Combination regimen of mixed herbal extract and anticancer agent for preventing or treating cancer

Abstract

The present invention relates to: a pharmaceutical composition for preventing or treating cancer, the composition comprising a mixed extract of Astragalus membranaceus, Angelica gigas, and Trichosanthes kirilowii, and an anticancer agent; and a method for preventing or treating cancer by using the pharmaceutical composition.

Description

Combination therapy of mixed herbal extracts and anticancer agents for the prevention or treatment of cancer

[0001] The present invention relates to a pharmaceutical composition for preventing or treating cancer, comprising a mixed extract of Astragalus membranaceus, Angelica gigas, and Cnidium rhizome; and an anticancer agent, an anticancer adjuvant, and a method for preventing or treating cancer using the pharmaceutical composition.

[0002]

[0003] Anticancer drugs are a general term for chemotherapeutic agents used to treat malignant tumors, excluding surgery and radiation therapy. Most agents exhibit anticancer activity primarily by inhibiting nucleic acid synthesis. For example, compounds such as paclitaxel and docetaxel have been approved by the U.S. Food and Drug Administration and are currently in clinical use. Their effects stem from excessive stabilization of cellular microtubules, inhibiting normal cell division.

[0004] However, despite their effectiveness in treating cancer, most anticancer drugs have limitations, including drug resistance, cancer recurrence, and side effects. For example, high doses of anticancer drugs are known to induce additional levels of toxicity, while low or intermediate doses may not exhibit significant antitumor activity in patients.

[0005] For example, docetaxel (Taxol), a semisynthetic taxoid produced from the needles of the European yew tree (Taxus baccata), and its derivative, paclitaxel, are anticancer agents used to treat solid tumors or highly metastatic malignancies, including breast cancer, ovarian cancer, non-small cell lung cancer, and colon cancer, and are among the most important new and active chemotherapeutic agents on the market. However, despite being an effective anticancer agent, docetaxel has the limitations described above, which precludes its use as a monotherapy for cancer. Therefore, there is a need for the development of combination therapies that can exhibit synergistic anticancer effects when administered together with anticancer agents such as docetaxel.

[0006] In addition, the development of anti-PD1 therapy as a treatment strategy to overcome resistance is also a very important task along with combination therapy.

[0007]

[0008] Meanwhile, Astragalus membranaceus is a perennial herb of the legume family in the Rosaceae order of dicotyledons, distributed in Korea, Japan, Manchuria, northeastern China, and eastern Siberia. It is commonly cultivated as a medicinal herb, and in oriental medicine, it is collected in the fall, the outer layer and fine roots are removed, and it is dried in the sun. It is called astragalus membranaceus of herbal medicine. It is said to be more effective when used as is without peeling. It has the effects of strengthening, invigorating, diuretic, and reducing swelling, so it is prescribed for physical weakness, fatigue, lethargy, and night sweats.

[0009] Angelica gigas (Dang-Gwi) is the dried root of a perennial plant in the Apiaceae family. It has a sweet and spicy flavor and a warm nature. Angelica gigas's efficacy includes its blood-replenishing properties, which help generate blood when blood is deficient. It also promotes coronary blood flow and stimulates red blood cell production. Furthermore, it has been reported that an Angelica gigas extract containing decursin and / or decursinol angelate can be used as an anticancer composition (Korean Patent No. 10-1245328).

[0010] Trichosanthes kirilowii Maximowicz is the peeled root of the perennial hanultari or yellow hanultari, a member of the Cucurbitaceae family. It is odorless, bitter and sour in taste, and cool in nature. It is used to treat exhaustion, boils, and pus when body fluids are damaged by heat. It primarily reduces heat in the lungs and stomach, produces body fluids, quenches thirst, and moisturizes the body.

[0011] In this regard, the inventors of the present invention have confirmed the anticancer effects of a mixed extract of Astragali, Angelica, and Gastrodia elata (Korean Patent Publication No. 2014-0145087). However, the synergistic effects of combined treatment with existing anticancer drugs or radiation therapy using the mixed extract of Astragali, Angelica, and Gastrodia elata have not been elucidated, and research on this topic is lacking.

[0012] Furthermore, in the development of therapeutic strategies to overcome resistance to anti-PD1 therapy, there is no research on a therapeutic strategy to overcome resistance to anti-PD1 therapy using a combination therapy using a mixed extract of Astragali, Angelica, and Cnidium officinalis.

[0013]

[0014] The present inventors have made diligent efforts to develop a novel anticancer agent that exhibits a synergistic effect in combination therapy with existing anticancer drugs or anticancer therapies such as radiation therapy, and as a result, have confirmed that by combining the herbal extracts of Astragali, Angelica, and Cnidium officinalis with existing anticancer drugs or anticancer therapies, an anticancer agent with remarkably excellent efficacy can be provided, thereby completing the present invention.

[0015] In addition, the combined effect of the mixed herbal extract SH003 of the present invention and docetaxel (DTX) was evaluated in an anti-PD1 resistant lung cancer model, confirming its potential as a second-line treatment, and it also exhibited an immunomodulatory effect in an immunosuppressed model, contributing to the enhancement of the immune response. In particular, the combined treatment using SH003 targets the EGFR / JAK / STAT3 signal pathway to reduce PD-L1 expression, which inhibits the mechanism related to immune evasion, confirming that the SH003-DTX combination therapy has a dual mechanism of action that overcomes resistance through immune system regulation as well as a direct anticancer effect. Therefore, the SH003-DTX combination therapy has completed the present invention by suggesting a new therapeutic possibility for lung cancer patients who do not respond to anti-PD1 therapy.

[0016]

[0017] One object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer containing a mixed extract of Astragalus membranaceus, Angelica gigas, and Cnidium rhizome, wherein the pharmaceutical composition is administered in combination with an anticancer agent or in combination with radiation treatment.

[0018] In the pharmaceutical composition for preventing or treating cancer of the present invention, in one specific example, the mixed extract may be a mixture of Astragalus membranaceus, Angelica gigas, and Cnidium officinalis extract extracted with at least one solvent selected from the group consisting of water, alcohols having 1 to 4 carbon atoms, and mixed solvents thereof.

[0019] In any one of the specific examples described above, the alcohol having 1 to 4 carbon atoms may be 20 to 40% (v / v) ethanol.

[0020] In the pharmaceutical composition for preventing or treating cancer of the present invention, as one specific example, the mixed extract of Astragali, Angelica gigas, and Cnidium officinalis may be mixed in a weight ratio of Astragali: Angelica gigas: Cnidium officinalis = 0.5 to 5:0.5 to 1:0.5 to 1.

[0021] In the pharmaceutical composition for preventing or treating cancer of the present invention, in one specific example, the cancer may include lung cancer, breast cancer, melanoma, ovarian cancer, brain cancer, head and neck cancer, pancreatic cancer, stomach cancer, or prostate cancer.

[0022] In any one of the above-described specific examples, the lung cancer may include non-small cell lung cancer, small cell lung cancer, or Anti-PD1 refractory lung cancer.

[0023] In any one of the specific examples described above, the non-small cell lung cancer may include EGFR mutant non-small cell lung cancer or EGFR wild-type non-small cell lung cancer (squamous cell lung cancer).

[0024] In any one of the specific examples described above, the small cell lung cancer may include EGFR wild-type small cell lung cancer.

[0025] In any one of the specific examples described above, the breast cancer may include HER2 negative breast cancer or HER2 positive breast cancer.

[0026] In any one of the specific examples described above, the head and neck cancer may include head and neck cancer and radiation-resistant cancer.

[0027] In any one of the above-described specific examples, the anticancer agent may include at least one selected from the group consisting of docetaxel (DTX), anti-PD1, gemcitabine, pembrolizumab, doxorubicin (Dox, Doxo), trastuzumab, dabrafenib, trametinib, paclitaxel, carboplatin, temozolomide, and FOLFIRINOX (5-FU, irinotecan, leucovorin, oxaliplatin).

[0028] In any one of the specific examples described above, the anticancer agent may include two types of anticancer agents.

[0029] In the pharmaceutical composition for preventing or treating cancer of the present invention, in one specific example, the composition may additionally comprise a pharmaceutically acceptable carrier, excipient or diluent.

[0030] In the pharmaceutical composition for preventing or treating cancer of the present invention, in any one specific example, the composition may be administered by intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, or rectal administration.

[0031] Another object of the present invention is to provide an anticancer adjuvant containing a mixed extract of Astragalus membranaceus, Angelica gigas, and Cnidium rhizome, characterized in that the adjuvant is administered in combination with an anticancer agent or in combination with radiation treatment.

[0032] Another object of the present invention is to provide a method for preventing or treating cancer, comprising administering the pharmaceutical composition to a subject.

[0033] Another object of the present invention is to provide a composition containing a mixed extract of Astragalus membranaceus, Angelica gigas, and Cnidium rhizome and an anticancer agent for the prevention or treatment of cancer.

[0034] Another object of the present invention is to provide a use of a mixed extract of Astragalus membranaceus, Angelica gigas, and Cnidium rhizome as an anticancer adjuvant.

[0035] Another object of the present invention is to provide a use for combined administration of a composition containing a mixed extract of Astragalus membranaceus, Angelica gigas, and Cnidium rhizome and an anticancer agent.

[0036] Another object of the present invention is to provide a combination therapeutic agent comprising a composition containing a mixed extract of Astragalus membranaceus, Angelica gigas, and Cnidium rhizome and an anticancer agent.

[0037] Another object of the present invention is to provide a method for preparing a composition comprising a mixed extract of Astragalus membranaceus, Angelica gigas, and Cnidium rhizome and an anticancer agent.

[0038]

[0039] The mixed extract of Astragalus membranaceus, Angelica gigas, and Cnidium rhizome of the present invention can be used as an excellent anticancer treatment when administered in combination with existing anticancer agents or with anticancer therapies such as radiation, demonstrating a synergistic anticancer effect. Furthermore, the SH003-DTX combination therapy may offer new therapeutic potential for lung cancer patients who do not respond to anti-PD1 therapy.

[0040]

[0041] Figure 1 shows the results of confirming the IC50 values ​​of each of SH003, a mixed extract of Astragali, Angelica, and Cnidium officinalis of the present invention, Docetaxel, and Gemcitabine in non-small cell lung cancer cell lines H1975 and HCC-827 with EGFR mutations.

[0042] Figure 2 shows the results of confirming cell survival rate according to combined treatment with SH003, a mixed extract of Astragali, Angelica, and Cnidium officinalis of the present invention, Docetaxel, and Gemcitabine in non-small cell lung cancer cell lines H1975 and HCC-827 with EGFR mutations.

[0043] Figure 3 shows the results of selecting the IC50 values ​​of SH003, Docetaxel, and anti-PD-1, each alone, and the optimal combined concentration in a combo of the two, in EGFR wild-type non-small cell lung cancer cell lines H460 and H358.

[0044] Figure 4 shows the results of confirming cell survival rate according to the combined treatment of SH003, a mixed extract of Astragali, Angelica, and Cnidium officinalis of the present invention, Docetaxel, and anti-PD-1 in EGFR wild-type non-small cell lung cancer cell lines H460 and H358.

[0045] Figure 5 shows the results of confirming the IC50 values ​​of each of SH003, a mixed extract of Astragali, Angelica, and Cnidium officinalis of the present invention, and Carboplatin and anti-PD-1 using the EGFR wild-type small cell lung cancer cell line H69, and confirming the optimal combined concentration of SH003 and carboplatin.

[0046] Figure 6 is a diagram confirming the results of combined treatment with SH003, a mixed extract of Astragali, Angelica, and Cnidium officinalis of the present invention, and Carboplatin and anti-PD-1 using EGFR wild-type small cell lung cancer cell line H69.

[0047] Figure 7 shows the results of confirming the anticancer efficacy of SH003, a mixed extract of Astragalus membranaceus, Angelica gigas, and Cnidium rhizome of the present invention, and anti-PD-1 combined treatment in a lung cancer tumor model.

[0048] FIG. 8 shows the results of combined treatment with a mixed extract of Astragali, Angelica, and Cnidium rhizome (SH003) of the present invention and DTX in inhibiting tumor growth and inducing apoptosis in an LLC1 allograft mouse model. (A) is the administration schedule of SH003 and DTX, (B, D) are the results of measuring tumor volume and body weight three times a week, (C) is the result of measuring the size of the tumor after sacrifice, (E) is the result of isolating the tumor into single cells, staining it with Annexin V / 7-AAD, and performing flow cytometry analysis, (F) is the result of formalin-fixed, paraffin-embedded (FFPE), and analyzing the tumor tissue under a microscope, and (G) is the result of quantifying the staining intensity as the average intensity compared to the number of DAPI-stained cells after staining the FFPE tumor tissue with granzyme B (GrB; red arrow) and perforin (white arrow), and using DAPI as a counterstain.

[0049] Figure 9 shows that the mixed extract of Astragali, Angelica, and Cnidium rhizome (SH003) of the present invention and DTX treatment activated the immune response through an increase in tumor-infiltrating immune cells. (A) is the result of flow cytometry analysis of CD8+ cells in the tumor, (B) is the result of immunohistochemical staining (IHC) analysis of tumor tissue, (C) is the result of staining NK1.1 (NK cell marker; red arrow) in FFPE tumor tissue, and quantifying the staining intensity as the average intensity per cell number after using DAPI as a counterstain, (D) and (E) are the results of homogenizing spleen tissue and measuring the cytokine level in the supernatant at 450 nm.

[0050] FIG. 10 is a diagram showing the results of confirming that the mixed extract (SH003) of Astragalus membranaceus, Angelica gigas, and Cnidium rhizome of the present invention restores immune function in CTX-induced immunosuppressed mice. (A) is a diagram showing the induction of an immunosuppressed mouse model and the SH003 administration schedule, (B) is the result of measuring and recording body weight changes at 2-4 day intervals, (C) is the result of calculating the spleen index indicating the spleen size relative to body weight, (D) is the result of measuring the subgroup of immune cells in the spleen by flow cytometry, and (E) is the result of collecting blood through cardiac puncture and performing hematological analysis through flow cytometry.

[0051] Figure 11 shows the results of the combined treatment of the mixed extract of Astragali, Angelica, and Cnidium rhizome (SH003) of the present invention and DTX synergistically inducing apoptosis in LLC1 cells. (A) LLC1 cells were treated with SH003 or DTX for 48 hours, and then cell viability was measured using an MTT assay. Statistical significance was confirmed using Student's t-test. (B) After 48 hours of combined treatment, the IC50 values ​​of each drug were calculated using Prism software, and the combined dose showing the synergistic effect was confirmed using CompuSyn software. (C) After 24 hours of combined treatment, apoptosis was evaluated using flow cytometry. (D) After 24 hours of combined treatment, the result of Western blot analysis was performed to confirm the expression of apoptosis-related proteins in cells.

[0052] Figure 12 shows the results of inducing apoptosis of LLC1 cells by inactivating the STAT3 / PD-L1 signaling pathway with a mixed extract of Astragali, Angelica, and Cnidium rhizome (SH003) of the present invention and DTX. (A) is the result of analyzing the correlation between STAT3 expression and activated CD8+ T cells in human lung cancer using the TISIDB database, (B) is the result of Western blot analysis performed on cells after 1 hour of combined treatment, (C) is the result of measuring PD-L1 expression in LLC1 cells by flow cytometry after 24 hours of combined treatment, (D-F) is the result of confirming apoptosis through flow cytometry after 24 hours of combined treatment in LLC1 cells transduced with p-CMV6-STTA3 vector. (G) is the result of analyzing STAT3 / PD-L1 signals and the apoptosis marker MCL-1 in tumor tissues by immunohistochemical staining (IHC).

[0053] Figure 13 shows the results showing the mechanism by which the combined treatment of the mixed extract of Astragali, Angelica, and Cnidium officinalis (SH003) of the present invention and DTX inhibits anti-PD1 refractory lung cancer. The combined treatment of SH003 and DTX increases tumor infiltration of cytotoxic immune cells in anti-PD1 refractory lung cancer, thereby enhancing the anticancer immune response. In addition, it induces apoptosis through granzyme B and perforin secreted by cytotoxic immune cells, and reduces intratumoral PD-L1 expression through inhibition of STAT3 signaling.

[0054] Figure 14 shows the results of analysis of T cell exhaustion and regulatory T cell populations between treatment groups, showing the expression levels of regulatory T cells (CD25+ / Foxp3+ / CD4+) and exhaustion markers (TIM-3, PD-1, LAG3) as the proportion of cells expressing each marker in CD8+ T cells.

[0055] Figure 15 shows the results of confirming the IC50 values ​​of the mixed extract of Astragali, Angelica, and Cnidium officinalis (SH003) of the present invention and doxorubicin and DTX alone in two HER2-negative breast cancer cell lines.

[0056] Figure 16 shows the results of confirming cell survival rate by combined treatment with the mixed extract of Astragalus membranaceus, Angelica gigas, and Cnidium rhizome (SH003) of the present invention, doxorubicin, and DTX in two HER2-negative breast cancer cell lines.

[0057] Figure 17 shows the results of confirming the IC50 values ​​of the mixed extract of Astragali, Angelica, and Cnidium officinalis (SH003) of the present invention and TSZ (Trastuzumab) and DTX alone in two HER2-positive breast cancer cell lines.

[0058] Figure 18 shows the results of confirming the cell survival rate by combined treatment with the mixed extract of Astragalus membranaceus, Angelica gigas, and Cnidium rhizome (SH003) of the present invention and TSZ and DTX in two HER2-positive breast cancer cell lines.

[0059] Figure 19 shows the results of confirming the cell viability and tumor formation inhibition ability in a melanoma tumor model by combined treatment of the mixed extract of Astragali, Angelica, and Cnidium officinalis (SH003) of the present invention, Dabrafenib, and Trametinib in a melanoma cell line.

[0060] Figure 20 shows the results of confirming the IC50 values ​​for the mixed extract of Astragali, Angelica, and Cnidium officinalis (SH003) of the present invention, Carboplatin, and Paclitaxel, respectively, in two ovarian cancer cell lines.

[0061] Figure 21 shows the results of confirming the cell survival rate according to the combined treatment of the mixed extract of Astragali, Angelica, and Cnidium officinalis (SH003) of the present invention, Carboplatin, and Paclitaxel in two ovarian cancer cell lines.

[0062] Figure 22 shows the results of confirming cell survival rate by combined treatment of the mixed extract of Astragali, Angelica, and Cnidium officinalis (SH003) of the present invention and Temozolomide in a brain cancer cell line.

[0063] Figure 23 shows the results of confirming the cell viability of the mixed extract of Astragali, Angelica gigas, and Gastrodia elata (SH003) of the present invention in various cell lines related to head and neck cancer, and the efficacy of the combined use of the mixed extract of Astragali, Angelica gigas, and Gastrodia elata (SH003) of the present invention and radiation on head and neck cancer and radiation-resistant cancer cell lines.

[0064] Figure 24 shows the results confirming that the combined use of the mixed extract of Astragali, Angelica, and Cnidium officinalis (SH003) of the present invention and radiation has a specific combined effect on radiation-resistant cancer cells against head and neck cancer and radiation-resistant cancer cell lines.

[0065] Figure 25 shows the results of confirming the individual IC50 values ​​of the mixed extract of Astragali, Angelica, and Cnidium officinalis (SH003) of the present invention and polfirinox in two pancreatic cancer cell lines.

[0066] Figure 26 shows the results of confirming the effect of combined treatment with the mixed extract of Astragali, Angelica, and Cnidium officinalis (SH003) of the present invention and polfirinox in two pancreatic cancer cell lines.

[0067] Figure 27 shows the results of measuring the Combination Index (CI) when the mixed extract of Astragali, Angelica, and Cnidium officinalis (SH003) of the present invention and docetaxel were administered alone or in combination in the prostate cancer cell line DU145.

[0068] Figure 28 shows the results of Western blot analysis to compare the efficacy of the mixed extract of Astragali, Angelica, and Cnidium officinalis (SH003) of the present invention and docetaxel on cell death when administered alone or in combination in the prostate cancer cell line DU145, in terms of identifying proteins related to cell death.

[0069] Figure 29 shows that when a mixed extract of Astragali, Angelica, and Gastrodia elata of the present invention (SH003) and docetaxel were administered together in a subcutaneous xenograft for 7 days, the tumor growth inhibition effect was relatively more effective than that of the single administration group (a), and the body weight of the mice was not changed, confirming the absence of toxicity (b), and the H&E staining method confirmed that cancer cell proliferation was reduced (c). In addition, the expression of Ki67, a cell proliferation indicator, was decreased, the expression of Cleaved caspase3, an apoptosis indicator, was increased, and the phosphorylation of EGFR was inhibited and the expression of STAT3 was also inhibited. Consequently, compared to the results of an in vitro study, these are the results confirming that apoptosis was induced by inhibiting EGFR-STAT3 signaling in the same manner (d to e).

[0070] Figure 30 shows the results of confirming cell survival rate when the mixed extract of Astragali, Angelica, and Cnidium officinalis (SH003) of the present invention and docetaxel were administered alone or in combination to the gastric cancer cell line AGS.

[0071] Figure 31 shows the results of confirming whether the mixed extract of Astragalus membranaceus, Angelica gigas, and Cnidium rhizome (SH003) of the present invention and docetaxel, when administered alone or in combination, induce cell death, which is one of the cancer cell inhibition mechanisms.

[0072]

[0073] This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in the present invention can also be applied to each other description and embodiment. In other words, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. Furthermore, the scope of the present invention should not be considered limited by the specific descriptions described below.

[0074]

[0075] One aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer containing a mixed extract of Astragalus membranaceus, Angelica gigas, and Cnidium rhizome, wherein the pharmaceutical composition is administered in combination with an anticancer agent or treated in combination with radiation.

[0076] The mixed extract of Astragalus membranaceus, Angelica gigas, and Cnidium rhizome of the present invention can be usefully used as a cancer treatment agent by co-administering with an existing anticancer agent or co-administering with an anticancer treatment such as radiation therapy, thereby exhibiting a synergistic effect in anticancer treatment.

[0077]

[0078] The term "Astragalus membranaceus" in the present invention refers to a plant with the scientific name "Astragalus membranaceus"; "Danggui" refers to a plant with the scientific name "Angelica gigas"; and "Cheonhwabun" refers to a plant with the scientific name "Trichosanthes kirilowiiMaximowicz".

[0079] In the present invention, the ethanol extracts of Astragalus membranaceus, Angelica gigas, and Cnidium rhizome are used interchangeably with “SH003” and have the same meaning.

[0080] In the present invention, the Astragalus membranaceus, Angelica gigas Nakai, or Gastrodia elata extracts may be purchased commercially or may be collected or cultivated in nature, but are not limited thereto. Furthermore, the Astragalus membranaceus, Angelica gigas Nakai, or Gastrodia elata extracts may be extracted from natural, hybrid, or mutant plants, or may be extracted from plant tissue cultures.

[0081] The term "extract" of the present invention includes the extract itself and all formulations that can be formed using the extract, such as an extract obtained by extracting a mixture of Astragalus membranaceus, Angelica gigas, and Cnidium officinalis, a diluted or concentrated extract, a dried product obtained by drying the extract, a conditioned or purified product of the extract, a fraction of the extract, or a mixture thereof.

[0082] In the pharmaceutical composition for preventing or treating cancer of the present invention, in one specific example, the mixed extract may be a mixture of Astragalus membranaceus, Angelica gigas, and Cnidium officinalis extract extracted with one or more solvents selected from the group consisting of water, alcohols having 1 to 4 carbon atoms, and mixed solvents thereof, specifically, ethanol extract, but is not limited thereto.

[0083] In any one of the specific examples described above, the alcohol having 1 to 4 carbon atoms may be, but is not limited to, 20 to 40% (v / v) ethanol, specifically 30% (v / v) ethanol.

[0084] In the mixed extract of Astragalus membranaceus, Angelica gigas Nakai, and Cnidium officinalis of the present invention, the method for extracting the mixture is not particularly limited, and extraction can be performed according to a method commonly used in the art. Non-limiting examples of the extraction method include hot water extraction, ultrasonic extraction, filtration, and reflux extraction, and these may be performed alone or in combination of two or more methods.

[0085] In addition, the above extract may be manufactured and used in the form of a dry powder after extraction, but this is not limited thereto.

[0086] In the pharmaceutical composition for preventing or treating cancer of the present invention, in one specific example, the mixed extract of Astragali, Angelica gigas, and Cnidium officinalis may be mixed in a weight ratio of Astragali: Angelica gigas: Cnidium officinalis = 0.5 to 5:0.5 to 1:0.5 to 1, specifically, a weight ratio of Astragali: Angelica gigas: Cnidium officinalis = 1 to 3:0.5 to 1:0.5 to 1, and even more specifically, a weight ratio of Astragali: Angelica gigas: Cnidium officinalis = 1:1:1, but is not limited thereto.

[0087]

[0088] The term "anticancer agent" of the present invention refers to a general term for known drugs used in conventional cancer treatment that act on various metabolic pathways of cancer cells and exhibit cytotoxicity or cytostatic effects on cancer cells, and includes all metabolic antagonists, plant alkaloids, topoisomerase inhibitors, alkylating agents, anticancer antibiotics, hormones, immunotherapy agents, and other drugs developed to date.

[0089] The anticancer agent that is included in the pharmaceutical composition according to the present invention and particularly shows a synergistic effect with the mixed extract of Astragali, Angelica, and Cnidium officinalis may include cisplatin such as cisplatin, carboplatin, and heptaplatin, gemcitabine, 5-fluorouracil, taxol such as docetaxel and its derivative paclitaxel, anti-PD1 immunotherapy such as pembrolizumab, and more specifically, docetaxel (DTX), anti-PD1, gemcitabine, pembrolizumab, doxorubicin (Dox, Doxo), trastuzumab, dabrafenib, trametinib, paclitaxel, carboplatin, It may be at least one selected from the group consisting of temozolomide and FOLFIRINOX (5-FU, irinotecan, leucovorin, oxaliplatin), and more specifically, it may include two selected from the group consisting of docetaxel (DTX), anti-PD1, gemcitabine, pembrolizumab, doxorubicin (Dox, Doxo), trastuzumab, dabrafenib, trametinib, paclitaxel, carboplatin, temozolomide, and FOLFIRINOX (5-FU, irinotecan, leucovorin, oxaliplatin).

[0090] These anticancer drugs can be manufactured by known methods or commercially available products can be used.

[0091] Docetaxel (Taxol), a semisynthetic taxoid produced from the needles of the European yew tree (Taxus baccata), and its derivative, paclitaxel, are anticancer drugs used to treat solid tumors and highly metastatic malignancies, and are among the most important new and active chemotherapeutic agents on the market.

[0092] Although docetaxel is an effective anticancer agent, it has limitations such as drug resistance, cancer recurrence, and side effects. For example, high doses are known to induce additional toxic reactions, and low to intermediate doses of docetaxel have been shown to not exhibit significant antitumor activity in patients. Therefore, docetaxel is not considered a monotherapy for cancer. Therefore, there is a need to develop combination therapies that can exhibit synergistic anticancer effects when administered in combination with docetaxel.

[0093] Gemcitabine is a chemotherapy drug sold under brand names such as Gemzar. It is used to treat cancers such as testicular cancer, breast cancer, ovarian cancer, non-small cell lung cancer, pancreatic cancer, and bladder cancer, and is administered intravenously. It suppresses tumor growth by upregulating interferon signaling and inhibits the replication of orthohepevirus A, the virus that causes hepatitis E.

[0094] Pembrolizumab is classified as an anti-PD1 and is an immune checkpoint inhibitor. It is an anticancer drug that activates the body's immune system to attack cancer cells. Current cancer treatments have focused on killing rapidly dividing cells, a characteristic of cancer cells. This has resulted in side effects due to the drug affecting not only cancer cells but also rapidly dividing normal cells. In contrast, immunotherapy drugs such as pembrolizumab utilize the patient's immune system to target cancer cells, thus having the advantage of virtually eliminating the typical side effects of existing anticancer drugs. Pembrolizumab is administered intravenously, and treatment is determined by the clinician, typically administered over 30 minutes every three weeks.

[0095] Doxorubicin (Dox, Doxo), also known by brand names such as Adriamycin, is a chemotherapy drug used to treat cancer. These include breast cancer, bladder cancer, Kaposi's sarcoma, lymphoma, and acute lymphoblastic leukemia. It is administered intravenously.

[0096] Trastuzumab, sold under the brand name Herceptin, is a monoclonal antibody used to treat breast and stomach cancer. It is specifically used for HER2 receptor-positive cancers and can be used alone or in combination with other chemotherapy drugs. Trastuzumab is administered by slow injection into a vein, just under the skin.

[0097] Dabrafenib and trametinib are anticancer drugs used to treat melanoma, used as preoperative chemotherapy for localized disease and for BRAF V600E mutation-positive disease.

[0098] Paclitaxel is a diterpenoid anticancer agent extracted from the bark of the yew tree. It has been used as an anticancer agent since its effectiveness against ovarian cancer, breast cancer, and non-small cell lung cancer was discovered. It is also effective in treating rheumatoid arthritis and Alzheimer's disease.

[0099] Carboplatin, brand name Paraplatin, is a chemotherapy drug used to treat various types of cancer. These include ovarian cancer, lung cancer, head and neck cancer, brain cancer, and neuroblastoma. Administered intravenously, it is a platinum-based antineoplastic agent that acts by interfering with DNA replication. It is an analogue of cisplatin, developed to reduce its toxicity.

[0100] Temozolomide is an anticancer drug belonging to the alkylating agent class. It exerts its anticancer effects by attacking specific groups within cellular DNA, inhibiting DNA, RNA, and protein synthesis. It is most active against tumor cells in the quiescent phase, making it a non-cell cycle-selective anticancer drug. It is primarily used to treat glioblastoma multiforme and anaplastic astrocytoma, but it can also be used in other tumor types.

[0101] FOLFIRINOX (5-FU, irinotecan, leucovorin, oxaliplatin, FOLFIRINOX) is an anticancer drug known to be more helpful in extending the survival time of pancreatic cancer patients compared to other chemotherapy regimens. In particular, compared to gemcitabine monotherapy, FOLFIRINOX is a pancreatic cancer anticancer drug that has been reported to significantly increase the median survival time.

[0102] Radiotherapy refers to exposing a target to radiation. Here, it refers to a type of anticancer treatment that utilizes the properties of radiation to treat cancer cells by irradiating them.

[0103]

[0104] In the present invention, the term "cancer" refers to a lump that has grown abnormally due to autonomous excessive growth of body tissue, also called a tumor, and may specifically include lung cancer, breast cancer, melanoma, ovarian cancer, brain cancer, head and neck cancer, pancreatic cancer, stomach cancer, or prostate cancer.

[0105] The term "lung cancer" of the present invention refers to cancer that occurs in the lung itself or metastasizes to the lung from another organ, and in the present invention, lung cancer may be non-small cell lung cancer, small cell lung cancer, or Anti-PD1 refractory lung cancer.

[0106] The term "non-small lung cancer" of the present invention may be used interchangeably with non-small cell lung cancer. Non-small cell lung cancer is a type of carcinoma and refers to all epithelial lung cancers other than small cell lung cancer, and accounts for approximately 85% to 90% of all lung cancers. Symptoms of non-small cell lung cancer include persistent coughing, chest pain, weight loss, nail damage, joint pain, and shortness of breath. However, since non-small cell lung cancer generally progresses slowly, it rarely exhibits symptoms in the early stages, making early detection and treatment difficult, and it is highly likely that it will be discovered only after it has metastasized to the bones, liver, small intestine, and brain, among other parts of the body.

[0107] The above non-small cell lung cancer may include EGFR mutant non-small cell lung cancer or EGFR wild-type non-small cell lung cancer (squamous cell lung cancer).

[0108] The above “EGFR mutant non-small cell lung cancer” refers to non-small cell lung cancer in which a mutation occurs in the epidermal growth factor receptor (EGFR) gene, which is the most common genetic mutation found in non-small cell lung cancer.

[0109] The above “EGFR wild-type non-small cell lung cancer” is also called squamous cell lung cancer and refers to non-small cell lung cancer in which the EGFR gene is wild-type without mutation.

[0110] The above “small cell lung cancer” refers to lung cancer excluding non-small cell lung cancer, which accounts for 80-85% of lung cancers, and is also called small cell lung cancer. The small cell lung cancer is generally highly malignant, so many cases have already metastasized to other organs, the opposite lung, or the mediastinum (the space between the two lungs where the heart, trachea, esophagus, aorta, etc. are located) through the lymphatic vessels or blood vessels at the time of discovery.

[0111] The above small cell lung cancer may include EGFR wild-type small cell lung cancer, and EGFR wild-type is as described above.

[0112] The term "breast cancer" of the present invention refers to a cancer in which abnormal tissue continues to grow in the breast or spreads to other organs, and specifically, it may be HER2-negative breast cancer or HER2-positive breast cancer.

[0113] The term "HER2" of the present invention refers to HER2 (Receptor tyrosine-protein kinase erbB-2), a protein that is generally present in the cell membrane and is encoded by the ERBB2 gene. ERBB is an abbreviation for erythroblastic oncogene B, a gene originally isolated from the avian genome. The human protein is often referred to as HER2 (human epidermal growth factor receptor 2) or CD340 (cluster of differentiation 340). HER2 belongs to the human epidermal growth factor receptor (HER / EGFR / ERBB) family. However, unlike other members of the ERBB family, HER2 does not bind directly to its ligand. HER2 activation occurs by heterodimerization with other ERBB members or by homodimerization when the concentration of HER2 is high, such as in cancer. Amplification or overexpression of this oncogene has been shown to play a key role in the development and progression of certain aggressive types of breast cancer. In recent years, this protein has become a key biomarker and therapeutic target for approximately 30% of breast cancer patients.

[0114] Invasive breast cancer, which is a type of breast cancer, is breast cancer that invades surrounding tissues and is divided into HER2-negative and -positive types. HER2-positive breast cancer is known to be more aggressive and more likely to metastasize.

[0115] The term "head and neck cancer" in the present invention refers to all types of malignant tumors that occur in the nose, paranasal sinuses, oral cavity, face, larynx, pharynx, salivary glands, thyroid gland, etc. Depending on the location of occurrence, it can be divided into oral cancer, laryngeal cancer, pharyngeal cancer, salivary gland cancer, thyroid cancer, paranasal sinus cancer, etc.

[0116] The above head and neck cancer may be head and neck cancer and radiation resistant cancer.

[0117] The above terms, “head and neck cancer and radiation-resistant cancer” are used interchangeably with radiation-resistant head and neck cancer and refer to cancer that is resistant to radiation chemotherapy used in the treatment of head and neck cancer and cannot be treated with radiation or has minimal therapeutic effect.

[0118] The term "brain cancer" of the present invention is a general term for cancer that occurs in the brain, also called a brain tumor, and refers to a tumor that occurs in brain tissue or the membrane surrounding the brain, and a tumor that has metastasized to brain tissue or the membrane from a site distant from the skull or surrounding structures.

[0119] The term "melanoma" in the present invention refers to a cancer that occurs in the skin, and mainly refers to one that arises from melanocytes scattered in the basal layer of the epidermis. Among them, malignant melanoma is a malignant tumor of melanocytes, and can arise anywhere melanocytes exist, but it most commonly occurs in the skin. Melanoma can be defined as a malignant transformation of these melanocytes or pre-existing nevus cells (cells that form moles), and is highly malignant.

[0120] The term "ovarian cancer" in the present invention is a general term for cancer that occurs in the ovaries, and refers to cancer that occurs in the ovaries, which play a crucial role in female reproduction and hormone secretion. Depending on the tissue in which the cancer occurs, ovarian cancer is largely classified into epithelial cell carcinoma, germ cell tumor, and sex cord stromal tumor. More than 90% of ovarian cancers are epithelial ovarian cancers that occur in the epithelial cells on the surface of the ovaries, and in fact, most cancers found around us are epithelial ovarian cancers.

[0121] The term "pancreatic cancer" in the present invention is a general term for cancer that occurs in the pancreas, and is a lump of cancer cells that develops in the pancreas. Such a lump is called a tumor. There are various types of pancreatic cancer, but more than 90% of cases arise from the exocrine cells of the pancreatic duct, so pancreatic cancer generally refers to pancreatic ductal adenocarcinoma. Adenocarcinoma refers to cancer that arises from glandular cells, i.e., glandular cells, and the symptoms of pancreatic cancer vary depending on the location of occurrence.

[0122] The term "stomach cancer" of the present invention is a general term for cancers occurring in the stomach. The cancer that accounts for the majority of stomach cancers is adenocarcinoma, and in addition, rarely, lymphoma occurring in the lymphatic tissue of the stomach, stromal tumor occurring in the stromal cells of the stomach (gastrointestinal tumor), sarcoma originating from non-epithelial tissue, and neuroendocrine tumor that secretes hormones may also occur.

[0123] The term "prostatic carcinoma" in the present invention refers to cancer arising in the prostate, most often adenocarcinoma arising from prostate cells. Types are categorized based on the degree of differentiation of the tumor tissue and the characteristics of the cells. A widely used classification system, proposed by pathologist Donald Gleason, ranges from Grade 1 (highest differentiation) to Grade 5 (least differentiation). It is reported that better differentiation leads to a better prognosis.

[0124]

[0125] In the present invention, the cancer is not particularly limited as long as the symptoms thereof can be alleviated, relieved, improved or treated by a pharmaceutical composition for preventing or treating cancer comprising a mixed extract of Astragalus membranaceus, Angelica gigas, and Cnidium rhizome provided in the present invention and an anticancer agent.

[0126]

[0127] The term "prevention" of the present invention means any act of inhibiting or delaying the onset of cancer by administering a pharmaceutical composition for preventing or treating cancer, including a mixed extract of Astragali, Angelica, and Cnidium officinalis; and an anticancer agent.

[0128] The term "treatment" of the present invention means any act in which the symptoms of a subject suspected of having or developing cancer are improved or beneficially changed by administration of the pharmaceutical composition.

[0129] In the present invention, the term “combined administration” means treatment by using simultaneously or sequentially.

[0130] In a specific embodiment of the present invention, a mixed extract of Astragalus membranaceus, Angelica gigas, and Cnidium rhizome; And when one or two kinds selected from the group consisting of docetaxel (DTX), anti-PD1, gemcitabine, pembrolizumab, doxorubicin (Dox, Doxo), trastuzumab, dabrafenib, trametinib, paclitaxel, carboplatin, temozolomide and FOLFIRINOX (5-FU, irinotecan, leucovorin, oxaliplatin) were selected for each cancer type and administered in combination or treated in combination with radiotherapy, it was confirmed that there was a synergistic anticancer effect, and in particular, when the mixed extract of the present invention was administered in combination with one or more anticancer agents or treated in combination with radiotherapy, the therapeutic effect was confirmed to be significantly increased, and among them, It was confirmed that when SH003, a mixed herbal extract of the present invention, and two types of anticancer drugs were used in combination, there was a significant synergistic effect compared to when only one type of anticancer drug was used in combination. In head and neck cancer, it was confirmed that when SH003 was used in combination with radiation, there was a significant synergistic effect specifically in head and neck cancer that was resistant to radiation.

[0131] In addition, in a specific embodiment of the present invention, the combined effect of the mixed herbal extract SH003 of the present invention and docetaxel (DTX) was evaluated in an anti-PD1 resistant lung cancer model to confirm its possibility as a second-line treatment, and it also showed an immunomodulatory effect in an immunosuppressed model, contributing to the enhancement of the immune response. In particular, the combined treatment using SH003 targets the EGFR / JAK / STAT3 signal pathway to reduce PD-L1 expression, which inhibits the mechanism related to immune evasion, confirming that the SH003-DTX combination therapy has a dual mechanism of action that overcomes resistance through immune system regulation as well as a direct anticancer effect. Therefore, the SH003-DTX combination therapy presents a new therapeutic possibility for lung cancer patients who do not respond to anti-PD1 therapy.

[0132]

[0133] In the pharmaceutical composition for preventing or treating cancer of the present invention, in one specific example, the pharmaceutical composition may reduce the expression level or activity of EGFR (Epidermal growth factor receptor), but is not limited thereto.

[0134] The epidermal growth factor receptor (EGFR) has been identified as a target for the treatment of numerous cancers, particularly solid tumors, because it is involved in regulating cellular functions critical to cancer cell proliferation and survival. Increased expression of EGFR has been observed in bladder cancer, breast cancer, glioblastoma, head and neck cancer, lung cancer, and gastric cancer.

[0135] In the present invention, the pharmaceutical composition may exhibit a synergistic effect on anticancer treatment by reducing the expression level or activity of EGFR (Epidermal growth factor receptor).

[0136]

[0137] In the pharmaceutical composition for preventing or treating cancer of the present invention, in one specific example, the composition may additionally include a pharmaceutically acceptable carrier, excipient or diluent, but is not limited thereto.

[0138] The above pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, excipient or diluent commonly used in the manufacture of pharmaceutical compositions, and the carrier may comprise a non-naturally occurring carrier.

[0139] The term "pharmaceutically acceptable" in the present invention means that the composition exhibits properties of being non-toxic to cells or humans exposed to the composition.

[0140] Specifically, the pharmaceutical composition may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterile injection solutions, each according to a conventional method.

[0141] In the present invention, carriers, excipients, and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. When formulating, the composition is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid dosage forms for oral administration include tablets, pills, powders, granules, and capsules, and these solid dosage forms are prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose or lactose, and gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid dosage forms for oral administration include suspensions, oral solutions, emulsions, and syrups, and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives may be included. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include witepsol, macrogol, Tween 61, cocoa butter, laurin, and glycerogelatin.

[0142] In the pharmaceutical composition for preventing or treating cancer of the present invention, in any one specific example, the composition may be administered by intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, or rectal administration, but is not limited thereto.

[0143]

[0144] Another aspect of the present invention provides an anticancer adjuvant containing a mixed extract of Astragalus membranaceus, Angelica gigas, and Cnidium rhizome, wherein the adjuvant is administered in combination with an anticancer agent or in combination with radiation treatment.

[0145] The above terms, astragalus membranaceus, angelica root, celery root powder, mixed extract, anticancer agent, combined administration, radiation treatment, etc. are as described above.

[0146] The above term, “adjuvant”, refers to something that plays a supplementary role to an anticancer drug used for the purpose of anticancer treatment, and may maximize the anticancer effect of the anticancer drug. The form or formulation of the adjuvant is not particularly limited.

[0147]

[0148] Another aspect of the present invention provides a method for preventing or treating cancer, comprising administering the pharmaceutical composition to a subject.

[0149] At this time, the definitions of cancer, prevention and treatment are as described above.

[0150] The term "administration" in the present invention means introducing a predetermined substance to an individual by an appropriate method.

[0151] The term "subject" in the present invention refers to any animal, including rats, mice, and livestock, including humans, that has developed or may develop cancer. Specific examples include, but are not limited to, mammals, including humans.

[0152] The method for preventing or treating cancer of the present invention may specifically include a step of administering to a subject a pharmaceutical composition for preventing or treating cancer, which comprises a mixed extract of Astragalus membranaceus, Angelica gigas, and Cnidium rhizome; and an anticancer agent, and a pharmaceutically effective amount of the pharmaceutical composition.

[0153] The term "pharmaceutically effective amount" of the present invention means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment and not causing side effects, and the effective dosage level can be easily determined by those skilled in the art based on factors including the patient's sex, age, weight, health condition, type and severity of the disease, activity of the drug, sensitivity to the drug, method of administration, time of administration, route of administration, and excretion rate, duration of treatment, drugs used in combination or simultaneously, and other factors well known in the medical field.

[0154] Specifically, the composition of the present invention can be administered at a daily dose of 0.0001 to 100 mg / kg of body weight, more specifically 0.001 to 100 mg / kg of body weight, based on solid content. The above recommended dosage can be administered once a day or divided into several doses.

[0155] In the method for preventing or treating cancer of the present invention, the route and method of administration for administering the composition are not particularly limited, and any route and method of administration may be followed as long as the composition containing the composition can reach the target site. Specifically, the composition may be administered through various routes, such as oral or parenteral, and non-limiting examples of the routes of administration include oral, rectal, topical, intravenous, intraperitoneal, intramuscular, intraarterial, transdermal, intranasal, or inhalation.

[0156]

[0157] Hereinafter, to aid understanding of the present invention, examples and other embodiments will be described in detail. However, the embodiments according to the present invention may be modified in various ways, and the scope of the present invention should not be construed as being limited to the following examples. The embodiments of the present invention are provided to more fully explain the present invention to those of average skill in the art.

[0158]

[0159] Experimental Example 1. Preparation of mixed herbal medicine ethanol extract (SH003) and preparation / cultivation of anticancer drugs and cancer cell lines.

[0160] Astragalus membranaceus (Am), Angelica gigas (Ag), and Trichosanthes kirilowii Maximowicz (Tk) were mixed in a weight ratio (w / w) of 1:1:1, placed in an extractor, and extracted with 30% (v / v) ethanol at 100°C for 3 hours. The extract was filtered, and the filtrate was concentrated under reduced pressure and dried to obtain a mixed extract of Astragalus membranaceus, Angelica gigas, and Trichosanthes kirilowii Maximowicz. The dried mixed extract was dissolved in 30% ethanol and stored at -80°C until use.

[0161] Combination anticancer drugs, docetaxel (DTX), anti-PD1, gemcitabine, pembrolizumab, doxorubicin (Dox, Doxo), trastuzumab, dabrafenib, trametinib, paclitaxel, carboplatin, temozolomide, radiotherapy, and FOLFIRINOX (5-FU, irinotecan, leucovorin, oxaliplatin), were purchased and stored from Sigma-Aldrich (St. Louis, MO, USA).

[0162] In addition, lung cancer, breast cancer, melanoma, ovarian cancer, brain cancer, head and neck cancer, pancreatic cancer, stomach cancer, and prostate cancer cell lines were obtained from the American Type Culture Collection (ATCC) and used in the experiments. In addition, each of the above cancer cells was cultured in a cell culture medium containing RPMI 1640, heat-inactivated 10% FBS (Fetal Bovine Serum) and 100 U / mL antibiotic-antimycotic (Invitrogen) in a cell incubator at 37°C and 5% CO2.

[0163]

[0164] Experimental Example 2. Analysis of the Cell Viability Inhibition Effect

[0165] The cell viability inhibition effect identified in the present invention was analyzed using a known MTT assay. Specifically, an MTT experiment was conducted with SH003 and standard anticancer drugs for each cancer type for 48 hours. The IC50 value of each drug (anticancer drug, SH003) was calculated, and then co-administration was performed at different ratios based on that value. The Compusyn program was then used to determine whether the drugs had a synergistic effect (CI value < 1).

[0166] To confirm the synergistic effect with anti-PD1, a co-culture experiment was conducted with PBMC. 2.5 × 10⁴ cells / well were seeded into a 48-well plate and co-cultured with PBMC. After 48 hours, cell viability was measured using the WST-8 assay.

[0167] For small cell lung cancer H69 cells, they were labeled with CFSE (5 μM) for 25 min and then co-cultured with PBMCs. Cell viability was then measured using a microplate reader.

[0168]

[0169] Experimental Example 3. Homogeneous LLC1 Tumor Model and Drug Treatment

[0170] C57BL / 6N male mice (4 weeks old) were purchased from Nara Biotech (Seoul, Korea), and experiments were performed after a 2-week acclimation period. LLC1 cells (5 × 10^4) were suspended in Matrigel and PBS (1:1 mixture, 100 μL) and injected subcutaneously. After tumor formation after 7 days, the mice were randomly divided into four groups (n = 4) and administered SH003 (oral administration) and DTX (intraperitoneally administration) at fixed concentrations and times for 2 weeks (Fig. 1A). Tumor volumes were measured three times a week using the formula length × (width)^2 / 2. All animal experiments were approved by the Institutional Animal Care and Use Committee of Kyung Hee University (IACUC, KHSASP-23-313), and mice were maintained under a 12-h day / night cycle in an SPF environment. The administration concentration of SH003 was determined by converting the clinical concentration and prepared by dissolving in 1% ethanol (w / w). DTX was obtained from docetaxel trihydrate (Sigma-Aldrich, MO, USA) and dissolved in a solution containing 10% DMSO, 40% PEG400M, 5% Tween-80, and 45% saline for use as an injection.

[0171]

[0172] Experimental Example 4. Preparation of a single cell suspension

[0173] After dissociation, tumor tissues were dissociated in serum-free DMEM medium containing collagenase D (1 mg / mL, Roche, Basel, Switzerland) and DNase I (0.1 mg / mL, Roche) for 30 minutes. The cells were then filtered through a 70 μm nylon mesh to prepare a single-cell suspension, which was then centrifuged at 1,500 rpm for 3 minutes. After treatment with RBC lysis buffer for 3 minutes to remove red blood cells, the cells were washed twice with serum-free medium. Afterwards, the cells were washed with PBS (containing 2% FBS) and filtered through a 45 μm filter to prepare for flow cytometry analysis.

[0174]

[0175] Experimental Example 5. Flow Cytometry

[0176] For apoptosis analysis, cells were stained with Annexin V (BD Biosciences, NJ, USA) and 7-AAD (Sigma-Aldrich, MO, USA) for 15 minutes each at room temperature. For surface marker staining, cells were reacted with fluorescently conjugated primary antibodies for 30 minutes at room temperature. For intracellular staining of FOXP3, cells were stained using the FOXP3 / Transcription factor staining set from eBioscience (Invitrogen, San Diego, CA) according to the manufacturer's protocol. The stained cells were fixed with 2% PFA. For PD-L1 cell surface analysis, after treatment with SH003 and / or DTX for 24 hours, cells were reacted with anti-PD-L1 antibody (1:100 dilution, PBS containing 5% BSA) at 37°C for 30 minutes. After washing with 1% BSA-PBS, the cells were reacted with Alexa Fluor 488-conjugated staining antibody (1:250, Thermo Fisher Scientific, MA, USA) at 37°C for 30 minutes. Finally, after fixation with 0.5% PFA for 10 minutes, the fluorescence signal was measured using CytoFLEX (Beckman Coulter, CA, USA) and analyzed with FlowJo software (Treestar, OR, USA). The list of fluorescent staining antibodies used is shown in Table 1 below.

[0177] MarkerFluorophoreCatalog NumberDilutionSurface / IntracellularMHC*PE12-5320-821:100SurfaceCD8AF48853-0081-821:100SurfaceCD25PE12-0251-821:10 0SurfaceCD4PE-Cy5.535-0042-821:100SurfaceFoxp3APC17-5773-821:100IntracellularPD-1PE12-9985-821:100SurfaceLag-3Per CP-EF71046-2231-821:100SurfaceTim-3APC17-5871-821:100SurfaceCD3V45075-0032-U1001:100SurfaceCD45RPE-Cy760-0452-U10 01:100SurfaceCD11cBV650416-0114-821:100SurfaceF4 / 80APC-Cy725-4801-U1001:100SurfaceNK1.1APC20-5941-U1001:100Surface

[0178] Experimental Example 6. CTX-induced immunosuppression mouse model

[0179] C57BL / 6 male mice (7 weeks old) were purchased from Nara Biotech (Seoul, Korea) and divided into control (n = 4), CTX treatment (n = 4), and CTX+SH003 treatment groups (n = 4). To induce immunosuppression, CTX (50 mg / kg) was administered intraperitoneally 4 days before and on the day of the experiment. The CTX+SH003 group was then administered SH003 (558 mg / kg) orally for 5 days. Body weights were measured every 2–4 days during the experiment, and spleen weights were measured after the experiment and analyzed for correlation with the immune response.

[0180]

[0181] Experimental Example 7. Histology and Immunohistochemistry (IHC)

[0182] Formaldehyde-fixed, paraffin-embedded tumor tissues were sectioned at 10 μm thickness and stained with H&E. For IHC analysis, 7 μm-thick sections were deparaffinized and hydrated, and antigen retrieval was performed in a pH 6.0 citrate buffer. The sections were then blocked with hydrogen peroxide for 1 hour and reacted with primary antibodies at 4°C. After 1 hour of secondary antibody treatment, DAB staining was performed and observation was performed under a microscope (Carl Zeiss, Germany). The antibodies used were anti-cleaved-caspase 3, p-STAT3 (Tyr705), MCL1 (Cell Signaling Technology, MA, USA), CD8, PCNA, PD-L1 (Abcam, UK), and CD206 (Santa Cruz Biotechnology, TX, USA).

[0183]

[0184] Experimental Example 8. Immunofluorescence (IF)

[0185] FFPE tumor tissue was sectioned into 4 μm thick sections and dried at 60°C for 45 minutes. The section slides were deparaffinized and hydrated, subjected to antigen retrieval, and incubated with fluorescently labeled primary antibodies in a darkroom at 37°C for 1 hour. After incubation, the slides were washed with Tris-buffered saline with 0.1% Tween 20 (TBS-T) and then mounted for identification. The primary antibodies used were Granzyme B (GrB, PE-Cy5.5), Perforin (APC), and NK1.1 (APC), purchased from Invitrogen (Thermo Fisher Scientific, MA, USA).

[0186]

[0187] Experimental Example 9. ELISA

[0188] After measuring the spleen weight, it was washed with 1x PBS to remove excess blood. After performing two freeze-thaw cycles, the tissue was homogenized at 4°C in lysis buffer containing 25 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% NP-40, 5% glycerol, and proteinase inhibitor (PI) and PMSF. The homogenized sample was centrifuged at 16,000 × g for 10 min, and the supernatant was stored at -80°C. After diluting the sample to the optimal concentration range, mouse cytokines such as interleukin 2 (IL-2), interleukin 6 (IL-6), tumor necrosis factor-alpha (TNF-α), and interferon gamma (IFN-γ) were detected using DuoSet ELISA Development kits (R&D Systems, Seoul, Korea). Cytokine concentrations were measured at 450 nm using an ELISA reader (Molecular Devices, CA, USA).

[0189]

[0190] Experimental Example 10. Hematology Analysis

[0191] From each group, 0.3 mL of blood was collected via cardiac puncture. The collected blood was stored in tubes containing an anticoagulant (K2-EDTA, Becton Dickinson, NJ, USA) and subjected to hematological analysis. White blood cell (WBC), lymphocyte (LYM), and monocyte (MONO) counts were measured by flow cytometry at Biotoxtech Co., Ltd. (Ochang, Korea).

[0192]

[0193] Experimental Example 11. Cell culture and cell viability

[0194] Lewis lung carcinoma (LLC1) cells were derived from the lung tissue of C57BL / 6 mice harboring LLC1 tumors and purchased from the American Type Culture Collection (ATCC, VA, USA). Cells were cultured in DMEM (WelGENE, Gyeongsan, Korea) supplemented with 10% unheated FBS (JR Scientific, CA, USA) and 1% penicillin / streptomycin (WelGENE, Korea). To measure cell viability, 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) solution was added after drug treatment and incubated for 2 hours. Absorbance was measured at 570 nm using an ELISA reader (Molecular Devices, CA, USA). CompuSyn program was used to determine the synergistic inhibitory concentration of SH003 and DTX.

[0195]

[0196] Experimental Example 12. Western Blotting

[0197] Cells were treated with SH003 (100 μg / mL) and DTX (100 nM) for 24 h and then lysed in RIPA buffer containing protease and phosphatase inhibitors. Protein concentration was measured using the Bradford assay, and proteins were then separated using SDS-PAGE, transferred to nitrocellulose membranes, and blocked in blocking buffer containing 1x PBS and 0.1% Tween-20. The membranes were incubated with primary antibodies overnight at 4°C and then with secondary antibodies for 1 h at room temperature. Proteins were detected using an EZ-western detection kit (Dogen-Bio, Seoul, Korea). Primary antibodies used were PARP, cleaved-caspase 3, EGFR, p-EGFR (Tyr 1068), p-EGFR (Tyr 1173), JAK1, JAK2, p-JAK1 (Tyr 1034 / 1035), p-JAK2 (Tyr 1007), STAT3, p-STAT3 (Tyr 705), GAPDH, MCL1 (Cell Signaling Technology, MA, USA), and PD-L1 (Abcam, Cambridge, UK).

[0198]

[0199] Experimental Example 13. Transfection

[0200] LLC1 cells were seeded at a density of 1 × 10^5 cells / well. Cells were transiently transduced with pCMV6-AC-GFP control vector (OriGene, PS100010, MD, USA) or STAT3 (OriGene, RC215836, MD, USA) using Lipofectamine 3000 reagent (Invitrogen, CA, USA) for 24 h. Cells were then treated with SH003 and / or DTX for 24 h, harvested, and analyzed.

[0201]

[0202] Experimental Example 14. Correlation Analysis between STAT3 Expression and Activated CD8+ T Cells (Using the TISIDB Database)

[0203] To investigate the association between STAT3 expression and activated CD8+ T cells in lung adenocarcinoma (LUAD), we utilized the TISIDB database (http: / cis.hku.hk / TISIDB / ). A scatter plot was generated to visualize the relationship between STAT3 expression levels and the abundance of activated CD8+ T cells. Spearman's correlation coefficient (r) was calculated, and the corresponding p-value was included to assess statistical significance.

[0204]

[0205] Experimental Example 15. Statistical Analysis

[0206] The data presented so far are expressed as mean ± standard deviation (SD). Statistical analysis was performed using GraphPad Prism software 8.0.2 (GraphPad Software, CA, USA), and p-values ​​were calculated using one-way ANOVA and multiple comparison tests. Pearson's correlation coefficient (r) analysis was also performed using GraphPad Prism software 8.0.2.

[0207]

[0208] Example 1. Analysis of the synergistic effect of combined administration of the mixed extract of the present invention and an anticancer agent in lung cancer.

[0209] Example 1-1. Synergistic effect of combined administration of SH003, Docetaxel, and Gemcitabine in non-small cell lung cancer with EGFR mutations.

[0210] To confirm the synergistic effect of combined administration of the mixed extract prepared in Experimental Example 1, docetaxel, and gemcitabine on non-small cell lung cancer cells with EGFR mutations, an MTT assay was performed according to a conventionally known method. H1975 and HCC-827 were used as non-small cell lung cancer cell lines with EGFR mutations.

[0211] First, the two cultured cell lines were treated with various concentrations of the mixed extract of Astragali, Angelica, and Gastrodia elata prepared in Experimental Example 1 (SH003), docetaxel, and gemcitabine for 48 hours, and an MTT experiment was performed. After calculating the IC50 value of each drug, co-treatment was performed at different ratios based on the value. Afterwards, the Compusyn program was used to confirm whether the drugs had a synergistic effect (CI value < 1).

[0212] As a result, as can be confirmed in Fig. 1, in the H1975 cell line, the IC50 values ​​were 54.97 nM for docetaxel, 1049 nM for gemcitabine, and 240 μg / mL for SH003, and in the HCC-827 cell line, the IC50 values ​​were 153.1 nM for docetaxel, 147.6 nM for gemcitabine, and 317.3 μg / mL for SH003.

[0213] Next, using the MTT assay method described above, the two cell lines were treated with a mixture (3 combi) of SH003, docetaxel, and gemcitabine, which is a mixed extract of the present invention, and compared with each alone and the mixture of two types of GEM+DTX, and the results are shown in Figure 2.

[0214] As can be confirmed in Fig. 2, it was confirmed that the SH003, docetaxel, and gemcitabine 3 combi treatment of the present invention had a significantly superior synergistic effect compared to each treatment alone or docetaxel + gemcitabine 2 treatments in both non-small cell lung cancer cell lines having two EGFR mutations. In particular, in the H1975 cell line, synergistic effects were observed at all concentrations of IC50 and 1.25*IC50, 1.5*IC50, 1.75*IC50, and 2*IC50, and among them, the best synergistic effect was confirmed at IC50 and 1.25*IC50, and in the HCC-827 cell line, the best synergistic effect was confirmed at the concentrations of IC50 and 2*IC50.

[0215]

[0216] Example 1-2. Synergistic effect of combined administration of SH003, Docetaxel, and anti-PD-1 in EGFR wild-type non-small cell lung cancer.

[0217] To confirm the synergistic effect of combined administration of the mixed extract prepared in Experimental Example 1, docetaxel, and anti-PD-1 to EGFR wild-type non-small cell lung cancer, an MTT assay was performed according to a previously known method. H460 and H358 were used as EGFR wild-type non-small cell lung cancer cell lines.

[0218] First, as confirmed in the above Example 1-1, the IC50 value of each drug was confirmed by MTT assay, and as can be seen in Fig. 3, in the H460 cell line, the IC50 value for SH003 was 133.4 μg / mL and for docetaxel, it was 82.83 nM, and in the H358 cell line, the IC50 value for DTX was 6.668 nM and for SH003, it was 1913 μg / mL.

[0219] Next, the optimal combination concentration of SH003 and docetaxel (hereinafter, combi) was confirmed using an MTT assay. As shown in Fig. 3, in the H460 cell line, the optimal combination concentrations were 0.21*IC50 and 0.24*IC50, and among them, the concentration of 0.21*IC50 was confirmed to be the most optimal combination concentration. In the H358 cell line, there was a synergistic effect at 0.0626*IC50, 0.125*IC50, 0.25*IC50, 0.5*IC50, and 1**IC50, and among them, the concentration of 0.25*IC50 was confirmed to be the optimal combination concentration.

[0220] Finally, in order to confirm the synergistic effect of 3combi, the effect of combined administration of the combi (mixture of two types) and anti-PD-1 was confirmed, and as a result, as can be seen in Fig. 4, when the combi and anti-PD-1 were treated in both H460 and H358 cell lines, it was confirmed that there was a synergistic effect in all EGFR wild-type non-small cell lung cancers, and among them, when T (target) and E (effector) were viewed as PBMC and cancer cells, it was confirmed that the best synergistic effect was found when the ratio of T:E was 1:1 to 1:4.

[0221]

[0222] Example 1-3. Synergistic effect confirmed by combined administration of SH003 + Carboplatin + anti-PD-1 in EGFR wild-type small cell lung cancer.

[0223] To confirm the synergistic effect of combined administration of the mixed extract prepared in Experimental Example 1, carboplatin, and anti-PD-1 to EGFR wild-type small cell lung cancer, an MTT assay was performed according to a previously known method. H69 was used as the EGFR wild-type small cell lung cancer cell line.

[0224] First, as confirmed in the above Example 1-1, the IC50 value of each drug was confirmed by MTT assay, and as can be seen in Fig. 5, the IC50 value for SH003 in the H69 cell line was 281.6 μg / mL, and that for carboplatin was 322.8 μM.

[0225] Next, the optimal combination concentration of SH003 and carboplatin (hereinafter, combo) was confirmed using an MTT assay, and as can be seen in Figure 6, the optimal combination concentration was confirmed to be 0.25*IC50.

[0226] Finally, in order to confirm the synergistic effect of 3com (SH003 + carboplatin + anti-PD-1), the effect of combined administration of the combo (mixture of 2 types) and anti-PD-1 (hereinafter, Com + aPD-1) was confirmed. As can be seen in Fig. 6, when the combo and anti-PD-1 were treated in the H69 cell line, a synergistic effect was confirmed in EGFR wild-type small cell lung cancer. In particular, when T (target) and E (effector) were viewed as PBMC and cancer cells, the best synergistic effect was confirmed when the ratio of T:E was 1:1.

[0227]

[0228] Example 1-4. Confirmation of the synergistic effect of SH003+anti-PD-1 in a lung cancer tumor model.

[0229] The synergistic effect of combined treatment of the mixed extract SH003 of the present invention and anti-PD-1 was confirmed using a lung cancer tumor model.

[0230] All animal experiments, including maintenance and euthanasia, were approved by the Animal Care and Use Committee of Kyung Hee University Animal Hospital (KHU-IACUC-KHSASP-21-211). Specifically, C57BL / 6N male mice (4 weeks old) were purchased from Nara Biotech (Seoul, Korea) and acclimated for 2 weeks. LLC1 mouse lung cancer cell line (5 × 10⁴ cells) was mixed with Matrigel and PBS (1:1, 100 μL) and implanted subcutaneously. Seven days after tumor formation, the mice were randomly divided into four groups, and SH003 (oral administration) and Anti-PD1 (intraperitoneally administration) were administered at the established concentrations and schedule for 2 weeks. Tumor size was measured three times a week, and the tumor volume was calculated using the formula (length × width² / 2). All animal experiments were approved by the Kyung Hee University IACUC (KHSASP-23-313). Mice were housed under SPF conditions with a 12-h day / night cycle. The SH003 dosing concentration was determined by converting it to a clinical concentration and prepared by dissolving it in 1% ethanol (w / w).

[0231] As a result, as can be confirmed in Fig. 7, combined administration of SH003 and anti-PD-1 significantly reduced tumor volume and weight compared to the monotherapy group, respectively. Meanwhile, single and combined administration of SH003 and anti-PD-1 did not affect body weight.

[0232] This suggests that the combined administration of the mixed extract of the present invention and anti-PD-1 synergistically inhibits tumor growth without causing toxicity in mice.

[0233] In summary, it was confirmed that the mixed extract of Astragalus membranaceus, Angelica gigas, and Cnidium rhizome of the present invention induces apoptosis of lung cancer cells in vitro and in vivo without side effects, and that the combined administration of anti-PD-1 significantly enhances the anticancer effect compared to each extract alone.

[0234]

[0235] Example 1-5. Evaluation of the combined efficacy of SH003 and docetaxel using a lung cancer tumor animal model.

[0236] The synergistic effect of combined treatment with the mixed extract SH003 of the present invention and docetaxel was confirmed using a lung cancer tumor model. All procedures of animal experiments, including maintenance and euthanasia, were approved by the Animal Care and Use Committee of Kyung Hee University Animal Hospital (KHU-IACUC-KHSASP-21-211). First, the test cell line was confirmed using an anti-PD1 refractory mouse lung cancer cell line (LLC1), and the treatment concentrations were 557.569 mg / kg for SH003 and 10 mg / kg for docetaxel. More specific experimental methods are the same as Experimental Examples 3 to 14, and the results are shown in Figures 8 to 14.

[0237] As shown in Figure 8, SH003 and DTX were confirmed to inhibit tumor growth by inducing apoptosis in an anti-PD1-resistant lung cancer model. Specifically, to evaluate the efficacy of SH003 and DTX in lung cancer showing anti-PD1 resistance, an LLC1 lung cancer syngeneic mouse model with confirmed anti-PD1 resistance was established. Combination treatment was performed for 2 weeks according to the schedule presented in Figure 8A. As a result, significant inhibition of tumor growth was confirmed in the combination treatment group compared to the control and single treatment groups (Figure 8B). Furthermore, tumor weight was reduced compared to the DTX monotherapy group (Figure 8C). This effect was confirmed to have no effect on mouse toxicity (Figure 8D). Furthermore, intratumoral apoptosis was significantly increased in the combination treatment group (Figure 8E). Immunohistochemical analysis also confirmed that the expression of cleaved-caspase 3, an apoptosis marker, increased and the expression of PCNA, a proliferation marker, decreased (Figure 8F). These results suggest that the anticancer effect of combination treatment is mediated through cell death.

[0238] Next, since cancer cells that are inherently resistant to immunotherapy have a strengthened immune evasion ability due to a reduced immune cell defense function, we analyzed cytotoxic lymphocytes such as T cells and NK cells that suppress tumors by secreting signaling molecules such as perforin and granzyme B (GrB). Perforin degrades the cell membrane of target cells, allowing granzyme B, a serine protease, to penetrate into the cells, which then activates the caspase-mediated apoptosis pathway. As shown in Figure 8G, immunofluorescence staining confirmed the movement of GrB and perforin within tumor cells after the combination treatment (Figure 8G), suggesting that the immune cell-mediated perforin / GrB-dependent apoptosis pathway was activated. In conclusion, combined treatment with SH003 and DTX exhibited potent anticancer effects by inducing cell death via the perforin / GrB pathway, suggesting its potential as an immunomodulatory strategy in anti-PD1-resistant lung cancer.

[0239] In addition, through Figure 9, it was confirmed that the combined treatment of SH003 and DTX enhanced the LLC1 tumor infiltration ability of cytotoxic immune cells.

[0240] Specifically, given that combination therapy is associated with immune cell-mediated tumor suppression responses, we further investigated whether these effects were due to the induction of immune activation. In particular, we analyzed the effects of T cell exhaustion and immunosuppressive regulatory T (Treg) cells, known as mechanisms promoting resistance to immune checkpoint inhibitors (ICIs). Specifically, we examined the expression levels of inhibitory immune checkpoint proteins LAG-3, TIM-3, and PD-1, whose expression increases during T cell exhaustion, as well as the proportion of CD4+CD25highFoxp3+ Treg cells within tumors. We confirmed that combination therapy did not restore the function of exhausted T cells or reduce the immunosuppressive effect of Treg cells (Figs. 13 and 14).

[0241] However, not only enhanced immune cell function but also increased infiltration of immune cells within tumors can induce a strong anti-tumor immune response, leading to the formation of so-called "hot tumors." Therefore, the effect of the combined treatment on immune cell infiltration within tumor tissue was investigated. As a result, as shown in Figure 9, a significant increase in intratumoral CD8+ cells was observed (Figures 9A and 9B). This suggests that cytotoxic T cell infiltration within the tumor microenvironment (TME) was promoted.

[0242] Additionally, analysis of other immune cell populations induced within the TME revealed a significant decrease in the levels of CD206, a marker of immunosuppressive M2 macrophages, and CD11b+ myeloid-derived suppressor cells (MDSCs) that promote tumor progression (Fig. 9B, bottom). Conversely, expression of NK1.1, a NK cell marker, increased, confirming enhanced anti-tumor immune function (Fig. 9C). These results suggest that the combination treatment modulates the TME into an anti-tumor immune environment.

[0243] Additionally, cytokines secreted from the spleen were analyzed to assess anticancer immune activity. IL-6, TNF-α, IL-2, and IFN-γ secreted by activated T cells served as key indicators of the anticancer immune response. However, combination treatment did not affect the secretion of IL-6 and IL-2, and rather, it was confirmed that the secretion of IFN-γ and TNF-α was reduced compared to the single treatment group (Fig. 9D). These cytokines increased in the initial T cell-mediated immune response, but tended to decrease during the sustained tumor suppression process. In particular, it was confirmed that overexpression of inflammatory cytokines can be converted to promote tumor proliferation in the long term. Therefore, combination treatment is likely to play a role in maintaining anticancer immunity by preventing excessive inflammation rather than promoting the initial inflammatory response.

[0244] In summary, this study confirmed that combined treatment with SH003 and DTX increases the infiltration of immune cells into the tumor microenvironment and induces an anti-tumor immune response through immunomodulatory effects that contribute to tumor suppression.

[0245]

[0246] Next, the immunomodulatory effect of SH003 was confirmed in an immunosuppressed mouse model, which is shown in Fig. 10. Specifically, it was confirmed that the combined treatment of SH003 and DTX of the present invention increased immune cell infiltration in an anti-PD1 resistant lung cancer model (Fig. 10A), and accordingly, the immunomodulatory effect of SH003 treatment alone in an immunosuppressed mouse model was confirmed. More specifically, immunosuppression was induced using cyclophosphamide (CHX), and then SH003 treatment was performed to determine the effect on immune cell populations, which is shown in Fig. 10. As a result, as can be confirmed in Figs. 10B and 10C, a significant increase in spleen index was confirmed in the SH003 treatment group compared to the CHX treatment alone group, which indicates an expansion of the immune system without a change in body weight (Figs. 10B and 10C). Furthermore, SH003 treatment significantly increased the numbers of macrophages, CD8+ T cells, and NK cells in the spleen (Figure 10D). Furthermore, SH003 was found to increase the numbers of white blood cells (WBCs), lymphocytes, and monocytes in the blood (Figure 10E). These results suggest that SH003 has the potential to enhance the efficacy of anticancer therapy by modulating various immune cell populations, even in an immunosuppressive environment.

[0247]

[0248] Next, we confirmed that the combined treatment of SH003 and DTX of the present invention induces apoptosis in LLC1 cells. Specifically, to further elucidate the apoptosis mechanism of the combined treatment observed in vivo, the effect on apoptosis in the LLC1 cell line was confirmed and shown in Fig. 11. As a result, as can be seen in Fig. 11, SH003, DTX, and the combined treatment significantly reduced cell viability (Figs. 11A and 11B), and it was confirmed that the combined treatment induced significantly higher apoptosis than the single treatment, especially at a synergistic concentration (SH003: 100 μg / mL, DTX: 100 nM, CI value < 1) (Fig. 11C). In addition, cleavage of PARP and caspase-3 was confirmed in the combined treatment group (Fig. 11D), indicating that the combined treatment effectively induced apoptosis in LLC1 cells. These results suggest that apoptosis serves as a major mechanism in the anticancer efficacy of the combined treatment.

[0249] Finally, we confirmed that the combined treatment induced apoptosis in LLC1 cells by targeting the EGFR / STAT3 / PD-L1 pathway. EGFR is frequently mutated in lung cancer and is a major factor in cancer growth, known to contribute to anticancer drug resistance. The JAK / STAT3 signaling pathway activated by EGFR is involved in immunosuppression and promotes resistance to anti-PD1 therapy. Analysis of the TISIDB database confirmed a negative correlation between STAT3 expression and activated CD8+ T cell infiltration in lung adenocarcinoma (LUAD) (Fig. 12A). This suggests that STAT3 suppresses immune cell function and promotes immune evasion. Therefore, in this study, we investigated whether STAT3, which mediates anti-PD1 resistance, was suppressed by the combined treatment. As a result, as can be confirmed in Figure 12A, the combined treatment inhibited the phosphorylation of EGFR / JAK / STAT3, and also, it was confirmed that the expression of PD-L1, which is regulated by STAT3, was significantly reduced after the combined treatment (Figure 12C), which means that the combined treatment inhibits the immune evasion mechanism of lung cancer cells. To confirm whether STAT3 inhibition mediates cell death by the combined treatment, the apoptotic effect was confirmed in cells overexpressing STAT3, and as a result, STAT3 overexpression suppressed the apoptotic signal by the combined treatment, reduced the cleavage of PARP and caspase-3 (Figures 12E and 12F), and it was confirmed that the combined treatment also reduced the expression of PD-L1 and anti-apoptotic protein MCL-1, which are regulated by STAT3 (Figures 12D and 12F). Even in tumor tissues, combined treatment reduced STAT3 activation and the expression of its downstream signaling proteins (Fig. 12G). These results suggest that combined treatment is important for inducing apoptosis by targeting STAT3 signaling, which may provide a strategy for overcoming anti-PD1 resistance in lung cancer.That is, these results suggest that in addition to simply inducing cancer cell death, it involves a mechanism that reduces immune evasion through downregulation of PD-L1.

[0250]

[0251] Therefore, these results suggest that combined administration of SH003 and Docetaxel is effective as a treatment for overcoming immunotherapy refractoriness and resistance.

[0252]

[0253] Example 2. Analysis of the synergistic effect of combined administration of the mixed extract of the present invention and an anticancer agent in breast cancer.

[0254] In order to confirm the synergistic effect of combined administration of the mixed extract prepared in Experimental Example 1, docetaxel, and doxorubicin or trastuzumab for breast cancer, an MTT assay was performed according to a conventionally known method.

[0255] The specific MTT assay method is the same as Experimental Example 2, and with regard to breast cancer cell lines, it was confirmed using MCF-7 and MDA-MB-231 cell lines as locally advanced and metastatic breast cancer HER2-negative cell lines, and HCC1419 and SK-BR-3 as locally advanced and metastatic breast cancer HER2-overexpressing (or positive) cell lines.

[0256] As in Example 1, the IC50 values ​​of the drugs alone were confirmed in each cell line. As can be seen in Fig. 15, in the case of HER2-negative breast cancer, the IC50 values ​​were confirmed to be 987.5 μg / mL for SH003, 0.7393 μg / mL for doxorubicin, and 0.09241 μM for DTX in the MDA-MB-231 cell line, and in the MCF-7 cell line, the IC50 values ​​were confirmed to be 894.7 μg / mL for SH003, 0.5086 μg / mL for doxorubicin, and 0.06906 μM for DTX.

[0257] Afterwards, the cell viability according to the combined treatment was confirmed, and the optimal concentration of the two anticancer drugs, doxorubicin and DTX, was selected, and the result of comparing the two-type treatment with the three-type combination treatment up to SH003 of the present invention was confirmed as shown in Fig. 16, and it was confirmed that the three-type combination treatment up to SH003 of the present invention had a significantly superior synergistic effect compared to the two anticancer drugs (doxorubicin + DTX) alone in both HER2-negative breast cancer cell lines.

[0258]

[0259] Next, in the HER2-positive breast cancer cell line, the IC50 value was first confirmed for each anticancer drug or SH003 alone, and is shown in Fig. 17. After that, the optimal concentration for the combined use of trastuzumab and DTX was selected for both HER2-positive breast cancer cell lines, and for direct comparison with this, the results of 3combi treatment up to SH003 were compared, and are shown in Fig. 18.

[0260] As a result, as can be confirmed in Figure 18, it was confirmed that the synergistic effect of the combination was significantly superior to the result of treating only two anticancer drugs in the 3 combi treatment with SH003+trastuzumab+DTX.

[0261]

[0262] Example 3. Analysis of the synergistic effect of combined administration of the mixed extract of the present invention and an anticancer agent in melanoma and ovarian cancer.

[0263] Similar to what was confirmed in the above Experimental Example 2 and Example 1, it was confirmed whether there was a synergistic effect of 3combi (SH003 + mixture of two anticancer drugs) compared to SH003 alone, anticancer drug alone, SH003 mixed with only one anticancer drug, or a mixture of two anticancer drugs in melanoma and ovarian cancer.

[0264] The experimental method was performed using the same process as Experimental Example 2 and Example 1.

[0265] First, in the case of melanoma, the effect of the combined use of SH003+Dabrafenib+Trametinib was confirmed, and the effect was confirmed using SK-MEL-28 cell line and A375 cell line as melanoma cell lines.

[0266] In addition, a melanoma xenograft model was established to verify whether SH003 exhibits a synergistic effect when administered in combination with Trametinib and Dabrafenib in a melanoma model. Specifically, SK-MEL-28 cells were subcutaneously transplanted into BALB / C nude male mice, and then randomly divided into five groups. SH003 (557.57 mg / kg), Dabrafenib (20 mg / kg), and Trametinib (0.2 mg / kg) were administered in combination, and each drug was administered orally three times a week for three weeks.

[0267] As a result, as can be confirmed in Fig. 19, in melanoma, it was confirmed that the synergistic effect was significantly better when SH003 was treated with two anticancer drugs, dabrafenib and trametinib, as a 3combi, compared to when only dabrafenib and trametinib were treated. In particular, in the melanoma tumor model, it was confirmed that the 3combi (triple combi) had a significantly better tumor formation inhibition ability compared to when SH003 alone or only dabrafenib and trametinib were treated.

[0268] In addition, the effect of the combined use of SH003+Carboplatin+Paclitaxel was confirmed in ovarian cancer, using SKOV3 cell lines and OVCAR-3 cell lines.

[0269] As a result, as can be confirmed in Fig. 20, the IC50 values ​​of SH003, carboplatin, and paclitaxel alone were confirmed in each cell line, and as a result of comparing the three types of 3combi treatment with SH003 alone, carboplatin alone, paclitaxel alone, and carboplatin and paclitaxel double anticancer drug treatment groups in Fig. 21, it was confirmed that the 3 combi treatment group had a significantly superior synergistic effect in both ovarian cancer cell lines. In particular, when the IC50 value was checked at various treatment concentrations, all were effective, and the SK0OV-3 cell line had the best synergistic effect at a concentration of 0.5*IC50, and the OVCAR-3 cell line had the best synergistic effect at a concentration of 2*IC50, confirming that the 3 combi combination treatment had an excellent effect at a concentration of 0.5*IC50 to 2*IC50.

[0270]

[0271] Example 4. Analysis of the synergistic effect of combined administration of the mixed extract of the present invention and an anticancer agent in brain cancer, head and neck cancer, and pancreatic cancer.

[0272] Similar to what was confirmed in the above Experimental Example 2 and Example 1, the synergistic effect of the combined administration of the mixed extract of the present invention and an anticancer agent was analyzed in brain cancer, head and neck cancer, and pancreatic cancer.

[0273] The results are shown in Figs. 22 to 26, respectively.

[0274] First, in brain cancer, it was confirmed that the combined treatment with SH003 had a significantly superior effect compared to the treatment with temozolomide alone in the U-373 brain cancer cell line (Fig. 22).

[0275] In head and neck cancer, it was confirmed by combining anticancer drugs with radiation treatment, and for head and neck cancer cell lines, it was confirmed using various cell lines such as HSC4, YD10B, CAL27, and HSC4RR to confirm head and neck cancer radiation resistance.

[0276] As a result, as can be confirmed in Fig. 23, the low cytotoxicity of SH003 for head and neck cancer cell lines (HSC4, YD10B, CAL27) was confirmed, and based on these concentrations, the efficacy of SH003 in combination with radiation for head and neck cancer and radiation-resistant cancer cell lines was confirmed, and it was confirmed that there was a significant cell viability inhibition effect in radiation-resistant cancer cell lines.

[0277] In particular, as shown in Fig. 24, when head and neck cancer and radiation-resistant cancer cell lines were treated with radiation, it was confirmed that radiation treatment was resistant, and when SH003 and radiation were combined, there was a slight effect of combination for head and neck cancer cell lines, but when treated for head and neck cancer radiation-resistant cancer cell lines, it was confirmed that SH003 and radiation combined treatment had a significantly superior effect, confirming that SH003 and radiation combined treatment has a combination effect specifically for radiation-resistant cancer cell lines.

[0278]

[0279] Finally, the effect of combined treatment with SH003+FOLFIFNOX was confirmed in pancreatic cancer, and AsPC1 and HPAC were used as pancreatic cancer cell lines.

[0280] First, the IC50 values ​​of SH003 and FOLFIRINOX were confirmed in each cell line (Fig. 25), and the effect of combined treatment was confirmed. As can be seen in Fig. 26, it was confirmed that there was a significantly superior synergistic effect compared to each treatment alone in both pancreatic cancer cell lines.

[0281]

[0282] Example 5. Analysis of the synergistic effect of combined administration of the mixed extract of the present invention and docetaxel in prostate cancer and stomach cancer.

[0283] Using a similar experimental method as confirmed in Experimental Example 2 and Example 1, the synergistic effect of combined administration of the mixed extract of the present invention and docetaxel was confirmed in gastric cancer and prostate cancer.

[0284]

[0285] Example 5-1. Analysis of the synergistic effect of combined administration of the mixed extract of the present invention and docetaxel in prostate cancer.

[0286] First, to verify the efficacy of the combined effect of the mixed extract SH003 of the present invention and Docetaxel in the prostate cancer cell line DU145, an MTT assay was performed for single treatment and combination treatment. The Combination Index (CI) was measured based on the results of the combination treatment.

[0287] As a result, as can be confirmed in Figure 27, it was confirmed that 1 nM Docetaxel and 300 μg / ml SH003 showed an excellent synergistic effect. Here, if the CI is less than 1, it can be interpreted as a synergistic effect, if it is 1, it can be interpreted as a cooperative effect, and if it is greater than 1, it can be interpreted as an antagonistic effect.

[0288] Based on these results, the concentration for subsequent experiments was selected to achieve cell viability close to 50%.

[0289] To compare the efficacy of the combined SH003 and Docetaxel treatment on apoptosis in the prostate cancer cell line DU145, Western blot analysis was performed to identify apoptosis-related proteins. The combination of concentrations obtained through the MTT assay most clearly demonstrated apoptosis. Flow cytometry also identified a relatively higher number of cells exhibiting apoptosis (Fig. 28).

[0290]

[0291] Finally, when subcutaneous xenograft was administered for 7 days, the group administered combination therapy showed relatively more effective tumor growth inhibition than the other groups. In addition, there was no change in the body weight of the mice, confirming that there was no toxicity. It was confirmed that cancer cell proliferation was reduced through H&E staining. In addition, the expression of Ki67, a cell proliferation indicator, was also reduced. The expression of Cleaved caspase 3, an apoptosis indicator, was increased, and it was confirmed that the phosphorylation of EGFR was inhibited and the expression of STAT3 was also inhibited. As a result, it was confirmed that apoptosis was induced by inhibiting EGFR-STAT3 signaling in the same way as the results of the in vitro study (Fig. 29).

[0292]

[0293] Experimental Example 5-2. Analysis of the synergistic effect of combined administration of the mixed extract of the present invention and docetaxel in gastric cancer.

[0294] Using a gastric cancer cell line, it was confirmed whether the mixed extract (SH003) of the present invention has a synergistic effect through combined administration in gastric cancer.

[0295] First, cell viability was confirmed by combined treatment. For reference, the specific experimental method is the same as that of Experimental Example 2 and Example 1.

[0296] In particular, for gastric cancer, an animal model of gastric cancer was established to confirm the effects of combined administration. Specifically, an AGS cell xenograft model was established using 5-week-old BALB / C nude male mice. After tumor formation following subcutaneous transplantation of AGS gastric cancer cells, the mice were randomly assigned to experimental groups. Docetaxel trihydrate (10 mg / kg) was administered intraperitoneally once a week, and SH003 was administered orally three times a week.

[0297] As a result, as can be confirmed in Fig. 30, it was confirmed that when SH003 and Docetaxel were combined and treated with the gastric cancer cell line AGS, the AGS cell survival rate was decreased in a concentration-dependent manner when each was administered alone. In particular, when the cell survival rate effect by the combination was confirmed, it was confirmed that the cell survival rate was reduced significantly more when the combination was treated than when each was administered alone.

[0298] Furthermore, the combination index (CI) was calculated to determine whether the decrease in cell viability due to combination treatment had a synergistic effect compared to single treatment. As a result, the CI value was less than 1, clearly confirming a synergistic effect. This indicates that the combination treatment of the mixed extract of the present invention and docetaxel has a synergistic effect.

[0299] Based on these results, additional experiments were conducted with a combination treatment concentration with a low CI value.

[0300]

[0301] Next, the cell death induction effect of the combined treatment of the mixed extract of the present invention and docetaxel was confirmed. In particular, it was intended to determine whether the combined treatment induced cell death, one of the cancer cell suppression mechanisms.

[0302] As a result, as can be confirmed in Figure 31, the cell death induction effect confirmed through Annexin V and 7-AAD staining confirmed that cell death increased when combined treatment was performed compared to the single treatment group. In addition, when the expression of PARP, cleaved caspase-3, and cleaved caspase-7, which are protein markers related to the cell death mechanism, was confirmed to be regulated, it was confirmed that all cell death-related proteins increased by combined treatment.

[0303] Furthermore, to confirm whether the apoptosis-induced mechanism by combination therapy is the primary mechanism of gastric cancer cell inhibition, changes in cell viability were observed by treating with a caspase inhibitor, an apoptosis-related protein. As a result, it was confirmed that the expression of PARP and cleaved-caspase 7, which had increased due to combination therapy, was reduced when treated with a caspase inhibitor, confirming that the cell viability inhibition effect induced by combination therapy was suppressed.

[0304] In conclusion, it was confirmed that the cell death induction effect by combined treatment with the mixed extract of the present invention and docetaxel was the main inhibitory mechanism for AGS gastric cancer cell line inhibition.

[0305]

[0306] In summary, it was confirmed that when the mixed extract of the present invention is administered in combination with an anticancer agent, the cancer treatment effect is significantly superior to when each is administered alone, and it was confirmed that the mixed extract of the present invention significantly enhances the anticancer effect of the anticancer agent without side effects.

[0307] In particular, it was confirmed that the mixed extract of the present invention has a significantly better cancer treatment effect when two anticancer drugs are administered together in a 3combi treatment than when one anticancer drug is administered together, compared to when each drug is administered alone or when two anticancer drugs are administered together. It was also confirmed that the anticancer effect is significantly improved without side effects even when administered together with an immuno-cancer drug.

[0308]

[0309] From the above description, those skilled in the art will understand that the present invention can be implemented in other specific forms without altering its technical spirit or essential characteristics. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be interpreted as encompassing all changes or modified forms derived from the meaning and scope of the following claims and their equivalent concepts, rather than the detailed description above.

Claims

1. A pharmaceutical composition for preventing or treating cancer containing a mixed extract of Astragalus membranaceus, Angelica gigas, and Cnidium rhizome, A pharmaceutical composition for preventing or treating cancer, wherein the pharmaceutical composition is administered in combination with an anticancer agent or in combination with radiation treatment.

2. A pharmaceutical composition according to claim 1, wherein the mixed extract is a mixture of Astragalus membranaceus, Angelica gigas, and Cnidium rhizome extracted with at least one solvent selected from the group consisting of water, alcohols having 1 to 4 carbon atoms, and mixed solvents thereof.

3. A pharmaceutical composition according to claim 2, wherein the alcohol having 1 to 4 carbon atoms is 20 to 40% (v / v) of ethanol.

4. A pharmaceutical composition in the first paragraph, wherein the mixed extract of Astragali, Angelica gigas, and Cnidium officinalis is mixed in a weight ratio of Astragali: Angelica gigas: Cnidium officinalis = 0.5 to 5: 0.5 to 1: 0.5 to 1.

5. A pharmaceutical composition according to claim 1, wherein the cancer is lung cancer, breast cancer, melanoma, ovarian cancer, brain cancer, head and neck cancer, pancreatic cancer, stomach cancer, or prostate cancer.

6. A pharmaceutical composition according to claim 5, wherein the lung cancer is non-small cell lung cancer, small cell lung cancer, or Anti-PD1 refractory lung cancer.

7. A pharmaceutical composition according to claim 6, wherein the non-small cell lung cancer is EGFR mutant non-small cell lung cancer or EGFR wild-type non-small cell lung cancer (squamous cell lung cancer).

8. A pharmaceutical composition according to claim 6, wherein the small cell lung cancer is EGFR wild-type small cell lung cancer.

9. A pharmaceutical composition according to claim 6, wherein the breast cancer is HER2-negative breast cancer or HER2-positive breast cancer.

10. A pharmaceutical composition according to claim 6, wherein the head and neck cancer is head and neck cancer and radiation-resistant cancer.

11. A pharmaceutical composition according to claim 1, wherein the anticancer agent is at least one selected from the group consisting of docetaxel (DTX), anti-PD1, gemcitabine, pembrolizumab, doxorubicin (Dox, Doxo), trastuzumab, dabrafenib, trametinib, paclitaxel, carboplatin, temozolomide, and FOLFIRINOX (5-FU, irinotecan, leucovorin, oxaliplatin).

12. A pharmaceutical composition according to claim 1, wherein the composition further comprises a pharmaceutically acceptable carrier, excipient or diluent.

13. A pharmaceutical composition according to claim 1, wherein the composition is administered by intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, or rectal administration.

14. As an anticancer supplement containing a mixed extract of Astragalus membranaceus, Angelica gigas, and Cnidium rhizome, An anticancer adjuvant, characterized in that the above adjuvant is administered in combination with an anticancer drug or in combination with radiation treatment.

15. A method for preventing or treating cancer, comprising administering to a subject a composition of any one of claims 1 to 13.

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