Patent Application
20100113590
The present invention relates to a novel ADAM inhibitor.
A group of the membrane type metalloproteases called ADAM (a disintegrin and metalloprotease) are enzymes which play important roles in interactions between cells and extracellular matrices and known to be greatly involved in the differentiation and proliferation of cells, extension and myelination of axons and the like (see, for example, Non-Patent Reference 1). In addition, ADAM as well as the matrix metalloprotease (MMP) has a function of cutting various growth factors including heparin-binding EGF-like growth factor (HB-EGF) and tumor necrosis factor (TNF), transforming growth factor; TGF, cytokines, amyloid precursor protein (APP) and many other membrane proteins on the cell surface and releasing them out of the cell. It is known that ADAM is highly expressed in the pathologic tissues of various malignant tumors (glioma, lung cancer, breast cancer, etc.), rheumatism, and in the cerebrum of patients with Alzheimer's disease, and promotes proliferation and infiltration of the cell or induces the angiogenesis essential for infiltration of cancer cells (see, for example, Non-Patent References 2-6). Therefore, suppression of ADAM activity is considered to be very important in preventing progression of the disease such as cancer, rheumatism, Alzheimer's disease and the like.
Non-Patent Reference 1: Yang P, Baker K A, Hagg T. The ADAMs family: Coordinators of nervous system development, plasticity and repair. Prog Neurobiol 2006; 79: 73-94.
Non-Patent Reference 2: Arribas J, Bech-Serra J J, Santiago-Josefat B. ADAMs, cell migration and cancer. Cancer Metastasis Rev 2006; 25: 57-68.
Non-Patent Reference 3: Kodama T, Ikeda E, Okada A et al. ADAM12 is selectively overexpressed in human glioblastomas and is associated with glioblastoma cell proliferation and shedding of heparin-binding epidermal growth factor. Am J Pathol 2004; 165: 1743-53.
Non-Patent Reference 4: Liu P C, Liu X, Li Y et al. Identification of ADAM10 as a Major Source of HER2 Ectodomain Sheddase Activity in HER2 Overexpressing Breast Cancer Cells. Cancer Biol Ther 2006; 5: 657-64.
Non-Patent Reference 5: Rocks N, Paulissen G, Quesada Calvo F et al. Expression of a disintegrin and metalloprotease (ADAM and ADAMTS) enzymes in human non-small-cell lung carcinomas (NSCLC). Br J Cancer 2006; 94: 724-30.
Non-Patent Reference 6: Asakura, M. et al. 2002, Cardiac hypertrophy is inhibited by antagonism of ADAM12 processing of HB-EGF: metalloproteinase inhibitors as a new therapy, Nat. Med. 8: 35-40.
While TAPI-1{N—(R)-(2-(Hydroxyaminocarbonylmethyl)-4-Methylpentanoyl-L-NaI-L-Alanine2-Aminoethyl amide(L-NaI:L-3-(2′-Naphthyl)alanine)} and TAPI-2{N—(R)-(2-(Hydroxyaminocarbonyl)methyl)-4-Methylpentanoyl-L-t-Butyl-Glycyl-L-Alanine2-Aminoethyl amide} are known as ADAM inhibitors, ADAM inhibitory ability of those substances is not sufficient and their safety as external medications has not been established. Therefore, there has been a need for developing a safer and more efficacious agent.
The present invention has been attained in view of the above situation and for the purpose of providing an ADAM inhibitor, which has an ADAM inhibiting action and can prevent or ameliorate the diseases caused by increase of ADAM activity.
ADAM is an enzyme which exists on the cell surface, and it releases the growth factors including HB-EGF, TNF-α, TGF-α, and cytokines, amyloid precursors, etc. out of the cell membrane, and activates them. One of the important mechanisms for induction or aggravation of diseases including cancers, rheumatism or Alzheimer's disease is considered that ADAM is activated or its expression is enhanced by the signal of malignant transformation or external stimuli leading to promotion of release/activation of the growth factors such as HB-EGF and cytokines as well as differentiation, proliferation and migration of the cell. Therefore, it is assumed that induction or progression of the conditions such as cancers, rheumatism or Alzheimer's disease could be prevented or ameliorated by suppressing ADAM activity leading to suppressing differentiation, proliferation or filtration of the cell. Further, the invention could not only be applied to the diseases caused by increase of ADAM activity but also could prevent or ameliorate the wrinkle by application to the wrinkle caused by hyperplasia of the epidermal cell.
In searching for a new compound which may inhibit ADAM, the inventors found that certain ascorbic acid derivatives and salts thereof have high ADAM inhibitory activity, thus leading to attainment of the invention.
The invention is an ADAM inhibitor characterized by comprising an ascorbic acid derivative represented by the general formula (I) described below or a salt thereof:
wherein, R represents a linear or branched alkyl group having 1 to 22 carbon atoms.
The ascorbic acid derivatives of the invention are those which prevent abnormal differentiation, proliferation and migration of the cell by inhibiting or suppressing ADAM activity.
Further, the invention provides a method for inhibiting ADAM characterized in that the ADAM inhibitor is applied to the skin to prevent or ameliorate the conditions caused by increase of ADAM activity.
The ADAM inhibitor of the invention can inhibit ADAM activity present in the tissue to suppress very effectively the release/activation of different growth factors and cytokines on the cell surface as well as differentiation, proliferation and migration of the cell.
According to the method of the invention for inhibiting ADAM, the conditions caused by increase of ADAM activity can be prevented or ameliorated effectively using an ADAM inhibitor described above.
The most preferred embodiments of the invention will be described below. An ascorbic acid derivative or a salt thereof used in the invention is represented as general formula (I) described below.
wherein, R represents a linear or branched alkyl group having 1 to 22 carbon atoms.
R in the ascorbic acid derivative (1) of the inventive compounds is a linear or branched alkyl group having 1 to 22 carbon atoms, preferably a linear or branched alkyl group having 4 to 18 carbon atoms, more preferably a linear or branched alkyl group having 12 to 18 carbon atoms. Such groups include, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl, heptyl, octyl, 2-ethylhexyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosane, heneicosane, docosane, etc., preferably, butyl, isobutyl, tert-butyl, pentyl, hexyl, heptyl, octyl, 2-ethylhexyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, etc., more preferably, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, etc.
Ascorbic acid derivatives of the invention include, for example, 2-O-methylascorbic acid, 2-O-ethylascorbic acid, 2-O-propylascorbic acid, 2-O-isopropylascorbic acid, 2-O-butylascorbic acid, 2-O-isobutylascorbic acid, 2-O-tert-butylascorbic acid, 2-O-pentylascorbic acid, 2-O-hexylascorbic acid, 2-O-heptylascorbic acid, 2-O-octylascorbic acid, 2-O-(2-ethylhexyl)ascorbic acid, 2-O-nonylascorbic acid, 2-O-decylascorbic acid, 2-O-undecylascorbic acid, 2-O-dodecylascorbic acid, 2-O-tridecylascorbic acid, 2-O-tetradecylascorbic acid, 2-O-pentadecylascorbic acid, 2-O-hexadecylascorbic acid, 2-O-heptadecylascorbic acid, 2-O-octadecylascorbic acid, 2-O-nonadecylascorbic acid, 2-O-eicosane ascorbic acid, 2-O-heneicosane ascorbic acid, 2-O-docosane ascorbic acid, etc. Among them, 2-O-dodecylascorbic acid and 2-O-octadecylascorbic acid are particularly preferable.
Ascorbic acid derivatives used in the invention include, besides free acids, medically acceptable salt forms thereof. These salts include, for example, but not limited to, alkali metal salts such as sodium and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, amine salts such as methylamine salts, dimethylamine salts, trimethylamine salts, methylpiperidine salts, ethanolamine salts, diethanolamine salts, triethanolamine salts and lysine salts, ammonium salts or basic amino acid salts.
Ascorbic acid derivatives and salts thereof may be produced according to the known producing methods.
The ADAM inhibitor comprising an ascorbic acid derivative or a salt thereof may be used as but not limited to dermatological external agents, powders, granules, ampules and injections.
In case of using an ADAM inhibitor of the invention in a dermatological external agent, the combination amount of an ascorbic acid derivative or a salt thereof varies depending on the its use aspect and product form, being, for example, but not particularly limited to preferably 0.001-10% by mass, more preferably 0.005-5% by mass, even more preferably 0.01-1% by mass based on the total amount.
In the specification here, the “dermatological external agents” include cosmetics, drugs and quasi drugs, etc. Its formulations include any such as an aqueous, solubilizing, emulsified, oil, gel, paste, ointment, aerosol, water-oil bilayer and water-oil-powder three layer systems. They also include those carried on the sheeted base.
The dermatological external agents can also adopt any product forms and uses. For example, it can be used as an external agent for the face, body or scalp including lotions, emulsions, creams and packs.
The dermatological external agent may mix properly as needed besides the above described ascorbic acid derivative or a salt thereof, any other components used in the dermatological external agent including ordinary cosmetics and drugs, and produced according to the conventional methods depending on the intended formulation. For example, an ascorbic acid derivative or a salt thereof may be mixed with one or two or more of the ingredients below to prepare a dermatological external agent.
Ultraviolet light absorbing agents include, for example, ultraviolet absorber of benzoic acid system such as para-aminobenzoic acid (hereinafter, abbreviated as PABA), PABA monoglycerin ester, N,N-dipropoxy PABA ethyl ester, N,N-diethoxy PABA ethyl ester, N,N-dimethyl PABA ethyl ester, N,N-dimethyl PABA butyl ester, and N,N-dimethyl PABA methyl ester, etc.; ultraviolet absorber of anthranilic acid system such as homomethyl-N-acetylanthranilate; ultraviolet absorber of salicylic acid system such as amyl salicylate, menthyl salicylate, homomethyl salicylate, octyl salicylate, phenyl salicylate, benzyl salicylate, p-isopropanol phenyl salicylate, etc.; ultraviolet absorber of cinnamic acid system such as octyl cinnamate, ethyl-4-isopropyl cinnamate, methyl-2,5-diisopropyl cinnamate, ethyl-2,4-diisopropyl cinnamate, methyl-2,4-diisopropyl cinnamate, propyl-p-methoxy cinnamate, isopropyl-p-methoxy cinnamate, isoamyl-p-methoxy cinnamate, octyl-p-methoxy cinnamate (2-ethylhexyl-p-methoxy cinnamate), 2-ethoxyethyl-p-methoxy cinnamate, cyclohexyl-p-methoxy cinnamate, ethyl-α-cyano-β-phenyl cinnamate, 2-ethylhexyl-α-cyano-β-phenyl cinnamate, glycerylmono-2-ethylhexanoyl-dipara-methoxy cinnamate, and methyl bis(trimethylsiloxane)silylisopentyl trimethoxy cinnamate, etc.; 3-(4′-methylbenzylidene)-d,1-camphor; 3-benzylidene-d,1-camphor; urocanic acid; urocanic acid ethyl ester, 2-phenyl-5-methylbenzoxazole, 2,2′-hydroxy-5-methylphenylbenzotriazole; 2-(2′-hydroxy-5′-t-octylphenyl)benzotriazole; 2-(2′-hydroxy-5′-methylphenylbenzotriazole; dibenzaladine, dianisoylmethane, 4-methoxy-4′-t-butyldibenzoylmethane; 5-(3,3-dimethyl-2-norbornylidene)-3-pentane-2-one; dimorpholinopyridazinone, etc. and any one or two or more may be used.
Ultraviolet light scattering agents include, for example, powders such as titanium oxide, particulate titanium oxide, zinc oxide, particulate zinc oxide, ferric oxide, particulate ferric oxide, and ceric oxide, etc.
For these ultraviolet light scattering agent, a spicular, spindle, spherical or granular powder is usually used. In addition, particulate powders which have the particle size of not more than 0.1 μm is preferable.
Also preferable are ultraviolet light scattering agents silicone-treated by methyl hydrogen polysiloxane or silane coupling agent; metallic soap-treated; fluorine-treated by perfluoroalkyl phosphate diethanolamine salt or perfluoroalkyl silane; hydrophobilized-treated by dextrin fatty acid ester treatment and the like.
Liquid oils include, for example, avocado oil, camellia oil, turtle oil, macadamia nuts oil, corn oil, mink oil, olive oil, rape seed oil, yolk oil, sesame oil, persic oil, wheat embryo oil, sasanqua oil, castor oil, flaxseed oil, safflower oil, cottonseed oil, perilla oil, soybean oil, peanut oil, tea seed oil, kaya oil, rice bran oil, shinagiri oil, Japan tung oil, jojoba oil, embryo oil, triglycerin, and the like.
Solid oils include, for example, cocoa butter, coconut oil, equine tallow, hydrogenated coconut oil, palm oil, beef tallow, mutton tallow, hydrogenated beef oil, palm kernel oil, lard, bovine bone tallow, Japan kernel oil, hydrogenated oil, bovine leg tallow, Japan wax, hydrogenated castor oil, and the like.
Waxes include, for example, beeswax, candelilla wax, cotton wax, carnauba wax, baybery wax, ibota wax, spermaceti wax, montan wax, rice wax, lanolin, kapok wax, lanolin acetate, liquid lanolin, sugarcane wax, lanolin fatty acid isopropyl, hexyl laurate, reduced lanolin, jojoba wax, hard lanolin, shellac wax, POE lanolin alcohol ether, POE lanolin alcohol acetate, POE cholesterol ether, lanolin fatty acid polyethylene glycol, POE hydrogenated lanolin alcohol ether, and the like.
Hydrocarbon oils include, for example, liquid paraffin, ozokerite, squalane, pristane, paraffin, ceresin, squalene, vaseline, microcrystalline wax, polyethylene wax, Fischer-Tropsch wax, etc.
Higher fatty acids include, for example, lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, oleic acid, undecylenic acid, tolic acid, linoleic acid, linolenic acid, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), etc.
Higher alcohols include, for example, straight chain alcohols (e.g., lauryl alcohol, cetyl alcohol, stearyl alcohol, behenyl alcohol, myristyl alcohol, oleyl alcohol, cetostearyl alcohol, etc.); branched chain alcohols (e.g., monostearyl glycerin ether (batyl alcohol), 2-decyltetradecynol, lanolin alcohol, cholesterol, phytosterol, hexyldodecanol, octyldodecanol, etc.) and the like.
Synthesized ester oils include isopropyl myristate, cetyl octanoate, octyldodecyl myristate, isopropyl palmitate, butyl stearate, hexyl laurate, myristyl myristate, decyl oleate, hexyldecyl dimethyloctanoate, cetyl lactate, myristyl lactate, lanolin acetate, isocetyl stearate, isocetyl isostearate, cholesteryl 12-hydroxystearate, ethylene glycol di-2-ethyl hexanoate, dipentaerythritol fatty acid ester, N-alkyl glycol monoisostearate, neopentyl glycol dicaprate, diisostearyl malate, glycerin di-2-heptylundecanoate, trimethylolpropane tri-2-ethyl-hexanoate, trimethylolpropane triisostearate, pentaerythritol tetra-2-ethylhexanoate, glycerin tri-2-ethyl hexanoate, glycerin trioctanoate, glycerin triisopalmitate, trimethylolpropane triisostearate, cetyl 2-ethyl hexanoate, 2-ethylhexyl palmitate, glycerin trimyristate, glyceride tri-2-heptylundecanoate, castor oil fatty acid methyl ester, oleyl oleate, acetoglyceride, 2-heptylundecyl palmitate, diisobutyl adipate, N-lauroyl-L-glutamic acid-2-octyldodecyl ester, di-2-heptylundecyl adipate, ethyl laurate, di-2-ethylhexyl sebacate, 2-hexyldecyl myristate, 2-hexyldecyl palmitate, 2-hexyldecyl adipate, 2-ethylhexyl succinate, triethyl citrate, polyoxyethylene-polyoxypropylene random polymer methyl ether, etc.
Silicone oils include, for example, chain polysiloxanes (e.g., dimethyl polysiloxane, methylphenylpolysiloxane, diphenylpolysiloxane, etc.); cyclic polysiloxanes (e.g., octamethylcyclotetrasiloxane, decamethylcyclopentasiloxane, dodecamethylcyclohexasiloxane, etc.), silicone resins which form three dimensional network structures, silicone gums, a wide variety of denatured polysiloxanes (amino-denatured polysiloxane, polyether-denatured polysiloxane, alkyl-denatured polysiloxane, fluorine-denatured polysiloxane, etc.) and the like.
Others include, for example, humectants such as polyethylene glycol, glycerin, 1,3-butyleneglycol, erythritol, sorbitol, xylitol, maltitol; thickeners such as cellulose, hydroxyethylcellulose, hydroxypropylcellulose, methylhydroxypropylcellulose, methylcellulose, carboxymethylcellulose, quinceseed, carrageenan, pectin, mannan, curdlan, chondroitin sulfate, starch, galactan, dermatan sulfate, glycogen, acacia gum, heparan sulfate, hyaluronic acid, sodium hyaluronate, tragacanth gum, keratan sulfate, chondroitin, xanthan gum, mucoitin sulfate, hydroxyethyl guar gum, carboxymethyl guar gum, guar gum, dextran, kerato sulfate, locust bean gum, succinoglucan, caronic acid, chitin, chitosan, carboxymethylchitin and agar, etc.; lower alcohols such as ethanol; antioxidants such as butylhydroxytoluene, tocopherol, and phytin; antibacterial agents such as benzoic acid, salicylic acid, sorbic acid, paraoxybenzoic acid alkyl ester, and hexachlorophene; organic acids such as acyl sarcosine (e.g., sodium lauloyl sarcosine), glutathione, citric acid, malic acid, tartaric acid and lactic acid; vitamin A and its derivatives, vitamin Bs such as vitamin B6 hydrochloride, vitamin B6 tripalmitate, vitamin B6 dioctanoate, vitamin B2 and its derivatives, vitamin B12, and vitamin B15 and its derivatives, vitamin Cs such as ascorbic acid, ascorbic acid sulfate ester (salt), ascorbic acid phosphate ester (salt), and ascorbic acid dipalmitate, vitamin Es such as α-tocopherol, β-tocopherol, δ-tocopherol, and vitamin E acetate; vitamins such as vitamin Ds, vitamin H, pantothenic acid, and pantethine; saponins such as nicotinamide, benzyl nicotinate, γ-orizanol, allantoin, glycyrrhizic acid (salt), glycyrrhetic acid and its derivatives, hinokitiol, bisabolol, eucalyptone, thymol, inositol, saikosaponin, ginseng saponin, Luffa cylindrica saponin, and soapberry saponin; various drugs such as pantothenylethyl ether, ethynylestradiol, tranexamic acid, arbutin, cepharanthin, and placenta extract, plant extracts such as Rumex japonicus, Clara, Nuphar japonicum DC, orange, sage, Achillea alpina, Malva sylvestris, Swertia japonica, thyme, Japanese angelica, bitter orange peel, birch, Field Horsetail, Luffa cylindrica, common horsechestnut, Saxifragaceae, arnica, lily, sagebrush, paeoniae radix, aloe, Gardenia jasminoides, Sawara cypress, Hawthorn extract, Hypericum perforatum extract, iris-inextract, Gambir extract, ginkgo leaf extract, ibukijyakou extract, Foeniculum vulgare extract, Oolong tea extract, water-lily extract, Rosae Multiflorae Fructus extract, Plectranthus japonicus extract, Scutellariae Radix extract, Phellodendri Cortex extract, Lamium album extract, G. uralensis extract, Gardenia jasminoides extract, black tea extract, Tamarix chinensis Lour extract, Potentilla Erecta extract, rose extract, Luffa cylindrica extract, peppermint extract, rosemary extract, and royal jelly extract, pigment; non-ionic surfactants such as sorbitan monolaurate, sorbitan monopalmitate, sorbitan sesquioleate, sorbitan trioleate, polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monostearate, polyethylene glycol monooleate, polyoxyethylene alkyl ether, polyglycol diether, lauroyldiethanol amide, fatty acid isopropanolamide, maltitolhydroxy fatty acid ether, alkylated polysaccharide, alkyl glucoside, and sugar ester, cationic surfactants such as stearyltrimethylammonium chloride, benzalkonium chloride, and laurylamine oxide, anionic surfactants such as sodium palmitate, sodium laurate, sodium lauryl acid, potassium lauryl sulfate, alkylsulfuric acid triethanolamine ether, turkey-red oil, linear dodecylbenzene sulfate, polyoxyethylene hydrogenated castor oil maleic acid, and acylmethyl taurine; ampholytic surfactants; neutralizers; antioxidants such as δ-tocopherol and butylhydroxytoluene, and preservatives such as phenoxyethanol and paraben.
The above described ingredients are only illustrative and the invention is not limited to them. Additionally, these ingredients may be mixed being combined appropriately according to description depending on the desired form.
The ADAM inhibitor of the invention may be used as a drug intended to prevent or ameliorate the conditions caused by increase of ADAM activity.
In order to formulate the ADAM inhibitor of the invention in such uses, for example, powders, granules, ampoules, injections, isotonic solutions and the like are prepared according to ordinary methods. In the case of preparing oral solid preparations, added are excipients and if needed binders, wetting agents, disintegrators, surfactants, lubricants, dispersing agents, flavors and correctives followed by preparation as tablets, coated tablets, granules, capsules or the like according to conventional methods.
The excipients used include, for example, lactose, glucose, sorbitol, cornstarch and mannitol, etc.; the binders include, for example, polyvinyl alcohol, polyvinyl ether, ethylcellulose, acacia gum, gelatin, hydroxypropylcellulose and polyvinylpyrrolidone, etc; the disintegrators include calcium carbonate, calcium citrate, dextrin, starch, gelatin powder, etc.; the lubricants include calcium carbonate, calcium citrate, talc, polyethyleneglycol, etc.; the coloring agents include cocoa powder, mint flavoring acid and mint oil, etc. These tablets and granules may be coated properly if needed by sugar, gelatin and others.
In the case of preparing the injections, added are if needed pH adjusting agents, buffers, surfactants, solubilizers, solvents, stabilizers, preservatives and the like and prepared as subcutaneous, intramuscular or intravenous injections.
In the method of the invention for inhibiting ADAM, the ascorbic acid derivatives or salts thereof may be applied in any forms singly or in combination with any other ingredients as long as they can be applied to the skin and can attain the purpose of the invention. In addition, the location of the skin where they are applied to is not limited but includes the skin of all over the body surface including the scalp. In addition, they may be applied by oral administration or by injection.
Further, use of the ADAM inhibitor comprising an ascorbic acid derivative or a salt thereof is not limited to treatment or prevention of different diseases adversely affected by growth or differentiation of the skin cell due to the activation of ADAM as described above, but includes any uses which can exert the effect by inhibiting the ADAM activity. The invention for example, may not only be applied to the diseases caused by increase of the ADAM activity, but also applied to the wrinkle caused by hyperplasia of the epidermal cells thereby the wrinkle may be prevented or ameliorated.
Examples will now be given to describe the present invention in more detail. The invention is not intended to be limited to them. The combination amounts herein are % by mass.
1. Exploring a New ADAM Inhibitor
For the ADAM inhibiting effect, the TNF-α which is cut out to be released from the membrane of the U937 cell derived from the human lymphoma through the action of ADAM was used as an index, and the inhibiting effect on the TNF-α release by the agents was assessed. A new ADAM inhibitor was explored by screening different compounds according to the present method.
The U937 cells derived from the human lymphoma were seeded to the 48-well plate adjusting the number of the cells to 1×105 cells/0.25 ml and cultured at 37° C. overnight. The medium which contained 5 μg/ml or 30 μg/ml of an ascorbic acid derivative was added and incubated at 37° C. for 30 min. to be pretreated. 10 nM PMA (phorbol ester: 12-O-tetradecanoylphorbol-acetate; Sigma P8139) was then added and cultured at 37° C. for 6 hours. After completion of the treatment, the supernatant of the culture was collected, centrifuged and the cell-free supernatant was cryopreserved at −20° C. The assay of the released hTNF-α was performed using the hTNF-α Duo-Set ELISA kit from R & D Ltd. Each supernatant of the culture was diluted 5-folds with the medium, then 100 ml each was added to the 96-well plate and measured according to the protocol of R & D Ltd. The absorbance at 450-570 nm of each well was measured by the measurement kit and concentrations of the samples were obtained from the standard curve. The inhibition rates were calculated according to the following equation wherein A0 is the absorbance of 0% inhibiting control (the medium containing only PMA), A100 is the absorbance of 100% inhibiting control (only the medium) and AS is the absorbance of the sample.
The inhibition rate (%)=(A0−AS)/(A0−A100)×100
Consequently, a high suppressing effect on HB-EGF release was noted for 2-O-dodecyl ascorbic acid and 2-O-octadecyl ascorbic acid which have particularly greater number of carbon atoms among the inventive ascorbic acid derivatives suggesting that they inhibit the ADAM activity. The release suppressing rate of each ascorbic acid derivative is shown in Table 1.
Examples of preparations containing ascorbic acid derivatives or salts thereof according to the invention will be shown below, but the invention is not to be limited to them.
100 mg of finely powdered 2-O-dodecyl ascorbic acid was dissolved in 5 ml of chloroform. 10 mg of Tween-80 was added and mixed, then filtrated through a millipore filter followed by evaporation to dryness with a sterile evaporator. 10 ml of sterile distilled water was added and mixed vigorously, then sonicated and filled into an ampoule to prepare an injection.
Carboxy vinyl polymer is dissolved in a little amount of the purified water (phase A). Polyethylene glycol 1500, triethanolamine and 2-O-dodecylascorbic acid are added to the remaining purified water, dissolved with heating, then maintained at 70° C. (aqueous phase). Other ingredients are mixed, melted with heating, then maintained at 70° C. (oil phase). The oil phase is added to the aqueous phase to pre-emulsify, then phase A is added to emulsify homogeneously with a homomixer. After emulsification, it is cooled to 30° C. while mixing well to obtain an emulsion.
Citric acid, sodium citrate and 2-O-octadecylascorbic acid were dissolved in the purified water to make an aqueous phase. On the other hand, other ingredients were dissolved with stirring. This was added to the aqueous phase, and made homogeneous to obtain a lotion.
2-O-dodecylascorbic acid, 2-O-octadecylascorbic acid, 1,3-butylene glycol, trisodium edetate and sodium glycyrrhetinate were added to the purified water and maintained at 70° C. to make an aqueous phase. On the other hand, other ingredients were heated with stirring, and melted then maintained at 70° C. to make an oil phase. The oil phase was added to the aqueous phase to pre-emulsify, then emulsified homogenously with a homomixer followed by cooling to 30° C. to obtain a cream.
Rehrnannia root extract
Rehrnannia root extract
Curcuma extract
Pyrola japonica extract
Lempuyang extract
Clara extract
Gambir extract
Peony root extract
Belamcanda extract
Scutellaria extract
Saxifraga stolonifera extract
Origanum majorana extract
Scutellaria extract
Macadamia nuts oil
Loquat leaf extract
Foeniculum vulgare extract
Hamamelis japonica extract
Phellodendron bark extract
Rehmannia glutinosa extract
Eucalyptus oil
Foeniculum vulgare extract
Hamamelis japonica extract
Phellodendron bark extract
Rehmannia glutinosa extract
Eucalyptus oil
Macadamia nuts oil
Any injection or dermatological external agents of the preparation Examples 1-29 described above had high ADAM inhibiting effect.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2007-016536 | Jan 2007 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP2007/075121 | 12/27/2007 | WO | 00 | 7/24/2009 |
