JP6176655B2 - Matrix metalloprotease activity inhibitor, skin external preparation, anti-aging skin external preparation, anti-wrinkle skin external preparation, rheumatoid arthritis and other diseases treatment or prevention agent - Google Patents

Description

  The present invention inhibits matrix metalloprotease, prevents skin wrinkles, anti-aging skin external agents and rheumatoid arthritis, arteriosclerosis, osteoarthritis, periodontal disease, ectopic angiogenesis, tumor invasion and metastasis , Ulceration, bone disease, vascular reocclusion, vascular restenosis, HIV infection, or diabetic complications.

Changes due to aging of the skin, that is, disappearance of wrinkles and texture, decreased elasticity, etc. are accelerated by some external factors such as aging, ultraviolet irradiation, significant air drying, excessive skin washing, etc. To do.
When these changes are seen microscopically, dermal matrix components such as collagen and elastin are reduced, and the involvement of matrix proteases has been pointed out as a factor inducing this change.
Matrix metalloproteinase (matrix metalloproteinase) is a general term for metalloenzymes that degrade protein components of extracellular matrix, and is generally classified into the following five groups.
The collagenase group includes MMP-1 (interstitial collagenase), MMP-8, MMP-13, and the like. MMP-13 has a degrading action such as type I collagen.
The gelatinase group includes MMP-2, MMP-9 and the like. These MMP-2 and 9 are known as enzymes that degrade base membrane components such as type IV collagen and laminin, and dermal matrix component elastin.
MMP-2 is also activated by MMP-1 whose expression is greatly increased by ultraviolet irradiation, and skin aging further proceeds (Non-patent Document 1). The strome lysin group includes MMP-3, MMP-10 and the like. These MMP-3 and 10 are known as enzymes that degrade proteoglycan, which is a basement membrane component, type IV collagen, laminin, and other fibronectin.
The membrane-bound matrix metalloprotease group includes MMP-14, MMP-15, MMP-16, and MMP-17.
Other groups include MMP-7, MMP-11, and MMP-12.
MMP-12, also called macrophage elastase (HME) expressed in macrophages, is a member of the matrix metalloprotease family and degrades elastin. In addition to elastin, it decomposes matrix and non-matrix components such as type IV collagen, fibronectin, laminin and vitronectin.
In addition, the relationship between these matrix metalloproteinases and various diseases has been gradually clarified, such as rheumatoid arthritis, arteriosclerosis, osteoarthritis, periodontal disease, ectopic angiogenesis, tumor invasion and Matrix metalloproteases are deeply involved in the treatment and prevention of metastasis, ulceration, bone disease, vascular reocclusion, vascular restenosis, HIV infection, or diabetic complications. (Patent Document 1)

As the matrix metalloprotease inhibitors, flavones and anthocyanidins, esculetin derivatives, sulfonylamino acid derivatives, TIMPs and the like are known. However, considering activity, absorption in the body, toxicity, etc., new matrix metalloprotease inhibitors are desirable. It is. (Patent Documents 2 to 5)
On the other hand, bivalve shells are used for calcium preparations and road pavement materials, but only a small part of the enormous shells generated from pearl and scallop farming are used.

JP 2000-344672 A JP-A-8-104628 JP-A-8-183785 JP-A-9-309875 Japanese Patent Laid-Open No. 10-17492 Development of skin aging, whitening and moisturizing cosmetics issued by CMC Co., Ltd. P54, 2001

  The object of the present invention is a topical skin preparation that inhibits matrix metalloproteinases and rheumatoid arthritis, arteriosclerosis, osteoarthritis, periodontal disease, ectopic angiogenesis, neoplastic invasion and metastasis, ulceration, bone disease, The present invention relates to a therapeutic and preventive agent for vascular reocclusion, vascular restenosis, HIV infection, or diabetic complications.

As a result of intensive studies by the present inventors, it has been found that a component that inhibits a matrix metalloproteinase exists in the ligament and mantle (central part) of a bivalve shell that has never been used for a topical skin preparation. We found a new application to increase the use of bivalve shells, which were not highly used.
The component that inhibits this matrix metalloprotease was a protein of about 13.6 kDa (hereinafter also referred to as PfTIMP), and was confirmed to exist in the center of the mantle gorge in addition to the ligament of the shell.
This will be described in detail below.
The ligament and / or mantle is removed from the bivalve shell, as fresh as possible, using a knife or the like. It is not necessary to be concerned only with the ligament and / or the mantle (center), and it is only necessary to obtain a peripheral part including the ligament and mantle (center).
Although it grind | pulverizes as needed, when grind | pulverizing, it grind | pulverizes, performing cooling etc. depending on the case so that it may not be heated.
It can be used as a matrix metalloproteinase inhibitor or as a skin external preparation.
Furthermore, a ligament or mantle (center) of a bivalve shell may be used as an extract.
An aqueous acid solution is added to obtain an extract. The acid used is at least one of inorganic acid and organic acid such as hydrochloric acid, sulfuric acid, nitric acid, formic acid, acetic acid, lactic acid, citric acid, etc., but the acid concentration depends on the type of acid, reaction time, ligament, or Depending on the method of collecting the ligament and mantle (center), some shells may also be mixed, and since calcium carbonate is present, a part of the acid is consumed in this, so excluding the consumed part, 0 .1 to 20 mol, preferably 0.3 to 5 mol.
Extract with a solution of acid added to the ligaments and / or mantle (center) of bivalve shells, stirring as necessary.
This extract can be used as it is or after neutralization and desalting as necessary. Furthermore, it can also be used after purification. The method includes methods such as ultrafiltration, dialysis, and chromatography, and purification is performed using one or more of them.
In the case of the pearl oyster, the present inventors have confirmed that a 13.6 kDa protein inhibits matrix metalloproteinase in the acid lysate.
Of course, this protein can be produced in a large amount by known genetic engineering using a host cell selected from any one of Escherichia coli, yeast, and sputum. For example, it can be produced by a method described in International Publication No. WO96 / 35786, and a protein obtained by such a method can also be used.

In addition to this protein and ligaments and / or mantles (center) of bivalve shells containing this protein or extracts thereof, preparations are made together with raw materials that can be used for pharmaceuticals, foods, cosmetics, or existing pharmaceuticals, foods These can also be used in cosmetics.
Examples of available raw materials
Avocado oil, almond oil, fennel oil, egoma oil, olive oil, orange oil, orange rafa oil, sesame oil, cacao butter, chamomile oil, carrot oil, cucumber oil, beef tallow fatty acid, kukui nut oil, safflower oil, shea butter, liquid shea Fat, soybean oil, camellia oil, corn oil, rapeseed oil, persic oil, castor oil, cottonseed oil, peanut oil, turtle oil, mink oil, egg yolk oil, palm oil, palm kernel oil, owl, coconut oil, beef tallow, lard , Squalene, squalane, pristane or various fats and oils such as hydrogenated products (hardened oil, etc.) of these fats and oils.
Waxes such as beeswax, carnauba wax, whale wax, lanolin, liquid lanolin, reduced lanolin, hard lanolin, candelilla wax, montan wax, shellac wax, rice wax;
Mineral oils such as liquid paraffin, petrolatum, paraffin, ozokelide, ceresin, microcrystalline wax.
Fatty acids such as lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, oleic acid, linoleic acid, linolenic acid, docosahexaenoic acid, eicosapentaenoic acid, 12-hydroxystearic acid, undecylenic acid, tall oil, and lanolin fatty acid.
Alcohols such as ethanol, isopropanol, lauryl alcohol, cetanol, stearyl alcohol, oleyl alcohol, lanolin alcohol, cholesterol, phytosterol, phenoxyethanol, 2-hexyldecanol, isostearyl alcohol, 2-octyldodecanol.
Ethylene glycol, diethylene glycol, triethylene glycol, ethylene glycol monoethyl ether, ethylene glycol monobutyl ether, diethylene glycol monomethyl ether, diethylene glycol monoethyl ether, polyethylene glycol, propylene glycol, polypropylene glycol, 1,3-butylene glycol, pentyl glycol, glycerin, Polyhydric alcohols such as pentaerythritol, threitol, arabitol, xylitol, galactitol, sorbitol, lactitol, and maltitol.

Isopropyl myristate, isopropyl palmitate, butyl stearate, hexyl laurate, myristyl myristate, oleyl oleate, decyl oleate, octyldodecyl myristate, hexyldecyl dimethyloctanoate, cetyl lactate, myristyl lactate, diethyl phthalate, phthalate Esters such as dibutyl acid, lanolin acetate, ethylene glycol monostearate, propylene glycol monostearate, propylene glycol dioleate.
Metal soaps such as aluminum stearate, magnesium stearate, zinc stearate, calcium stearate and zinc palmitate.
Gum arabic, guaiac fat, karaya gum, tragacanth gum, quince seed, agar, casein, lactose, fructose, sucrose or ester thereof, trehalose or derivative thereof, dextrin, gelatin, pectin, starch, carrageenan, carboxymethyl chitin or chitosan, ethylene oxide Hydroxyalkyl (C2 to C4) chitin or chitosan to which alkylene (C2 to C4) oxide is added, etc., low molecular chitin or chitosan, chitosan salt, sulfated chitin or chitosan, phosphorylated chitin or chitosan, alginic acid or a salt thereof, Hyaluronic acid or its salt, chondroitin sulfate or its salt, heparin, ethyl cellulose, methyl cellulose, carboxymethyl cellulose, carboxyethyl cellulose, carboxyethyl cellulose sodium, hyaluronan Droxyethyl cellulose, hydroxypropyl cellulose, nitrocellulose, crystalline cellulose, polyvinyl alcohol, polyvinyl methyl ether, polyvinyl pyrrolidone, polyvinyl methacrylate, polyacrylate, polyalkylene oxide such as polyethylene oxide and polypropylene oxide, or a crosslinked polymer thereof, carboxy Gum, saccharides or water-soluble polymer compounds such as vinyl polymer and polyethyleneimine.

  Alkyl carboxylate, alkyl sulfonate, alkyl sulfate ester salt, alkyl phosphate ester salt, alkyl amine salt, alkyl quaternary ammonium salt, carboxylic acid type amphoteric surfactant, sulfate ester type amphoteric surfactant, sulfonic acid type Amphoteric surfactant, phosphate ester type amphoteric surfactant, ether type nonionic surfactant, ether ester type nonionic surfactant, ester type nonionic surfactant, block polymer type nonionic surfactant, nitrogen-containing Anionic surfactants such as type nonionic surfactants, cationic surfactants, amphoteric surfactants, nonionic surfactants.

Retinol, retinal, dehydroretinal, carotene, lycopene, thiamine hydrochloride, thiamine sulfate, riboflavin, pyridoxine, cyanocobalamin, folic acid, nicotinic acids, pantothenic acids, biotins, choline, inositols, vitamin C or derivatives thereof, ergocalci Ferrol, cholecalciferol, dihydrotaxosterol, vitamin E or its derivatives, various vitamins such as ubiquinones or their derivatives.
Valine, leucine, isoleucine, threonine, methionine, phenylalanine, tryptophan, lysine, glycine, alanine, asparagine, glutamine, serine, cysteine, cystine, tyrosine, proline, hydroxyproline, aspartic acid, glutamic acid, hydroxylysine, ornithine, histidine, etc. And various amino acids such as their sulfates, phosphates, nitrates, citrates, or amino acid derivatives such as pyrrolidone carboxylic acid.

  Red buds, ground yellow, woody, gallbladder, monomedicine, ginkgo, oolong tea, fennel, turmeric, summer hay, Chen bark, Ezokogi, Akemiko, auren, white birch, giant crape myrtle, okra, ginseng or tochibaninjin (carrot), olive, oregano , Orange, chamomile or roman chamomile, cam come, guarana, magnolia, gorye, gooseberry, clara, cranberry, grapefruit, clove, kobushi, magnolia, mausoleum, sakura, hemp, sanchinin carrot, yam, yam, sandscon, Sikkunshi, Shiso, Peonies, Honeysuckle, Horse Chestnut, Celery, Cicada, Senburi, Daio, Tachikakuso, Chikutsujinjin, Tengusa, Kocha, Tendaiyaku, Tokusa, Dokudami, Tochu, Toneriko, Jujube, Keishin, Pyramid, Rose, wild rose, pearl barley, rose, rhododendron, loquat, blackberry, prune, bowfish (windproof), honoki, bodaiju, button, hop, jojoba, maoh, maca, macadamia nut, mulberry bark, ginseng, garlic, bamboo berry, Mirobaran, Mukuroji, Murasaki, Protozoa, Eurasian primrose, Melissa, Merirot, Mokko, Yacon, Eucalyptus, Yukinoshita, Yucca, Yuzu, Lily, Wormweed, Artemisia, Lavender, Lemon, Lemongrass, Rosemary, Ground, Chlorella, Kombu, Wakame , Giant kelp, hijiki, hibamata, giraffe, asakusa nori, susabinori, spirulina, ishige, iroro, fukuronori and other plants, seaweed and algae extracts.

Horse or pig placenta extract, silk protein and its degradation products or their derivatives, milk, casein and its degradation products or their derivatives, skim milk powder and its degradation products or their derivatives, lactoferrin or its degradation products, egg component Animal-derived materials such as fish meat degradation products and nucleic acid-related substances (ribonucleic acid, deoxyribonucleic acid).
Marine products such as deep seawater, sea salt, dried sea water, dead sea or Atlantic or Pacific sea water, sea mud, pearl powder, pearl conchiolin hydrolyzate.
Yeast extract, Bacteria metabolite, Bacteria extract, Mold or actinomycete metabolite, Mold or actinomycetes extract, Natto metabolite, Natto extract, Rice fermented extract, Rice bran (red rice cake, White rice) fermented extract, Fungi-derived substances such as lactic acid fermentation products of raw milk or skim milk powder, lactic acid bacteria fermentation products of legumes, and lactic acid bacteria fermentation products of coconut palm plants.
Α-hydroxy acids such as glycolic acid, citric acid, malic acid, tartaric acid and lactic acid.
Silica, magnesium silicate, talc, kaolin, bentonite, mica, mica titanium, bismuth oxychloride, zirconium oxide, magnesium oxide, zinc oxide, titanium oxide, calcium carbonate, magnesium carbonate, yellow iron oxide, red iron oxide, black iron oxide , Inorganic pigments such as Gunjo, chromium oxide, chromium hydroxide, carbon black, and calamine.
UV absorbers or blocking agents such as benzophenone derivatives, paraaminobenzoic acid derivatives, methoxycinnamic acid derivatives, salicylic acid derivatives, urocanic acid derivatives.

Paraaminobenzoic acid derivative, salicylic acid derivative, anthranilic acid derivative, coumarin derivative, amino acid compound, benzotriazole derivative, hydroquinone or derivative thereof, nicotinic acid derivative, kojic acid or derivative thereof, oxybenzone, benzophenone, arbutin, guaiazulene, shikonin, baicalin , Whitening agents such as baicalein, berberine, placenta extract, ellagic acid, lucinol.
Ictamol, indomethacin, kaolin, salicylic acid, sodium salicylate, methyl salicylate, acetylsalicylic acid, diphenhydramine hydrochloride, d-camphor, dl-camphor, hydrocortisone, guaiazulene, camazulene, chlorpheniramine maleate, glycyrrhizic acid or its salt, glycyrrhetinic acid or its Salt anti-inflammatory agent.
Antibacterial, bactericidal and antiseptics such as acrinol, sulfur, calcium gluconate, chlorhexidine gluconate and triclosan.
Musk, Civet, Castorium, Ambergris, Ylang Ylang Essential Oil, Iris Essential Oil, Fennel Essential Oil, Orange Essential Oil, Cardamom Essential Oil, Geranium Essential Oil, Copaiba Balsam Essential Oil, Cedarwood Essential Oil, Jasmine Essential Oil, Rose Essential Oil, Bergamot Essential Oil, Lavender Essential Oil, Lemongrass Essential Oil Animal and plant fragrances such as lemon essential oil and rosemary essential oil, and other synthetic fragrances.
Photosensitive element 101, photosensitive element 201, photosensitive element 401, photosensitive element 301, hinokitiol, pantothenic acid or derivatives thereof, allantoin, pentadecanoic acid glyceride, urea, guanidine and other various drugs.
Other hormones, sequestering agents, pH adjusters and the like can be mentioned.

  From these, various selections are made and used in any form. Examples of forms include tablets, capsules, powders, granules, solids, liquids, gels, bubbles, emulsions, creams, ointments, sheets and the like.

EXAMPLES Next, an Example is given and this invention is demonstrated in detail.
Example 1
The pearl oyster shells collected at Mikimoto Takoku Farm in Hamashima-cho, Shima City, Mie Prefecture, were washed thoroughly and the ligaments of the shells were cut out with a knife. After drying, it was pulverized to 300 mesh or less.

Example 2 (PfTIMP)
The pearl oyster shells collected at Mikimoto Takoku Farm in Hamashima-cho, Shima City, Mie Prefecture, were washed thoroughly and the ligaments of the shells were cut out with a knife. To 0.5 g of this ligament part, 10 ml of 1M acetic acid aqueous solution was added and left for 24 hours with stirring. The supernatant was desalted and concentrated by ultrafiltration.
This was separated by reverse phase HPLC under the following conditions.

1. Column PEGASIL-300 C4P (Senshui Science)
2. Linear gradient from 0.05% trifluoroacetic acid aqueous solution to 0.05% trifluoroacetic acid / 80% acetonitrile The reverse phase HPLC elution pattern is shown in FIG. In addition, the part of the arrow was fractionated.
The result of SDS-PAGE of this fraction is shown in FIG.

Using this Example 2 as a test product, the inhibition of matrix metalloprotease activity was tested.
The method used MMP Inhibitor Profiling Kit (# AK-308) (Enzo Life Sciences) to measure the fluorescence intensity for 2 to 20 minutes at an excitation wavelength of 532 nm and a fluorescence wavelength of 580 nm. (Variable Image Analyzer (Typhoon
9400))
The results of the test conducted at a final concentration of 0.088 μg / 100 μl are shown in FIG.
The matrix metalloprotease activity inhibition rate calculated from the results of FIG. 3 is shown in FIG.
Further, for MMP-13, the final concentrations were 0.0167 μg / 100 μl and 0.00167 μg / 100 μl. The result is shown in FIG.

The N-terminal amino acid sequence of the purified 13.6 kDa protein was analyzed using a protein sequencer. 51 amino acid residues were determined from the N-terminus. Based on this sequence, a search was performed from a database to obtain the corresponding base sequence. A primer was prepared based on this base sequence, and RT-PCR was performed to confirm the entire 13.6 kDa protein cDNA sequence.
SEQ ID NO: 1 is the amino acid sequence of a protein obtained from the pearl oyster ligament.

In order to confirm where the protein of the example was expressed in the body tissue of the pearl oyster, site-specific expression analysis using RT-PCR was performed.
RNA was extracted from each tissue and reverse transcription was performed to synthesize cDNA. On the other hand, the expression state of the gene in each tissue was confirmed by performing PCR using a primer based on the protein sequence of 13.6 kDa.
The following 7 sites were confirmed.
1. mantle isthmus (center)
2. mantle isthmus (edge)
3. mantle edge
4). mantle pallium
5. gill
6). muscle
7). gonad

The results are shown in FIG. As a result, it was found that it was highly expressed in the mantle isthmus (central part) and the expression level was small in the mantle edge and membrane.
To summarize the above results, the 13.6 kDa protein present in the ligaments and mantle of the bivalve shells inhibits matrix metalloproteinases-2, -9, -12 and -13 in small amounts, and matrix metalloproteinases Inhibitor, external skin preparation, rheumatoid arthritis, arteriosclerosis, osteoarthritis, periodontal disease, ectopic angiogenesis, neoplastic invasion and metastasis, ulceration, bone disease, vascular reocclusion, vascular restenosis, It has been found useful as a treatment and prevention agent for HIV infection or diabetic complications.

  The examples can be used as they are as external preparations for skin and for the treatment and prevention of various diseases. However, it is also possible to prepare other preparations for the preparation of skin and for the treatment and prevention of various external diseases.

External preparation 1
Olive oil 0.5
Polyoxyethylene (20E.O.) sorbitan monostearate 2.0
Polyoxyethylene (60E.O.) hydrogenated castor oil 2.0
Ethanol 10.0
Purified water 80.0
Example 1 0.3
Methyl paraoxybenzoate 0.2

External preparation 2
Olive oil 0.5
Polyoxyethylene (20E.O.) sorbitan monostearate 2.0
Polyoxyethylene (60E.O.) hydrogenated castor oil 2.0
Ethanol 10.0
Purified water 80.2
Example 2 0.1
Methyl paraoxybenzoate 0.2

External preparation 3
A
Squalane 20.0
Olive oil 2.0
Mink oil 1.0
Jojoba oil 5.0
Beeswax 5.0
Cetostearyl alcohol 2.0
Glycerol monostearate 1.0
Sorbitan monostearate 2.0
Example 1 0.0001
B
Purified water 51.6999
Polyoxyethylene (20E.O.) sorbitan monostearate 2.0
Polyoxyethylene (60E.O.) hydrogenated castor oil 1.0
Glycerin 5.0
L-ascorbic acid-2-glucoside 2.0
Methyl paraoxybenzoate 0.1

External preparation 4
A
Squalane 20.0
Olive oil 2.0
Mink oil 1.0
Jojoba oil 5.0
Beeswax 5.0
Cetostearyl alcohol 2.0
Glycerol monostearate 1.0
Sorbitan monostearate 2.0
B
Purified water 51.69
Example 2 0.01
Dipropylene glycol 2.0
Glycerin 3.0
Polyoxyethylene (20E.O.) sorbitan monostearate 2.0
Polyoxyethylene (60E.O.) hydrogenated castor oil 1.0
L-ascorbic acid-2-glucoside 2.0
Methyl paraoxybenzoate 0.1

The elution pattern of PfTIMP in reverse phase HPLC (RP-HPLC) is shown. The result of SDS-PAGE of PfTIMP is shown. The result of the PfTIMP inhibition of matrix metalloprotease activity (final concentration 0.088 μg / 100 μl) is shown. It is a result of the matrix metalloprotease activity inhibition rate (final concentration 0.088 microgram / 100 microliter) of PfTIMP. It is a result of the test of matrix metalloproteinase-13 activity inhibition of PfTIMP. It is the result of RT-PCR in each site | part of PfTIMP.

Claims (4)

  A matrix metalloprotease activity inhibitor comprising a 13.6 kDa protein as an active ingredient in an acid lysate of a pearl oyster ligament and / or mantle (center).   A skin external preparation containing a 13.6 kDa protein as an active ingredient in an acid lysate of pearl oyster ligament and / or mantle (center).   A matrix metalloprotease activity inhibitor comprising a protein having at least 90% identity with the amino acid sequence of SEQ ID NO: 1 as an active ingredient.   A skin external preparation comprising as an active ingredient a protein having at least 90% identity with the amino acid sequence of SEQ ID NO: 1.