
R1 and R2 may be the same or different, and/or a salt thereof.
[b] A lymphedema ameliorant comprising the tranexamic acid amide derivative and/or salt thereof described in [a].
[c] A wrinkle ameliorant comprising the tranexamic acid amide derivative and/or salt thereof described in [a].
[d] An obesity ameliorant comprising the tranexamic acid amide derivative and/or salt thereof described in [a].
[e] A lymphatic vessel neogenesis and/or function promoter comprising the tranexamic acid amide derivative and/or salt thereof described in [a].
[f] A VEGFC production promoter comprising the tranexamic acid amide derivative and/or salt thereof described in [a].
[g] The lymphatic vessel neogenesis and/or function promoter described in [e] that prevents or inhibits bloating.
[h] The VEGFC production promoter described in [f] that prevents or inhibits bloating.
[i] The lymphatic vessel neogenesis and/or function promoter described in [e] that prevents or inhibits lymphedema.
[j] The VEGFC production promoter described in [f] that prevents or inhibits lymphedema.
[k] The lymphatic vessel neogenesis and/or function promoter described in [e] that prevents or inhibits wrinkle formation.
[l] The VEGFC production promoter described in [f] that prevents or inhibits wrinkle formation.
[m] The lymphatic vessel neogenesis and/or function promoter described in [e] that prevents or inhibits obesity.
[n] The VEGFC production promoter described in [f] that prevents or inhibits obesity.
[o] A method for inhibiting bloating comprising applying the lymphatic vessel neogenesis and/or function promoter described in [e] to the skin.
[p] A method for inhibiting bloating comprising applying the VEGFC production promoter described in [f] to the skin.
[q] A method for inhibiting wrinkle formation comprising applying the lymphatic vessel neogenesis and/or function promoter described in [e] to the skin.
[r] A method for inhibiting wrinkle formation comprising applying the VEGFC production promoter described in [f] to the skin.
Definitions: The term “bloating” in the present description is used with the same meaning as “edema”, and refers to a state in which tissue fluid or lymph accumulates in cells, intercellular space or body cavities for some reason, and which is observed in subcutaneous tissue. Moreover, the term “lymphedema” in the present description refers to a state in which tissue fluid accumulates in peripheral portions of the body due to occlusion of lymphatic vessels.
Screening of drugs that induce lymphatic vessel activating factor in the form of VEGFC was carried out as described in detail in the following examples by allowing various candidate drugs to act on cell line HaCat, which consists of cultured cells derived from human epidermis, quantifying VEGFC, and selecting drugs having VEGFC induction activity as effective drugs by using the amount of VEGFC as an indicator of VEGFC induction activity.
As a result, a tranexamic acid derivative and/or salt thereof was found to induce VEGFC.
The tranexamic acid amide derivative as claimed in the present invention is a compound represented by the following general formula (I):
wherein, R1 and R2 represent hydrogen atoms, linear or branched alkyl groups having 1 to 18 carbon atoms, cycloalkyl groups having 5 to 8 carbon atoms, benzyl groups or groups represented by the following general formula (II):
wherein, X represents a lower alkyl group, lower alkoxy group, hydroxy group, amino group or halogen atom and n=0 to 3, and
R1 and R2 may be the same or different.
The tranexamic acid amide derivative represented by general formula (I) as claimed in the present invention is a known substance, and can be easily synthesized by a known method such as the method described in ActPharm. Suecica, 7, 441 (1970) or J. Med. Chem., 15, 247 (1972). Namely, after protecting the amino group of tranexamic acid with an appropriate protecting group such as a benzyloxycarbonyl group, an amide form of protected tranexamic acid is produced by allowing an amine component to react with the protected form or reactive derivative of the protected form. An acid halide such as an acid chloride or acid bromide or a mixed acid anhydride and the like are preferable as a reactive derivative of the protected form. Subsequently, the protecting group is eliminated by contact reduction and the like to produce an amide derivative of tranexamic acid.
In addition, the tranexamic acid amide derivative as claimed in the present invention can be converted to an inorganic salt or organic salt by a known method. Although there are no particular limitations on the salt used in the present invention, examples include inorganic salts such as hydrochlorides, sulfates, phosphates, hydrobromides, sodium salts, potassium salts, magnesium salts, calcium salts or ammonium salts. Examples of organic salts include acetates, lactates, maleates, fumarates, tartrates, citrates, methanesulfonates, p-toluenesulfonates, triethanolamine salts, diethanolamine salts and amino acid salts. Hydrochlorides are used preferably.
The tranexamic acid amide derivative and/or salt thereof as claimed in the present invention is extremely effective for promoting the formation and function of lymphatic vessels. Examples of symptoms associated with lymphatic vessel dysfunction include not only bloating and lymphedema, but also photoaging of skin accompanying exposure to ultraviolet light (such as wrinkle formation) and obesity. Thus, the tranexamic acid amide derivative and/or salt thereof as claimed in the present invention is effective for preventing and inhibiting bloating and lymphedema as well as photoaging of skin accompanying exposure to ultraviolet light and obesity. Moreover, the tranexamic acid amide derivative and/or salt thereof is also considered to be effective for treatment of congenital lymphedema.
Photoaging of skin typically refers to changes in skin appearance and function observed as a result of repeated exposure to sunlight. Ultraviolet (UV) light, which is a composite element of sunlight, and particularly intermediate UV light (referred to as UVB having a wavelength of 290 to 320 nm) is the main cause of photoaging. The level of exposure to UVB required for causing photoaging is currently unknown. However, repeated exposure to UVB at a level that causes erythema or sunburn normally leads to photoaging. Photoaging can be identified clinically by rough skin, wrinkle formation, colored spots, soil-colored skin, formation of sagging skin, onset of angiotelectasia, mole formation, onset of purpura, increased susceptibility to skin injury, hypertrophy, formation of areas of fibrotic depigmentation, and the occurrence of premalignant and malignant tumors. Photoaging normally occurs in skin routinely exposed to sunlight, such as skin of the face, ears, head, neck and hands.
The tranexamic acid amide derivative and/or salt thereof in the bloating ameliorant, lymphedema ameliorant, wrinkle ameliorant, obesity ameliorant, lymphatic vessel neogenesis and/or function promoter or VEGFC production promoter of the present invention is incorporated at 20 to 0.001% by weight, and preferably at 7 to 0.01% by weight, based on the total weight of the drug.
In addition, a component other than the aforementioned essential component routinely used in cosmetics, pharmaceuticals and other skin preparations for external use, such as whiteners, moisturizers, antioxidants, oily components, ultraviolet absorbers, surfactants, thickeners, alcohols, powdered components, colorants, aqueous components, water or various types of skin nutrients, can be suitably incorporated in the bloating ameliorant, lymphedema ameliorant, wrinkle ameliorant, obesity ameliorant, lymphatic vessel neogenesis and/or function promoter or VEGFC production promoter of the present invention as necessary.
In addition, metal chelating agents such as disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate or gluconic acid, drugs such as pantothenyl ethyl ether, caffeine, tannin, verapamil, glycyrrhiza extract, glabridin, rosewood fruit hot water extract, various types of herbal medicines, yeast extract, tocopherol acetate, glycyrrhizic acid, and derivatives or salts thereof, whiteners such as allantoin, vitamin C, magnesium ascorbyl phosphate, ascorbyl glucoside, albutin, kojic acid or potassium 4-methoxysilicylate, sugars such as Glucose, fructose, mannose, saccharose or trehalose, vitamin A substances such as retinoic acid, retinol, retinol acetate or retinol palmitate, L-serine, hyaluronic acid and derivatives thereof can also be suitably incorporated.
The bloating ameliorant, lymphedema ameliorant, wrinkle ameliorant, obesity ameliorant, lymphatic vessel neogenesis and/or function promoter or VEGFC production promoter of the present invention may be used in any type of conventional skin preparation for external use, examples of which include ointments, creams, milky liquids, lotions, packs or bath oils, may be used for aesthetic purposes, and there are no particular limitations on the form thereof.
HaCaT cells, which are cultured cells derived from human epidermis (German Cancer Research Center, Heidelberg, Germany), were cultured in DMEM containing 10% FBS and seeded into a 24-well plate at 15×104 cells per well. After about 24 hours, the medium was replaced with serum-free DMEM followed by culturing for 24 hours. Subsequently, a solution of a candidate drug dissolved in water to a concentration of 7% by weight was added in an amount equal to 1/100, 1/1000 or 1/10000 the amount of culture liquid followed by additional culturing. 48 hours after adding the drug, the culture supernatant was recovered and the amount of VEGFC in the culture supernatant was quantified by ELISA (R&D Systems) in accordance with the protocol. In addition, Alamar Blue (TREK Diagnostic Systems) was added to the cells following recovery of culture supernatant followed by measurement of the number of cells as fluorescence intensity (Ex: 544 nm, Em: 590 nm). The amount of VEGFC expressed per cell was determined by dividing the amount of VEGFC in the culture supernatant by the fluorescence value of the Alamar Blue. A solvent in the form of 70% EtOH was added to the cells instead of drug for use as a control, and the value thereof was assigned a value of 100. In addition, 5% FBS was used as a positive control.
On the basis of the above screening results, tranexamic acid amide derivative and/or salt thereof, and particularly tranexamic acid methylamide hydrochloride, demonstrated dose-dependent VEGFC induction activity that exceeded that of the control (
Melilotus extract
Lamium Album extract
Sophora Angustifolia extract
Iris Florentina root extract
Crataegus Cuneata fruit extract
Rehmannia root extract
Mortierella oil
Iris Florentina root extract
Crataegus Cuneata fruit extract
Uncaria gambir extract
Melilotus extract
Lamium Album extract
Scutellaria root extract
Melilotus extract
Crataegus Cuneata fruit extract
Lamium Album extract
Melilotus extract
Iris Florentina root extract
Lamium Album extract
Saxifraga sarmentosa extract
Iris Florentina root extract
| Number | Date | Country | Kind |
|---|---|---|---|
| 2008-010675 | Jan 2008 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP2009/050580 | 1/16/2009 | WO | 00 | 7/20/2010 |
