KR100811744B1 - Pharmaceutical composition containing arazyme for the prevention and treatment of cancer - Google Patents

Abstract

A composition comprising arazyme produced by Aranicola proteolyticus is provided to increase body weight, inhibit tumor cells and show the infiltration reduction when administered to a human lung cancer cell line transplanted mouse and decrease the expression amount of MMP-9, NF-kappaB and PCNA, thereby being usefully used as a pharmaceutical composition for preventing and treating cancer. A pharmaceutical composition for preventing and treating cancer comprises arazyme as an effective ingredient. In the composition, the arazyme is a polypeptide having an amino acid sequence of SEQ ID : NO. 1 and is isolated from a culture solution of Aranicola proteolyticus which is Aranicola proteolyticus HY-3(deposition no. KCTC 0268BP). Further, the cancer is lung cancer.

Description

Pharmaceutical composition containing arazyme for the prevention and treatment of cancer}

1 is a graph showing changes in body weight after administration of arazyme to nude mice implanted with human lung cancer cell line (A549):

    Control: arazyme 0 mg / kg, oral administration; T1: arazyme 150 mg / kg, administered orally; T2: arazyme 300 mg / kg, orally administered; And T3: arazyme 600 mg / kg, orally administered.

2 is a graph showing the tumor size after administration of arazyme to a nude mouse transplanted with a human lung cancer cell line (A549):

Control: arazyme 0 mg / kg, oral administration; T1: arazyme 150 mg / kg, administered orally; T2: arazyme 300 mg / kg, orally administered; T3: arazyme 600 mg / kg, orally administered; ^* : p <0.05; And ^** : p <0.01.

Figure 3 is a histological picture after administration of arazyme to nude mice implanted with human lung cancer cell line (A549) (13 and 33):

    Control: arazyme 0 mg / kg, oral administration; T1: arazyme 150 mg / kg, administered orally; T2: arazyme 300 mg / kg, orally administered; And T3: arazyme 600 mg / kg, orally administered.

Figure 4 is an immunohistograph of MMP-9 after administration of arazyme to nude mice implanted with human lung cancer cell line (A549) (33 and 66):

    Control: arazyme 0 mg / kg, oral administration; T1: arazyme 150 mg / kg, administered orally; T2: arazyme 300 mg / kg, orally administered; And T3: arazyme 600 mg / kg, orally administered.

5 is a graph showing the degree of immunohistochemical expression of MMP-9 after administration of arazyme to nude mice transplanted with human lung cancer cell line (A549):

    Control: arazyme 0 mg / kg, oral administration; T1: arazyme 150 mg / kg, administered orally; T2: arazyme 300 mg / kg, orally administered; And T3: arazyme 600 mg / kg, orally administered.

FIG. 6 is an immunohistograph of NF-κB after administration of arazyme to nude mice implanted with human lung cancer cell line (A549) (33-fold and 66-fold):

    Control: arazyme 0 mg / kg, oral administration; T1: arazyme 150 mg / kg, administered orally; T2: arazyme 300 mg / kg, orally administered; And T3: arazyme 600 mg / kg, orally administered.

7 is a graph showing the immunohistochemical expression level of NF-κB after administration of arazyme to nude mice transplanted with human lung cancer cell line (A549):

    Control: arazyme 0 mg / kg, oral administration; T1: arazyme 150 mg / kg, administered orally; T2: arazyme 300 mg / kg, orally administered; And T3: arazyme 600 mg / kg, orally administered.

8 is an immunohistograph of PCNA after administration of arazyme to nude mice implanted with human lung cancer cell line (A549) (66 and 132 times):

    Control: arazyme 0 mg / kg, oral administration; T1: arazyme 150 mg / kg, administered orally; T2: arazyme 300 mg / kg, orally administered; And T3: arazyme 600 mg / kg, orally administered.

9 is a graph showing the degree of immunohistochemical expression of PCNA after administration of arazyme to nude mice implanted with human lung cancer cell line (A549):

    Control: arazyme 0 mg / kg, oral administration; T1: arazyme 150 mg / kg, administered orally; T2: arazyme 300 mg / kg, orally administered; And T3: arazyme 600 mg / kg, orally administered.

The present invention relates to a pharmaceutical composition for preventing and treating cancer comprising arazyme as an active ingredient, and more particularly to preventing lung cancer containing arazyme (arazyme) produced by Aranicola proteolyticus . It relates to a therapeutic pharmaceutical composition.

Lung cancer has a very poor prognosis, and the incidence rate is increasing rapidly around the world, and it is already the second largest cancer death rate in Korea (Bae JM, et. al ., Cancer Res Treat , 34: 319-325, 2002). Among them, non-small cell lung cancer, which accounts for more than 75% of lung cancers, is already found to be inoperable in over 65% when diagnosed (Scientific Committee of Korean Academy of Tuberculosis and Respiratory disease, Tubere). Respir Dis , 46: 455-465. 1999). Recent advances in surgical and radiotherapy, the development of new anti-cancer therapies, and the combined application of several therapies have also improved the survival rate of some cancer patients. However, radiation therapy has a direct effect on the skin and can cause radiation dermatitis. Most of the previously developed anticancer drugs have a strong anti-cancer effect, but the side effects due to toxicity are high and the risk of recurrence is known. Therefore, researches are actively conducted to find a substance having little or no side effects and a large therapeutic effect of cancer, and for this purpose, many studies at home and abroad have been conducted on the biological activity of natural products.

Genetic predisposition is also deeply involved in the development of cancer, but environmental factors also have a significant impact. In advanced countries, the incidence of cancer is increasing. Possible causes include: increased use of pesticides and pesticides, increased residues in foods, increased consumption of processed foods with added food preservatives, preservatives, and colorants, increased water quality, soil, air pollution, increased stress in modern people, and increased activity. Reduction and obesity through abundant diets have been reported (kim HJ, et. al ., Liv Sci of Tj Univ . 3: 99-130, 1997; Jacobs MM, Nutr Tod , 28 (3): 19-23, 1993) .

Neovascularization is essential for tumor growth and metastasis (Folkman J, J Natl). Cancer Inst , 82: 4-6, 1990). Angiogenesis not only supplies nutrients and oxygen to the proliferating tumors, but also allows the penetration of tumor cells through the vascular wall from the primary path and metastases to distant organs (Kim KJ, et al ., Nature , 362: 841-844). , 1993). Lung cancer occurs histologically as cancer develops through changes in hyperplasia, metaplasia, dysplasia, and carcinoma in situ (Mulshine JL, et al. al ., Oncology , 5: 25-32, 1991) It is well known that progressively more alleles are lost (Wistuba II, et. al ., Oncogene , 18: 643-650, 1991).

On the other hand, the present inventors have tried to develop a new proteolytic enzyme, a new microorganism Aranicolaa Proteoricicus ( Aranicola ) from the Nephila clavata of Korea proteolyticus ) HY-3 strain (Accession No .: KCTC 0268BP; WO 01/57222) was isolated, and arazyme (arayzme), a novel protease, was isolated from the strain. Arazyme not only showed stable proteolytic activity at low temperatures and high salt concentrations, but also showed the highest activity at human body temperature of 37 ° C and stable activity over a wide pH range. Genes were identified (WO 01/57222).

The inventors have found that Aranicola Investigation of the effects of arazyme derived from proteolyticus strains revealed that arazyme causes weight gain and tumor cell suppression in nude mice transplanted with human lung cancer cell line (A549). The present invention has been completed by confirming that the expression patterns of -κB and PCNA are reduced and revealing that they can be usefully used as pharmaceutical compositions for preventing and treating cancer.

An object of the present invention is to provide a new use of arazyme (arazyme) produced by Aranicola proteolycius as an active ingredient in the pharmaceutical composition for preventing and treating lung cancer.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing and treating cancer containing arazyme (arazyme) as an active ingredient.

In addition, the present invention provides a health food for cancer prevention, containing the culture solution of aranicola proteoricicus or arazyme isolated therefrom as an active ingredient.

The present invention also provides a composition for preventing and inhibiting cancer metastasis containing arazyme as an active ingredient.

In addition, the present invention provides a cancer metastasis prevention health food containing the culture solution of aranicola proteoricicus or an arazyme isolated from it.

In addition, it provides an MMP-9 expression inhibitor containing arazyme as an active ingredient.

In addition, it provides an NF-κB expression inhibitor containing arazyme as an active ingredient.

In addition, it provides a PCNA expression inhibitor containing arazyme as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for preventing and treating cancer containing arazyme as an active ingredient.

The cancer is characterized in that lung cancer.

The arazyme of the present invention is an enzyme produced by the aranicola proteoricicus strain, comprising the steps of: 1) culturing the aranicola proteoricicus strain to obtain a culture solution; 2) filtering the culture solution to obtain a supernatant; And 3) purifying the arazyme contained in the supernatant using a resin (WO 01/57222). It is preferable to use aranicoli proteoricicus strain as a microorganism producing arazyme, and Aranicola proteoricicus HY-3 of Accession No. KCTC 0268BP, deposited on July 29, 1996 with the Korea Biotechnology Research Institute Gene Bank. More preferably, but is not limited thereto. Aranicola Proteoricicus HY-3 strain is an aerobic Gram-negative bacterium isolated from the intestine of the spider's intestine, and has a spherical and motility of 0.5 to 0.8 mm in size, positive for catalase, and negative for oxidase. Reaction (Korean Patent No. 0220091). In the present invention, arazyme obtained by the above method was used.

Arazyme of the present invention, (a) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1; (b) a polypeptide having at least 70% homology with SEQ ID NO: 1 and having a biologically equivalent function as the polypeptide of (a); And (c) has a polypeptide sequence consisting of an amino acid sequence in which one or a plurality of amino acids in the amino acid sequence set forth in SEQ ID NO: 1 is substituted, deleted, added and / or inserted, but which is biologically equivalent to the polypeptide of (a) The protein may be used.

To assay the anticancer efficacy of the arazyme against lung cancer, nude mice transplanted with human lung cancer cell line (A549) were treated with a control (administered 0 mg / kg of arazyme), T1 (arazyme 150 mg / Kg administration), T2 (administered 300 mg / kg of arazyme) and T3 (administration of arazyme 600 mg / kg) were divided into four groups, and oral administration of arazyme was repeated (see Table 1).

Arazyme was dissolved in sterile water for injection and subjected to forced administration using a zonde for oral administration six times a week. Body weight and general symptoms were observed after arazyme administration. Tumor incidence was observed once a week, its size was measured, and autopsy was performed 12 weeks after arazyme administration. In gross pathological findings, no abnormal abnormalities were observed in all organs, and even in microscopic observation, no abnormal findings of parenchyma by arazyme administration were observed in all individuals. Body weight was increased in the groups administered arazyme 300 mg / kg (T2) and 600 mg / kg (T3) (see Table 2 and FIG. 1). 300 mg / kg (T2) as a result of immunohistochemical observation by tumor size measurement and hematoxylin-eosin staining (H & E staining) obtained from nude mice transplanted with human lung cancer cell line (A549) In the group administered 600 mg / kg (T3) of arazyme, the arazyme concentration-dependent inhibition of tumorigenicity and tumor cell infiltration were observed (see FIGS. 2 and 3).

Matrix metalloproteinases (MMPs) are proteolytic enzymes that degrade the extracellular matrix (ECM) (Matrisan LM, Trends Genet , 6: 121-125, 1990) Increased production of MMPs is known to increase inflammation and invasion and metastasis in many cancer types (Stearns MA, et. al ., Cancer Res , 53: 878-883, 1993). During the tumor metastasis, collagen type IV, the most important structure of the vascular basement membrane, must be degraded before it can be transferred into the bloodstream. The degradation of collagen type IV is mainly caused by MMP-9 and MMP-2. (Liotta LA, et al ., Cell , 64: 327-336, 1991). MMP is also recognized as a necessary material for the migration and germination of vascular endothelial cells in angiogenesis (Fisher C, et. al ., Devel Biol , 162: 499-510, 1994). The activity of MMPs is 1) to regulate transcription of the MMP gene; 2) activity of precursors; 3) differences in lipid specificity; And 4) MMP inhibitors such as a-macroglobulins and tissue matrix metalloproteinases inhibitors (TIMPs) and the like (Birkedal-Hansen. H, J periodontol , 64: 474). -484, 1993). Higher signaling mechanisms that regulate the expression of MMP-9 involve growth factors including mitogen-activated protein kinases (MAPKs), cytokines, and tumor promoters. Among them, p38 kinase and extracellular signal-regulated protein kinase (ERK) regulated by extracellular signals are activated by inflammatory cytokines or apoptosis related signals and growth factors. Activated MAPKs move into the nucleus and react with transcription factor binding sites around the MMP primers to regulate the expression of downstream target proteins by activating various transcription factors. Most cancers are metastasized from primary to secondary by processes such as matrix invasion of cancer cells, migration from primary to vascular system, migration from vascular to secondary lesions, and formation of secondary tumors. Invasion of cancer cells requires the breakdown of extracellular matrix by protease. MMPs are the most important enzymes that break down extracellular matrix. Among the various MMPs, MMP-2 and MMP-9 are 72 and 92 kDa collagenases, which break down the collagen that forms the cell's basement membrane to promote cancer invasion and metastasis (Himelstein BP, et. al ., Invasion Metastasis , 14: 246-258, 1994).

In the present invention, it was confirmed that the expression of MMP-9, which promotes cancer invasion and metastasis, was reduced by arazyme (see FIGS. 4 and 5), indicating the anticancer efficacy of arazyme.

Nuclear transcription factor-kappaB (NF-κB) is an induced transcription factor that is activated in response to various environmental stimuli (Baeuerle, PA and Baichwal, VR, Adv). Immunol 65: 111-137, 1997; Auphan, N, et al ., Science , 270: 286-290, 1995). The tentative form of NF-κB is bound to the inhibitory protein, I-κB, in the cytoplasm. Stimulation of various NF-κB activators, such as tumor necrosis factor, IL-1, lipopolysaccharides, and UV (ultraviolet), caused phosphorylation and ubiquitin of the inhibitory protein I-κB. Degradation is induced through ubiquitination. This process transfers the NF-κB separated from I-κB into the nucleus to allow binding to the NF-κB-binding site at the promoter site of the target gene, thereby reducing the expression of the target gene. (Baldwin, AS, Annu Rev Immunol , 14: 649-683, 1996; Huxford T, et al ., Cell , 95: 759-770, 1998). NF-κB is another influencer of drug refractory. It is an important regulator of cytokine synthesis, and when activated as a transcription factor that increases the expression of various genes involved in immune or inflammatory reactions, it moves into the nucleus and increases the expression of inflammatory genes.

In the present invention, the expression of such NF-κB was reduced by administration of arazyme (see FIGS. 6 and 7), which means the reduction of tumor cells by arazyme.

PCNA does not exist in the G0 phase of the cell cycle, but begins to increase during the G1 phase, most synthesized in the S phase and decreased in the G2 phase, 36 kDa As a supplementary protein of DNA polymerase delta, it is known to be essential for cell DNA synthesis and cell proliferation (Miyagawa S, et. al ., J Invest Dematol , 93: 678-681, 1989). In general, tissue immunofluorescence of PCNA acts as a measure of how much of the tumor tissue is involved in growth. PCNA expression has been associated with histopathological grades of keratinocyte proliferation, gliosis, pigmented skin lesions, and non-small cell carcinoma of the lung (Underhill C, J Cell). Sci , 103: 293-298, 1992).

It was confirmed that the expression of PCNA decreased by the administration of arazyme (see FIGS. 8 and 9), which means that the growth of tumor tissues was reduced.

When arazyme of the present invention was administered to nude mice transplanted with a human lung cancer cell line (A549), the weight gain and tumor volume were reduced, and the anticancer effect was shown. Weak infiltration was observed and immunohistochemically, the expression of MMP-9, NF-κB and PCNA was decreased.

The above results indicate that arazyme has potential for anticancer and cancer prevention efficacy. Toxicity experiments were performed by oral administration of arazyme to female Wistar rats. There were no abnormalities in general symptoms and gross pathological findings at concentrations of 0, 1250, 2500 and 5000 mg / kg. As described above, the 50% lethal dose (LD ₅₀ ) by the oral administration toxicity test is considered to be a safe substance of at least 5,000 mg / kg or more. Therefore, the arazyme of the present invention may be provided as a pharmaceutical composition for preventing and treating cancer.

In addition to the arazyme of the present invention, it may contain one or more active ingredients exhibiting the same or similar functions. For administration, it may be prepared further comprising one or more pharmaceutically acceptable carriers. Pharmaceutically acceptable carriers may be used in combination with saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components, if necessary And other conventional additives such as bacteriostatic agents can be added. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to formulate main formulations, powders, tablets, capsules, pills, granules or injections, such as aqueous solutions, suspensions, emulsions and the like. Furthermore, it may be preferably formulated according to each disease or component by a suitable method in the art or using a method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).

The pharmaceutical composition for preventing and treating cancer of the present invention may be administered parenterally (for example, intravenously, subcutaneously, intraperitoneally, or topically) or orally according to a desired method. The range varies depending on age, sex, health condition, diet, administration time, administration method, excretion rate and severity of disease. The daily dose of arazyme according to the present invention is 0.01 to 5000 mg / kg, preferably 0.01 to 10 mg / kg, and more preferably administered once to several times a day.

In addition, the present invention provides a health food for cancer prevention, containing the culture solution of aranicola proteoricicus or arazyme isolated therefrom as an active ingredient.

When using the culture solution of aranicola proteoricicus of the present invention or the arazyme separated from it as a food additive, the culture medium of aranicola proteoricicus or the arazyme as described above is used as it is or used with other food or food ingredients. It may be used and may be appropriately used according to a conventional method. Arazyme is obtained and used in the same manner as described above. The mixed amount of the active ingredient may be appropriately determined depending on the purpose of use (prevention, health or therapeutic treatment). In general, in the preparation of food or beverage, the culture solution of aranicola proteoricicus of the present invention or arazyme separated therefrom is added in an amount of 0.01 to 10 parts by weight, preferably 0.05 to 1 part by weight based on the raw materials. . However, in the case of long-term intake for health and hygiene or health control, the amount may be below the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety. .

There is no particular limitation on the kind of food. Examples of the food to which the substance can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, drinks, Alcoholic beverages and vitamin complexes, etc., includes all of the health food in the conventional sense.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates, etc. as additional components, as in the general beverage. The natural carbohydrates described above are glucose, monosaccharides such as fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin, cyclodextrin, sugar alcohols such as xylitol, sorbitol, and erythritol. As the sweetener, natural sweeteners such as tautin and stevia extract, synthetic sweeteners such as saccharin, aspartame, and the like can be used. The proportion of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 ml of the beverage composition of the present invention.

In addition to the above, Aranicola proteoricicus culture medium or arazyme isolated therefrom is a variety of nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH It can be added to regulators, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks and the like. In addition to the pulp for the production of natural fruit juices, fruit juice beverages and vegetable beverages can be added to the culture solution of aranicola proteoricicus or arazyme separated therefrom. These components can be used independently or in combination. The ratio of such additives is not critical, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the culture solution of aranicola proteoricicus of the present invention or arazyme separated therefrom.

The present invention also provides a pharmaceutical composition for preventing and inhibiting cancer metastasis containing arazyme as an active ingredient.

In addition, the present invention provides a cancer metastasis prevention health food containing the culture solution of aranicola proteoricicus or an arazyme isolated from it.

In addition, it provides an MMP-9 expression inhibitor containing arazyme as an active ingredient.

In addition, it provides an NF-κB expression inhibitor containing arazyme as an active ingredient.

In addition, it provides a PCNA expression inhibitor containing arazyme as an active ingredient.

Hereinafter, the present invention will be described in detail by Examples, Experimental Examples and Formulation Examples.

However, the following Examples, Experimental Examples, and Formulation Examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following Examples, Experimental Examples, and Formulation Examples.

< Example  1> Arazyme ( arazyme Manufacturing

In order to prepare arazyme (arazyme), the active ingredient of the present invention, Aranicola proteoricicus HY-3 strain (KCTC 0268BP) was cultured in a culture medium (bacto tryptone 0.5%, yeast extract 0.5%, sodium chloride 0.1%, 0.05% potassium chloride, 0.02% calcium chloride, 0.02% magnesium sulfate) was incubated at 22 ° C. for 18 hours. The culture solution was separated from the cells and the supernatant using 2 μm membrane filtration, and the separated supernatant was concentrated using membrane filtration of 10 kDa. Since the arazyme of the present invention basically has anion properties, the ion exchange resin and the ion exchange resin using DEAE-cellulose, which have been pretreated with 50 mM tris-hydrochloric acid buffer (pH 7.6), and 20 mM Purification was carried out using a gel filtration exchange resin using Sephadex G-75 pretreated with Tris-HCl buffer (pH 7.6). The purified enzyme solution was subjected to electrophoresis with 10% SDS-PAGE (Sodium dodecyl sulfate-polyacrylamide gel), and the band tendency was confirmed. As a result, the arazyme of the present invention was a monomer having no subunit and was approximately 51.5 kDa. It was confirmed that a band having a molecular weight of? However, Aranicola proteoritikkusu HY-3 strain (KCTC 0268BP) for the production of arazyme can be produced in a variety of industrial media, and the produced culture solution can also be isolated and purified by various methods. The arazyme of the present invention has a polypeptide sequence as set out in SEQ ID NO: 1 and a polynucleotide sequence as set out in SEQ ID NO: 2.

< Experimental Example  1> Test group  Composition and Administration

In the present invention, an average body weight of 15 g of five-week-old specific pathogen-free (SPF) nude mice (Orient, Korea) of CAnN.Cg-Foxn1nu / CrljBgi strain was used.

Mice were quarantined and purified for about 7 days in the animal room after acquisition, and only healthy animals were selected and used for testing by observing general symptoms during the acclimation period.

In the case of the mouse used in the present invention, the temperature of 22 ± 3 ℃, relative humidity 50 ± 10%, lighting time 12 hours (08:00 light ~ 20:00 light) in the animal room equipped with an automatic temperature and humidity control device is set and Breeding Environmental conditions were measured regularly (once every three months). As a result of environmental measurements, no fluctuations that are expected to affect the test were accepted. Less than 5 animals were housed in polycarbonate breeding boxes [240 W x 390 L x 175 H (mm)] during the entire test period. Individual identification was done using the skin marker method using oily magic pen and the individual identification card marking method by breeding box. Feed was freely ingested by irradiation (13.2 kGy) sterilization of experimental animal solid feed (PMI Nutrition International, 505 North 4th Street Richmond, in 47374, USA). Water was freely ingested by tapping the tap water purified through a carbon filter.

After a week of acclimatization, human Lung Adenocarcinoma cells, A549 cell line, were transplanted into 1x10 ⁷ cells subcutaneously in the left armpit of the mouse to induce cancer and at the same time in four groups (control, T1, After separation into T2, T3 and Table 1), arazyme was administered for 12 weeks at 0 mg / kg (control), 150 mg / kg (T1), 300 mg / kg (T2) and 600 mg / kg (T3). Oral administration with a zonde six times a week. Body weights and general symptoms were observed during the entire experimental period, and necropsy was performed 12 weeks later.

The composition of the experimental group is shown in Table 1 below.

Experimental  Configuration group Animal Xenograft (S.C.) Arazyme oral administration Control 1-10 A549 1 × 10 ⁷ cells per mouse 0 mg / kg T1 11-20 A549 1 × 10 ⁷ cells per mouse 150 mg / kg T2 21-30 A549 1 × 10 ⁷ cells per mouse 300 mg / kg T3 31-40 A549 1 × 10 ⁷ cells per mouse 600 mg / kg

< Experimental Example  2> Arazyme  Investigate the effects of dosing on weight change

As in Experiment 1, the human lung cancer cell line (A549) was transplanted into nude mice, and then body weight was measured by measuring weight once a week, and general symptoms of animals were observed.

The body weight gain of the arazyme group was increased over the entire experimental period (12 weeks), especially in the 300 mg / kg administration group (T2) and 600 mg / kg administration group (T3). And FIG. 1).

Mouse weight Mouse weight change (g) Week 10 control groups 10 T1 10 T2 10 T3 weight weight weight weight One 18.05 ± 0.95 18.15 ± 0.93 18.26 ± 1.08 18.96 ± 0.57 2 17.52 ± 1.63 18.62 ± 0.93 19.14 ± 1.07 19.03 ± 0.96 3 18.57 ± 1.02 18.63 ± 1.06 19.30 ± 1.24 19.11 ± 0.78 4 19.04 ± 1.29 19.03 ± 0.88 19.92 ± 1.15 19.54 ± 0.93 5 19.30 ± 1.17 19.78 ± 0.98 20.34 ± 1.23 20.33 ± 1.22 6 19.75 ± 1.27 20.08 ± 1.00 20.33 ± 1.21 20.25 ± 1.24 7 19.97 ± 1.58 19.72 ± 0.94 20.74 ± 1.15 20.67 ± 1.20 8 20.05 ± 1.49 20.42 ± 0.79 21.33 ± 1.18 21.00 ± 1.32 9 20.16 ± 1.53 21.00 ± 1.10 21.17 ± 2.17 21.27 ± 1.64 10 20.16 ± 1.50 20.92 ± 1.75 21.01 ± 2.65 21.66 ± 1.75 11 19.78 ± 1.56 19.91 ± 1.56 19.77 ± 2.42 20.58 ± 2.19 12 19.67 ± 1.58 19.74 ± 1.77 19.41 ± 2.42 20.27 ± 2.26

Control: arazyme 0 mg / kg orally,

T1: arazyme 150 mg / kg oral administration,

T2: oral administration of arazyme 300 mg / kg,

T3: oral administration of arazyme 600 mg / kg.

< Experimental Example  3> Arazyme  Effect of dosing on tumor growth

As in Experiment 1, human lung cancer cell line (A549) was transplanted to nude mice, and then tumor size was visually confirmed from the 5th week, and then each mouse was used once a week for 8 weeks using a caliper. The size of the tumor at was measured. The volume of the tumor was calculated as shown in Equation 1 by measuring the long and short diameters of the tumor.

<Equation 1>

Tumor volume = (long diameter × short diameter ² ) / 2

As a result, the tumor size was reduced and a statistical significance was observed in the arazyme administered 300 mg / kg (T2) and 600 mg / kg (T3) groups compared to the control group for the entire experimental period (FIG. 2).

Experimental Example 4 Histopathological Observation of Lung Cancer Tissues

Twelve weeks after transplanting human lung cancer cell lines to nude mice, anesthesia was sacrificed by ethyl ether, and tumors and parenchyma were collected. The tumor tissues were weighed and organs such as heart, liver, lung, pancreas, kidney and spleen were collected. The collected tumors and organs were fixed in 10% neutral formalin for 24 hours and then treated with an automatic tissue processor. After treatment with tissues, tumors and organs were cut to 4 μm thickness after embedding paraffin and subjected to hematoxylin-eosin staining (H & E staining).

As a result, the arazyme-administered group had a weaker tumor cell infiltration than the control group (FIG. 3).

Experimental Example 5 Immunohistochemical Observation of MMP-9

For immunohistochemical observation of MMP-9, paraffin embedded tissues fixed in formalin were cut to 4 μm thickness and deparaffinized, followed by 30 minutes in 3% H ₂ O ₂ to inhibit endogenous peroxidase activity. After treatment and washing with 0.01 M phosphate-buffer saline (PBS), and then treated with 0.01 M citrate buffer to increase the antigen expression of the tissues, and was subjected to high heat treatment with microwave (microwave). In order to inhibit the nonspecific reaction, 20 g / ml proteinase K was treated for 10 minutes at 37 ° C., and then washed with phosphate buffered saline and then blocked for 1 hour. Subsequently, the anti-rabbit primary antibody MMP-9 (1: 100, Santa Cruz Bio, USA) was treated overnight at 4 ° C., then washed three times with PBS buffer for 5 minutes, followed by biotin and Combined. After washing the primary antibody bound with biotin in buffer, attach HRP (Horse-Rradish Peroxidase) conjugated with streptavidin, and at 37 ° C with Histostantin-plus bulk kit (Zymed Laboratories Inc, USA). After sufficient reaction and washing for 3 minutes, the solution was developed with 3,3-diaminobenzidine tetrahydrochloried (DAB, Zymed Laboratories Inc, USA) solution, washed with distilled water, and then washed with Meyer's hematoxylin (Research genetics, USA). Control staining was carried out and observed with an optical microscope.

As a result, the expression pattern of MMP-9 was reduced in the arazyme administered group compared to the control group (Figs. 4 and 5).

Increased production of matrix metalloproteases (MMPs) is known to increase inflammation and invasion and metastasis in many cancers. MMPs are also mainly made up of MMP-9 and MMP-2. In particular, higher signaling mechanisms that regulate the expression of MMP-9 include growth factors, cytokines, and tumor promoters including MAPKs. Decreased expression of MMP-9 means that cancer invasion and metastasis are suppressed.

< Experimental Example  6> NF Immunohistochemical Observation of -κB

For immunohistochemical observation of NF-κB, In the same manner as in Experimental Example 5, the primary antibody was NF-κB (1: 200, Cell Signaling Technology Inc, USA), which is an anti-rabbit primary antibody instead of MMP-9.

As a result, the expression of NF-κB was reduced in the arazyme administered group compared to the control group (FIGS. 6 and 7).

 NF-κB is an induced transcription factor that is activated in response to various environmental factors. It is also another factor of drug refusal. It is an important factor in cytokine synthesis, and when activated as a transcription factor that increases the expression of various genes involved in immune or inflammatory reactions, it moves into the nucleus and increases the expression of inflammatory genes. Decreased expression of NF-κB means that cancer is inhibited.

Experimental Example 7 Immunohistochemical Observation of PCNA

For immunohistochemical observation of PCNA, The same procedure as in Experiment 5 was carried out, and the primary antibody was an anti-mouse primary antibody PCNA (1: 800, Santa Cruz Bio, USA) instead of MMP-9.

As a result, the expression pattern of PCNA was reduced in the arazyme administered group compared to the control group (Figs. 8 and 9).

PCNA is a complementary protein of the DNA polymerase delta. PCNA expression has been known to be associated with histopathological grades of keratinocyte proliferation, neurogliosis, pigmented skin lesions and lung non-small cell cancer. Tissue immunofluorescence of PCNA acts as a measure of how much of the tumor tissue is involved in growth. Reduction of the expression of PCNA means that the growth of tumor tissue is inhibited.

Experimental Example 8 Acute Toxicity Test of Arazyme

In order to determine the acute toxicity for arazyme of the present invention, the following experiment was performed.

Four rats of 9-week-old SPF female rats (Orient Co., Ltd., Seoul, Korea) of Wistar system were divided into four groups, with temperature 22 ± 3 ℃, humidity 55 ± 10%, and illumination. It was bred in an animal room at 12L / 12D conditions. After the rats were obtained, they were quarantined and purified for one week in the animal room. Up to five animals were housed in a polycarbonate breeding box [240 W X 390 L X 175 H (mm)] for the entire testing period. For individual identification, skin marker using oil-based magic pen and individual identification card marking method by breeding box were used. Solid animal feed (PMI Nutrition International, USA) was sterilized and water was freely ingested using tap water.

Arazyme of the above example was dissolved in sterile injectable water for injection at concentrations of 0, 1250, 2500 and 5000 mg / kg, prepared at a dose of 10 ml / kg, and orally administered to rats using a zonde for oral administration. It was. After a week-long purifying period, the rats were divided into four groups, administered to arazyme concentrations in 10-week-old rats, followed by general symptoms observation, and autopsies were performed 24 hours later to evaluate the toxicity of arazyme to the organs. .

Oral administration of arazyme to female rats was performed to observe the behavior, appearance and function of animals in order to accurately observe the general symptoms and to evaluate toxicity.

As a result, no abnormal symptoms were observed in the activity, gait, temperament and spasms of the animals after arazyme administration, and the appearance and breathing of the animals such as the skin condition, periphery, ears, genitals, limbs, tail, etc. No abnormal symptoms were observed in the observation of status, saliva secretion, feces, and vomiting. An autopsy was performed 24 hours after arazyme administration. In the gross autopsy findings, no abnormalities were observed in the long-term. Arazyme chisarang (LD ₅₀ ) is believed to be a high concentration of 5000 mg / kg or more. Microscopic observations showed no pathological abnormalities in parenchymal organs such as liver, heart, lung and pancreas.

Examples of preparations for the compositions of the present invention are illustrated below.

< Formulation example  1> Preparation of Pharmaceutical Formulations

<1-1> Powder  Produce

2 g of arazyme

1 g lactose

After mixing the above components, the airtight cloth was filled to prepare a powder.

<1-2> Preparation of Tablet

Arazyme 100 mg

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

<1-3> Preparation of Capsule

Arazyme 100 mg

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, the capsule was prepared by filling in gelatin capsules according to the conventional method for producing a capsule.

<1-4> Preparation of the ring

1 g of arazyme

Lactose 1.5 g

1 g of glycerin

Xylitol 0.5 g

After mixing the above components, it was prepared to be 4 g per ring in a conventional manner.

<1-5> Preparation of Granules

Arazyme 150 mg

Soy extract 50 mg

Glucose 200 mg

Starch 600 mg

After mixing the above components, 100 mg of 30% ethanol was added and dried at 60 ° C. to form granules, which were then filled into fabrics.

<1-6> Preparation of Injection Solution

Arazyme 10 μg / ml

Dilute hydrochloric acid BP until pH 7.6

Main sodium chloride BP up to 1 ml

Arazyme was dissolved in a suitable volume of main sodium chloride BP, the pH of the resulting solution was adjusted to pH 7.6 with dilute hydrochloric acid BP, and the volume was adjusted with a main volume of sodium chloride BP and thoroughly mixed. The solution was filled into a 5 ml Type I ampoule made of clear glass, encapsulated under an upper grid of air by dissolving the glass, and sterilized by autoclaving at 120 ° C. for at least 15 minutes to prepare an injection solution.

< Formulation example  2> Manufacture of food

The powders, tablets, capsules, pills, and granules of Formulation Example 1 may be applied to foods, and foods containing the culture solution of aranicola proteoritus or arazyme separated therefrom are prepared as follows. It was.

<2-1> Preparation of Flour Food

Foods for health promotion by adding 0.1 to 10.0 parts by weight of the culture solution of Aranicola proteoriticus or the arazyme separated therefrom to flour, and preparing breads, cakes, cookies, crackers and noodles using the mixture in a conventional manner. Was prepared.

<2-2> soup  And juicy ( gravies Manufacturing

0.1-1.0 parts by weight of the culture solution of Aranicola proteoriticus or arazyme isolated therefrom was added to soups and broth to prepare meat products for health promotion, soups of noodles and broth in a conventional manner.

<2-3> ground Ground beef  Produce

10 parts by weight of the culture solution of Aranicola proteoricicus or arazyme isolated therefrom was added to the ground beef to prepare ground beef for health promotion in a conventional manner.

<2-4> dairy products ( dairy products Manufacturing

0.1-1.0 parts by weight of the culture solution of Aranicola proteoricicus or arazyme isolated therefrom was added to milk, and various dairy products such as butter and ice cream were prepared in a conventional manner using the milk.

<2-5> Linear  Produce

Brown rice, barley, glutinous rice, and yulmu were alphad by a known method, and then dried and roasted to prepare a powder having a particle size of 60 mesh.

Black beans, black sesame seeds, and perilla were also steamed and dried by a known method, and then ground to a powder having a particle size of 60 mesh.

Araniacola proteoricicus medium or arazyme separated therefrom was decompressed and concentrated in a vacuum concentrator, dried by spraying and drying with a hot air dryer, and then pulverized with a particle size of 60 mesh to obtain a dry powder.

The grains, seeds and the culture solution of Aranicola proteoriticus prepared above or the dry powder of arazyme separated therefrom were combined in the following ratio to prepare a conventional method.

Cereals (30 parts by weight brown rice, 15 parts by weight brittle, 20 parts by weight of barley),

Seeds (7 parts by weight perilla, 8 parts by weight black beans, 7 parts by weight black sesame seeds),

Aranicola proteoricicus culture or dry powder of arazyme isolated therefrom (1 part by weight),

Ganoderma lucidum (0.5 parts by weight),

Foxglove (0.5 part by weight)

< Formulation example  3> Manufacture of beverage

A beverage comprising a culture solution of Aranicola proteoricicus or arazyme isolated therefrom was prepared as follows.

<3-1> Health drink  Produce

Substances such as liquid fructose (0.5 parts by weight), oligosaccharides (2 parts by weight), sugar (2 parts by weight), salt (0.5 parts by weight), water (75 parts by weight), and a culture medium of Aranicola proteoricicus or After the separate arazyme (0.5 parts by weight) homogeneously blended for instant sterilization, it was packaged in small packaging containers such as glass bottles and plastic bottles to prepare a healthy beverage.

<3-2> Vegetable juice  Produce

A culture solution of aranicola proteoricicus or 0.5 g of arazyme isolated therefrom was added to 1,000 ml of a vegetable juice such as tomato or carrot to prepare a vegetable juice for health promotion in a conventional manner.

<3-3> Fruit juice  Produce

A culture solution of Aranicola proteoricicus or 0.1 g of arazyme isolated therefrom was added to 1,000 ml of fruit juices such as apples or grapes to prepare fruit juice for health promotion in a conventional manner.

As described above, arazyme produced by Aranicola proteolyticus of the present invention increases the weight of nude mice transplanted with human lung cancer cell lines and reduces tumor size and invasion. Since it reduces the expression of MMP-9, NF-κB and PCNA, it can be usefully used as a pharmaceutical composition for preventing and treating lung cancer.

Attach sequence list electronic file  

Claims (13)

A pharmaceutical composition for preventing and treating cancer, containing arazyme as an active ingredient. The pharmaceutical composition for preventing and treating cancer of claim 1, wherein the arazyme is a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 1. The method of claim 1, wherein the arazyme is Aranicola proteolyticus ) pharmaceutical composition for cancer prevention and treatment, characterized in that isolated from the culture. The method of claim 3, wherein the Aranicola proteolyticus ( Aranicola proteolyticus ) is Aranicola proteolyticus ) HY-3 (Accession No .: KCTC 0268BP), characterized in that the pharmaceutical composition for preventing and treating cancer. The pharmaceutical composition for preventing and treating cancer according to claim 1, wherein the cancer is lung cancer. Cancer foods for cancer prevention containing aranico liquor or arazyme isolated from the culture medium of Aranicola proteoricicus. The method according to claim 6, wherein the Aranicola proteoritis ( Aranicola) proteolyticus ) is Aranicola proteolyticus ) HY-3 (Accession No .: KCTC 0268BP) is a health food for cancer prevention, characterized in that. The health food for cancer prevention according to claim 6, wherein the arazyme is a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 1. Cancer metastasis prevention and inhibition pharmaceutical composition containing arazyme as an active ingredient. Cancer metastasis prevention health food containing arazyme as an active ingredient. The pharmaceutical composition of claim 1, wherein the composition inhibits MMP-9 expression. The pharmaceutical composition of claim 1, wherein the composition inhibits NF-κB expression. 2. The pharmaceutical composition for preventing and treating cancer according to claim 1, which inhibits PCNA expression.

Images