KR100673565B1 - - - Medicament improving the anti-tumour action of interferon-gamma and anti-cancer composition comprising interferon-gamma and the medicament - Google Patents

Abstract

The present invention relates to an anti-cancer effect-improving agent of interferon-gamma and an anti-cancer composition comprising the anti-cancer effect-improving agent and interferon-gamma as an active ingredient, more specifically, phytohemagglutinin and interferon- which are lectins derived from kidney beans. It relates to a pharmaceutical composition for anticancer drugs containing gamma as an active ingredient.

Phytohemagglutinin according to the present invention is contained in interferon-gamma preparations, which are widely used in cancer treatment, etc., to effectively raise anticancer effects by interferon-gamma even at low doses without side effects. Combination products with a small amount of / 30 interferon-gamma plus a small amount of phytohemagglutinin have a greater anticancer effect than interferon-gamma alone, thus alleviating many side effects from excessive interferon-gamma administration. The treatment effect of cancer by interferon-gamma can be improved and a small amount of interferon-gamma administration can greatly ease the medical burden on the patient.

Anticancer adjuvant effective against lectin, phytohemagglutinin, interferon-gamma, interferon-gamma derived from kidney beans, pharmaceutical composition for anticancer agent

Description

Interferon-gamma improving anticancer action and anti-cancer composition containing the anticancer action improving agent and interferon-gamma as an active ingredient {Medicament improving the anti-tumour action of interferon-gamma, and anti-cancer composition comprising interferon-gamma and the medicament }

1 shows the concentration dependence (a and b) and time dependence (c and d) of phytohemagglutinin on the production of inducible nitric oxide synthase (iNOS) mediating tumor killing from macrophages. The effect is shown.

a and c: result of immunoblotting;

b and d: light intensity measurement analysis of the data

FIG. 2 shows the concentration dependent (a and b) and time dependent (c and d) effects of interferon-gamma on the production of inducible nitric oxide synthase (iNOS) from macrophages.

a and c: result of immunoblotting;

b and d: light intensity measurement analysis of the data

Figure 3 shows the concentration-dependent (a) and time-dependent (b) effect of phytohemagglutinin on the production and secretion of nitric oxide (NO), a tumor killing substance from macrophages.

4 shows the effect of concentration dependent (a) and time dependent (b) effects of interferon-gamma on induction of production and secretion of nitric oxide (NO) from macrophages.

Figure 5 shows the effect of phytohemagglutinin on the mRNA expression of inducible nitric oxide synthase (iNOS) gene from macrophages by interferon-gamma.

a: result of reverse transcriptase-polymerase chain reaction (RT-PCR);

b: Light density measurement analysis results of the data

Figure 6 shows the enhancing effect of phytohemagglutinin on the production of inducible nitric oxide synthase (iNOS) from macrophages by interferon-gamma.

a: result of immunoblotting;

b: Light density measurement analysis results of the data

Figure 7 shows the enhancing effect of phytohemagglutinin on the production of nitric oxide (NO) from macrophages by interferon-gamma.

FIG. 8 compares the potentiating effect of phytohemagglutinin on the production of nitrogen oxide (NO) from macrophages by interferon-gamma and the effect of high concentrations of interferon-gamma.

Figure 9 shows the enhancing effect of phytohemagglutinin on the killing of sarcoma-180 sarcoma-180 from macrophages by interferon-gamma.

Figure 10 shows the toxic effect of phytohemagglutinin on the splenocytes of the experimental animals in a graph of survival rate according to the concentration.

Figure 11 graphically shows the toxicity (GOT, GPT) of phytohemagglutinin to liver tissue of experimental animals.

Figure 12 graphically shows the toxicity (CRE, BUN) of phytohemagglutinin to kidney tissue of experimental animals.

Figure 13 shows the effect of phytohemagglutinin on the growth of immature experimental animals by weight change.

Figure 14 shows the effect of phytohemagglutinin on the growth of mature experimental animals by weight change.

The present invention relates to an anti-cancer effect improving agent of interferon-gamma and an anti-cancer composition comprising the anti-cancer effect improving agent and interferon-gamma as an active ingredient, and more particularly, phytohemagglutinin derived from kidney beans (phytohemagglutinin; PHA ") and interferon-gamma as an active ingredient, the present invention relates to a pharmaceutical composition for anticancer drugs.

Interferon-gamma is a type of cytokine, an immunomodulator, that clinically enhances the activity of lymphocytes and other immune cells (Wu L. et al., Science, 309 (5735). ): 774 ~ 7, July 29, 2000), is widely used to suppress acute inflammation such as hepatitis B and C, or to treat various cancers and bacteria or fungal diseases including multiple myeloma. . However, the therapeutic effect of interferon-gamma is not reported to be high yet. For example, it is known that the treatment efficiency is only 29% for chronic hepatitis C and about 20% for multiple myeloma (Japan Medical Information Center, 2003 Japanese Medicine Collection, Yakukyo Governor, 2002).

In particular, interferon-gamma causes numerous side effects such as fever, diarrhea and coma due to its short half-life (half life), which requires frequent or long-term oral or vascular injections at high doses. In view of the problems of interferon-gamma, many studies have been conducted on adjuvants that alleviate the side effects to enhance the therapeutic effect of interferon, but are still in the development stage with no results.

Immune enhancers have been first introduced by Roman in the concept (Roman G., Bull. Soc. Centr. Med. Vet. , 101, 277, 1925), and have shown cellular and humoral immunity to antigens. Refers to all substances that can be elevated (Rajesh K. Gupta et. Al., Vaccine , 11: 293, 1993). As such, the mechanism of action of an immune enhancer that raises various immune actions in vivo against antigens typically includes activation of antigen-providing cells (eg, macrophages, etc.) and specific activity effects on lymphocytes. (Waksman BH, Springer Semin, Immunopathol. , 2: 5, 1979; Bomford, R. , Clin.Exp . Immunol. , 39: 435, 1980).

Phytohemagglutinin (PHA), a lectin derived from kidney beans, is known as a mitogen of T lymphocytes, is involved in other immune responses, and has been reported to have an immune boosting effect (Gerlag DM et. al., J. Immunol. , 165 (3): 1652-8, August 1, 2000; Linderoth A. et al., J. Pediatr.Gastroenterol Nutr. , 41 (2): 195-203 , 2005 August).

Therefore, the present inventors have continued to develop an immune enhancer to enhance the efficacy of the existing immune activator, as a result, phytohemaggluti which is a type of lectin, a hemagglutinin derived from kidney beans, interferon-gamma, which is also used as an anticancer agent In combination with nin, it has been confirmed that the production of anti-tumor substances and the anti-cancer effect are greatly increased even at a concentration of 1/30 lower than that alone, without the side effects, and it contains active ingredients of phytohemagglutinin. The present invention was successfully completed by providing an anticancer adjuvant and a pharmaceutical composition for anticancer agent containing interferon-gamma.

 An object of the present invention is to provide an anticancer adjuvant comprising an active ingredient of phytohemagglutinin (PHA) and a pharmaceutical composition for an anticancer drug containing interferon-gamma.

In order to achieve the above object, the present invention provides a pharmaceutical composition for anticancer drugs containing phytohemagglutinin and interferon-gamma as active ingredients.

Phytohemagglutinin of the present invention may include phytohemagglutinin and / or derivatives thereof, which are hemagglutinin derived from kidney beans.

The interferon-gamma used in the present invention may include natural interferon-gamma, interferon-gamma and / or interferon-gamma derivatives having the activity of interferon-gamma produced by genetic recombination.

The present invention provides a pharmaceutical composition for anticancer drugs that increases the production of tumor killing substances containing the phytohemagglutinin and interferon-gamma as active ingredients.

The present invention also provides the use of the pharmaceutical composition for the treatment of tumors, including sarcoma cancer.

Hereinafter, the present invention will be described in detail.

The present invention provides an anticancer adjuvant that enhances the action of an immunoactive agent containing an active ingredient of phytohemagglutinin (PHA).

Phytohemagglutinin used in the present invention includes hematocrit phytohemagglutinin, which is a lectin derived from kidney beans, and / or phytohemagglutinin derivatives having an immune enhancing activity.

Preferably, the present invention provides an anticancer adjuvant containing phytohemagglutinin derived from kidney beans as an active ingredient. At this time, the phytohemagglutinin can be used by purchasing the product Sigma (USA).

 The present invention also provides an anticancer adjuvant effective for interferon-gamma containing an active ingredient of phytohemagglutinin to enhance the effects of anticancer and antitumor agents.

The interferon-gamma used as an anticancer agent in the present invention includes natural interferon-gamma, interferon-gamma and / or interferon-gamma derivatives having the activity of interferon-gamma produced by genetic recombination.

Specifically, in the present invention, phytohemagglutinin, a type of lectin derived from kidney beans, produces interferon-gamma on macrophages to produce tumor killing substances including nitric acid (hereinafter referred to as "NO"). It is confirmed to increase the anti-cancer effect by increasing the stability of the drug (see FIGS. 1 to 9). In addition, it was verified that the phytohemagglutinin did not exhibit cytotoxicity, hepatotoxicity and kidney toxicity, but also did not show any inhibitory activity on its growth in immature and mature experimental animals (see FIGS. 10 to 14). Therefore, the anticancer adjuvant of the present invention can be used as an active ingredient in an anticancer composition that is safe for humans and has no side effects and at the same time effectively raises the anticancer activity of interferon-gamma.

In addition, the present invention provides a pharmaceutical composition for an anticancer agent containing the anticancer adjuvant and the active ingredient of the anticancer agent and further comprising a pharmaceutically acceptable carrier, stabilizer and buffer.

In the present invention, the anticancer adjuvant may include phytohemagglutinin, which is a lectin derived from kidney beans, and / or phytohemagglutinin derivatives having an immune enhancing activity, and preferably, hemagglutinates derived from kidney beans. Phosphorus phytohemagglutinin is used.

In the present invention, as the anticancer agent, interferon-gamma and the like are used, wherein the interferon-gamma includes an interferon-gamma derivative having activity of natural interferon-gamma, genetically produced interferon-gamma and / or interferon-gamma. .

Preferably, the anticancer pharmaceutical composition of the present invention may include the anticancer adjuvant and the anticancer or antitumor agent in a weight ratio of 1,000: 1 to 100,000: 1.

The present invention also provides a use of the anticancer pharmaceutical composition as an antitumor agent.

The pharmaceutical composition for an anticancer agent of the present invention may be provided as a preparation such as tablets, capsules or infusions for oral administration.

In addition, the pharmaceutical composition for an anticancer agent of the present invention may be provided as a preparation such as a solution or suspension for intravenous or subcutaneous injection that can be parenterally administered.

In addition, the pharmaceutical composition for an anticancer agent of the present invention may be provided as a preparation such as a liquid droplet inhalation spray or powder for inhalation that can be administered intranasally.

In addition, the present invention may provide a method for searching for other anticancer or antitumor adjuvants, including phytohemagglutinin.

In addition, the present invention may provide a method for searching for an anticancer agent. Preferably, a method of searching for an anticancer agent or an antitumor agent containing phytohemagglutinin or a similar substance and interferon-gamma or a similar anticancer active substance may be provided.

Specifically, the present invention performs the following efficacy tests to show that phytohemagglutinin has anti-cancer synergistic effect in inducing interferon-gamma to produce oncolytic substances in macrophages. (1) Effects of phytohemagglutinin and interferon-gamma on the production of inducible nitric oxide synthase (hereinafter abbreviated as "iNOS") in macrophages, primary immune-responsive cells; (2) the effect of phytohemagglutinin and interferon-gamma on the production of nitric oxide (hereinafter referred to as "NO") in macrophages; (3) synergistic effect of phytohemagglutinin on the induction of iNOS gene expression by interferon-gamma; (4) synergistic effect of phytohemagglutinin on iNOS production induction by interferon-gamma; (5) synergistic effect of phytohemagglutinin on induction of NO production by interferon-gamma; (6) synergistic effect of phytohemagglutinin on antitumor action by interferon-gamma; (7) test for cytotoxicity of phytohemagglutinin; (8) histotoxicity tests to investigate the effects of phytohemagglutinin on liver and kidney, which are sensitive to organs; And (9) safety tests investigating the effects of phytohemagglutinin on changes in weight gain in immature or mature experimental animals.

The anticancer adjuvant and the anticancer composition of the present invention may be used independently or as a pharmaceutical additive together with a pharmaceutically acceptable carrier or excipient according to the intended use, or in any other form suitable for administration to humans. Carriers that may be included in the anticancer adjuvant and anticancer composition of the present invention may include extenders, high fiber additives, encapsulating agents, lipids, and the like, examples of which are well known in the art.

The anticancer adjuvant and the pharmaceutical composition for the anticancer agent of the present invention may be administered by varying the dose depending on the extent of disease progression, age, sex, physical condition, administration period, administration method, weight of the patient, meal, rate of discharge, and the like.

Preferably, the phytohemagglutinin in the pharmaceutical composition for anticancer drugs may be administered parenterally or orally in the range of 0.01 to 10 μg per kg of body weight and interferon-gamma in the range of 1 to 10,000 IU per kg of body weight. have.

Hereinafter, the present invention will be described in more detail with reference to Examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Examples of the present invention include lipopolysaccharides (lipopolysaccharides) which are endotoxins of bacteria against phytohemagglutinin (Sigma, USA) and interferon-gamma (R & D Systems, USA) which are main experimental reagents; The presence or absence of contamination was examined by the residual amount of the abbreviation "LPS". As a result, the residual amount of lipopolysaccharide was found to be 20 endotoxin units (EU) / ml. For reference, in the European countries, the standard allowable range of LPS residual amount for the test reagent is 350 EU / ml (Scheer, Arzenimittelforschung, 43 (7): 975 to 800, July 1993), and the phytohemaggle used in the present invention. Rutinin and interferon-gamma were found to have an endotoxin LPS residual of less than 1/18 of the residual flow tolerance compared to the baseline tolerance.

In the embodiment of the present invention, to investigate the anticancer synergistic effects of phytohemagglutinin and interferon-gamma, macrophage cells, which are primary immune response cells and representative antigen-providing cells, were used. To test the toxicity on growth, kidney and growth, experimental animals Balb / c mice (provided by Daejeon Chemical Research Institute, Korea) were used.

Example 1 Investigation of Efficacy of Phytohemagglutinin and Interferon-gamma on Induction of Tumor Killing Mediator iNOS Production in Macrophages

The present invention is to investigate the effects of PHA and interferon-gamma on iNOS production capacity, one of the tumor killing mediators from macrophages, a major cell in the primary immune response of the immune system, as described below (1) mouse abdominal cavity Isolation of macrophages from (2) macrophage stimulation (3) immunoblotting was performed to analyze iNOS production induction.

(1) Isolation of Macrophages from Mouse Abdominal cavity

Experimental animals bred to extract macrophages were used 6 _to 10 week old Balb / c mice, 4 days after the administration of thioglycollate medium (Gibco, USA) cultured for 2 months or more Mice were then sacrificed by cervical dislocation. Incomplete RPMI-1640 medium (produced by Gibco, USA) with 100 U / ml penicillin-streptomycin added to the abdominal cavity of the sacrificed mice was extracted and centrifuged into macrophages in the abdominal cavity. Aliquots were made at 10 ⁶ cells / well concentration.

After incubation in a CO ₂ incubator for about 1 hour, cells and other suspensions that did not adhere to the plate were washed twice with incomplete RPMI-1640 medium. Then inject complete RPMI-1640 medium with 1.5 mL / well of 100 U / mL penicillin-streptomycin and 10% fetal bovine serum and incubate CO ₂ incubator (100% humidity, 37 ° C.) for 3 days. , 5% CO ₂ ).

Purity of macrophages was measured by separating adherent cells, attaching them to a slide glass using Cytospin-III (500 rpm, 5 minutes), and staining them with Wright and Giemsa staining. Purity was above 95%.

(2) macrophage stimulation

The macrophages obtained from the mouse macrophage isolation process described above were plated at 3 x 10 ⁶ cells / well in 6 well plates (Falcon, USA) and completely supplemented with 100 U / ml penicillin-streptomycin and 10% fetal bovine serum. Cultured for 3 days using RPMI-1640 medium (produced by Gibco, USA). After the incubation, the culture medium was removed and macrophages were stimulated by concentration and time by addition of PHA and interferon-gamma under fresh incomplete RPMI-1640 medium conditions. In this case, a well-known lipopolysaccharide (10 µg / ml; manufactured by Sigma, USA) was used as a positive control group.

Immediately after incubation for stimulation, the cells were washed three times with cold PBS buffer, and the cells were lysed using a lysis buffer containing various protease inhibitors. After cell lysis, the lysis samples were examined for the amount of iNOS present in the cells by immunoblotting.

(3) iNOS analysis using immunoblotting

A brief description of the immunoblotting process follows. Separation gel and top gel of 0.1% SDS polyacrylamide electrophoresis phase used 16% and 3%, respectively, and the electrophoresis current was 55mA (Powersupply; Pharmacia, Sweden), temperature cooling system (Sweden LKB) Company) was run for about 4 _to 5 hours while maintaining 4 ℃. The electrophoretic system used here was Hoefer SE 600 (manufactured by Hoefer-Pharmacia, USA). Each sample was electrophoresed by loading the same total protein amount into the gel.

The electrophoretic gel was pre-wet a nitrocellulose membrane in transfer buffer (39 mM glycine, 2.9 g, 48 mM tris-base, 5.8 g, 0.037% SDS, 20% methanol; pH 8.3). , NC) put on the fiber pad (fiber pad) and assembled. The immunoblotting transfer cassette was then placed in the transfer chamber with the NC membrane in the positive charge direction and the gel in the negative charge direction. The current was supplied at 1 A, and the temperature was transferred for 1 hour and 30 minutes while maintaining a temperature of 4 DEG C using a cooling system.

The NC paper is about 200ml of TBS-T buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05% Tween-20) in blocking buffer (5% nonfat dry milk). Lock for 1 hour at Red-Rocker (manufactured by Hoefer, USA) to block non-specific binding of the antibody, and then once again for 15 minutes with TBS-T buffer for 10 minutes. After washing twice, 3 ml TBS-T buffer was dispensed into a siliconeized bottle, to which a rabbit anti-murine iNOS polyclonal primary antibody; 500 μg / 1.5 30 ml (1: 300 seats) was added to the solution, and the NC paper was placed in a silicone container at 12 ° C. in a 4 ° C hybridization incubator (Robins, USA) for 12 hours. Reacted for a while.

After incubation, the cells were washed in a hybridization incubator three times for 20 minutes with 70 ml TBS-T buffer at 12 rpm, followed by a secondary antibody, ie, goat anti-rabbit IgG horseradish. 30 μl of affinity isolated antibody (goat anti-rabbit IgG horseradish peroxidase conjugated affinity purified antibody; stock solution dissolved in 0.02 mg / 4 ml concentration in PBS buffer) using peroxidase binding in 3 ml TBS-T buffer (1: 20,000 dilution) was added and incubated for 3 hours in a hybridization incubator at 4 ° C, and washed 4 times with 70 ml TBS-T buffer for 4 minutes each. After washing, the NC paper was placed in a thin transparent vinyl, and 3 ml of 50% of ECL (peroxidase substrate) reaction solution 1 and 2 were dispensed into NC paper and incubated for 5 minutes. After that, the X-ray film was placed in a cassette containing NC in a dark room (10 W safety lamp (manufactured by Kodak, USA)) and exposed to light for 5 or 10 minutes or more, and then the X-ray film was developed in a developing box. solution) and reacted for about 3 minutes.

After the above process, the X-ray film was washed again in water for 2 minutes, and then reacted for 3 minutes in a fixing solution (fixing solution) and then washed for 2 minutes in water to analyze the band (band).

As a result, PHA started producing iNOS protein at 0 µg / ml concentration and showed the highest iNOS production at 300 µg / ml concentration (see FIGS. 1a and b). It started to show the highest iNOS production 8 hours after stimulation, after which production decreased (see FIGS. 1 c and d).

1 shows the concentration dependent (a, b) and time dependent (c, d) effects of phytohemagglutinin on the production of inducible nitric oxide synthase (iNOS) from macrophages. Panels a and c show immunoblotting. And panels b and d are the results of the light density measurement analysis of the data. In this case, the stimulation time was 8 hours in the concentration dependent effect test, and 300 µg / ml of phytohemagglutinin was used in the time dependent effect test. Endotoxin LPS (1 µg / ml) used here was used as a positive control.

 In the case of interferon-gamma, iNOS production began to appear at 1 ng / ml concentration, followed by peak iNOS production at 10 ng / ml concentration (see FIGS. 2 a and b), and iNOS production started 2 hours after stimulation. The highest iNOS production was seen at 8 hours later and thereafter decreased (see FIGS. 2 c and d).

Figure 2 shows the concentration dependent (a, b) and time dependent (c, d) effects of interferon-gamma on the production of inducible nitric oxide synthase (iNOS) from macrophages, with panels a and c of immunoblotting The panels b and d are the light concentration measurement analysis results of the data. At this time, the stimulation time was 8 hours in the concentration dependent effect test, and 10ng / ml of interferon-gamma was used in the time dependent effect test. LPS (1 μg / ml) used here was used as a positive control.

Therefore, as shown in FIGS. 1 and 2, it was found that PHA and interferon-gamma acted to produce a large amount of iNOS from macrophages, and the results showed that PHA and interferon-gamma were concentration-dependent and stimulation time-dependent in iNOS production. It indicates that there is an effect.

Example 2: Investigation of the efficacy of phytohemagglutinin and interferon-gamma on the induction of oncolytic NO production in macrophages

The present invention confirms that PHA and interferon-gamma produce a large amount of iNOS, one of antitumor mediators, from macrophages that are the major immune response to the immune system, leading to the production of antitumor NO. To determine whether or not they were: (1) macrophage stimulation (2) nitrogen oxide (NO) measurements were performed to analyze NO production as described below.

(1) macrophage stimulation

Macrophages obtained by the isolation of mouse macrophages described above were plated at a concentration of 3 x 10 ⁶ cells / well on a 6-well plate (product of Falcon, USA) to 100 U / ml penicillin-streptomycin and 10% fetal bovine. Serum was incubated for 3 days using complete RPMI-1640 medium (produced by Gibco, USA). After incubation, the culture medium was removed and the macrophages stimulated PHA and interferon-gamma by concentration and time, respectively, under fresh incomplete RPMI-1640 medium conditions. At this time, a well-known lipopolysaccharide (manufactured by Sigma, USA) was used as a positive control at a concentration of 10 µg / ml.

Immediately after the cultivation for stimulation, each cell culture was collected and centrifuged (1000 g, 4 ° C., 5 minutes) to collect the culture supernatant, and the amount of NO produced was measured as follows.

(2) Quantitative analysis of NO

Since NO produced from activated macrophages reacts with oxygen and is converted into nitrite, NO production was examined by measuring nitrite in culture. Nitrite is measured using a grease reagent (Green LCs et al., 1982, Anal Biochem., 126 (1): 131-138). The reaction was mixed with 100 μl culture supernatant and 100 μl grease reaction solution and incubated at room temperature for 10 minutes. After the incubation, the absorbance was measured at 540 nm using an ELISA reader. Reagent for was used sodium nitrite (Sigma, USA).

As a result, NO production was observed from macrophages in the range of 0 to 1000 μg / ml and 0 to 16 hours for PHA (see FIGS. 3A and 3B).

Figure 3 shows the concentration dependent (a) and time dependent (b) effects of phytohemagglutinin on the production of nitric oxide (NO) from macrophages. In the concentration dependent effect test, the stimulation time was 8 hours, and in the time dependent effect test, phytohemagglutinin 300 µg / ml was used. Endotoxin LPS was used as a positive control at a concentration of 1 ㎍ / ㎖.

 In the case of interferon-gamma, NO production began at 1 ng / ml, followed by peak NO production at 10 ng / ml (see FIG. 4A), and 3 hours after stimulation. It showed the highest NO production at time and then decreased (see FIG. 4B).

4 shows the concentration dependent (a) and time dependent (b) effects of interferon-gamma on NO production secretion from macrophages. In the concentration dependent effect test, the stimulation time was 8 hours, and in the time dependent effect test, the interferon-gamma concentration was 10 ng / ml. Endotoxin LPS was used as a positive control at a concentration of 1 ㎍ / ㎖.

The results showed that PHA had no effect on NO production, whereas interferon-gamma had a concentration-dependent and stimulation time-dependent effect on NO production from macrophages.

Example 3: Investigation of the synergistic effect of phytohemagglutinin on iNOS mRNA gene expression by interferon-gamma

The present invention confirms that PHA and interferon-gamma act to produce a large amount of iNOS, one of antitumor mediators, and NO, an antitumor, from macrophages, a major cell in the immune system. In order to examine whether the gene is due to mRNA expression, it was analyzed by (1) macrophage stimulation and isolation of total RNA (2) reverse transcriptase polymerase chain reaction (RT-PCR) as described below.

(1) Macrophage Stimulation and Total RNA Isolation

A brief description of the experimental process follows. Macrophages obtained according to the above-described mouse macrophage isolation process are plated at 3 x 10 ⁶ cells / well concentration in 6 well plates (Falcon, USA) and 100 U / ml penicillin-streptomycin and 10% fetal bovine serum It was incubated for 3 days using the complete RPMI-1640 medium (produced by Gibco, USA). After incubation, the culture medium was removed and macrophages were stimulated with PHA and interferon-gamma under appropriate medium conditions with incomplete RPMI-1640 medium (produced by Gibco, USA) with fresh 100 U / mL penicillin-streptomycin.

After the stimulation, the cells were washed twice with cold PBS buffer, PBS buffer was removed, and then 1 ml of Trizol was injected into each well and incubated at room temperature for 5 minutes. After incubation, the cell solution dissolved in Trizol was pipetted three times and then placed in an E-tube, followed by addition of 0.2 ml chloroform, vortexed for 10 seconds, mixed well, and centrifuged. Isolated (12,000 rpm / 60 min, 4 ° C.). Only the supernatant, the entire RNA layer, was floated and transferred to the E-tubes, placed in cold 100% isopropanol and allowed to settle for at least one day. After centrifugation for about 20 minutes under the same conditions, the supernatant was discarded, and this process was repeated to separate pure whole RNA. Then, add 75% ethanol, shake by hand, mix well, centrifuge, and after centrifugation, discard supernatant again, add 0.1% DEPC-H ₂ O, heat at 65 ℃ for 10 minutes, and use at -20 ℃. Stored until.

(2) Reverse Transcription Polymerase Chain Reaction (RT-PCR)

In order to perform reverse transcriptase-polymerase chain reaction, all the primers described below were ordered by Bioneer Co., Ltd. (Cheongwon, Chungbuk). Specifically, iNOS forward primer (5'-TGCCCCTTCAATGGTTGGTA-3 ') and reverse primer (iNOS reverse primer; 5'-ACTGGAGGGACCAGCCAAAT-3') and beta-actin forward primer (β-actin forward primer; 5'-CAAAGAAAATGGACGCCGCCGAACTTGG-3 ') and β-actin reverse primer (5'-CCTGCTTGCTTGCTGATCCACATCTGCTGG-3') were used.

To synthesize cDNA by polymerase chain reaction, 5 × reverse transcriptase buffer, 2.5 mM dNTP, 500 ng primer / μl, 200 U / μl MMLV reverse transcriptase (MMLV resverse trancriptiase; manufactured by Takara, Japan) The reaction mixture was prepared using secondary distilled water treated with, total RNA and 0.1% DEPC. The primer used at this time was a reverse primer. Then, 30 minutes of incubation at 42 ° C and 30 minutes of incubation at 75 ° C yielded cDNA. RT mixture with cDNA generated during Reverse transcription reaction, 10x Taq polymerase buffer, 2.5 mM dNTP, forward primer (25 pmol / μl), reverse primer (25 pmol / μl), Taq polymerase (2.5 units / μl; Takara Co., Ltd.) and secondary distilled water were mixed well, and cDNA was amplified by repeating the reaction conditions for 30 seconds at 95 ° C for 30 seconds at 55 ° C for 30 minutes at 1 minute 72 ° C. Then, cDNA was electrophoresed to confirm the difference in mRNA expression of the iNOS gene.

As a result, as shown in Figure 5, it was found that there is a synergistic effect of PHA on the expression ability of the iNOS gene by interferon-gamma. When interferon-gamma was stimulated together at a low concentration of 1 ng / ml at 1/30 of the highest concentration of 30 ng / ml and PHA at a low concentration of 10 μg / ml (1/30 of the highest concentration), It was confirmed that the mRNA expression effect of the iNOS gene, which is one of the anti-tumor mediators of macrophages, was stronger than or nearly equal to the highest concentration of 30 ng / ml (see FIGS. 5A and 5B).

5 shows the potentiating effect of phytohemagglutinin on the induction of mRNA expression of iNOS gene from macrophages by interferon-gamma, panel a is the result of reverse transcriptase-polymerase chain reaction (RT-PCR), panel b is a light density measurement analysis result of the data. At this time, the concentration of interferon-gamma (IFN) was 1 ng / ml, the stimulation time was 4 hours, the concentration of phytohemagglutinin (PHA) was 10 μg / ml, the stimulation time was 4 hours, and IFN + PHA was IFN 1. ng / ml and 10 µg / ml of PHA were administered together and the stimulation time was 4 hours. MA (medimu alone) was stimulated with saline instead of phytohemagglutinin and was used as a negative control. Endotoxin LPS was used as a positive control at a concentration of 1 μg / ml.

Therefore, interferon-gamma and PHA co-administration showed the same high efficacy as the interferon-gamma high concentration alone even at low interferon-gamma concentrations. This synergistic effect of PHA shows that the side effects of high concentrations of interferon-gamma can be reduced.

Example 4: Investigation of the synergistic effect of phytohemagglutinin on iNOS production induction by interferon-gamma

As described in Example 3, although there is a synergistic effect of PHA on the iNOS gene expression ability by interferon-gamma, to investigate whether the synergistic effect of PHA on iNOS gene expression ability leads to iNOS production capacity INOS analysis using (1) macrophage stimulation (2) immune blotting was performed as described.

(1) macrophage stimulation

Macrophages were isolated and cultured in mice by the procedure described in Example 1 above. After the incubation was completed, the culture medium was removed and the macrophages were treated with interferon-gamma (1 ng / ml), PHA with incomplete RPMI-1640 medium (produced by Gibco, USA) with fresh 100 U / ml penicillin-streptomycin. (10 μg / ml), Interferon-gamma (1 ng / ml) + PHA (10 μg / ml) was stimulated with an optimal stimulation time of 8 hours.

Immediately after incubation for stimulation, the cells were washed three times with cold PBS buffer, and the cells were lysed using a lysis buffer containing various protease inhibitors. After lysis, the lysis samples were measured by immunoblotting as follows to determine the amount of iNOS present in the cells.

(2) Analysis of iNOS using immunoblotting

In the same manner as described in Example 1, iNOS analysis using immunoblotting was performed to investigate whether there is a synergistic effect of PHA on iNOS production by interferon-gamma.

As a result, as shown in Figure 6, it was clearly demonstrated that there is a synergistic effect of PHA on iNOS production capacity by interferon-gamma. When interferon-gamma was stimulated with a low concentration of 10 μg / ml (1/30 of the highest concentration) of PHA at a low concentration of 1 ng / ml, 1/30 of the highest concentration (30 ng / ml), the highest concentration of interferon-gamma was 30. The effect of increasing iNOS production of macrophages was confirmed to be stronger or equal to ng / ml (see FIGS. 6A and 6B).

Figure 6 shows the potentiation effect of phytohemagglutinin in inducing iNOS production from macrophages by interferon-gamma, panel a is the result of immunoblotting, panel b is the light concentration measurement analysis of the data . Interferon-gamma (IFN) concentration was 1 ng / ml, stimulation time was 8 hours, PHA concentration was 10 μg / ml, stimulation time was 8 hours, IFN + PHA was IFN 1 ng / ml and PHA 10 ㎍ / ml Was administered together and the stimulation time was 8 hours. MA (medimu alone) was stimulated with saline instead of phytohemagglutinin and was used as a negative control. Endotoxin LPS was used as a positive control at a concentration of 1 μg / ml.

Therefore, as shown in the expression effect of the iNOS gene shown in Figure 5, when the interferon-gamma and PHA administered together showed the same high iNOS production efficacy as the high concentration of interferon-gamma alone even at a low concentration of interferon-gamma It was. The synergistic effect of iNOS production, one of the anti-tumor mediators of PHA, demonstrates that the side effects of high doses of interferon-gamma can be reduced.

Example 5: Investigation of the Synergistic Effect of Phytohemagglutinin on Induction of NO Production by Interferon-gamma

As described in Examples 3 and 4, the synergistic effect of PHA on the expression and production capacity of the iNOS gene by interferon-gamma was confirmed, but the synergistic effect of PHA on the expression and production capacity of this iNOS gene was NO. (1) Macrophage stimulation (2) NO analysis was performed as described below to investigate whether it leads to production capacity.

(1) macrophage stimulation

Macrophages were extracted from mice and cultured in the same manner as described in Example 1 above. After the incubation was completed, the culture medium was removed and macrophage cells were interferon-gamma (1 ng / ml) under suitable medium conditions with incomplete RPMI-1640 medium (produced by Gibco, USA) with fresh 100 U / ml penicillin-streptomycin. PHA (10 μg / ml), interferon-gamma (1 ng / ml) + PHA (10 μg / ml) was stimulated with an optimal stimulation time of 8 hours. As described in Example 2, each culture solution was taken immediately after the incubation for the stimulation reaction, and centrifuged (1000 g, 4 ° C., 5 minutes) to collect the culture supernatant, and the NO amount was measured as follows.

(2) Quantitative analysis of NO

The analysis was performed by the same procedure described in Example 2 above. That is, 100 μl culture supernatant and 100 μl Grease reagent were mixed together and incubated at room temperature for 10 minutes. After the incubation, the absorbance was measured at 540 nm using an ELISA reader. At this time, sodium nitrite (manufactured by Sigma, USA) was used as a reagent for the standard curve.

As a result, as shown in Figure 7 and 8, it was confirmed that there is a synergistic effect of PHA on the NO production capacity by interferon-gamma. Interferon-gamma peaked at a low concentration of 1 ng / ml, 1/30 of the highest concentration (30 ng / ml) and PHA at a low concentration of 10 µg / ml (1/30 of the highest concentration). The concentration of 3 to 30 ng / ml showed a stronger effect on NO production of macrophages than when administered alone (see FIGS. 7 and 8).

Figure 7 shows the potentiating effect of phytohemagglutinin on NO secretion from macrophages by interferon-gamma. The concentration of interferon-gamma (IFN) was 1 ng / ml, the stimulation time was 8 hours, the PHA concentration was 10 µg / ml, the stimulation time was 8 hours, and IFN + PHA was IFN 1 ng / ml and PHA 10 µg / ml. It was administered together and the stimulation time was 8 hours. MA (medimu alone) was stimulated with saline instead of phytohemagglutinin and was used as a negative control. Endotoxin LPS was used as a positive control at a concentration of 1 μg / ml.

FIG. 8 compares the effect of phytohemagglutinin enhancement and high concentration interferon-gamma on NO secretion from macrophages by interferon-gamma. The concentration of interferon-gamma (IFN) is 1-30 ng / ml, the stimulation time is 8 hours, the phytohemagglutinin (PHA) concentration is 10 μg / ml, the stimulation time is 8 hours, and IFN + PHA is IFN 1 ~ 30 ng / ㎖ and PHA 10 ㎍ / ㎖ was administered with the stimulation time was 8 hours.

Thus, as shown in the synergistic effects of expression and production of iNOS genes shown in FIGS. 5 and 6, the present results indicate that the administration of interferon-gamma and PHA together produces the same high NO production as the high concentration of interferon-gamma alone, even at low concentrations of interferon-gamma. Demonstrated efficacy. The synergistic effect for NO production, one of the antitumor substances of PHA, demonstrates that the side effects of high concentrations of interferon-gamma can be reduced.

Example 6: Investigation of the Synergistic Effect of Phytohemagglutinin on Antitumor Activity by Interferon-gamma

As shown in Examples 3, 4 and 5, the synergistic effect of PHA on the iNOS gene expression and production ability and NO production ability by interferon-gamma was confirmed, but the production of such an anti-tumor substance NO To investigate whether the synergistic effect of PHA on neutrophils also leads to antitumor activity, (1) co-culture and stimulation of ⁵¹ Cr-labeled Sarcoma-180 tumor cells and macrophages, as described below, (2) ⁵¹ Cr secretion measurements were performed and analyzed.

(1) Simultaneous Culture and Stimulation of ⁵¹ Cr-labeled Sarcoma-180 Tumor Cells and Macrophages

Macrophages were isolated and cultured in mice by the procedure described in Example 1 above. After incubation, the culture medium was removed and macrophages were incubated for 2 hours prior to the experiment under suitable medium conditions with incomplete RPMI-1640 medium (produced by Gibco, USA) with fresh 100 U / ml penicillin-streptomycin. At the same time, ⁵¹ Cr (manufactured by Amersham, USA) was added to Sarcoma-180 tumor cells at a concentration of 500 μCi / 5 × 10 ⁶ cells, and then cultured for 1 hour, and washed three times with PBS buffer. Add 100 μl of ⁵¹ Cr-labeled Sarcoma-180 cells to macrophages and add interferon-gamma (1 ng / ml), PHA (10 μg / ml), interferon-gamma (1 ng / ml) + PHA (10 μg / Ml) was added to each other to stimulate the optimal stimulation time of 8 hours.

Immediately after the cultivation for stimulation, each culture was collected and centrifuged (1000 g, 4 ° C., 5 minutes) to collect the culture supernatant, and the amount of ⁵¹ Cr secreted was measured as follows.

(2) ⁵¹ Cr secretion measurement

500 μl of each culture supernatant was collected and measured for cpm using a gamma spectrophotometer (manufactured by Pharmacia, USA), followed by% cytotoxicity according to the following formula. Toxicity (%) = 100 x (experimental cpm-natural cpm) / (total cpm-natural cpm). Total cpm is the amount of isotope obtained after lysing ⁵¹ Cr labeled Sarcoma-180 cells with 100 μl DMSO.

As a result, as shown in Figure 9, it was confirmed that there is a synergistic effect of PHA on sarcoma-180 killing ability of sarcoma cancer cells by interferon-gamma. The highest concentration of interferon-gamma when stimulated with interferon-gamma at a low concentration of 10 μg / ml (1/30 of the highest concentration) of PHA with a low concentration of 1 ng / ml, 1/30 of the highest concentration (30 ng / ml) The antisarcoma cancer effect was shown to be stronger or equal to 30 ng / ml of phosphorus (see FIG. 9).

9 shows the potentiating effect of phytohemagglutinin on sarcoma-180 killing of sarcoma cancer from macrophages by interferon-gamma. The concentration of interferon-gamma (IFN) was 1 ng / ml, the stimulation time was 8 hours, the concentration of phytohemagglutinin (PHA) was 10 μg / ml, the stimulation time was 8 hours, and IFN + PHA was IFN 1 ng / ml. ㎖ and PHA 10 ㎍ / ㎖ was administered together and the stimulation time was 8 hours. MA (medimu alone) was stimulated with saline instead of phytohemagglutinin and was used as a negative control. NMMA is a NO production inhibitor.

Therefore, the administration of interferon-gamma and PHA together showed the same killing ability of Sarcoma-180 sarcoma-180 sarcoma cancer cells even at low concentrations of interferon-gamma. The synergistic effect of this anti-tumor on PHA is to reaffirm the side effects of high doses of interferon-gamma. Finally, since the tumor killing effect is reduced to NMMA, a NO production inhibitor, the synergistic effect of the present invention becomes more effective since NO is closely related to tumor killing.

Example 7 Example 5 Test for Cytotoxicity of Phytohemagglutinin

The present invention, even if phytohemagglutinin, an adjuvant adjuvant of interferon-gamma, which is an immune activator, is excellent in immune activity and toxic in vivo, it cannot be used pharmaceutically, as follows: (1) splenocyte extraction and culture; And (2) MTT test for chemical cytotoxicity assay to investigate the safety against cytotoxicity.

(1) Splenocyte Extraction and Culture

To investigate the cytotoxicity of phytohemagglutinin of the present invention on normal cells, mouse spleen cells were used. Specifically, 7-week-old Balb / c mice (Daejeon Chemical Research Institute) were sacrificed by cervical spinal bone, the spleen was recovered aseptically, and cold incomplete RPMI-1640 medium containing only 100 U / ml of penicillin-streptomycin was added. Washed twice with Gibco Co., Ltd., USA, and injected with cold incomplete RPMI-1640 medium. Each cell was made using a Daunce cell homogenizer (manufactured by Wheaton, USA) and incubated in ice water for 10 minutes.

After incubation, only the cell supernatant was collected, centrifuged (1000 g, 5 min, 4 ° C.), and the supernatant was again poured out to obtain cells. Splenocytes were dispensed in 96-well plates at a concentration of 1.5 × 10 ⁵ cells / well and cultured in complete RPMI-1640 medium (produced by Gibco, USA) with 100 U / ml penicillin-streptomycin and 10% fetal calf serum. It was. Then, phytohemagglutinin was added to each well by concentration, and cultured for 2 days to perform MTT analysis.

(2) MTT assay for chemical cytotoxicity test

In the present invention, the cytotoxicity of phytohemagglutinin to splenocytes was verified using the MTT method. MTT analysis was performed according to the method of Francois D and Lita L., J. Immunological methods, 89, 271-277, 1986. When the culturing of splenocytes was completed, 50 μl of MTT reaction solution (concentration 1 mg / ml in HBSS buffer solution) was added to each well and incubated again at 37 ° C. for 4 hours. The culture supernatant was then removed and stirred for 15 minutes by adding 100 μl of dimethylsulfoxide (DMSO) to each well to dissolve the precipitated insoluble formazan. ELISA reader; USA Pharmacia) was used to measure the absorbance (OD) at 540 nm wavelength to determine the% cytotoxicity according to the following formula.

×100

× 100

As a result, as shown in Figure 10, phytohemagglutinin of the present invention did not have a toxic effect on the splenocytes even at a high concentration of 300 ㎍ / ㎖, rather showed a higher survival rate compared to the control (Fig. 10).

10 is a graph showing the viability of the phytohemagglutinin of the present invention on the spleen cells of the experimental animals, wherein the concentration of phytohemagglutinin is in the range of 10 ~ 300 ㎍ / ㎖ and the stimulation time is 2 days MA (medimu alone) was stimulated with saline instead of PHA and as a negative control, endotoxin LPS was used as a positive control at a concentration of 1 ㎍ / ㎖.

These results confirm that phytohemagglutinin has no cytotoxicity and is safe as an immune enhancing adjuvant for interferon-gamma.

Example 8 Long-term Toxicity Testing of Phytohemagglutinin

In the present invention, since the cytotoxicity test of phytohemagglutinin described in Example 7 alone cannot be assured of safety, the effects of phytohemagglutinin on liver and kidney, which are the main organs sensitive to metabolism and toxicity of living body, are as follows. Was examined. Kidney function is examined by measuring blood levels of blood creatin (CRE) and blood urea nitrogen (BUN), and liver function is measured by GOT (glutamic-oxalacetate transaminase) and GPT (glutamic pyruvic) transaminase) was tested by measuring blood levels of both enzymes.

Using 7-week old mice (Balb / c; Daejeon Chemical Research Institute, Korea), 10 ㎍ and 300 ㎍ of phytohemagglutinin were dissolved in 2 ml of 0.01M PBS buffer solution and administered 2 ml once daily to the vein. In the normal group, only 2 ml of PBS buffer solution was administered once daily, and the same procedure was performed. After 1, 2, 5, and 10 days of intravenous injection, 10 μl of serum obtained from the eye of the mouse was placed on a multilayer film, and the Fuji Dry Chem System manufactured by Fugi Photo Film of Japan. ), And measured CRE, BUN, GOT and GPT values, respectively.

As a result, as shown in Figs. 11 and 12, in the experimental group administered with 10 ㎍ of phytohemagglutinin, the blood concentration of GOT, GPT, CRE and BUN was almost the same as the normal group, and 300 ㎍ of phytohemagglutinin was administered. Was slightly higher than the normal group, but there was no significant difference (FIGS. 11 and 12). Therefore, it can be confirmed that phytohemagglutinin does not have harmful toxicity in both liver and kidney.

11 is a graph showing the hepatotoxic effect of phytohemagglutinin. 10 μg and 300 μg of phytohemagglutinin used were administered to mice daily, and blood toxicity was examined (GOT, GPT) on 5 and 10 days a day, and the control group (Con) was phytohemaggle. Stimulated with saline instead of rutinin.

12 is a graph showing the renal toxicity effect of phytohemagglutinin. 10 μg and 300 μg of phytohemagglutinin used were administered to mice daily, and blood samples were collected at 5 days and 10 days a day, and tested for toxicity to kidney tissues (CRE, BUN). Stimulated with saline instead of rutinin.

Example 9 Weight Change Test of Experimental Animals with Phytohemagglutinin

In order to confirm the safety of phytohemagglutinin of the present invention, the effect on body development of immature or mature rats was investigated by weight change. Phytohemagglutinin was dissolved in 0.01 M PBS buffer to concentrations of 10 μg / ml and 300 μg / ml and injected subcutaneously daily into immature mice at day 1 and intraperitoneally to mature mice at 7 weeks of age. The control group was injected subcutaneously or intraperitoneally with 0.01 M PBS buffer.

As a result, as shown in FIG. 13 (for immature mice) and 14 (for immature mice), the concentrations of 10 μg / ml and 300 μg / ml of phytohemagglutinin in both immature and mature mice There was no significant difference in comparison (FIGS. 13 and 14). Thus, it was found that phytohemagglutinin was safe without causing harmful toxicity to body development in experimental animals.

FIG. 13 and FIG. 14 show the effect of phytohemagglutinin of the present invention on the weight of each immature and mature mouse by weight change. At this time, 10 μg and 300 μg of PHA were administered daily to mice, followed by 0, 2, The mice are weighed on days 4, 6, 8 and 10.

As discussed above, the effect of phytohemagglutinin alone on the expression and production of the iNOS gene, one of the antitumor mediators, and on the production of the antitumor nitrogen oxides (NO) was not clear. However, when phytohemagglutinin and interferon-gamma are administered together, the expression and production of iNOS gene of interferon-gamma, NO production ability of antitumor substance, and sarcoma-180 killing ability of sarcoma cancer cells are elevated by PHA. there was. Thus, phytohemagglutinin exhibits the same sarcoma killing effect as that administered alone at a high concentration even when interferon-gamma is administered at low concentrations. Thus, the synergistic effect of phytohemagglutinin is caused by the high concentration of interferon-gamma. Proven that side effects can be reduced. In addition, animal experiments using mice confirmed that phytohemagglutinin was not toxic to cells, tissues, and body development.

As described above, the present invention relates to a pharmaceutical composition for anticancer drugs comprising phytohemagglutinin (PHA) and interferon-gamma as active ingredients, and phytohemagglutinin is used for interferon-gamma used in cancer treatment and the like. It can be effectively promoted anticancer effect even at low concentrations without side effects. In fact, when a small amount of phytohemagglutinin is used in combination with interferon-gamma, anti-cancer activity is significantly increased even with an interferon-gamma concentration of 1/30 than when interferon-gamma alone is used. Even a small amount of interferon-gamma can have the same effect as a large amount of interferon-gamma while eliminating existing side effects. Thus, phytohemagglutinin is used as a new anticancer and anti-tumor agent for effective interferon-gamma, which greatly improves the therapeutic effect of cancer and tumors, and at the same time, due to the small dose instead of the conventional high dose of interferon-gamma. It can relieve the patient of much medical burden.

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A pharmaceutical composition for an anticancer agent comprising phytohemagglutinin and interferon-gamma as active ingredients. delete delete delete

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