WO2007111038A1

WO2007111038A1 - Aggrecanase production inhibitor

Info

Publication number: WO2007111038A1
Application number: PCT/JP2007/051248
Authority: WO
Inventors: Akira Ito; Keisuke Imada; Takashi Sato; Ayana Tsuchida
Original assignee: Tokyo University Of Pharmacy & Life Sciences
Priority date: 2006-03-24
Filing date: 2007-01-26
Publication date: 2007-10-04
Also published as: JP2007254411A

Abstract

Disclosed is an aggrecanase production inhibitor containing at least one carotenoid selected from the group consisting of compounds represented by the general formula (1), the general formula (2) and the general formula (3) shown below, and their therapeutically acceptable salts, ethers, esters and isomers. [Chemical formula 1] (1) [Chemical formula 2] (2) [Chemical formula 3] (3) (In the formulae, R1 represents a hydrogen, an alkyl group, a cycloalkyl group, an aryl group, a heteroaryl group, an aralkyl group, a halogen, a hydroxy group or an alkoxy group, which may be substituted by a heteroatom; R2 and R2' respectively represent a hydrogen, a hydroxy group or an alkoxy group; and an alkene hydrogen in the structural formula may be substituted by a halogen atom.)

Description

Specification

Aggrecanase production inhibitor

Technical field

[0001] The present invention relates to an aggrecanase inhibitor. More specifically, the present invention relates to an aggrecanase production inhibitor containing a natural product-derived rotenoid pigment.

Background art

[0002] According to the results of the 2004 National Life Basic Survey, “stress in shoulders” and “back pain” followed by “pain in joints of limbs” are the third most common symptoms of female complainants (per 1,000 people) 72. 7), and it can be said that so many people complain of joint pain.

[0003] Examples of joint diseases mainly include rheumatoid arthritis and osteoarthritis. In particular, degenerative arthropathy is the most common joint disease since middle age. For these diseases, non-steroidal anti-inflammatory drugs and steroid preparations are generally used to suppress inflammation, and intraarticular injection of hyaluronic acid preparations is used to suppress joint pain. It ’s difficult to say that the drug therapy is still satisfactory.

[0004] The cartilage covering the joint surface is composed of type II collagen and proteodarican aggregates formed mainly by hyaluronic acid and aggrecan, and this structure is thought to contribute to the smooth movement of the joint . In rheumatoid arthritis and osteoarthritis, the degradation of this articular cartilage is enhanced, and joint movement is restricted with severe pain. The ability of the matrix meta-oral protease represented by collagenase to be an enzyme deeply involved in the degradation of articular cartilage. In the joint fluid of rheumatoid arthritis and osteoarthritis, it is rather a degradation product of aggrecan by proteolytic enzymes other than the matrix meta-oral protease. Is detected a lot

(See, for example, Non-Patent Document 1) Therefore, a new enzyme that degrades aggrecan is considered to be deeply involved in these diseases!

[0005] In 1999, two types of aggrecanases that specifically degrade aggrecan were discovered (see, for example, Non-Patent Document 1 and Non-Patent Document 2). Two types of aggrecanases (adalidanase 1 and aggrecanase-2) both belong to an enzyme group called A disintegrin and metalloproteinase with thrombospondin motifs (AD AMTS) and are generally aggrecan. Nase 1 is called ADAMTS-4 and aggrecanase-2 is called ADAMTS-5. The activity of both aggrecanases is inhibited by Tissue inhibitor of metalloproteinases (TIMP) -3 produced simultaneously (for example, see Non-Patent Document 3). ADAMTS-5 has recently been demonstrated to play a central role in joint destruction in osteoarthritis and arthritis in experiments using ADAMTS-5 knockout mice (for example, Non-Patent Document 4 and Non-Patent Document 4). (See Patent Document 5).

[0006] Aggrecanase degrades not only aggrecan but also brevican, a constituent proteolycan of the brain, and is deeply involved in invasion and metastasis of glioblastoma cells, which are cancer cells of the cranial nervous system. (For example, see Non-Patent Document 6).

[0007] On the other hand, several drugs that suppress the activity of these aggrecanases have already been reported. For example, Patent Document 1 describes (2R, 3R) 1- [4- (2,4-dichloro-benzyloxy) -benzenesulfonyl] -3-hydroxy-3-methyl-piperidine-2-carboxylic acid hydroxy Aggrecanase inhibitor power that also has carboxylic acid hydroxyamide derivative power such as amide In Patent Document 2, fibronectin or aggrecanase inhibitor power that also has a fragment power of about 40 kilodalton located at the COOH terminus of fibronectin 3 includes N-hydroxy-Na-methyl-Na- (4-phenoxybenzenesulfol) -2- [2- (pyrimidine-2,4-dione-1-yl) ethyl] Aglycanase inhibitors that also have the ability to sulphonamide derivatives such as glycinamide have been described. However, none of these aggrecanase inhibitors have been applied to pharmaceutical treatment or prevention. In addition, since these diseases progress gradually over many years, it is necessary to continue treatment for a long time including prevention, and high safety is required.

Patent Document 1: Japanese Patent Application Laid-Open No. 2001-114765

Patent Document 2: JP 2004-256436 A

Patent Document 3: Japanese Patent Laid-Open No. 2001-163885

Non-Patent Document 1: Tottorella MD, Burn TC, Pratta MA et al .; Science; vol. 284: 1664-16 66; 1999

Non-Patent Document 2: Abbaszade I, Liu RQ, Yang F et al .; J Biol Chem; vol. 274: 23443-23 450; 1999 Non-Patent Document 3: Kashiwagi M, Tortorella M, Nagase H et al .; J Biol Chem; vol. 276: 12 501-12504; 2001

Non-Patent Document 4: Glasson SS, Askew R, Sheppard B et al .; Nature; vol.434: 644-648; 20 05

Non-Patent Document 5: Stanton H, Rogerson FM, East CJ et al .; Nature; vol. 434: 648-652; 2 005

Non-Patent Document 6: Nakada M, Miyamori H, Kita D et al .; Acta Neuropathol (Berl); vol. 1 10: 239-246; 2005

Disclosure of the invention

Problems to be solved by the invention

Accordingly, an object of the present invention is to provide a drug that inhibits the production of aggrecanase.

Means for solving the problem

[0010] The present inventors have found that a natural product-derived carotenoid pigment specifically inhibits cartilage matrix aggrecan in human synovial cells and chondrocytes, aggrecanase-1 (ADAMTS-4) and aggrecanase-2. It was discovered that the expression of (ADAMTS-5) is extremely effectively suppressed, and the present invention has been completed. That is, as a means for solving the above-mentioned problems, the present invention provides an aggrecanase production inhibitor containing a natural product-derived carotenoid pigment.

[0011] The aggrecanase production inhibitor of the present invention can be used not only as a preventive and Z or therapeutic agent for diseases such as rheumatoid arthritis and osteoarthritis, which may be caused by the degradation of aggrecan by aggrecanase, but also as a cancer cell. Inhibition of metastasis / metastasis and lung cancer, cervical cancer, esophageal cancer, bladder cancer, colon cancer, skin cancer, etc. Furthermore, the functional food containing the aggrecanase production inhibitor of the present invention is also useful for preventing these diseases.

Brief Description of Drawings [Fig. 1] Characteristic diagram explaining the effect of | 8-cryptoxanthin on ADAMTS-4 mRNA expression in human synovial cells, where lane 1 is untreated (control) and 2 is IL-1 | 8 (10ngZmL), 3 is IL—l j8 (10ng / mL) + β cryptoxanthin (: L molZL), 4 is IL—l jS (lOngZmD + jS cryptoxanthin (5 molZL), 5 is IL—l jS (1 Ong / mL) + β-Talyptoxanthine (lO / z molZL) 6 represents IL-1 j8 (10 ng / mL) + Dexamethasone (1 μmol / L), respectively.

[Fig. 2] Characteristic diagram explaining the effect of cryptoxanthin on ADAMTS-5 mRNA expression in human synovial cells, where lane 1 is untreated (control) and 2 is IL-1 | 8 (10 ngZmL) , 3 is IL—l j8 (1 Ong / mL) + β cryptoxanthin (: L molZL), 4 is IL—l jS (lOngZmD + jS cryptoxanthin (5 molZL), 5 is IL—l jS (1 Ong / mL) + β-Taliptoxanthin (10 / z molZL) 6 represents IL-1 j8 (1 Ong / mL) + Dexamethasone (1 μmol / L), respectively.

[Fig. 3] Characteristic diagram explaining the effect of cryptoxanthin on ADAMTS-4 mRNA expression in human articular chondrocytes, where lane 1 is untreated (control), 2 is IL-1 β (10 ngZmL), 3 Is IL—l j8 (1 Ong / mL) + β cryptoxanthin (: L molZ L), 4 is IL 1 β (1 Ong / mL) + β—talhipoxanthin (5 μmol / L), 5 is IL — 1 β (10 ng / mL) + j8 Cryptoxanthine (10 molZL), 6 indicates IL— 1 j8 (10 ng / mL) + dexamethasone (1 μmol / L).

[Fig. 4] Characteristic diagram explaining the effect of cryptoxanthin on ADAMTS-5 mRNA expression in human articular chondrocytes, where lane 1 is untreated (control), 2 is IL-1 β (10 ngZmL), 3 Is IL—l j8 (1 Ong / mL) + β cryptoxanthin (: L molZ L), 4 is IL 1 β (1 Ong / mL) + β—talhipoxanthin (5 μmol / L), 5 is IL — 1 β (10 ng / mL) + j8 Cryptoxanthine (10 molZL), 6 indicates IL— 1 j8 (10 ng / mL) + dexamethasone (1 μmol / L).

[Fig.5] j8-cryptoxaxin for prostaglandin (PG) E production in human synovial cells

2

In the figure, lane 1 is untreated (control), 2 is IL 1 β (10 ngZmL), 3 is IL—l j8 (1 Ong / mL) + β cryptoxanthin (: L molZL), 4 is IL—l j8 (10 ng / mL) + j8—Talipoxanthin (5 molZL), 5 is IL—1 j8 (lOng / mL) + β cryptoxanthin (10 molZL), 6 indicates IL—1 j8 (10 ng / mL) + dexamethasone (1 μmol / L), and ## in lane 2 is in lane 1. In contrast, ** in Lanes 3 to 6 indicates that there is a significant difference (p <0.01) compared to Lane 2 (P <0.01).

[Fig. 6] Characteristic diagram explaining the effect of β cryptoxanthin on cycloxygenase (COX) -1 and -2 production in human synovial cells, in which lane 1 is untreated (reference), 2 is IL—1 j8 (10 ngZmL), 3 is IL—1 j8 (10 ng / mL) + β-Talipoxanthin (: L mol / L), 4 is IL 1 β (10 ng / mL) + β-Talyptoxanthin (5 μmol / L), 5 is IL—l j8 (10 ng / mL) + j8—Talipoxanthin (10 molZL), 6 is IL—1 j8 (lOng / mL) + dexamethasone (1 μmol / L) Respectively.

[Fig. 7] Characteristic diagram explaining the effect of β-cributoxanthin on human synovial cell viability, where lane 1 is untreated (control) and 2 is | 8-cryptoxanthin (1 / z molZL), 3 is j8—cryptoxanthin (5 / ζ πιοΐΖ, 4 is j8—cryptoxanthin (10 / z molZ L), 5 is j8—cryptoxanthin (20 / ζ πιοΐΖυ, 6 is j8—cryptoxanthin (50 / zmol / L), and ** in lane 6 indicates a significant difference (p <0.001) from lane 1.

BEST MODE FOR CARRYING OUT THE INVENTION

[0013] The aggrecanase production inhibitor of the present invention contains a natural product-derived carotenoid pigment.

Carotenoid pigments are contained in various vegetables and fruits, especially fruits such as Wenzhou mandarin oranges, papaya, persimmons, persimmons, persimmons, and green peppers (especially red bell peppers), tomatoes, carrots, strength Abundant in green and yellow vegetables.

[0014] The aggrecanase production inhibitor of the present invention has the following general formulas (1), (2) and (3),

[Chemical 1]

[Chemical 2]

(In the above formulas, R ¹ represents hydrogen, an alkyl group, a cycloalkyl group, an aryl group, a heteroaryl group, an aralkyl group, a halogen, a hydroxy group or an alkoxy group, which are substituted with a hetero atom. R ² and R ² ′ represent hydrogen, a hydroxy group or an alkoxy group. The alkene hydrogen in the structural formula may be substituted with a halogen atom.) The resulting salts, ethers, esters and isomers contain at least one carotenoid selected.

[0015] The term "isomer" in the definitions of the general formulas (1), (2) and (3) includes stereoisomerism, geometric isomerism, optical isomerism, plane isomerism, coordination isomerism, coordination position isomerism, etc. Used to mean all isomers.

[0016] Typical examples of the compound represented by the general formula (1) will be given.

R ^{1 =} CH, R ² = H, IT = H: a

Three

R ^{1 =} CH ² , R ² = OH, R ² '= H: a-Talyptoxanthin (a-carotene-3-ol);

Three

R ^{1 =} CH, R ² = OH, R ² '= OH: Rutin (a-carotene-3,3, -diol)

Three

[0017] Typical examples of the compound represented by the general formula (2) are given.

R ^{1 =} CH, R ² = H, R ² '= H: β-carotene;

Three

R ^{1 =} CH ² , R ² = OH, R ² '= H: β-Taliptoxanthine (j8-Carotene-3-ol); R ^{1 =} CH, R ² = OH, 1 ^ ² '= 011: Zeaxanthin (| 8, j8—Strength 3, 3, 1

Three

Le)

[0018] Typical examples of the compound represented by the general formula (3) will be given.

R ^{1 =} CH, R ² = H, 1 ^ ² '= 11: Canthaxanthin (| 8, β-strength 4, 4, dione);

Three

R ^{1 =} CH, R ² = OH, R ² '= OH: Fastaxanthin (3, 3, dihydroxy- β, β—

Three

Carotene 4, 4 'dione)

[0019] The carotenoid pigment compounds represented by the general formulas (1) to (3) are all derived from natural products and have the advantage of extremely low cytotoxicity. More plant groups such as vegetables and fruits can be easily extracted and separated.

[0020] Whereas conventional drugs merely have a pharmacological effect that suppresses the enzymatic activity of aggrecanase, the drugs of the present invention that have carotenoid pigment compound powers represented by the general formulas (1) to (3) It has a pharmacological effect that suppresses the expression of aggrecanase-1 (ADAMTS-4) and -2 (ADAMTS-5) genes that specifically degrade cartilage matrix aggrecan in human synovial cells and chondrocytes. It is a period. That is, the conventional drug is a therapeutic drug that suppresses the enzymatic activity of aggrecanase that has already occurred, whereas the drug of the present invention is a radical drug that blocks the expression of aggrecanase itself. Clearly different.

[0021] The drug of the present invention having carotenoid pigment compound power represented by the general formulas (1) to (3) is also useful for suppressing invasion and metastasis of cancer cells in addition to diseases such as rheumatoid arthritis and osteoarthritis. It is. In particular, it exerts a significant carcinogenic inhibitory effect on lung cancer, cervical cancer, esophageal cancer, bladder cancer, colon cancer, and skin cancer. In addition, osteoarthritis, joint damage, juvenile rheumatoid arthritis, reactive arthritis, psoriatic arthritis, neuropathic arthropathy (Charco joint), hemophilia arthropathy, infectious arthritis, ankylosing spondylitis, lighter It is also useful for diseases such as syndrome, gout, acute pyrophosphate arthritis (pseudo-ventilation), inflammatory bowel disease, Crohn's disease, periodontal disease, osteoporosis and loosening of artificial joint implants. When functional foods are used, they can be expected to prevent these diseases.

[0022] When the aggrecanase production inhibitor of the present invention is used as a pharmaceutical, it is represented by a pharmacologically effective amount of the above general formulas (1) to (3) for the purpose of treatment and Z or prevention of the disease. The carotenoid pigment compound can be formulated with other pharmaceutically acceptable ingredients. Other pharmaceutically acceptable ingredients include, for example, carriers, diluents, excipients, lubricants, binders, solubilizers, tonicity agents, antioxidants, preservatives, surfactants, Such as dairy, coloring, flavoring and sweetening agents.

[0023] When the aggrecanase production inhibitor of the present invention is used as a pharmaceutical, the compounding amount of the carotenoid pigment compound represented by the general formulas (1) to (3) as an active ingredient can be pharmaceutically acceptable. Generally, it is in the range of 0.01 to 15% by weight, preferably 0.1 to 5% by weight, based on the total weight including other components.

[0024] When the aggrecanase production inhibitor of the present invention is used as a pharmaceutical product, it can be used in any dosage form. Examples include dosage forms such as granules, fine granules, tablets, capsules, pills, ointments, gels, pastes, creams, sprays, solutions and suspensions. As administration routes, all administration routes including oral, subcutaneous, intramuscular and intravaginal injection can be used in addition to oral, enteral, nasal and transdermal administration.

[0025] When the aggrecanase production inhibitor of the present invention is used as a pharmaceutical product, it is usually 0.05 mg in terms of the amount of the carotenoid pigment compound represented by the general formulas (1) to (3) as active ingredients. ~: A dose of LOOOmg can be administered once to several times a day. For example, in the case of β-strength 10 mg / day, the serum concentration of 21 mg / z gZdl, and in the case of β-talhiptoxanthin, the dosage of 0.0 mg / day , 12. 3- 13. 3 μg / dl serum concentration is obtained. In particular, β cryptoxanthin is preferable because it is absorbed and a high serum concentration can be obtained even at a low dose. This dose can be appropriately changed according to the age, weight, and sex of the patient, and can be appropriately changed according to the administration route and the degree of the patient's medical condition.

[0026] When the aggrecanase production inhibitor of the present invention is used as a functional food, the shape of the food may be any solid, semi-solid, liquid, suspension, gel, glue or paste known to those skilled in the art. , Powdered and granular foods.

Example 1

[0027] The whole form of Satsuma mandarin was ground with a mixer and mixed with acetone, and the upper layer was obtained as a crude carotenoid pigment compound extract. This is concentrated and dried by a rotary evaporator. Then, redissolve in 50% ethanol, reverse phase column using octadecyl silica gel as carrier, methanol ZlOmM phosphoric acid (4: 6 → 6: 4) as eluent, UV detector (wavelength: Sorting was performed while monitoring at 340 nm). The obtained fraction was concentrated and dried to obtain the desired carotenoid pigment compound.

[0028] α—Strengthen Ten:

Melting point: 187.5 ° C

[0029] β Cryptoxanthine (j8—Strength 3—Honore):

Melting point: 158 ° C ~ 159 ° C

[0030] Zeaxanthin (β, β-strength 3, 3, diol):

Melting point: 207 ° C

[0031] Canthaxanthin (j8, j8—Strengthen 4, 4, dione):

Melting point: 217 ° C

[0032] Fastaxanthin (3, 3, dihydroxy-13, β-strength 4, 4, dione): Melting point: 182 ° C ~ 183 ° C

Example 2

[0033] The effect of / 3 cryptoxanthin on the expression of aggrecanase-1 (ADAMTS-4) and aggrecanase 2 (ADAMTS 5) in human synovial cells and human articular chondrocytes was examined.

[Experimental materials and experimental methods]

1. Culture equipment and reagents

Plastic device for cell culture is manufactured by Asahi Techno Glass; Dulbeco's modified Eagle's medium (DMEM) is manufactured by Invirtogen; Ca and Mg -free phosphate buffered saline (PBS (-)) is manufactured by Nissui Pharmaceutical; fetal bovine serum ( FBS) is manufactured by BioWhittaker; penicillin G is manufactured by Manyu Pharmaceutical; streptomycin sulfate is manufactured by Meiji Seika; trypsin is DIFCO Laboratori es 0;; lactalbumin hyarolysate (LAH), bovine serum aloumin (BSA), peroxydase-conjugated goat anti- (sheep IgG) IgG, dimethyl sulphoxide (DMSO) and diethyl pyrocarbonate (DEPC) i made by Sigma; Isogen ί ま二ツブン ~~ ん社; mouse anti— [huma n cvclooxygenase (し OX— 1) ] monoclonal antibody, mouse anti— (human COX— 2) mon Each oclonal ant¾ody manufactured by Cayman was purchased and used.

Recombinant human interleukin-1β, rhIL-1j8, 2χ10 'units / mg) were provided by Otsuka Pharmaceutical.

All other reagents used special grade reagents.

2. Culture and drug treatment of human synovial cells and chondrocytes

Human synovial cells (purchased from Applied Cell Biology Research Institute) and chondrocytes (purchased from Cambrex Bio Science Walkersville) were seeded in 6-multiwell plates and confluent in D MEM / 10% (v / v) FBS. Until cultured. After washing the cells with DMEM / 0.2% (w / v) LAH, the drug was added to DMEM / 0.2% (w / v) LAH and added to the cell culture system. All j8-cryptoxanthin was added as a DMSO solution so that the final concentration of DMSO was 0.1% (v / V). The same concentration of DMSO was added to the j8-cryptoxanthin-free group. After incubation for 24 hours at 37 ° C in 5% CO -95% air gas phase,

2

The cell fraction was collected and stored at −20 ° C. until use in experiments.

3.Ryanoletime RT-PCR method

1) Total RNA extraction

Total RNA was extracted using Isogen according to the package insert! To prevent Ribonuclease contamination, the operation was performed wearing clean disposable gloves. Instruments that can be dry-heat sterilized were dry-heat sterilized at 180 ° C for over 9 hours, and those that were not possible were treated with an unused one three times in an autoclave. The water used was a 0.2% DEPC solution treated three times in an autoclave.

2) Reverse transcription reaction

Follow the package insert using Quantitect Reverse Transcription kit (Qiag en) to synthesize cDNA from total RNA (1 μg)!ヽ Reverse transcription reaction was performed.

3) Ryanoretime PCR method

A polymerase specific for ADA MTS-4 mRNA, ADAMTS—5 mRNA and glyceraldehyde—3—phospate dehydrogenase (GAPDH) mRNA using cDNA obtained by reverse transcription (total RNA equivalent to 25 ng) ABI Prism sequence detection 7000 (Applied) using a primer (quantitec t Primer Assay, Qiagen) for chain reaction (PCR) PCR reaction and data analysis were performed by Biosystems). Data are expressed as relative values with the gene expression level of the untreated group taken as 1, after correcting for the GAPDH mRNA expression level.

4.Measurement of prostaglandin E level

2

Prostaglandin E EIA kit-Monoclonal (C

twenty two

ayman) according to the package insert.

5. Western blotting

Add 20% (w / v) trichloroacetic acid (TCA) solution to the cytoplasmic fraction (10 μg protein) to a final concentration of 3.3% (w / v), and leave it at 4 ° C for 12 hours or longer. And centrifuged at 8,000 xg for 5 minutes. The resulting precipitate was washed once with diethylether, air-dried and then sample solution (63 mM Tris-HCl (pH 6.8) / 2% sodium dodecylsulfate (SDS) / 10% glycerol / 1% 2-- mercaptoe thanol / 0.001% bromophenol blue ). SDS—After separating the protein in the sample by polyacrylamide gel electrophores is (PAGE), transfer the gel to a buffer for transcription (20 mM Tris / 150 mM glycine / 20% (w / v) methanol / 0.1% (w / v) SDS (Protein was immersed in pH 9.2) and transferred to protein using a semi-dry type transfer device at 2 mA / cm ² for 1 hour. After the transfer, the nitrocellulose membrane was removed from the blocking solution [10% (w / v) fatty-free dry mil k / 10 mM Tris-HCl / 0.9% NaCl / 0.02% NaN (pH 7.5)] and block for 10 minutes

Three

went. Subsequently, the -trocellulose membrane was washed several times with ion-exchanged water and PBS-T buffer [0.1% Tween 20 / PBS-), and diluted with 1% (w / v) BSA / PBS (-) 1 Next antibody was soaked in a solution of [mouse anti- (human COX-1) monoclonal antibody or mouse anti- (human COX-2) monoclonal antibody] at 4 ° C for 12 hours or more. After binding of the primary antibody, blocking and washing were performed in the same manner, and immersed in peroxydase-conjugated goat anti- (mouse Ig) IgG for 1 hour at f¾. After binding with the secondary antibody, it was washed several times with PBS-T buffer, immersed in ECL-Western blot detection reagents (manufactured by Amersham), and reacted exactly for 1 minute. Immediately after the reaction was completed, detection was performed using Image Analyzer LAS-1000 plus (Fuji Photo Film).

6.Measure the number of living cells

Cell proliferation ability was measured using Cell counting kit-8 (Dojindo) according to the attached operation method. That is, human synovial cells were placed in a 96-well plate with a cell count of 1 x 10 ⁴ cells / 100 L / seeded well, DMEM / 10% (v / v) FBS in 5% CO-95% air in gas phase at 37 ° C for 24 hours

2

After incubation, the cells were treated with DMEM / 10% (v / v) FBS containing j8 cryptoxanthin for 24 hours. Next, add 10 μL / well of Cell couting kit-8, 5% CO-95% air in gas phase at 37 ° C for 2 hours.

2

After incubation, the absorbance at 430 nm was measured.

[0035] [Result]

In human synovial cells, ADAMTS-4 mRNA expression (see FIG. 1) and ADAMTS 5 mRNA expression (see FIG. 2) were promoted by IL 1 j8 (10 ng / mL). In contrast, β cryptoxanthin (j8-CPX) suppressed ADAMTS-4 mRNA expression and ADAMTS-5 mRNA expression in a concentration-dependent manner from 10 / ζ ζ. In cartilage cells, ADAMTS-4 mRNA expression was stimulated by IL-1 j8, and β-CPX suppressed ADAMTS-4 mRNA expression in a concentration-dependent manner (see Fig. 3). Unlike the synovial cells, ADAMTS-5 mRNA expression was unaffected by IL-1 18, but β-CPX inhibited ADAMTS-5 mRNA expression in a concentration-dependent manner (see Figure 4). In addition, for PGE production and COX-1 and COX-2 expression in human synovial cells | 8— CPX

2

As a result of the examination of the effects of J8-CPX on the promotion of PGE production by IL 118,

2

(8) CPX inhibited COX-2 production induced by IL--β without affecting COX-1 production (see Figure 6). . On the other hand, as a result of examining the cytotoxicity of βCPX, the j8-CPX of M showed no cytotoxicity between 1 μΜ and 20 力 which showed cytotoxicity (see Fig. 7).

[0036] [Discussion]

Based on the above results, the natural product-derived carotenoid pigments depend on the concentration of aggrecanase-1 (ADAMTS-4) and 2 (ADAMTS 5), which specifically degrade cartilage matrix aggrecan, in human synovial cells and chondrocytes. Turned out to be suppressed. Because AD AMTS-4 and ADAMTS-5 are closely related to cartilage substrate destruction in rheumatoid arthritis and osteoarthritis, natural product-derived carotenoid pigments are effective as joint destruction protective agents. Can be confirmed.

As described in Non-Patent Document 6 described above, aggrecanase decomposes not only aggrecan but also brevican, which is a constituent proteoglycan of the brain, and is a cancer cell of the cranial nervous system. It is also deeply involved in glioblastoma cell invasion and metastasis. Therefore, it can be confirmed that the aggrecanase production inhibitor of the present invention is effective as a drug for suppressing invasion / metastasis of glioblastoma cells which are cancer cells of the cranial nervous system.

It also suppresses the production of PGE, an inflammatory mediator, in a concentration-dependent manner.

2

found. These facts prove that the natural product-derived carotenoid pigment according to the present invention is effective as a joint destruction preventing substance.

Furthermore, the aggrecanase production inhibitor of the present invention can selectively inhibit the production of COX 2 without inhibiting the production of COX-1. COX-1 is constitutively produced in normal tissues and is involved, for example, in the production of prostaglandins that act to protect the gastrointestinal mucosa, whereas COX-2 is inflamed tissues (such as osteoarthritis and rheumatoid arthritis ) And in cancer tissues, it is suggested that selective inhibition of COX-2 production is an anti-inflammatory drug with low side effects such as gastrointestinal disorders.

Industrial applicability

In addition to diseases such as rheumatoid arthritis and osteoarthritis, the aggrecanase production inhibitor of the present invention suppresses invasion / metastasis of glioblastoma cells, which are cancer cells of the cranial nervous system, and lung cancer, cervical cancer, esophagus It is also useful for reducing the risk of cancer such as cancer, bladder cancer, colon cancer, and skin cancer. In addition, osteoarthritis, joint damage, juvenile rheumatoid arthritis, reactive arthritis, psoriatic arthritis, neuropathic arthropathy (Charco joint), hemophilic arthropathy, infectious arthritis, ankylosing spondylitis, It is also useful for diseases such as Reiter's syndrome, gout, acute pyrophosphate arthritis (pseudo-ventilation), inflammatory bowel disease, Crohn's disease, periodontal disease, osteoporosis and loosening of artificial joint implants. Furthermore, it is useful for the prevention of these diseases as a functional food.

Claims

(In the above formulas, R ¹ represents hydrogen, an alkyl group, a cycloalkyl group, an aryl group, a heteroaryl group, an aralkyl group, a halogen, a hydroxy group or an alkoxy group, which are substituted with a hetero atom. R ² and R ² represent hydrogen, a hydroxy group or an alkoxy group, wherein the alkene hydrogen in the structural formula may be substituted with a halogen atom.), A therapeutically acceptable compound thereof Group power consisting of salts, ethers, esters, and isomers. An aggrecanase production inhibitor containing at least one selected carotenoid.

The carotenoid force a a force mouth ten, α-cryptoxanthin, rutin, j8—force mouth ten The aggrecanase production inhibitor according to claim 1, wherein j8 is selected from the group consisting of cryptoxanthin, zeaxanthin, canthaxanthin and wastaxanthin.

[3] The aggrecanase production inhibitor according to claim 1 or 2, wherein the carotenoid is j8-cryptoxanthin.

[4] The aggrecanase production inhibitor according to any one of claims 1 to 3, which is used as a therapeutic and Z or prophylactic agent for aggrecanase-related diseases.

[5] Aggrecanase-related diseases include rheumatoid arthritis, osteoarthritis, cancer, osteoarthritis, joint damage, juvenile rheumatoid arthritis, reactive arthritis, psoriatic arthritis, neuropathic arthropathy (Charcot joint), hemophilia Pathologic arthropathy, infectious arthritis, ankylosing spondylitis, Reiter syndrome, gout, acute pyrophosphate arthritis (pseudo-ventilation), inflammatory bowel disease, Crohn's disease, periodontal disease, osteoporosis and prosthetic joint The aggrecanase production inhibitor according to claim 4, wherein the group power is selected.