KR101322478B1 - Abalone intestine gastro-intestinal digests having effects of inhibiting MMP activity and a pharmaceutical composition containing the same as an active ingredient - Google Patents

Abstract

The present invention is abalone intestine gastro-intestinal digests (AIGID) and MMP-2, MMP- containing it as an active ingredient having an inhibitory effect on MMP (Matrix metalloproteinases: hereinafter, 'MMP') activity 9 A pharmaceutical composition for inhibiting activity, which has a beneficial effect of preventing or treating diseases associated with MMP activity, such as cancer, neurodegenerative, cardiovascular and various inflammatory diseases.

Description

Abalone intestine gastro-intestinal digests having effects of inhibiting MMP activity and a pharmaceutical composition containing the same as an active ingredient

The present invention is an abalone intestine gastro-intestinal digests (hereinafter referred to as 'AIGID') having an inhibitory effect on MMP (Matrix metalloproteinases: hereinafter, 'MMP') activity and containing it as an active ingredient The present invention relates to a pharmaceutical composition for inhibiting MMP-2 and MMP-9 activity. More specifically, it is found that AIGID has an inhibitory effect against MMP-2 and MMP-9 activity, and it is contained as an active ingredient, thereby preventing cancer, neurodegenerative diseases, It is to provide a pharmaceutical composition effective for the prevention and treatment of cardiovascular and inflammatory diseases.

Matrix metalloproteinases (MMPs) are a type of zinc-dependent endopeptidase that play an important role in various biological and pathological processes such as extracellular matrix (ECM) degradation and tumor infiltration and metastasis. Zinc-dependent endopeptidase has more than 28 endopeptidase that can collectively degrade virtually all extracellular matrix components. MMPs are membrane-bound MMPs as well as minimal domain MMPs, simple hemopexin domain-containing MMPs, gelatin-binding MMPs, furin-activated secretion MMPs, vitronectin-like insertion MMPs It is broadly classified into eight subgroups of transmembrane MMPs, glycosyl-phosphatidyl inositol (GPI) -linked MMPs and type II transmembrane MMPs. MMPs regulate the synthesis and secretion of cytokines, growth factors, hormone receptors and cell adhesion molecules. They are also involved in cancer and a series of physiological and pathological pathways, including growth and expression, morphogenesis, tissue remodeling, angiogenesis, cardiovascular, allergy, neurodegenerative diseases. Thus, the development of matrix metalloproteinase inhibitors (MMPIs) has a wide range of uses in treating some important diseases including cancer, neurodegenerative, cardiovascular and various types of inflammatory diseases.

The activity of gelatinases (MMP-2 and MMP-9) plays an important role in cancer infiltration and metastasis, which is constantly found in malignant tissues. The metalloproteinase subgroup was originally described as type IV collagenase because of its ability to cleave type IV collagen. They have gelatin-binding domains with motifs similar to fibronectin.

One of these, MMP-2 (gelatinase-A, 72 kDa IV type collagenase), was originally purified in high metastatic murine tumors. MMP-2 binds to type I collagen through the fibronectin domain and is stabilized from autolysis, resulting in controlled activity. MMP-2 expression depends on extracellular matrix metalloproteinase derivatives, growth factors, cytokines and hormones. Pro-MMP-2 activation requires MT1-MMP and TIMP-2 distributions. After one cochlear ablation, the distribution of MMP-2 changes and increases in the ventral cochlear nucleus. Low concentrations of MMP-2 are generally considered a favorable prognosis in patients with high risk hormone receptor-negative tumors. MMP-2 is a zymogen that requires proteolytic activation for catalytic activity and is widely involved in infiltration and metastasis of many cancer model systems, including human breast cancer.

MMP-9 (92 kDa IV type collagenase, gelatinase B) is produced in human macrophages and multinuclear leukocytes. It is also localized in synovial fibroblasts in endothelial cells and rheumatoid arthritis synovial membranes. MMP-9 is expressed by keel cells in human normal bone tissue and appears to play a role in bone deformation. Adult fully dentinocytes also express MMP-9 and are found in dental caries lesions and saliva. However, MMP-9 is not expressed by human gingival fibroblasts. Like MMP-2, MMP-9 may be present in ECM bound to type I collagen, gelatin or laminin.

In the present invention to use the abalone in order to suppress the MMP-2, MMP-9 activity, as a conventional industrial use using the abalone, Republic of Korea Patent No. 1009025390000 'Functional beverage composition containing abalone extract and its manufacturing method ', Republic of Korea Patent Application Publication No. 1020100011580' immune strengthening composition as an active ingredient in abalone viscera extract 'and Republic of Korea Patent No. 1010274470000' cosmetic composition containing abalone extract and its preparation method 'is known, but MMP inhibitory effect It is not known at all about using it for anticancer purposes.

1. Republic of Korea Patent No. 1009025390000 (Registration date: June 05, 2009, Publication No. 1020090032562, Publication Date: April 1, 2009) 2. Korean Patent Application Publication No. 1020100011580 (published: 2010.02.03) 3. Republic of Korea Patent Registration No. 1010274470000 (Registration Date: 2011.03.30)

In the present invention, it is intended to elucidate the inhibitory effect of AIGID MMP-2, MMP-9 activity.

In addition, the present invention is to provide a pharmaceutical composition for inhibiting MMP-2, MMP-9 activity, comprising AIGID as an active ingredient.

The abalone used in the present invention is a marine gastropod molluse that inhabits the temperate and tropical waters of the northern and southern hemispheres of the earth. The abalone in the present invention is not particularly limited to the type and place of production, but is preferably Wando abalone in Korea, more preferably Haeotis discus hannai . Veterans abalone is an important species in Korea.

According to the present invention, the by-products after boiling abalone, that is, the hydrothermal extract is classified into an ultramembrane system with various ultramembrane systems, and various molecular weight fractions of abdominal gut digestive hydrolysates AIGID, AIGID I (> 100 kDa), AIGID II (100-10 kDa), AIGID III (10-5 kDa).

According to the present invention, denatured type I collagen resolution of MMP-2 and MMP-9 is utilized as a simple and reliable method for detecting whether MMP-2 and MMP-9 are present in a biological sample. This method, also known as gelatin zymography, confirms gelatin soluble activity in biological samples using a sodium dodecyl sulfate (SDS) -polyacrylamide gel impregnated with gelatin.

The human fibrosarcoma cell line HT1080 used in the present invention is widely used for the study of extracellular matrix proteins involved in attachment, infiltration and metastasis. In the present invention, HT1080 cells are used for both MMP-2 and MMP-9 enzymes. Use the characteristics you create.

Infiltration of tumor cells into ECM is an important step in the process of tumor metastasis and involves the attachment of tumor cells to ECM. Tumor cell migration and infiltration depend on gelatinase activity. MMP-9 and MMP-2 are important cells in the process of tumor metastasis resulting from ECM degradation. MMPs are highly expressed in human cancers, and the direct relationship between cancer progression and MMP expression is well established in many papers. Interaction between pepsinated human placental type IV collagen and human HT1080 fibrosarcoma cells activates proMMP-2 on the cell surface. According to the artificial substrate and gelatin zymography experiments of the present invention, AIGID II and AIGID III have MMP-2 and MMP-9 inhibitory effects. As a result, in PMA-treated HT-1080 cells, AIGID II and AIGID III inhibited the transcriptional activation of the MMP-9 promoter, thereby dose-dependently inhibiting increased expression and secretion of MMP-2 and MMP-9. (See FIGS. 5A and 5B).

The MMP-2 and MMP-9 promoters also contain an NF-κB site for transcription factors. Since tumors show high concentrations of MMP activity, MMP specific inhibitors are being sought. While human fibroblast cell line HT1080 produces large amounts of MMP-9 enzymes, normal human fibroblasts have been reported to secrete little or no enzyme. Although HT1080 cells enhance gymography based on the activity of both MMP-2 and MMP-9 compared to the PMA-treated blank group, MMP-2 expression in HT1080 cells after treatment with PMA was untreated. Lower than MMP-9 (see FIGS. 2, 3A and 3B).

In addition, the NF-κB family of transcription factors is present in most cell types, and specific NF-κB binding sites, denoted as κB sites, have been identified in the promoters of very many inducible genes. NF-κB regulates key pathways in cancer, including invasion, metastasis, apoptosis, angiogenesis, growth and proliferation. The transcription factor of NF-κB triggers MMP-2 and MMP-9 gene transcription in response to chemical stimuli such as phorbol 12myristate 13-acetate (PMA) in HT1080 cells. NF-κB activity is tightly controlled through several mechanisms. According to the present invention, phosphorylation of NF-κB and degradation of p65 protein in PMA-treated HT1080 cells are dose dependently inhibited (see FIGS. 6A and 6B). The inhibition clearly follows a dose dependent pattern. However, they did not inhibit PMA-induced translocation of p50, a component of NF-κB (see FIGS. 6A and 6B), so that AIGID II and AIGID III transcription of NF-κB in HT1080 human fibrosarcoma cells. Downregulation suggests inhibition of MMP-2 and MMP-9.

The pharmaceutical composition of the present invention may be formulated into a preparation for oral or parenteral administration according to the route of administration as described above.

In the case of preparations for oral administration, the pharmaceutical compositions of the present invention are formulated using methods known in the art as powders, granules, tablets, pills, dragees, capsules, solutions, gels, syrups, slurries, suspensions, and the like. Can be. For example, an oral preparation can be obtained by combining the active ingredient with a solid excipient, then milling it, adding suitable auxiliaries, and then processing the mixture into a granular mixture. Examples of suitable excipients include, but are not limited to, sugars including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol and maltitol, and starches including corn starch, wheat starch, rice starch and potato starch, Cellulose such as methylcellulose, sodium carboxymethylcellulose and hydroxypropylmethyl-cellulose and the like, fillers such as gelatin, polyvinylpyrrolidone and the like.

In the case of a preparation for parenteral administration, it can be formulated by a method known in the art in the form of injection, cream, lotion, external ointment, oil, moisturizer, gel, aerosol and nasal aspirate.

The AIGID of the present invention may preferably be included in an amount of 0.01 to 99.9% by weight based on the total weight of the pharmaceutical composition.

AIGID according to the present invention and a pharmaceutical composition comprising the same as an active ingredient has a beneficial effect of preventing or treating diseases associated with MMP activity, such as cancer, neurodegenerative, cardiovascular and various inflammatory diseases.

Figure 1 is a graph showing the effect on the HT1080 cell viability of AIGID, AIGID I, AIGID II, AIGID III at 10, 50, 100, 200, 400 mg / L concentration.
2 is a photograph showing gelatin zymography showing the effects of AIGID, AIGID I, AIGID II, and AIGID III on HT1080 cell MMP-2 and MMP-9 activation and expression inhibition.
Figure 3a is a gelatin zineography of HT1080 intracellular MMP-2 and MMP-9 treated with AIGID II, AIGID III at 10, 100, 200 mg / L concentration, Figure 3b is 10, 100, 200 mg It is a graph which shows the relative MMP-2 and MMP-9 expression rates (%) in AIGID II and AIGID III of / L concentration.
Figure 4 is a photograph showing the effect on HT1080 cell migration of AIGID II, AIGID III of 100, 200 mg / L concentration.
FIG. 5A is a Western blot of MMP-2, MMP-9 and β-actin protein expression in HT1080 when treated with AIGID II and AIGID III at 10, 100 and 200 mg / L concentrations. FIG. It is a graph which shows the relative MMP-2, MMP-9 expression rate (%) in AIGID II and AIGID III of 100, 200 mg / L concentration.
Figure 6a is an electrophoresis picture showing the p50 and p65 protein expression concentration to confirm the inhibition of NF-κB protein expression when treated with AIGID II, AIGID III of 10, 100, 200 mg / L concentration, Figure 6b is 10, It is a graph which shows the relative p50, p65 expression rate (%) in AIGID II and AIGID III of 100, 200 mg / L concentration.

The present invention is described in more detail by the following specific examples, but the following examples are intended to illustrate the present invention, and the scope of the present invention is not limited thereto.

material

Haliotis discus hannai was collected from Wando, Korea. Dulbecco's Modified Eagle's Medium (DMEM), Trypsin-EDTA, Fetal Bovine Serum (FBS) were obtained from Gibbco BRL, Life Technologies (Grand Island, NY, USA). HT1080 cells were obtained from the American Type of Culture Collection (Manassas, Va.). Primary and secondary antibodies used in Western blot analysis were MMP-2 (8B4, sc-13595), MMP-9 (H-129: sc-10737), NF-κB p65 (F-6: sc-8008), NF -κB P50 (D-6: sc-166588), β-actin (N-21: sc-130656), goat anti-rabbit IgG-HRP (sc-2004) and goth anti-mouse IgG1 -HRP (sc-2060) and purchased from Santa Cruz Biotechnology, INC (California, USA). MTT reagent, gelatin, phorbol 12-myristate 13-acetate (PMA) was purchased from Sigma Chemical Co. (St. Louis, MO). All other materials needed for cell culture were purchased from Gibco Bialel, Life Technologies.

Manufacturing example  One: UF  Preparation of AIGID Using Membrane Bioreactor

Abalone visceral gastrointestinal digestive hydrolyzate (AIGID) solution was prepared with 550 mL of 1 mol / L lactic acid, dispersed in distilled water under stirring, and made up to 10 L with distilled water. The pH of the solution was adjusted to 5.5 using saturated NaHCO ₃ solution. AIGID, AIGID I, AIGID II, and AIGID III were prepared by continuously hydrolyzing AIGID by molecular weight in an UF membrane reactor system connected to an enzyme reactor. The abalone obtained by the ultra-membrane system with various UF membranes was boiled and the MW 100 kDa, 10 kDa and 5 kDa fractions of the remaining byproduct (hydrothermal extract) were lyophilized and stored at -20 ° C.

AIGID II and AIGID III in the samples were digested with HCl at 110 ° C. for 24 hours under nitrogen atmosphere. An automatic amino acid analyzer was used to identify and quantify amino acids derived from phenylisothiocyanates. Peptides with increased hydrophobicity among peptides derived from many protein sources are known to be associated with antioxidant properties, and several amino acids, including histidine, proline, alanine and leucine, have been reported to be involved in eliminating free radicals.

AIGID II (100-10 kDa) and AIGID III (10-5 kDa) by in vitro GI degradation model (SHGD) were shown in Table 1 below.

As can be seen from the above, the abdominal visceral gastrointestinal hydrolyzate contains 18 amino acids in AIGID II and AIGID III and has a different composition. AIGID II is also rich in glutamic acid (Glu), proline (Pro), alanine (Ala), histidine (His), lysine (Lys) and aspartic acid (ASP), and AIGID III is glutamic acid (Glu), proline (Pro), Alanine (Ala), Leucine (Leu) and Aspartic Acid (Asp).

Manufacturing example  2: cell culture

Human fibrosarcoma cell line HT1080 was cultured as a monolayer in a 10 cm culture dish under 5% CO ₂ and 37 ° C. humidified atmosphere. Dulbecco's Modified Eagle's Medium (DMEM) was used as culture medium for HT1080 cells and contained 10% fetal bovine serum. Cells were passaged for 2 days with trypsin-EDTA (0.05%) and used for the experiment after 5 passages.

Experimental Example  One: MTT  Determination of Cell Viability by Analysis

The MTT colorimetric assay demonstrates the ability of live cells to convert soluble tetrazolium salt [3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide] (MTT) into an insoluble formazan precipitate. Decide To determine the cytocompatible effects of AIGID, AIGID I, AIGID II, and AIGID III, HT1080 cells were seeded in 96-well plates at a density of about 1 × 10 ⁴ cells / well and serum present Incubated with samples of different concentrations (10, 50, 100, 200, 400 mg / L) for 24 hours. After incubation, 50 μL of MTT reagent was added to each well and incubation continued for another 4 hours. Mitochondrial succinate dehydrogenase in living cells converts MTT into visible formazan crystals during incubation. The formazan crystals were then dissolved in DMSO and the optical density was measured at 540 nm using a microplate reader. Relative cell viability was calculated by comparison with the untreated blank group. The data are expressed as mean values of three or more separate experiments and p <0.05 was considered significant.

As a result, as can be seen in Figure 1, all used AIGID used in this experiment did not show any cytotoxic effect even at the highest concentration tested (400 mg / L).

Experimental Example  2: MMP -2, MMP Determination of Gelatinase Activity of -9

In this experimental example, gelatinase activity of MMP-2 and MMP-9 is determined by gelatin zymography.

HT1080 cells were cultured in serum free DMEM medium on 24-well plates. After 1 hour stimulation, PMA (final concentration 10 mg / L) was added to HT1080 cells treated with AIGID, AIGID I, AIGID II, and AIGID III at 200 mg / L. After 36 hours, the conditioned medium was harvested and centrifuged at 3,000 rpm for 10 minutes to remove cell debris. Concentrated medium was electrophoresed on 10% sodium dodecyl sulfate (SDS) -polyacrylamide gel impregnated to 0.15% final concentration gelatin without heating under non-reducing conditions. After electrophoresis, the SDS was removed from the gel by washing with 2.5% Triton X-100 solution for 1 hour 30 minutes, and the developed buffer [50 mmol / L Tris-HCl buffer (pH 7.5), 200 mmol / L NaCl, 5 mmol / L CaCl ₂ H ₂ O, 0.02% Brif-35]. The gel was then stained with a blue R-250 solution of 1% Coomassie containing 45% methanol and 10% glacial acetic acid. After 30 minutes the gel was decolorized with 10% glacial acetic acid solution, 40% methanol. The cleaved portion appears transparent on a blue background, indicating the gelatinase position.

As can be seen in Figure 2, AIGID II and AIGID III showed a significant inhibitory effect on MMP-2 and MMP-9 activation in HT1080 compared to the PMA-only treatment group. However, in the case of AIGID and AIGID I, no significant inhibitory effect was observed for both MMP-2 and MMP-9 activation. Thus, AIGID II and AIGID III were used in subsequent experiments.

In addition, gelatin zymography tested whether AIGID II and AIGID III affected the gelatinolytic activity of MMP-2 and MMP-9 secreted from HT1080 cells at various concentrations of 10, 100 and 200 mg / L. It was.

As can be seen in FIG. 3, MMP-2 and MMP-9 activity was reduced in the presence of AIGID II and III AIGID at 100 and 200 mg / L concentrations, in particular AIGID III at 200 mg / L concentration was MMP activity than AIGID II. Significantly suppressed.

Experimental Example  3: analysis of cell migration ( migration assay )

Migration capacity is a prerequisite for cell infiltration through the basement membrane. In this experimental example, it is confirmed whether AIGID II and AIGID III inhibit tumor cell migration by dose-dependent inhibition of HT1080 cell migration in a damaged space.

First, the cells were cultured to divide the cells into 6-well plates. After washing with PBS it was treated with various concentrations of AIGID II and III AIGID and photographed at the first time point. After 24 hours, cells migrated to complete medium. The results were observed on the microscope at the designated subsequent time points.

As shown in FIG. 4, the movement occurs across the injury site. To clarify this, damage transfer assays were performed using cells pretreated with mitomycin C. Both AIGID II and AIGID III at 200 mg / L concentrations appeared to block HT1080 cell migration. From the above, it can be concluded that the non-toxic range of AIGID II and III AIGID significantly reduces MMP activity, cell migration, motility and invasiveness.

Experimental Example  4: NF for determining κB activity Western Blot  analysis

The effect of AIGID II and AIGID III on NF-κB activity was confirmed through p50 and p65. p50 and p65 are two subunits of the transcription factor NF-κB containing the transactivation domain required for gene induction. Western blot studies were conducted to confirm down regulation of the gene expression.

To this end, cultured cells are harvested, followed by RIPA buffer (50 mmol / L Tris-HCl, pH 7.5), 150 mmol / L NaCl, 1% Triton X-100, 1% sodium deoxyclate, 2 mmol / Lysing in L EDTApH 8.0) and 0.1% SDS), the protein concentration of the cell lysate was determined by the bicinchoninic acid (BCA) method. Samples were separated by electrophoresis on 10% SDS-polyacrylamide gels and transferred to nitrocellulose membranes. The membrane was blocked with 5% skim milk in TBST (20 mmol / L Tris-HCl, pH 7.6, 136 mmol / L NaCl and 0.1% Tween-20) and then the primary antibody in the blocking formulation at 4 ° C. overnight. 1: 500 dilution). After washing with TBST, the membrane was incubated with secondary antibody (1: 5,000 dilution) at room temperature for 2 hours. The membrane was then washed three times with TBST. Bands were visualized by enhanced chemiluminescence and LAS-4000 imaging system (Fujifilm, Japan). Basal levels of MMP-2 and MMP-9 proteins were normalized by analyzing β-actin protein concentrations.

As can be seen in Figure 6, AIGID II and AIGID III treatment induces downregulation of p65 protein, which was observed in the presence of AIGID II and AIGID III at concentrations of 10, 100 and 200 mg / L. In addition, AIGID III was shown to inhibit p65 activity more than AIGID II at a concentration of 200 mg / L. However, no significant inhibitory effect on p50 expression was observed at 10, 100, 200 mg / L concentrations. These results demonstrate that AIGID II and AIGID III downregulate MMP-2 and MMP-9 through transcriptional down regulation of NF-κB.

Experimental Example  5: MMP -2 and MMP -9 for expression determination Western Blot  analysis

Western blot studies were conducted to confirm downregulation of the gene expression. To study the binding effects of AIGID II and AIGID III, protein expression concentrations and activities of both MMP-2 and MMP-9 can be inhibited at various concentrations of 10, 100 and 200 mg / L in HT1080 cells. Conditioned medium was used for MMP-2 and MMP-9 protein concentration determination by Western blot as described in the 'Materials and Methods' column. As shown in Figures 5a and 5b, the protein expression concentration of MMP-2 was relatively low compared to the MMP-9 and PMA-only treatment group. In addition, significant inhibitory effects on MMP-2 and MMP-3 proteins were similar between the 200 mg / L AIGID II and AIGID III treatment groups.

Formulation example  1: Preparation of tablets

25 mg of AIGID, 26 mg of lactose, 3.5 mg of Avicel (microcrystalline cellulose), 1.5 mg of sodium starch glyconate and 8 mg of L-HPC (low-hydrosyprophylcellulose) were added to a U-type mixer and mixed for 20 minutes. After the mixing was completed, 1 mg of magnesium stearate was further added and mixed for 3 minutes. Tablets were prepared by tableting and film coating after a quantitative test and a humidity test.

Claims (2)

A matrix obtained by dispersing abalone intestine and gastrointestinal tract in lactic acid and distilled water to prepare a digestive hydrolyzate solution, adjusting to pH 5.5 with Na ₂ HCO ₃ and hydrolyzing by molecular weight in an ultrafiltration membrane (UF) system connected to an enzyme reactor. Metalloproteinase (MMP) -2 or MMP-9 activity of the abdominal visceral gastrointestinal digestive digestion, characterized in that it has an inhibitory effect and comprises a peptide of molecular weight size (MW) 100 ~ 5kD. A pharmaceutical composition for the prevention and treatment of MMP-2 or MMP-9 activity-related inflammatory diseases, comprising the abalone viscera gastrointestinal digestive hydrolyzate of claim 1 as an active ingredient.

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