KR20240013352A - A pharmaceutical composition for preventing or treating cancer comprising deoxyshikonin or a pharmaceutically acceptable salt thereof as an active ingredient - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating cancer containing Deoxyshikonin, a low-molecular-weight compound derived from natural products, or a pharmaceutically acceptable salt thereof as an active ingredient, and more specifically, to a pharmaceutical composition for the prevention or treatment of cancer, including IL-2 and receptor. It was discovered by screening for low-molecular-weight compounds derived from natural products that inhibit IL-2Rα (CD25) binding, and the screened deoxycyconine inhibits the binding of IL-2 to its receptor, IL-2Rα (CD25), at a low concentration (IC ₅₀ = 12.269 μM), increases CD8 ⁺ T cells, which are immune cells involved in anti-cancer immunity, and decreases regulatory T cells that function to regulate immune responses, so it can be useful as an immuno-anticancer agent.

Description

A pharmaceutical composition for preventing or treating cancer comprising deoxyshikonin or a pharmaceutically acceptable salt thereof as an active ingredient}

The present invention relates to providing a pharmaceutical composition for preventing or treating cancer containing deoxyshikonin, a low-molecular-weight compound derived from natural products, or a pharmaceutically acceptable salt thereof, as an active ingredient.

Interleukin 2 (IL-2) was the first growth factor described for T-cells. Since the discovery of T-cell proliferation and survival in vitro (Smith, K A. (1988) Science. 240, 1169-76) and immune responses in the context of T viral infection (Blattman, J N, et al. (2003) Nat Med 9 , 540-7) or vaccines (Fishman, M., et al. (2008) J Immunother. 31, 72-80, Kudo-Saito, C., et al. (2007) Cancer Immunol Immunother. 56, 1897-910; Lin, C T., et al. (2007) Immunol Lett. 114, 86-93).

IL-2 acts on immune cells by binding to a receptor (IL-2R) consisting of IL-2Rα (CD25), IL-2Rβ (CD122), and IL-2Rγc (CD132), and has conflicting biological activities depending on the receptor it binds to. has This selective binding can enhance the powerful antitumor immune response of IL-2 by activating CD8+ T cells and NK cells that mainly express IL-2Rβγ, a dimeric receptor, while IL-2Rα (CD25) As it stimulates regulatory T cells (Treg cells) that are expressed at high levels and have an immunosuppressive function, thereby weakening the anti-tumor efficacy, it is important to suppress the function of regulatory T cells by blocking the binding to IL-2Rα (CD25). It is emerging as an attractive approach in cancer treatment strategies.

Republic of Korea Patent Publication No. 10-2018-0027576 (published on March 14, 2018)

In order to solve the above problems, the present invention aims to provide a pharmaceutical composition for preventing or treating cancer containing deoxycyconine, a low-molecular-weight compound derived from natural products, or a pharmaceutically acceptable salt thereof, as an active ingredient.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating cancer containing deoxycyconine or a pharmaceutically acceptable salt thereof as an active ingredient.

Additionally, the present invention provides an anti-cancer immunological agent comprising deoxycyconine or a pharmaceutically acceptable salt thereof as an active ingredient.

According to the present invention, a pharmaceutical composition containing deoxycyconine or a pharmaceutically acceptable salt thereof as an active ingredient binds directly to IL-2 and selectively inhibits the binding of IL-2 to IL-2Rα (CD25). By doing so, it has the characteristic of showing effects such as increasing the number of CD8 ⁺ T cells and decreasing the number of regulatory T cells. In addition, since the deoxycyconine is a natural product, it can reduce side effects that frequently occur with chemical substances. Accordingly, the pharmaceutical composition is expected to be useful as an anti-cancer immunotherapy agent.

Figure 1 is a graph showing the results of screening low-molecular-weight compounds derived from natural products.
Figure 2 is a graph of enzyme-linked immunosorbent assay (ELISA) results to determine the inhibitory effect of IL-2:IL-2Rα (CD25) binding and the effect on IL-2:IL-2Rβ binding depending on the concentration of deoxycyconine.
Figure 3 is a graph showing the results of evaluating the direct binding of deoxycyconine to IL-2 using Surface Plasmon Resonance (SPR) analysis.
Figure 4 is a graph evaluating the ability of deoxycyconine to inhibit proliferation of IL-2-dependent growth cell lines.
Figure 5 is a graph showing the results of evaluating the ability of deoxycyconine to inhibit the IL-2-mediated signaling pathway using a Western blot test.
Figure 6 is a graph analyzing the inhibition of IL-2 downstream signaling in immune cells by deoxycyconine.
Figure 7 is a graph evaluating the anticancer efficacy of deoxycyconine in a melanoma cancer animal model.
Figure 8 shows the results of evaluating the anticancer efficacy mechanism of deoxycyconine.
Figure 9 is a graph evaluating the pattern of immune cell changes caused by deoxycyconine in the tissues of melanoma cancer animals.

Hereinafter, the present invention will be described in more detail.

The present inventors worked diligently to find a compound useful as an anti-cancer immunotherapy agent. The compound binds directly to IL-2 and selectively inhibits the binding of IL-2 to IL-2Rα (CD25), thereby suppressing CD8 ⁺ T cells, which are immune cells. The present invention was completed by discovering deoxycyconine, a compound that increases and decreases regulatory T cells.

The present invention provides a pharmaceutical composition for preventing or treating cancer containing deoxycyconine or a pharmaceutically acceptable salt thereof as an active ingredient.

The deoxycyconine may have the structure of Formula 1 below.

[Formula 1]

Deoxycyconine may be isolated from a natural product, for example, Lithospermum erythrorhizon . The separation can be performed, for example, by extracting the borage with lower alcohol, suspending it in distilled water, and then adding an organic solvent such as n-hexane, ethyl acetate, and n-butanol to separate the borage fraction to produce a borage fraction. there is. Afterwards, the deoxycyconine can be separated using adsorption chromatography, for example, silica gel column chromatography. The method of obtaining the deoxycyconine of the present specification is not limited, and it may be purchased and used commercially.

The deoxycyconine may be included in the composition as its pharmaceutically acceptable salt. The salt may be an acid addition salt or a base addition salt. The acid addition salt includes salts with inorganic acids or organic acids. Examples of the inorganic acid salts include salts with hydrochloric acid, hydrobromic acid, phosphoric acid, or sulfuric acid, and the organic acid salts include acetic acid, trifluoroacetic acid, propionic acid, maleic acid, fumaric acid, malic acid, citric acid, tartaric acid, and lactic acid. , benzoic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, or naphthalenedisulfonic acid, but is not limited thereto.

In the present invention, it is construed that deoxycyconine or a pharmaceutically acceptable salt may exist in any form selected from the group consisting of any crystalline form and amorphous form thereof, and hydrates, solvates, and co-crystals thereof.

In the present invention, “prevention” refers to any action that inhibits or delays the onset of cancer by administering the composition according to the present invention, and “treatment” refers to suppressing or delaying the worsening of cancer by administering the composition according to the present invention. It refers to all actions that heal and recover from cancer. Furthermore, the prevention and/or treatment of cancer includes not only the effect of suppressing the growth of cancer cells, but also the effect of suppressing the worsening of cancer through migration, invasion, metastasis, etc.

In addition, the pharmaceutical composition of the present invention may further include suitable carriers, excipients, or diluents commonly used in the preparation of pharmaceutical compositions in addition to the effective ingredients described above for administration.

Specifically, the pharmaceutical composition is formulated in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterile injection solutions according to conventional methods. can be used Carriers, excipients and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium Silicates, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The composition may be characterized by directly binding to IL-2 and selectively inhibiting the binding of IL-2 to IL-2Rα (CD25).

In the present invention, IL-2 represents a protein (15.4 kDa) of 133 amino acids that has no substantial sequence homology to any other factor.

In the present invention, IL-2Rα (CD25) exists as a high-affinity IL-2 receptor (high-affinity IL-2R) and a low-affinity IL-2 receptor (low-affinity IL-2R) depending on the affinity, and CD25 is a chain that does not exist in the low-affinity IL-2 receptor, but only exists in the high-affinity IL-2 receptor.

The composition may be characterized by binding to IL-2 and inhibiting the function of IL-2.

The composition may be characterized in that it does not bind to CD25 and IL-2Rβ.

The composition may be characterized by reducing phosphorylation of the IL-2-mediated signaling pathway, JAK-1, JAK-3, and STAT5.

The composition may be characterized by increasing the number of CD8 ⁺ T cells.

The composition may be characterized by reducing the number of regulatory T cells.

The above cancers include breast cancer, colon cancer, rectal cancer, brain tumor, lung cancer, liver cancer, skin cancer, esophagus cancer, testicular cancer, kidney cancer, stomach cancer, bladder cancer, ovarian cancer, bile duct cancer, gallbladder cancer, uterine cancer, cervical cancer, prostate cancer, head and neck cancer, pancreatic cancer, and squamous cancer. It may be characterized as being any one selected from the group consisting of epithelial cell carcinoma.

Additionally, the present invention provides an anti-cancer immunological agent comprising deoxycyconine or a pharmaceutically acceptable salt thereof as an active ingredient.

In the present invention, “immune anticancer agent” refers to a therapeutic agent that induces immune cells to selectively attack only cancer cells by injecting artificial immune proteins into the body to stimulate the immune system, unlike existing anticancer agents that attack the cancer itself. It can be said to be a drug that restores or strengthens the tumor recognition or destruction ability of the immune system in order to overcome the immunosuppression or immune evasion mechanism acquired by cancer cells. The immuno-anticancer drugs can be divided into those used in passive immunotherapy and those used in active immunotherapy. Passive immunotherapy includes immune checkpoint inhibitors, immune cell therapy, and therapeutic antibodies, while active immunotherapy includes cancer treatment vaccines and immunomodulators. modulating agents), etc., but are not limited thereto.

The immuno-anticancer agent may be administered in combination with an immune checkpoint inhibitor, and each component may be administered simultaneously or sequentially. In the case of sequential administration, the order may be changed.

In the present invention, an “immune checkpoint inhibitor” is a drug that targets cancer cells by activating immune cells by blocking the activity of a target immune checkpoint protein involved in suppressing immune cells.

At this time, the immune checkpoint inhibitor may be characterized as one or more selected from the group consisting of anti-PD-1 antibody, anti-CTLA4 antibody, and anti-PD-L1 antibody, but is not limited thereto.

The administration may be performed by methods known in the art. Administration may be administered directly to the subject by any means, such as, for example, intravenous, intramuscular, transdermal, mucosal, intranasal, intratracheal or subcutaneous administration. .

The subject may be a mammal, such as a human, cow, horse, pig, dog, sheep, goat, or cat.

The administration includes 0.1 mg to 1,000 mg of deoxycyconine, for example, 0.1 mg to 500 mg, 0.1 mg to 100 mg, 0.1 mg to 50 mg, 0.1 mg to 25 mg, or 0.1 mg to 1 kg of subject. This may be administering 5 mg.

Hereinafter, the present invention will be described in more detail through the following examples. However, the present invention is not limited to these examples.

<Example>

1. Statistical analysis

All data were analyzed using GraphPad Prism 8 software and analyzed as mean ± standard deviation (SD) or standard error of the mean (SEM). Statistical significance was analyzed using Dunnett's or Sidak's multiple comparisons test of T-test, one-way ANOVA, or two-way ANOVA, and statistical significance was judged to be present in the case of P <0.05. Statistical significance was indicated by *, **, and ***, meaning * P < 0.05, ** P < 0.01, and *** P < 0.001, respectively.

2. ELISA for screening small molecule compounds derived from natural products

An ELISA test was performed to find low-molecular-weight compounds derived from natural products that can inhibit the interaction between IL-2 and IL-2Rα (CD25). 20 ng of mouse recombinant protein IL-2 was coated on a 96 well plate for 16 hours at 4°C, washed with PBST (PBS containing 0.05% Tween-20), and incubated with PBSA (PBS containing 0.1% BSA) at room temperature. Blocking was performed by processing for 1 hour. After blocking, 20 μM of natural product compound and 40 ng of mouse recombinant protein IL-2Rα (CD25) were treated and reacted at room temperature for 2 hours, and IL-2Rα (CD25) was incubated using an anti-his-tag antibody and an HRP-conjugated antibody. IL-2Rα (CD25) bound to 2 was captured. After binding the antibody, color was developed by treatment with TMB solution containing the HRP substrate, the reaction was stopped with 2N hydrochloric acid solution, and the absorbance was measured at 450 nm with an EPOCH microplate spectrophotometer. The degree of inhibition of IL-2 and IL-2Rα (CD25) binding of the compounds was measured by calculating the percentage based on the control group (well to which no compound was added), and compounds showing binding inhibition of more than 33% were selected as candidates. The plate was washed three times with PBST between all processes including IL-2 coating, blocking, natural product compound and IL-2Rα (CD25) treatment, anti-his-tag antibody treatment, and HRP-conjugated antibody treatment.

Figure 1 is a graph showing the results of screening low-molecular-weight compounds derived from natural products. Referring to Figure 1, as a result of performing an ELISA test in three steps using a library containing over 500 types of IL-2 and natural compounds, it was found that IL-2:IL-2Rα (CD25) binding was inhibited by more than 50%. Deoxycyconine, which has been shown to be effective, was discovered as a candidate material.

<Experimental example>

1. ELISA using deoxycyconine

Deoxycyconine selected through screening was verified to inhibit binding to human recombinant proteins IL-2 and IL-2Rα (CD25) and mouse recombinant proteins IL-2 and IL-2Rα (CD25), and its effect on binding to IL-2Rβ was verified. ELISA was performed while treating with various concentrations of deoxycikonine to confirm that it was not given. First, a 96-well immunoplate was treated with 200 ng of human IL-2, coated at 4°C for 16 hours, washed with PBST, and blocked with PBSA for 1 hour at room temperature. After blocking, 0∼200 μM of deoxycyconine was treated for 2 hours at each concentration, and 20 ng of IL-2Rα (CD25) or IL-2Rβ was treated for 1 hour each. To confirm whether IL-2Rα (CD25) or IL-2Rβ bound to plate-coated IL-2, HRP-bound antibody was treated at room temperature for 1 hour, and TMB solution containing HRP substrate was added and color development reaction was performed. After this, the reaction was stopped with 2N HCl solution and the absorbance was measured at 450 nm with an EPOCH microplate spectrophotometer. The degree of inhibition of IL-2 and IL-2Rα (CD25) or IL-2 and IL-2Rβ binding of the compounds was measured by calculating the percentage based on the control group (well to which no compound was added). The plate was washed three times with PBST (PBS containing 0.1% BSA and 0.05% Tween-20) between all processes except developing color with TMB substrate and terminating the reaction with 2N HCl solution.

Figure 2 is a graph of ELISA results to determine the inhibitory effect of IL-2:IL-2Rα (CD25) binding and the effect on IL-2:IL-2Rβ binding depending on the concentration of deoxycyconine. Referring to Figures 2A and 2B, as the treatment concentration of deoxycikonine increased, the degree of inhibition of IL-2:IL-2Rα (CD25) binding increased (IC50 = 12.269 μM), and referring to Figure 2C, deoxycikonine It can be seen that it does not affect IL-2:IL-2Rβ (CD122) binding.

2. Surface Plasmon Resonance (SPR) experiment

To determine whether deoxycyconine binds to IL-2, IL-2Rα, or IL-2Rβ, SPR analysis was performed using Biacore T200 equipment. First, human IL-2 (2,060.7 RU), human IL-2Rα (1,051.6 RU), and human IL-2Rβ (813.7 RU) were each immobilized on a CM5 sensor chip, and then deoxycyconine was added to the chip on which each protein was immobilized. was injected and the binding and degree of binding were analyzed. For analysis, 10X PBS-P+ buffer and DMSO (dimethyl sulfoxide) were used. Deoxycyconine was diluted starting from a concentration of 200 μM and flowed at a rate of 15 μl/min for 200 seconds at a concentration of 0 to 200 μM, and then separation was performed with running buffer (PBS-P+ buffer) for 400 seconds. The results were analyzed using Biacore T200 Evaluation software.

Figure 3 is a graph showing the results of evaluating the direct binding of deoxycyconine to IL-2 using SPR analysis. Referring to Figures 3a and 3b, deoxycyconine was flowed at increasing concentrations to the CM5 chip on which IL-2 was immobilized, and as a result, it was confirmed that binding to IL-2 increased in a concentration-dependent manner. You can. In addition, referring to Figures 3c and 3d, in order to examine the binding to the receptors IL-2Rα (CD25) and IL-2Rβ (CD122), IL-2Rα (CD25) and IL-2Rβ (CD122) were immobilized, respectively. As a result of flowing deoxycyconine onto the CM5 chip and analyzing it, it was confirmed that deoxycyconine did not bind to IL-2Rα (CD25) and IL-2Rβ (CD122). Through this, it can be seen that deoxycyconine selectively binds to IL-2.

3. CTLL-2 cell proliferation

CTLL-2 cells are cells that grow dependently on IL-2 and express IL-2Rα, IL-2Rβ, and IL-2Rγ. To verify whether deoxycyconine inhibits the proliferation of CTLL-2 cells, CTLL-2 cells were seeded at ^1.0 Cultured for 5 hours in RPMI-1640 media containing % FBS and 1% P/S. 20 ng of murine recombinant protein IL-2 and 0.5 to 5 μM of deoxycyconine were added to CTLL-2 cells after nutrient starvation, respectively, and cultured for 48 hours. Afterwards, the CCK solution was added and cultured for about 4 hours. When color development was sufficiently achieved by the CCK solution, the degree of cell proliferation was measured by measuring the absorbance at 450 nm with an EPOCH microplate spectrophotometer.

Figure 4 is a graph evaluating the ability of deoxycyconine to inhibit proliferation of IL-2-dependent growth cell lines. Referring to Figure 4, it can be seen that CTLL-2 proliferation is inhibited as the concentration of deoxycikonine treated with cells increases. This is because deoxycikonine binds to IL-2 and interferes with the role of IL-2. This may be the result.

4. IL-2 mediated signaling

To determine whether deoxycikonine can inhibit IL-2-mediated signaling in the IL-2-dependent-growing cell line CTLL-2, IL-2 alone or IL-2 and deoxycikonine were added to CTLL-2. After processing, the protein was separated and Western blot was performed. After dispensing CTLL-2 cells at 1.5x10 ⁶ /well in a 12 well cell culture plate, complete RPMI-1640 media (containing 10% FBS and 1% P/S) without T-STIM for starvation. cultured in RPMI-1640 media) for 5 hours. After starvation, 20 ng of murine recombinant protein IL-2 and 0∼5 μM of deoxycyconine were added to CTLL-2 cells and incubated for 15 minutes. Afterwards, cells were collected, washed with PBS, and cells were washed with RIPA buffer. The protein was separated by lysis. The separated proteins were quantified using the BCA protein assay kit, and then the proteins were separated by size through SDS-PAGE and transferred to a PVDF membrane for IL-2-mediated signaling proteins (p-STAT5/STAT5, p-JAK1/JAK1, p -JAK3/JAK3) was analyzed using an antibody against.

Figure 5 is a graph showing the results of evaluating the ability of deoxycyconine to inhibit the IL-2-mediated signaling pathway by Western blot testing. Referring to Figure 5, as the treatment concentration of deoxycyconine increased, the phosphorylation of JAK-1, JAK-3, and STAT5 decreased, which was related to the IL-2:IL-2Rα (CD25) binding inhibitory ability of deoxycykonine. This result indicates that inhibition of intracellular signaling is proportionally increased, and this may be a result showing that CTLL-2 proliferation is suppressed.

5. Analysis of IL-2 signaling using mouse splenocytes

To determine whether IL-2-mediated signaling in immune cells present in the spleen of mice is affected by Deoxyshikonin, cells were isolated from the spleen and IL-2 and Deoxyshikonin ) was processed and then flow cytometry was performed. The spleen was isolated from a C57BL/6 mouse, the tissue was dissociated with a 70 μm cell strainer, and red blood cells were removed using RBC lysis buffer. Cells isolated from the spleen were cultured in a water bath at 37°C for 30 minutes, then treated with IL-2 alone or a mixture of IL-2 and Deoxyshikonin, and cultured in a water bath at 37°C for 90 minutes. As soon as the culture was over, the tube containing the cells was transferred to ice, and then the cells were fixed and permeabilized using the FoxP3 transcription factor staining buffer set, and then stained with anti-FoxP3-PE-antibody. Afterwards, additional fixation and permeabilization were performed using 4% PFA (paraformaldehyde) and 90% methanol, and then cells were stained with anti-pSTAT5-AlexaFluor647 antibody to evaluate the degree of phosphorylation of STAT5, an IL-2-mediated signaling pathway in cells. Staining was performed with anti-CD3-FITC, anti-CD4-PE-Cy7, anti-CD8-PE, and anti-NK1.1-PE to gating regulatory T cells, CD8 ⁺ T cells, and NK cells. . The stained cells were analyzed using a flow cytometer, and the mean fluorescence intensity (MFI) was analyzed for the degree of STAT5 phosphorylation in each cell type.

Figure 6 is a graph analyzing the inhibition of IL-2 downstream signaling in immune cells by deoxycyconine. Referring to Figure 6a, cells isolated from the mouse spleen were treated with IL-2 or a mixture of IL-2 and deoxycyconine, and after incubation for 90 minutes, the degree of STAT5 phosphorylation was measured in regulatory T cells, CD8 ⁺ T cells, and NK cells. It is analyzed using a flow cytometer. Referring to Figure 6b, when regulatory T cells were treated with deoxycyconine at 1 μM and 5 μM, STAT5 phosphorylation was significantly decreased, and Figure 6c, in CD8 ⁺ T cells, deoxycykonine was significantly reduced. When treated with 5 μM, STAT5 phosphorylation significantly increased, and no changes were observed in NK cells. This shows that deoxycyconine is involved in IL-2-mediated signaling of regulatory T cells and CD8 ⁺ T cells, which are immune cells that play a role in anticancer activity.

6. Animal testing

All animal experiments were conducted in compliance with the experimental methods and research ethics approved by the Catholic University of Korea Animal Experiment Ethics Committee (IACUC) (permit numbers: CUK-IACUC-2019-044, CUK-IACUC-2019-044-01). 6-week-old male C57BL/6 mice and BALB/C nude mice were used and purchased from Orient Bio. In the breeding environment, light and dark were controlled in a 12-hour cycle, temperature (22 ± 3°C) and humidity (40-60%) were maintained, and food and water were freely available. After animal testing, euthanasia was performed by injecting CO ₂ gas at 30-70% chamber volume/min.

7. Melanoma mouse model

To create a melanoma cancer model, 2.0x10 ⁵ B16F10 cell lines were injected subcutaneously into the right flank of C57BL/6 or BALB/C nude mice. Deoxycikonine was used dissolved in 10% DMSO, and from the time the cancer began to be felt, 0.5 mg/kg or 1 mg/kg of deoxycikonine or 10% DMSO as a solvent was administered intraperitoneally every two days. The size of the cancer was measured with a vernier caliper. Dissection was performed before the cancer volume reached approximately 2,000 mm ³ and all experiments were completed, and the experimental animals were euthanized using CO ₂ gas.

Figure 7 is a graph evaluating the anticancer efficacy of deoxycyconine in a melanoma cancer animal model. Referring to Figure 7a, it can be observed that tumor growth was significantly inhibited in both groups administered 0.5 mg/kg and 1 mg/kg of deoxycyconine to a level similar to that of S4B6 used as the positive control group. Referring to Figure 7b, The results of measuring the actual tumor weight after dissection also showed that tumor growth was significantly suppressed in both groups administered deoxycikonine. Additionally, referring to Figure 7c, there was no significant difference in mouse body weight between the control group (vehicle) and the drug administration group, indicating that there was no drug toxicity.

Figure 8 shows the results of evaluating the anticancer efficacy mechanism of deoxycyconine. Referring to Figure 8a, there was no difference in tumor volume between the group administered deoxycyconine at a concentration of 1 mg/kg and the control group not administered the drug, and referring to Figure 8b, the actual volume measured after dissection. There was no difference in the size and weight of the tumor between the deoxycikonine administration group and the control group. Additionally, referring to Figure 8c, there was no significant difference in mouse body weight between the control group and the drug administration group, indicating that there was no drug toxicity.

8. Tissue analysis of melanoma mouse model

To determine changes in immune cells caused by deoxycyconine in the tissues of mice injected with melanoma cancer, lymph nodes were obtained from the mice and analyzed. Lymph node cells were obtained by grinding the tissue in a 70 μm cell strainer, and the cells were fixed and permeabilized using FoxP3 transcription factor staining buffer set, anti-CD3-FITC, anti-CD4-PE-Cy7, anti -Stained with CD8-PE, anti-NK1.1-PE, and anti-FoxP3-PE antibodies. The stained cells were analyzed using flow cytometry.

Figure 9 is a graph evaluating the pattern of immune cell changes caused by deoxycyconine in the tissues of melanoma cancer animals. Referring to Figure 9a, as a result of analyzing the patterns of regulatory T cells and CD8 ⁺ T cells, the number of CD8 ⁺ T cells, which suppress cancer by stimulating the immune response, increased in the group administered 1 mg/kg of deoxycikonine (p =0.099), and referring to Figure 9b, it can be observed that regulatory T cells that interfere with anticancer activity decrease (p=0.095). Through this, when calculating the ratio of CD8 ⁺ T cells and regulatory T cells, which are indicators of the distribution of immune cells with anti-cancer activity, it can be confirmed that it increases in both the deoxycikonine 0.5 mg/kg and 1 mg/kg administration groups. It can be seen that the anti-cancer effect of deoxycyconine is the result of stimulation of the immune response by immune cells.

As the specific parts of the present invention have been described in detail above, it is clear to those skilled in the art that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. something to do. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (11)

A pharmaceutical composition for preventing or treating cancer containing Deoxyshikonin or a pharmaceutically acceptable salt thereof as an active ingredient. In claim 1,
The composition is characterized in that it binds directly to IL-2 and selectively inhibits the binding of IL-2 to IL-2Rα (CD25). In claim 1,
The composition is characterized in that it binds to IL-2 and inhibits the function of IL-2. In claim 1,
The composition is characterized in that it does not bind to IL-2Rα (CD25) and IL-2Rβ. In claim 1,
The composition is characterized in that it reduces phosphorylation of JAK-1, JAK-3, and STAT5, which are IL-2-mediated signaling pathways. In claim 1,
The composition is characterized in that it increases the number of CD8 ⁺ T cells. In claim 1,
The composition is characterized in that it reduces the number of regulatory T cells (Treg). In claim 1,
The above cancers include breast cancer, colon cancer, rectal cancer, brain tumor, lung cancer, liver cancer, skin cancer, esophagus cancer, testicular cancer, kidney cancer, stomach cancer, bladder cancer, ovarian cancer, bile duct cancer, gallbladder cancer, uterine cancer, cervical cancer, prostate cancer, head and neck cancer, pancreatic cancer, and squamous cancer. A composition, characterized in that it is any one selected from the group consisting of epithelial cell carcinoma. An immunotherapy agent containing Deoxyshikonin or a pharmaceutically acceptable salt thereof as an active ingredient. In claim 9,
The immuno-anti-cancer agent is characterized in that it is administered in combination with an immune checkpoint inhibitor. In claim 10,
The immune checkpoint inhibitor is an immuno-anticancer agent, characterized in that at least one selected from the group consisting of anti-PD-1 antibody, anti-CTLA4 antibody, and anti-PD-L1 antibody.