Description MATRIX METALLOPROTEINASE-9 INHIBITOR

CONTAINING CHLOROGENIC ACID

Technical Field

[1] The present invention relates to a matrix metalloproteinase-9(MMP-9) inhibitor which contains chlorogenic acid.

Background Art

[2] MMPs play an important role in degradation of various components in extracellular matrix (ECM). Examples of MMPs-mediated diseases include arteriosclerosis, restenosis, MMP-mediated osteopenia, inflammatory disease in central nerve system, Alzheimer's disease, skin aging, rheumatoid arthritis, osteoarthritis, septic arthritis, ker- atohelcosis, malunion of wound, bone disease, proteinuria, abdominal aortic aneurysms, cartilage-degenerative loss owing to traumatic joint damages, de- myelinative diseases in nerve system, hepatic cirrhosis, glomerulus disorders, premature rupture of fetal membrane, inflammatory bowel diseases, periodontal disease, aging-related macular degeneration, diabetic retinopathy, proliferative vitreo- retinopathy, immature retinopathy, opthalmia, conical cornea, Sjogren syndrome, short sightedness, ophthalmic tumor, corneal transplant rejection, vascularization, invasion and metastasis of carcinoma and the like.

[3] Particularly, MMP-2 and MMP-9 among MMPs are widely known to have specific activities on type IV collagen, thereby playing a key role in diseases such as inflammation, stroke, tumor growth and metastasis.

[4] It has been reported that MMP-9 level in cerebrospinal fluid is related to multiple sclerosis and other neurotic diseases [Beeley, N.R.A. et al., supra.; Miyazaki, K. et al., Nature 362, 839-841(1993)], and contributes to degradation and accumulation of amyloid b -protein [Backstrom JR, et al., J neurosci 16(24), 7910-9 (1996)].

[5] Further, it is also known that MMP-9 has an important role in invasion and metastasis of hepatocarcinoma. The metastasis and invasion of tumor are fundamental characteristic feature of malignant tumor cells. In the initial stage of cancer, occurred is the invasion of a tumor to blood vessel as well as its metastasis. Tumor cells go through several important steps during the invasion and metastasis process. The tumor cells are surrounded by extracellular matrix and basement membrane which act as a barrier against the invasion of tumor cells, accordingly, the substantial initial step in those processes is degradation of the extracellular matrix which constitutes the basement membrane. In the degradation process, various proteinases are involved, and then the external barrier, i.e. the extracellular matrix(ECM) and basement membrane,

is finally degraded (Woessner, J. F. Jr. FASEB J., 5; 2145-2154, 1991). A number of proteolytic enzymes participate in these processes to degrade environmental barriers such as extracellular matrix (ECM) and basement membrane (Chung et al., 2004a; Chung et al., 2004b). MMP-9 of MMP-family has been known to degrade type IV collagen, which is major constituent of the basement membrane in cancer invasion and metastasis (Basset et al., 1997; Nelson et al., 2000). In addition, the expression of MMP-9 may be associated with progression and invasion of human hepatocarcinoma cells (Chung et al., 2002, Chung et al., 2004b). During the process, secreted from cells is an inactive proMMP-9, which is activated by a series of enzymatic activation processes including the generation of an active type of plasminogen (plasmin) by a urokinase-type plasminogen activator (uPA). The activated MMP-9 promotes the invasion potential of tumor cells (registered Korean Patent No. 2002-71674).

[6] These observations have led to the development of MMP-9 inhibitors to prevent tumor metastasis. Several novel MMP-9 inhibitors have been developed and are currently being investigated in clinical trials (Suguta, 1999). Since some broad- spectrum MMP inhibitors (i.e. Batimastat/BB-94 and Marimastat/BB-2516) may have serious muscloskeretal side effects such as arthralgia, myalgia, stiffness, and edema (Brown, 2000), selective (narrow-spectrum) MMP-9 inhibitors, such as BAY12-9566 (Gatto et al., 1999), and MMI- 166 (Katori et al., 2002, have been developed to reduce the risk of such side effects.

[7] The relation between the invasion and metastasis of tumor and the expression of

MMP-9 which has great effects thereon was investigated by using several analytic methods such as zymography, Northern blot and Western blot with MMP-9 being expressed on tumor cells. The MMP-9 expression on tumor cells was very high. The test on invasion using a matrigel showed plasmin-dependent increase in the degree of invasion. The results suggest that the transcription activity of MMP-9 plays important role in the invasion and metastasis of tumor cells. The MMP-9 expression has also important role in the invasion and metastasis of brain tumor, tumors of the nervous system and breast cancer (Rao et al, 1993; Rao et al, 1996 Scorilas et al, 2001). Further, the promoter region of MMP-9 has binding sites for AP-I and NF-κB (Sato, H., and Seiki, M. Oncogene, 8: 395-405, 1993), therefore stimulating elements such as tumor cells, cytokines thereof or PMA (phorbol 12-myristate 13-acetate) control the MMP-9 expression by regulating the activation of transcription factors like AP-I and NF-KB, etc. through Ras/Raf/ERK, JNK and PI-3K/AKT signal transduction pathways (Arai, K. et al., Glia. 43: 254-264, 2003; Sato, H., and Seiki, M.. Oncogene, 8: 395-405, 1993; Gum, R et al., Oncogene, 14: 1481-1493, 1997; Eberhardt, W., et al., J. Immunol., 165: 5788-5797, 2000; Abiru, S. et al., Hepatology, 35: 1117-1124, 2002; Kim, D et al., FASEB J., 15: 1953-1962, 2001; Sato, T. et al., Cancer Res., 62:

1025-1029, 2002). NF- KB controls the expression of various genes which are directly involved in the onset of cancer (Pahl H. L., Oncogene, 18: 6853-6866, 1999; Garg A., Aggarwal B. B., Leukemia (Baltimore), 16: 1053-1068, 2002). Examples of the various genes include antiapoptosis genes, etc. such as survivin, TRAF, bcl-2 and the like. Besides, NF- KB is a key transcription factor which is involved in the activation of inflammatory-cytokine genes such as tTNF-α or IL- lβ. The NF- KB also induces the activation of MMP-9, COX-2 and iNOS genes. Therefore, several substances which inhibit the activation of NF-κB are suggested to be potent to inhibit the onset and metastasis of cancer, and researches thereon for developing them as a therapeutic agent have been actively conducted. Disclosure of Invention

Technical Solution

[8] Therefore, an object of the present invention is to provide a novel use of chlorogenic acid (CHA, 5-caffeoylquinic acid)as a direct inhibitor to the enzymatic activity of MMP-9.

[9] In order to achieve the above object, the inventors of the present invention have intensively studied and developed an MMP-9 inhibitor contained chlorogenic acid which is isolated from Euonymus alatus extract and identified MMP-9 inhibitory effect.

[10] The present invention relates to an MMP-9 inhibitor having chlorogenic aicd as an active ingredient.

[11] The present invention is further characterized in that the inhibitor suppresses the generation of MMP-9 enzyme.

[12] The present invention is also characterized in that the inhibitor suppresses the transcription for MMP-9 expression.

[13] The present invention is further characterized in that the inhibitor suppresses the activity of an MMP-9 promoter.

[14] Further, the present invention provided a pharmaceutical composition which contains chlorogenic acid for inhibiting MMP-9 activity.

[15] The present invention also is characterized in that the composition is used to rheumatoid arthritis, osteoarthritis, osteoporosis and diseases related with the metastasis, invasion or development of tumor.

Description of Drawings

[16] Fig. 1 is a bar graph showing the results of XTT assay for estimating the effect of the proliferation of a hepatocellular carcinoma cell line, Hep3B.

[17] To examine the effect of CHA on Hep3B cells growth, Hep3B cells were treated with various concentrations of CHA for 24hr. Cell proliferation was determined by an XTT assay. The absorbance was measured on an ELISA reader (Molecular Devices,

USA) at a wavelength of 490 nm. The results are expressed as the percentage of cell proliferation of the control at 0 concentration of CHA. The data shown are the mean + SD of three independent experiments.

[18] Fig. 2 is a results showing the effects of CHA on the gelatinolytic activity of MMP-

9.

[19] (A) is showing results of the effects of CHA on MMP-9 activity. The data shown are the mean + SD of three independent experiments.

[20] (B) is showing results of the effects of CHA(30 m g/ml), TIMP-I(IOO nM) and

EGCG (50 m g/ml) on MMP-9 activity.

Mode for Invention

[21] This invention provids an MMP-9 inhibitor having chlorogenic aicd as an active ingredient. In preferred embodiments, chlorogenic aicd can be isolated from Euonymus alatus by solvent extraction and chromatography procedures .

[22] The present invention also provids pharmaceutical composition which contains chlorogenic acid. The pharmaceutical composition can be used as MMP-9 inhibitor, particularly used for the treatment and prevention of rheumatoid arthritis, osteoarthritis, osteoporosis and diseases related with the metastasis, invasion or development of tumor.

[23] The composition includes a therapeutically effective amounts of chlorogenic acid within/without a pharmaceutically acceptable delivery vehicle. Chlorogenic acid may be formulated with a pharmaceutical vehicle or diluent for oral, intravenous, subcutaneous, intranasal, intrabronchial or rectal administration. The pharmaceutical composition can be formulated in a classical manner using solid or liquid vehicles, diluents and additives appropriate to the desired mode of administration. Orally, the compounds can be administered in the form of tablets, capsules, granules, powders and the like. The compounds of the present invention may also be employed with sodium lauryl sulfate or other pharmaceutically acceptable detergents to enhance oral bioavailability of such compounds.

[24] Chlorogenic acid may be administered in a dosage ranges of about

0.01~1000mg/kg, preferably 0.1-500 mg/kg, and may be administered 0.5-3 times a day.

[25] Hereinafter, the present invention is further described in detail with referencing the examples provided below. However, those examples are only provided for illustrative purposes, by no means limiting the scope of the present invention.

[26]

[27] Materials

[28] (-)-Epigallocatechin gallate (EGCG) and tissue inhibitor of metalloproteinase- 1

(TIMP-I) were obtained from Aldrich-Sigma Chemical Co. (St. Louis, Mo, USA).

Other chemicals were of analytical or HPLC grade.

[29]

[30] Cell culture

[31] Human hepatocellular carcinoma cell line (Hep3B) was obtained from the Korean cell line bank and cultured in Dulbecco's modified eagle's medium (DMEM, Gibco- BRL, USA) containing 10% heat inactivated fetal bovine serum (FBS, Gibco-BRL, USA) supplemented with penicillin (100 units/ml), streptomycin (100 μg/ml) and sodium bicarbonate (2.2 g/L) at 37 ⁰ C in 5% CO -air. Cells were grown to sub- confluence and were rinsed with phosphate-buffered saline (PBS) and then incubated in serum-free medium for 24h. The serum-free medium contained gelatinase such as MMP-9. The amount of gelatinase in the conditioned media were estimated and quantified by cell numbers.

[32]

[33] Example 1: Isolation of chlorogenic acid

[34] Five hundred grams of the fresh weight of E. alatus were extracted with MeOH and concentrated to aqueous phase in vacuo. The aqueous residue was adjusted to pH 2 and partitioned with n-BuOH. The n-BuOH fraction was partitioned with 2OmM carbonate buffer(pH 9.5). The buffer fraction was adjusted to pH 2 and extracted with n-BuOH again. The acidic n-BuOH fraction was subjected to a silica gel column and eluted 10% stepwise with MeOH in EtOAc. The fractions eluted with 0 and 10% MeOH in EtOAc were passed through an ODS Sep-Pak cartridge(Waters) and then purified by ODS-HPLC using the following conditions: column, Develosil ODS-5 column (10 mm f x 150 mm, Nomura Chemicals); solvent, 0-7 min, MeOH/H 0/AcOH (25:70:5), 7-40 min, linear gradient of MeOH/H /AcOH (25:70:5)-MeOH; flow rate, 5.0 niL/min; column temperature, 4O ⁰ C; UV detection, 254 and 300 nm.

[35]

[36] Example 2: Determination of chlorogenic acid

[37] Chlorogenic acid was determined by high-performance liquid chro- matography(HPLC) after extraction from the plant. The HPLC conditions for chlorogenic acid are as follows: column, CAPCELL PAK C18 MG (5 μm, 4.6 x 150 mm, Shiseido, Japan); mobile phase, methanol/water/acetic acid (20:78:2,v/v/v) containing 5OmM lithium acetate; and flow rate, 0.8 mL/min (analysis B). Elution was monitored with an electrochemical detector (Coulochem II, ESA, Bedford, MA) with the first electrode potential of + 100 mV and second electrode potential of +800 mV.

[38] Chlorogenic acid (retention volume: 4.8 mL in analysis B) was quantitatively determined by an external standard method. The detection limits for chlorogenic acid was 0.05 μM with linear detector response up to 20 mM. When necessary, samples were diluted with mobile phase before HPLC analysis. The final yield of CHA was 0.5

mg. A purified compound (5 mg) was isolated, which showed the activity. The structure of the active compound(formula 1) was determined as chlorogenic acid (Buckingham, 1984). C H O , melting point 205 ⁰ C to 206 ⁰ C, alpha D-33.25 (H O).

16 18 9 2

Its identity was confirmed by comparing its physical data as well as its infrared (IR), nuclear magnetic resonance ( H NMR, C NMR) and mass spectral data with those of an authentic sample. Sodium chlorogenate (0.112 g) was prepared by the reaction of chlorogenic acid(O.lg) with sodium hydrogen carbonate(0.24g) in water (5mL) followed by lyophilization of the resulting solution.

[39] The FAB-MS data were obtained with a JEOL JMS-SX102A system. ¹ H and ¹³ C NMR, ¹ U- ¹ H COSY, ¹ H- ¹³ C COSY, and HMBC data were obtained with JEOL JNM- LA500 and JMN-EX270 systems in DMSO-J at 35 ⁰ C.

[40] FAB-MS: mlz 354. [41] ¹ H NMR (270 MHz, DMSO-J ): delta 7.42( ¹ H, d, /= 16 Hz, H-7 caffeoyl), 7.03 (* H, d, /= 2 Hz, H-2 caffeoyl), 6.98 ( ¹ U, dd, /= 2 and 8 Hz, H-6 caffeoyl), 6.77 (IH, d, /= 8 Hz, H-5 caffeoyl), 6.15 ( ¹ H, d, / = 16 Hz, H-8 caffeoyl), 5.08 ( ¹ H, br d, / = 5 Hz, H-3 quinic), 3.94 ( ¹ H, br s, H-5 quinic), 3.55 ( ¹ H, br d, /= 4 Hz, H-4 quinic), 1.7-2.1 (2H, m, H-6 quinic), 1.98 (2H, br d, / = 5 Hz, H-2 quinic).

[42] ¹³ C NMR (125 MHz, DMS0-J6): delta 174.9 (C-7 quinic), 165.7 (C-9 caffeoyl), 148.3 (C-4 caffeoyl), 145.5 (C-3 caffeoyl), 144.8 (C-7 caffeoyl), 125.6 (C-I caffeoyl), 121.2 (C-6 caffeoyl), 115.7 (C-5 caffeoyl), 114.7 (C-2 caffeoyl), 114.3 (C-8 caffeoyl), 73.6 (C-I quinic), 70.9 (C-3 quinic), 70.6 (C-4 quinic), 68.3 (C-5 quinic), 37.2 (C-6 quinic), 36.5 (C-2 quinic).

[43] H NMR and C NMR data of the compound was identical to the authentic 5-caffeoylquinic acid (chlorogenic acid).

[44] [45] Formula 1 [46]

[47] [48] Example 3: Effect of CHA on the cell viability

[49] Hep3B cells were treated with various concentrations of CHA and cell viability was measured using the XTT assay.

[50] Cell proliferation was investigated using a commercially available proliferation kit

II (XTT, Boehringer Mannheim, Germany). Briefly, Hep3B cells were subcultured into 96-well culture plates at a density of 10,000 cells per well in 100 μl of DMEM medium and incubated for 24hr at 37 ⁰ C. Following 24hr incubation, the old medium was removed and the cells were replenished with 100 m 1 of new medium and were treated with various concentrations of 0, 10, 20, 50, 100, 200, 300 μg/ml CHA. The plates were incubated in a 37 ⁰ C humidified incubator under an atmosphere of 5% CO for 24hr. At the end of the incubation, the medium was discarded, and the cells were washed with PBS. 50 μl of XTT test solution (sodium

3'-[l-(phenyl-aminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy- 6 -nitro) ben- zenesulfonic acid hydrate and N-methyl dibenzopyrazine methyl sulfate; mixed in proportion 50:1) prepared by mixing 5ml of XTT-labeling reagent and 100 μl of electron coupling reagent, was then added to each well. After 4hr of incubation in a 37 ⁰ C and 5% CO incubator, the absorbance was measured on an ELISA reader (Molecular Devices, USA) at a test wavelength of 490 nm. The results showed in Fig. 1. All determinations were confirmed using replication in at least three identical experiments. The data shown are for only one experiment, but representative for all replications.

[51] As shown in Fig. 1, the resulting survival curve shows that CHA showed cytotoxic effect on the proliferation of cells. The addition of 100 μg/ml of CHA did not change the viability, while treatment with 200 μg/ml of CHA to the cells reduced 22% of the cell viability only when compared to control.

[52]

[53] Example 4: Inhibitory effects of CHA on the MMP-9 activity

[54] The effect of CHA on the MMP-9 activity has been examined using Zymographic method.

[55] Zymography was performed as described previously (Choi et al., 2001; Chung et al., 2002; Demeule 2000). Culture supernatants were resuspended in a sample buffer containing 62.5 mM Tris-HCl (pH 6.8), 10% glycerol, 2% SDS, and 0.00625% (w/v) bromophenol blue and loaded without boiling in 7.5% acrylamide:bisacrylamide (29.2:0.8) separating gel containing 0.1% (w/v) gelatin. Electrophoresis was carried out at a constant voltage of 100V. After electrophoresis, the gels were soaked in 0.25% Triton X-100 (2 x 30 min) at room temperature and rinsed in NanoPure water. The gel was cut into slices corresponding to the lanes and then put in different tanks containing the incubation buffer [50 mM Tris-HCl (pH 7.5), 200 mM NaCl, 2.5 mM CaCl ] with 30 μg/ml CHA, 50 μg/ml EGCG and 10OnM TIMP-I (tissue inhibitor of metallo-

proteinase- 1). The gel treated with CHA, EGCG and TIMP-I was incubated at 37°C for 24hr. Bands corresponding to activity were visualized by negative staining using Coomassie Brilliant Blue R-250 (Bio-Rad, USA) and molecular weights were timated by reference to prestained SDS-PAGE markers.

[56] The results showed in Fig 2. As shown in Fig. 2A, CHA dramatically inhibited proteolytic activity of MMP-9 in a dose-dependent manner. Additionally, TIMP-I, which is known as MMP-9-specific inhibitor, suppressed enzymatic activity of MMP- 9, and EGCG, a MMP-9 inhibitor isolated from green tea, also inhibited MMP-9 activity(Fig. 2B). These results show that CHA inhibits enzymatic activity of MMP-9 protein that is secreted from Hep3B cells.

Industrial Applicability

[57] The prevent invention provides an inhibitor which can directly inhibit the MMP-9, by using chlorogenic acid as active ingredients. Therefore, the inhibitor of the this invention can be suitably used as a therapeutic agent of various MMP-mediated diseases such as MMP-9 dependant tumors or rheumatoid arthritis, etc.