JP2000226329A - Mmp inhibitor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an MMPs(matrix metaprotease) inhibitor useful in treating and preventing intractable diseases caused by MMPs activity regulation impotence by including a catechin compound. SOLUTION: This MMPs inhibitor is such one as to contain a catechin compound which can be afforded by subjecting tea leaves including green tea and black tea leaves to extraction with an aqueous solvent such as water or a lower alcohol or an organic solvent such as acetone or ethyl acetate, or by extraction and isolation from other plant(s), or by chemical synthesis, being e.g. theasinensin A, theasinensin F, theasinensin D, theasinensin G, oolongtheanin. The daily dose of this inhibitor is 0.1-500 (pref. 0.5-200) mg/kg body weight either singly or in two to four portions.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a matrix metalloprotease (matr) containing a catechin compound as an active ingredient.
ix metalloproteinases (MMPs). Furthermore, extracellular matrix (extracellular matrix: E
Rheumatoid arthritis, caused by the breakdown of CM)
The present invention relates to MMPs inhibitors useful for treating and preventing intractable diseases such as joint diseases such as osteoarthritis, metastasis of cancer cells, and gingivitis.

[0002]

2. Description of the Related Art MMPs are used to degrade and remodel connective tissue.
A group of enzymes involved in modeling, with Zn ²⁺ in the active site. To date, as many as 16 human MMPs have been identified (Na
gase, H .: Biol. Chem., 378: 151-160, 1997), which differ in primary structure and substrate specificity from collagenases, gelatinases, stromelysins, membranes (MT-M
MP) and other (matrilysin).

[0003] MMPs degrade constituent proteins of the extracellular matrix, for example, proteins present in joint lining, interstitial connective tissue, basement membrane, cartilage and the like. These proteins are collagen,
Laminin (laminin), elastin (elastin), fibronectin (fibronectin), proteoglycan (prote
oglycan), and the like.

[0004] Collagen is the major structural protein that makes up about one-third of mammalian tissue and is an essential component of many matrix tissues, including cartilage, bone, tendons, and skin. Interstitial collagenase (MMP-1) is native I, II,
Type III collagen molecules are specifically cleaved at 3: 1. When cleaved at one site by collagenase, collagen molecules that are stable in normal tissue will spontaneously denature at physiological temperature (body temperature) into single-chain gelatin and be degraded by various other proteases. become. As a result, the structural integrity of the matrix organization is lost. This process is irreversible.

[0005] Gelatinase (MMP-2) is a multifunctional of highly crosslinked macromolecules present in gelatin (denatured collagen), type IV collagen (basement membrane) and type V collagen, fibronectin (soft connective tissue and basement membrane). Denatures sex glycoproteins) and elastin (a structural protein that is a special component of elastic tissues such as arteries, tendons, and skin).

[0006] Stromelysin 1 (MMP-3) and 2 (MMP-3)
-10) degrades a wide range of matrix substrates including laminin, fibronectin, proteoglycans, and collagen (types IV and IX).

[0007] Matrilysin (MMP-7) also degrades a wide range of matrix substrates including proteoglycans, gelatin, elastin and laminin.

[0008] In normal tissues, the activity of MMPs is 1)
It is strictly regulated in three steps: production of a latent enzyme (pro-MMP), 2) activation of the latent enzyme, and 3) inhibition of the activated enzyme by an inhibitor. As a result, MM
Degradation of connective tissue by Ps and synthesis of new matrix tissue are in dynamic equilibrium.

However, in many pathological diseases, dysregulation of MMPs activity leads to enhanced MMPs activity,
Decomposition of M is accelerated. These pathological conditions include arthritis (eg, rheumatoid arthritis and osteoarthritis), periodontal disease, ectopic angiogenesis, neoplastic invasion and metastasis, tissue ulceration (eg, corneal ulcer, gastric ulcer, Or epidermal ulcers), bone diseases (eg, bone resorbable diseases such as osteoporosis and loosening after artificial joint replacement), vascular reocclusion and restenosis,
It is one of the major causes delaying the cure of intractable diseases such as HIV infection and diabetic complications. Therefore, substances having an inhibitory effect on MMPs are considered to be useful as agents for preventing and treating these intractable diseases.

[0010] For example, as a therapeutic agent for cancer metastasis by inhibiting angiogenesis or activation of MMPs, marimastat (3R- (2,2-dimethyl-1S-methylcarbamoyl-propylcarbamoyl) -2S) having a hydroxamic acid skeleton is used. -Hydroxy-5-methyl-hexano-hydroxamic acid) are in clinical development. However, since these anti-tumor metastasis drugs target the biological characteristics of tumor cells, they do not have a direct tumor shrinkage effect unlike anti-cancer drugs, so the evaluation criteria for anti-cancer drugs are applied as they are to make a judgment. It is difficult to do so, and clinical evaluation of its usefulness is yet to come.

Other MMPs inhibitors include flavonoid compounds (JP-A-8-104628), esculetin derivatives (JP-A-8-183785), sulfonyl amino acid derivatives (JP-A-9-309875), and TIMPs (JP-A-9-309875). JP-A-10-17492), and the like.

[0012]

As described above, regarding the clinical applications of many MMPs inhibitors reported so far, their clinical usefulness is still unknown, taking an antitumor metastatic drug as an example. Further, there is a need for the development of new candidates for MMPs inhibitors.

[0013]

Means for Solving the Problems In order to solve the above problems, the present inventors focused on various biological activities such as antioxidant activity and antiviral activity of catechin compounds, Assuming that it may have an inhibitory effect, as a result of examining the MMPs inhibitory activity of catechin, catechin shows an MMP inhibitory effect together with its various biological activities, resulting in dysregulation of MMPs activity It has been found that usefulness for treatment and prevention of intractable diseases can be expected.

That is, the present invention provides (1) a matrix metalloprotease (MMPs) inhibitor containing a catechin compound as an active ingredient, and (2) a catechin compound comprising
sinensin A, theasinensin F, theasinensin D, theasi
nensin G, oolongtheanin 3'-O-gallate, oolongtheani
n, assamicain A, assamicain C, oolonghomobisflavan
C, EGCG 6-6 EGCG, (-)-epigallocatechin 3,5-di-O-ga
llate, (-)-epigallocatechin 3,3'-and 3,4'-di-O-gal
late, 8-C-ascorbyl (-)-epigallocatechin 3-O-gallat
e), epitheaflagallin 3-O-gallate, theaflavin 3'-O
-gallate, theaflavin 3,3'-di-O-gallate, (+) catechi
n, (-) catechin, (-) epicatechin, (±) gallocatechi
n, (-) epigallocatechin, (-) catechin-3-O-rha, (-) ep
igallocatechin-3-O-gallate, epicatechin-3-O-gallat
e, (-) catechin (-3-O-rhamnopyranosyl)-(4-8) (-) catec
hin, a matrix metalloprotease (MMPs) inhibitor according to (1), (3) an agent for treating and preventing intractable diseases caused by dysregulation of matrix metalloprotease (MMPs) activity, comprising a catechin compound as an active ingredient;
(4) Intractable diseases include rheumatoid arthritis, osteoarthritis,
Periodontal disease, ectopic angiogenesis, neoplastic invasion and metastasis, ulceration, bone disease, vascular restenosis, vascular restenosis, HIV infection,
Or a therapeutic and prophylactic agent for (3), which is a diabetic complication,
(5) The catechin compound is theasinensin A, theasinens
in F, theasin-ensin D, theasinensin G, oolongthean
in 3'-O-gallate, oolongtheanin, assamicainA, assam
icain C, oolonghomobisflavan C, EGCG 6-6 EGCG, (-)-
epigallocatechin 3,5-di-O-gallate, (-)-epigallocat
echin 3,3'-and 3,4'-di-O-gallate, 8-C-ascorbyl (-)-
epigallocatechin 3-O-gallate), epitheaflagallin 3
-O-gallate, theaflavin 3'-O-gallate, theaflavin 3,
3'-di-O-gallate, (+) catechin, (-) catechin, (-) epic
atechin, (±) gallocatechin, (-) epigallocatechin,
(-) catechin-3-O-rha, (-) epigallocatechin-3-O-galla
te, epicatechin-3-O-gallate, (-) catechin (-3-O-rham
(3) or (4), which is a nopyranosyl)-(4-8) (-) catechin.

[0015]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Catechin is a water-soluble polyphenol that is often contained in the wood core of woody plants and is considered to be a precursor of condensed tannin. Tea contains several types of catechins, which are astringent components unique to tea. The catechin content in green tea is 10-15%, with epigallocatechin gallate accounting for more than half. Catechin has various biological activities such as antioxidant activity, antiviral activity, etc., and enhances anticancer by combined use with anticancer drugs (JP-A-10-036260), and has the effect of inhibiting influenza virus infection (Tadakatsu Shimamura: Nippon Medical Corporation) Shinpo, No. 3737: 126-127, Heisei 29, Heisei 9). However, it has not been known that catechin has an inhibitory effect on MMPs.

In the present invention, tea leaves such as green tea and black tea are used.
Aqueous solvent such as water or lower alcohol, or acetone,
A catechin compound obtained by extraction with an organic solvent such as ethyl acetate, and further, a catechin compound obtained by fractionating and purifying these can be used, and those extracted and isolated from other plants (commercially available) ) Or chemically synthesized (Nonaka, G. et al .: Chem. Pharm. Bull. 34:
61-65, 1986; Hashimoto, F. et al .: Chem. Pharm. Bu
ll. 36: 1676-1684, 1988; Hashimoto, F. etal .: Che
m. Pharm. Bull. 37: 3255-3263, 1989). For example, the
asinensin A (1), theasinensin F (2), theasinen
sin D (3), theasinensin G (4), oolongtheanin
3'-O-gallate (5), oolongtheanin (6), assamicai
n A (7), assamicain C (8), oolonghomobisflavan
C, EGCG 6-6 EGCG (9) 、 (-)-epigallocatechin 3,5-d
iO-gallate (10), (-)-epigallocatechin 3,3'-and
3,4'-di-O-gallate (11), 8-C-ascorbyl (-)-epigal
locatechin 3-O-gallate) (12), epi-theaflagalli
n 3-O-gallate (13), theaflavin 3'-O-gallate (1
4), the-aflavin 3,3'-di-O-gallate (15), (+) ca
techin (16) 、 (-) catechin (17) 、 (-) epicatechi
n (18), (±) gallocatechin (19), (-) epigalloc
atechin (20), (-) catechin-3-O-rha (21), (-) e
pigallocatechin-3-O-gallate (22), epicatechin-3
-O-gallate (23), (-) catechin (-3-O-rhamnopyranos
yl)-(4-8) (-) catechin (24), and the like,
Other catechin compounds having the MMPs inhibitory activity of the present invention are also included in the present invention. In the present invention, these catechin compounds can be used alone or in combination.

In the present invention, it was revealed that the catechin compound has an inhibitory effect on MMPs as shown in the Examples. MMP-2 whose active form has been detected in cancer tissues
(Davies, B. et al .: Cancer Res. 53: 5365-5369, 19
93; Emonard, HP et al .: Cancer Res. 52: 5845-584
8, 1992), MT that interacts with latent MMP-2 on the surface of cancer cells
1-MMP and MMP-7 (Mori, M. et al .: Cancer, which is not expressed in normal epithelial cells but is specifically upregulated in epithelial cancer cells colon cancer and gastric cancer) , 75: 1516-151
9, 1995; Kataoka, H. Oncol. Res. 9: 101-109, 199
It is noteworthy that catechin compounds exhibit an inhibitory effect at extremely low concentrations in contrast to 7). The presence of activated MMP-2 in cancer tissues correlates very well with the degree of invasion of gastric and breast cancers (Polette, M. et al .: Virc
hows Arch. 428: 29-35, 1996), further highlighting the role of MMP-2 in cancer invasion. MT1-MMP
Has been demonstrated to be an activator of latent MMP-2 on the cell membrane (Sato, H. et al .: Nature, 370: 61-6
5, 1994), and MT1-MMP has been shown to be expressed on the membranes of various cancer cells (Yamamoto, M. et al .:
Cancer Res. 56: 384-392, 1996; Okada, A. et al .:
Proc. Natl. Acad. Sci. USA 92: 2730-2734, 1995; Oh.
tani, H. et al .: Int.J. Cancer, 68: 565-570, 199
6). In addition, MT1-MMP itself has been reported to degrade I, II, III collagen and fibronectin (Ohuc
hi, E. et al .: J. Biol. Chem. 272: 2466-2451, 199
7), It is suggested that expression of this enzyme promotes ECM degradation from two directions. By combining epigallocatechin with other catechins, it is expected that the spectrum of the inhibitory action of MMPs will be expanded, an additive effect, or a synergistic effect.

The preparation form of the MMPs inhibitor containing the catechin of the present invention as an active ingredient may be a general form. Either catechin alone or a mixture of catechin and a pharmaceutically acceptable simple substance or diluent can be used as a preparation. The amount of the active ingredient in the preparation is not limited.

The MMPs inhibitor of the present invention can be administered either orally or parenterally. The dose depends on age, individual difference, medical condition and the like, and is not particularly limited, but is 0.1 to 500 mg / kg (body weight) per day, preferably 0.5 to 200 mg / day.
kg (weight). Usually, the daily dose is administered once or in 2 to 4 divided doses.

[0020]

EXAMPLES Hereinafter, the present invention will be described specifically with reference to Examples, but the present invention is not limited to these Examples.

Example 1 Matrilysin (MMP-7),
Membrane-type MMP (Membrane-type MMP) and cDNA of gelatinase A (MMP-2) (Nagase, H .: Biol. Chem., 378)
: 151-160, 1997; Sato, H. et al .: Nature, 370: 6
1-65, 1994; Crit. Rev. Oral. Biol. Med., 4: 197-
250, 1993) was expressed as a recombinant latent enzyme (proMMP) having six histidine residues at the C-terminus using an Escherichia coli mass expression system and activated. The expression vector pTH-72 contains a ribosome binding site, a translation initiation codon, a multiple cloning site, and a tandem repeat downstream of the T7 promoter.
It contains the sequence encoding the hexahistidine tag and the translation stop codon. Human MT1-MMP (pro domain-catalytic domain) cDNA was converted to 5 'PCR primer: 5'-ggcggatccat
gctcgcctccctcggctcg-3 ′ (SEQ ID NO: 1) and 3 ′ PCR
Primer: 3'-gccgtcgacgttcccgtcacagatgttggg-5 '
Using (SEQ ID NO: 2), a PCR reaction was performed using pME18S-MTMMP containing a 3.5 kb cDNA fragment of MT1-MMP as a template. The obtained 0.8 kb PCR fragment encoding from ¹⁸ Leu to ³²² Asn was digested with Bam HI / Sal I, and the pTH-72 expression vector (pTH-MT
1MMP-PC) at the Bam HI / Sal I site. Human M
MP-2 (pro domain-catalytic domain) cDNA was used for 5 ′ PCR primer: 5′-ggcggatccatggcgccgtcgcccatcatc-3 ′ (SEQ ID NO: 3) and 3 ′ PCR primer: 3′-gccgtcgacta
Using caatgtcctgtttgcagat-5 '(SEQ ID NO: 4), MMP-
PCR was performed using pSG-GelA containing the 3.3 kb cDNA fragment as a template. The obtained 1.3 kb PCR fragment encoding ³⁰ Ala to ⁴⁷⁴ Val was digested with Bam HI / Sal I, and cloned into the Bam HI / Sal I site of the pTH-72 expression vector (pTH-MMP2-PC). Human MMP-7 was prepared by the method described in the literature (Itoh, M. et a
l .: J. Biochem., 119: 667-673, 1996). Expression, purification, and refolding of human recombinant MMPs are performed by methods described in the literature (eg, Yoshifumi Nishimura, Shigeo Ohno, supervised by: Protein Experiment Protocol, Cell Engineering Separate Volume, Experimental Protocol Series 2, Structural Analysis, 1997). ). That is, an expression vector containing MMPs (pTH-MMP-7, pTH-MT1-MMP-PC,
TH-MMP2-PC) was transfected into E. coli BL21 (DE3) strain, and the expression was induced with IPTG. Expression protein is Ni-NTA
After affinity purification using a resin (QIAGEN INC., USA), refolding was performed.

MMP-7 and MT1-MMP were combined with trypsin at 37 ° C.
After reacting for 5 minutes, DIFP (diisopropylphosphofluoridate) and trypsin inhibitor (Tris-HCl 50 mM, NaCl 150 mM, CaCl ₂
(10 mM, NaN ₃ 0.02%, Brij 35 0.05%) was added to transfer to the active form. On the other hand, MMP-2 is p-APMA (p-aminophenylmerucuric
acetate) at 37 ° C for 15 minutes to transfer to the active form.

Using these as enzyme samples, the activity of cleavage of the fluorescent peptide substrate (MOCAc / DNP peptide) was measured by fluorescence intensity using a fluorescent microplate reader (excitation wavelength: 340 nm, fluorescence wavelength: 400 nm) to determine the enzyme activity. Catechin compounds are represented by the general formula (I) (wherein R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R
⁶ and R ⁷ have the chemical formulas corresponding to Tables 1 to 4, respectively. rha indicates L-rhamnose. ).

[0024]

Embedded image

[0025]

[Table 1]

[0026]

[Table 2]

[0027]

[Table 3]

[0028]

[Table 4]

That is, theasinensin A (1), theasi
nensin F (2), theasinensin D (3), theasinensin
G (4), oolongtheanin 3'-O-gallate (5), oolong
theanin (6), assamicain A (7), assamicain C
(8) oolonghomobisflavan C, EGCG 6-6 EGCG
(9), (-)-epigallocatechin 3,5-di-O-gallate (1
0), (-)-epigallocatechin 3,3'-and 3,4'-di-O-gall
ate (11), 8-C-ascorbyl (-)-epigallocatechin 3-O-
gallate) (12), epi-theaflagallin 3-O-gallate
(13), theaflavin 3'-O-gallate (14), the-afl
avin 3,3'-di-O-gallate (15), (+) catechin (1
6), (-) catechin (17), (-) epicatechin (1
8), (±) gallocatechin (19), (-) epigallocatech
in (20), (-) catechin-3-O-rha (21), (-) epigal
locatechin-3-O-gallate (22), epicatechin-3-O-ga
llate (23), (-) catechin (-3-O-rhamnopyranosyl)-
(4-8) (-) catechin (24).

The catechin compound was dissolved in 50% ethanol to make 10 mM, and then dissolved in water at 1 mM, 300 μM, 100 μM, 30 μM, 10 μM.
M, diluted to 3 μM. Measurement of MMPs inhibitory activity
40 μl, catechin 20 μl, assay buffer 20 μl (Tri
s-HCl pH7.5 500mM, NaCl1.5M, CaCl ₂ 100mM, ZnSO ₄ 50
0μM, NaN ₃ 30mM, Brij35 0.05%), pre-incubation at 37 ° C for 15 minutes, and then MOCAc / DNP peptide 120μl
(4.16 μM) and reacted at 37 ° C. for 2 hours every 15 minutes. Table 5 shows the 50% enzyme inhibitory concentration (IC ₅₀ ) after 2 hours.

[0031]

[Table 5]

From Table 5, it is clear that the catechin compound has an inhibitory effect on MMPs.

[0033]

Industrial Applicability According to the present invention, catechin compounds are converted to MMPs
Have an inhibitory effect on MMPs
MMPs inhibitors containing as an active ingredient are intractable diseases caused by dysregulation of MMPs activity, for example, arthritis (for example,
Rheumatoid arthritis and osteoarthritis), periodontal disease, ectopic angiogenesis, neoplastic invasion and metastasis, tissue ulceration (eg, corneal ulcer, gastric ulcer, or epidermal ulcer), bone disease (eg, It can be expected as a therapeutic and preventive agent for osteoporosis and bone resorbable diseases such as loosening after artificial joint replacement, vascular reocclusion and restenosis, HIV infection and diabetic complications.

[Sequence list]

 SEQUENCE LISTING <110> MEIJI MILK PRODUCTS CO., LTD. TOSHIFUMI AKISAWA <120> matrix metalloproteinase inhibitor <130> P99006 <140> <141> <150> JP P1998-359996 <151> 1998-12-04 <160> 4 <210> 1 <211> 30 <212> DNA <213> HUMAN <400> 1 ggcggatcca tgctcgcctc cctcggctcg 30 <210> 2 <211> 30 <212> DNA <213> HUMAN <400> 2 gggttgtaga cactgccctt gcagctgccg 30 <210 > 3 <211> 30 <212> DNA <213> HUMAN <400> 3 ggcggatcca tggcgccgtc gcccatcatc 30 <210> 4 <211> 30 <212> DNA <213> HUMAN <400> 4 tagacgtttg tcctgtaaca tcagctgccg 30

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61K 31/00 619 A61K 31/00 619A 629 629A 631 631H 635 635 637 637C 643 643D 643A 31/70 613 31 / 70 613 // C07D 311/62 C07D 311/62 407/14 407/14 (72) Inventor Shoji Yamada 1-26-11 Midori, Sumida-ku, Tokyo Meiji Dairies Co., Ltd. Nutrition and Pharmaceutical Development Co., Ltd. (72) Inventor Yukie Suma Meiji Dairies Co., Ltd., Nutrition and Pharmaceutical Development Dept. 1-26-1, Midori, Sumida-ku, Tokyo (72) Inventor Tetsuya Kono 540 Narita, Odawara-shi, Kanagawa Meiji Dairies Co., Ltd. Katsuyuki 540 Narita, Odawara-shi, Kanagawa Meiji Dairies Co., Ltd. Inside the Russ Science Institute (72) Inventor Yukio Oshiba 540, Narita, Odawara-shi, Kanagawa Meiji Dairies Co., Ltd. F-term in the Health Sciences Laboratories Co., Ltd. ZA96 ZB15 ZB26 ZC20 ZC55

Claims (5)

[Claims] 1. A matrix metalloprotease (MMPs) inhibitor comprising a catechin compound as an active ingredient. 2. A catechin compound comprising: theasinensin A, thea
sinensin F, theasinensin D, theasinensin G, oolong
theanin 3'-O-gallate, oolongtheanin, assamicain
A, assamicain C, oolonghomobisflavan C, EGCG 6-6 EG
CG, (-)-epigallocatechin 3,5-di-O-gallate, (-)-epi
gallocatechin 3,3'-and 3,4'-di-O-gallate, 8-C-asco
rbyl (-)-epigallocatechin 3-O-gallate, epitheaflaga
llin 3-O-gallate, theaflavin 3'-O-gallate, theafla
vin 3,3'-di-O-gallate, (+) catechin, (-) catechin,
(-) epicatechin, (±) gallocatechin, (-) epigallocate
chin, (-) catechin-3-O-rha, (-) epigallocatechin-3-O
-gallate, epicatechin-3-O-gallate, (-) catechin (-3-
The matrix metalloprotease (MMPs) inhibitor according to claim 1, which is O-rhamnopyranosyl)-(4-8) (-) catechin. 3. An agent for treating and preventing intractable diseases caused by inability to regulate matrix metalloprotease (MMPs) activity, comprising a catechin compound as an active ingredient. 4. The intractable disease is rheumatoid arthritis, osteoarthritis, periodontal disease, ectopic angiogenesis, neoplastic invasion and metastasis, ulceration, bone disease, vascular reocclusion, vascular restenosis, HIV
The therapeutic or prophylactic agent according to claim 3, which is an infectious disease or a diabetic complication. 5. The method according to claim 5, wherein the catechin compound is theasinensin A, thea
sinensin F, theasinensin D, theasinensin G, oolong
theanin 3'-O-gallate, oolongtheanin, assamicain
A, assamicain C, oolonghomobisflavan C, EGCG 6-6 EG
CG, (-)-epigallocatechin 3,5-di-O-gallate, (-)-epi
gallocatechin 3,3'-and 3,4'-di-O-gallate, 8-C-asco
rbyl (-)-epigallocatechin 3-O-gallate), epitheafla
gallin3-O-gallate, theaflavin 3'-O-gallate, theafl
avin 3,3'-di-O-gallate, (+) catechin, (-) catechin,
(-) epicatechin, (±) gallocatechin, (-) epigallocate
chin, (-) catechin-3-O-rha, (-) epigallocatechin-3-O
-gallate, epicatechin-3-O-gallate, (-) catechin (-3-
The prophylactic and therapeutic agent according to claim 3 or 4, which is O-rhamnopyranosyl)-(4-8) (-) catechin.