JP2011231034A - Aggrecanase production inhibitor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an effective and highly safe aggrecanase production inhibitor capable of being taken daily and continuously, and to provide an arthritis-ameliorating agent.SOLUTION: The aggrecanase production inhibitor contains an extract of lindane. The dosage form can be selected from various kinds, and especially preferably is an internal agent. The dose is changed by purpose of use, age, body weight or the like, but is 0.005-300 mg per kg of body weight, especially preferably 0.03-100 mg per kg. The inhibitor is useful for a medicine, an unregulated drug, a food, a cosmetic or the like targeting the prevention and amelioration of arthritis and a disease associated with the arthritis, because of exhibiting excellent ameliorating effects on the arthritis.

Description

  The present invention relates to an aggrecanase production inhibitor and an arthritis ameliorating agent characterized by containing an extract of lindane.

  According to the results of the survey on subjective symptoms of health status in the Ministry of Health, Labor and Welfare “Heisei 16 National Life Survey”, the rate of female complainants is 350.5 per 1,000 population. Looking at the breakdown of the symptoms of the complainant, among women, “Stiff shoulders” and “Back pain” followed by “Hand and leg joints hurt” was the third place (72.7 against a population of 1,000), and many people It turns out that it has joint pain.

  Representative arthritis includes osteoarthritis and rheumatoid arthritis. In the treatment of these diseases, non-steroidal anti-inflammatory agents, steroid preparations, hyaluronic acid preparations, etc. are used for the purpose of suppressing pain and swelling, but all are symptomatic treatments, and their effects are sufficiently satisfactory It's hard to say.

  Articular cartilage has a very high water spilling ability, thereby imparting viscoelastic properties to the cartilage. This cartilage is characterized by an abundant extracellular matrix composed mainly of aggrecan and type II collagen. Aggrecan is a typical large proteoglycan present in cartilage tissue. Aggrecan core protein is covalently bonded to hyaluronic acid to form huge aggregates, absorbs a large amount of water, and fills the network skeleton of type II collagen fibers to maintain a structure unique to cartilage tissue is doing. Therefore, in osteoarthritis and rheumatoid arthritis, cartilage loss due to increased degradation of these extracellular matrix components is thought to lead to a decrease in joint function.

  Among the extracellular matrix components, the degradation of type II collagen includes matrix metalloproteases (MMP) typified by collagenase, and many MMP inhibitors that prevent or ameliorate joint diseases by inhibiting MMP have been proposed. (For example, Patent Document 1). However, it has become clear that many aggrecan degradation products by proteolytic enzymes other than MMP are detected in the joint fluid of osteoarthritis and rheumatoid arthritis (Non-patent Document 1). Enzymes are attracting attention.

  In recent years, two types of proteases belonging to the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family have specifically demonstrated aggrecanases (aggrecanase-1: ADAMTS-4, aggrecanase-2: ADAMTS-5). It was identified (Non-Patent Document 1, Non-Patent Document 2). Degradation products cleaved to aggrecanase have been detected in the joint fluid of human arthritis patients (Non-patent Document 3), and aggrecan degradation precedes type II collagen degradation in a mouse arthritis model (Non-patent Document 3) From literature 4), it can be said that degradation of aggrecan by aggrecanase is closely related to cartilage destruction and is more important than MMP.

  For this reason, in order to prevent degradation of aggrecan, several aggrecanase activity inhibitors and aggrecanase production inhibitors have been proposed. However, the effects of the aggrecanase activity inhibitors described in Patent Document 2 and Patent Document 3 are not satisfactory, and have not yet been applied to drug treatment or prevention. Patent Document 4 describes an aggrecanase production inhibitor by carotenoids. However, carotenoids are not suitable because they are widely known to be toxic when taken in large quantities or for long periods of time. In order to obtain a sufficient effect for the purpose of preventing and treating joint diseases for many years, since it is necessary to ingest daily and continuously, high safety is required.

The linden related to the present invention is a linden Takagi that grows mainly from the central and central Asia to Europe, and its scientific name is Tilia corda Mill. That's it. Linden is also called rainbow sage, small leaf linden, scallop linden, linden, and is generally said to be effective for mental relaxation, nervous strain relief, insomnia prevention, etc. There is a track record. Although linden has been proposed to eliminate depression by monoamine oxidase inhibition (Patent Document 5), hepatoprotective effect (Patent Document 6), etc., no aggrecanase production inhibitory effect has been reported. Patent Document 7 describes MMP inhibitors based on various plant extracts including lindens, and Patent Document 8 describes tryptase activity inhibitors based on a large number of plant extracts including jujube. However, MMP in Patent Document 7 described are collagen and elastin, proteoglycans, proteases degrade laminin such, the surface of the site of action and specifically degrades aggrecanase between aggrecan core protein ^Glu 372 ^{-Ala 373} Is completely different. The tryptase described in Patent Document 8 is a neutral serine protease localized in mast cells, and is completely different from aggrecanase mainly present in synovial fluid in terms of structure and localized cells.

JP 2008-104394 A JP 2004-256436 A Japanese Patent Laid-Open No. 2003-40800 JP 2007-254411 A JP 2000-256206 A JP 2005-154390 A JP 2001-192316 A JP 2007-186457 A Tortorella M.M. D. et al. "Purification and cloning of aggregation-1: a member of the ADAMTS family of proteins.", Science. , 284, 1664-1666, 1999 Abbaszade I.M. et al. , “Cloning and Characterization of ADAMTS11, an Aggrecanase from the ADAMTS Family”, J. Am. Biol. Chem. , 274, 23443-23450, 1999 Sandy J. et al. D. et al. , “The Structure of Aggrecan Fragments in Human Synthetic Fluid”, J Clin. Invest. 89, 1512-1516, 1992 Van Meurs J.H. B. et al. , “Kinetics of Aggrecanase- and Metalloproteinase-Induced Neoepitopes in Various Stages of Cartridge Deduction in Murine Arritris, Arthritis. , 42, 1128-1139, 1999

  An object of the present invention is to provide an aggrecanase production inhibitor and an arthritis ameliorating agent that can be used on a daily basis and are effective and highly safe.

  As a result of intensive studies to solve the above problems, the present inventors have found that a lindane extract exhibits an excellent aggrecanase production inhibitory effect, and has completed the present invention.

  The extract of lindane related to the present invention is obtained by extracting from a flower part of lindane with a solvent. The extraction method is not particularly limited, and for example, heating extraction, room temperature extraction, or the like can be selected as appropriate. A commercially available product can also be used as the lindane extract.

  Examples of the solvent to be extracted include water, lower alcohols (methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, etc.), liquid polyhydric alcohols (1,3-butylene glycol, propylene, etc. Glycol, glycerin, etc.), ketones (acetone, methyl ethyl ketone, etc.), acetonitrile, esters (ethyl acetate, butyl acetate, etc.), hydrocarbons (hexane, heptane, etc.), ethers (ethyl ether, tetrahydrofuran, propyl ether, etc.) Is mentioned. These solvents may be used alone or in combination of two or more. A polar solvent such as water or a lower alcohol is preferable, and water-containing ethanol is particularly preferable.

  The above extract may be used as it is, or may be used after treatment such as concentration, dilution, filtration, decolorization with activated carbon, deodorization, ethanol precipitation or the like, if necessary. Furthermore, the extracted solution may be subjected to a treatment such as concentration to dryness, spray drying, freeze drying, etc., and used as a dried product.

  The above-mentioned extract may be used as it is for the aggrecanase production inhibitor and the arthritis ameliorating agent according to the present invention, and other components may be blended within the range that does not impair the effect of the extract. Other components may be solid, liquid, or semi-solid, and examples include the following. That is, excipients, extenders, binders, wetting agents, disintegrants, surfactants, lubricants, dispersants, buffers, fragrances, preservatives, solubilizers, solvents, and the like. Specifically, lactose, sucrose, sorbit, mannitol, starch, precipitated calcium carbonate, heavy magnesium oxide, talc, calcium stearate, magnesium stearate, cellulose or derivatives thereof, amylopectin, polyvinyl alcohol, gelatin, surface activity Agents, water, physiological saline, ethanol, glycerin, propylene glycol, cacao butter, olive oil, petrolatum, paraffin, higher alcohols and the like.

  The aggrecanase production inhibitor and the arthritis ameliorating agent according to the present invention can be used for any of pharmaceuticals, quasi drugs, foods and cosmetics. Various preparation forms can be selected, but an internal preparation is particularly preferable. For example, powders, granules, tablets, sugar-coated tablets, capsules, syrups, pills, suspensions, liquids, emulsions and the like. Examples of parenteral dosage forms include lotions, creams, emulsions, bath preparations, injections, poultices, and suppositories.

  The amount of the extract according to the present invention varies depending on the preparation form, purpose of use, etc., but is usually 0.001% by weight or more, preferably 0.01 to 50.0% by weight as a dry solid content. The daily dose of the extract according to the present invention varies depending on the purpose of use, age, body weight and the like, but is 0.005 to 300 mg, particularly preferably 0.03 to 100 mg per kg body weight. If it is less than 0.005 mg / kg body weight, a sufficient effect is hardly expected. Even when administered in excess of 300 mg / kg body weight, it is difficult to recognize the enhanced effect, which is uneconomical. The addition method to the preparation may be added in advance or may be added during the production, and may be appropriately selected in consideration of workability.

  According to the present invention, an aggrecanase production inhibitor and an arthritis ameliorating agent that can be used on a daily basis and are effective and highly safe can be provided.

FIG. 1 is a diagram showing the arthritis improving effect of linden. ◯ indicates a control group, □ indicates a lindane 0.3% administration group, and ■ indicates a lindane 1.0% administration group.

  Examples are provided to describe the present invention in detail, but the present invention is not limited thereto. The part of the amount shown in the examples indicates part by weight, and% indicates% by weight.

Hot water extract of linden Add 2 kg of purified water to 100 g of dried linden product (flower part), extract at 95-100 ° C. for 2 hours, filter, concentrate the filtrate, freeze-dry and hot water extract Of 27 g was obtained.

50% ethanol extract of linden Add 100 kg of purified water and 1.0 kg of ethanol to 100 g of dried linden (flower part), extract at room temperature for 3 days, filter, concentrate the filtrate and freeze-dry 22 g of 50% ethanol extract was obtained.

Ethanol extract of Linden 2 kg of ethanol was added to 100 g of dried Linden (flower part), extracted at room temperature for 3 days, filtered, and the filtrate was concentrated and freeze-dried to obtain 17 g of ethanol extract.

50% 1,3-butylene glycol extract of linden After adding 500 g of purified water and 500 g of 1,3-butylene glycol (1,3-BG) to 100 g of dried linden (flower part), and extracting at room temperature for 7 days Filtration yielded 982 g of 50% 1,3-BG extract.

Powder formulation Formulation amount (part)
1. Linden 50% ethanol extract (Example 2) 20
2. Dried corn starch 30
3. Microcrystalline cellulose 50
[Production method] Components 1 to 3 are mixed to form a powder.

Tablet formulation (parts)
1. Linden hot water extract (Example 1) 1
2. Dried corn starch 29
3. Carboxymethylcellulose calcium 20
4). Microcrystalline cellulose 40
5). Polyvinylpyrrolidone 7
6). Talc 3
[Production Method] Components 1 to 5 are mixed, then 10% water is added as a binder, and after extrusion granulation, it is dried. Ingredient 6 is added to the formed granule, mixed and compressed. One tablet is 0.52 g.

Tablet confection formulation amount (parts)
1. Lindane ethanol extract (Example 3) 1.5
2. Dried corn starch 50.0
3. Erythritol 40.0
4). Citric acid 5.0
5). Sucrose fatty acid ester 3.4
6). Fragrance 0.1
[Manufacturing method] Components 1 to 4 are mixed, 10% water is added as a binder, and fluidized bed granulation is performed. Ingredients 5 and 6 are added to the formed granules, mixed and compressed. One tablet is 1.0 g.

Beverage prescription amount (parts)
1. Linden 50% ethanol extract (Example 2) 1.0
2. Stevia 0.05
3. Malic acid 5.0
4). Fragrance 0.1
5). Purified water 93.85
[Production Method] Components 1 to 4 are stirred and dissolved in a part of purified water of Component 5. The remaining purified water of Component 5 is then added and mixed, heated to 90 ° C. and filled into a 50 mL glass bottle.

Experimental Example 1 Confirmation of Aggrecanase Production Inhibitory Effect Using Chondrocytes Cartilage progenitor cells ATDC5 were suspended in DMEM / F-12 (1: 1) (GIBCO) + 5% FBS, and 5 × 10 ⁴ cells / 1 mL. Seeded in 12 well plate at concentration. Three days after cell seeding, the culture medium was replaced with a differentiation-inducing medium (DMEM / F-12 (1: 1) (GIBCO) + 5% FBS + 1% ITS (GIBCO)) to start differentiation. ITS refers to Insulin-Transferrin-Selenium-G Supplement (100 ×). On the 7th day from the start of differentiation, IL-1β (manufactured by Wako Pure Chemical Industries) 10 ng / mL and a hydrous ethanol extract of Linden (Bodaiju extract powder MF: manufactured by Maruzen Pharmaceutical) were added. For the control group, 10 ng / mL of IL-1β alone was added. 24 hours after the addition of the sample, total RNA was extracted using TRIZOL (manufactured by Invitrogen), and the mRNA expression level of aggrecanase was measured by RT-PCR method based on the total RNA. For the RT-PCR method, Super Script III Platinum Two-Step qRT-PCR Kit with SYBR Green (manufactured by Invitrogen) was used. The mRNA expression level of aggrecanase (ADAMTS-4, ADAMTS-5) was normalized by the expression level of GAPDH and compared. The primers used for measuring the expression of each gene are as follows.

ADAMTS-4 primer set Fw: 5′-CCCGGCAGGACCGTGT-3 ′ (SEQ ID NO: 1)
Re: 5′-TCTTCCCAAATACACAGCTCTCTA-3 ′ (SEQ ID NO: 2)
ADAMTS-5 primer set Fw: 5′-CAGCCCACCATCACAGAATTCC-3 ′ (SEQ ID NO: 3)
Re: 5′-GCTCTCCGTGTAGGTCTAGCA-3 ′ (SEQ ID NO: 4)
Primer set for GAPDH Fw: 5′-CAGCCCACCATCACAGAATTCC-3 ′ (SEQ ID NO: 5)
Re: 5'-GCTCTCCGTGTAGGTTCTAGCA-3 '(SEQ ID NO: 6)

  Quantification was carried out by the ΔΔCt method, and the expression level of aggrecanase mRNA when the lindane hydrous ethanol extract was added when the expression level of aggrecanase mRNA in the control group was taken as 100% was calculated. As shown in Tables 1 and 2, suppression of aggrecanase mRNA expression in cartilage progenitor cells ATDC5 was observed by adding a water-containing ethanol extract of lindane. Aggrecanase mRNA expression depends on the hydrated ethanol extract concentration of lindane to be added, and ADAMTS-4 mRNA expression is suppressed by about 50% when 0.3 mg / mL is added, compared to the control group, In addition, ADAMTS-5 mRNA expression was suppressed by about 60% when 0.3 mg / mL was added compared to the control group, and it was confirmed that the hydrous ethanol extract of linden inhibited aggrecanase production. .

  As a result of examining the aggrecanase production inhibitory effect in chondrocytes using the above experimental method, as shown in Table 1 and Table 2, the hydrous ethanol extract of linden according to the present invention inhibits aggrecanase mRNA expression in a concentration-dependent manner. As a result, it was revealed that it has an excellent aggrecanase production inhibitory effect.

Experimental Example 2 Confirmation of Arthritis Improvement Effect The method for inducing arthritis was performed as follows. First, an equal amount of Freund's complete adjuvant (CALBIOCHEM) was added to bovine joint-derived type II collagen (manufactured by Collagen Technology Workshop) and mixed to prepare a uniform emulsion solution. Subsequently, 0.1 mL of the emulsion solution prepared as described above was intradermally administered to 6-week-old male DBA / 1J mice. Twenty-one days after the first intradermal administration, an emulsion solution prepared in the same manner by replacing the above Freund's complete adjuvant with Freund's incomplete adjuvant (CALBIOCHEM) was intradermally administered to induce inflammation. The number of animals per group was 8.
The sample was administered by mixing a lindane hydrous ethanol extract (manufactured by Maruzen Pharmaceutical Co., Ltd .: Bodaiju extract powder MF) with the feed. That is, a feed prepared by mixing 0.3% or 1% of a lindane hydrous ethanol extract with a feed (Oriental Yeast Kogyo Co., Ltd .: MF for breeding mice and rats) was prepared and allowed to freely ingest by mice. For the control group, only feed was ad libitum. On the 21st, 25th, 27th, 29th, 32nd, 35th, 38th, 40th, 42th and 45th day after the first type II collagen administration, the degree of onset of arthritis in the limbs of mice is as follows: Visual evaluation was made according to the score.
0 points: No finger swelling 1 point: 1 to 2 fingers swollen 2 points: 3 to 5 fingers swollen or erythema 3 points: Strong swelling and erythema per limb A score of 0 to 3 points was given, and the total of the limbs was calculated as an arthritis score per individual.

  The result is shown in FIG. In the linden-administered group, the progression of arthritis was greatly suppressed after the 32nd day compared to the control group. On day 45, suppression of the arthritis score was confirmed in a concentration-dependent manner, about 10% in the linden 0.3% administration group and about 27% in the linden 1% administration group (FIG. 1). This shows that arthritis can be suppressed by administration of a hydrous ethanol extract of lindane.

The aggrecanase production inhibitor and the arthritis ameliorating agent characterized by containing the lindane extract according to the present invention exhibit an excellent improvement effect for the purpose of each agent. Therefore, it is useful for pharmaceuticals, quasi-drugs, foods, cosmetics and the like for the purpose of prevention and improvement of arthritis and diseases related to arthritis.

Claims (2)

  An aggrecanase production inhibitor comprising a lindane extract. An arthritis ameliorating agent comprising the aggrecanase production inhibitor according to claim 1.

Images