Pharmaceutical combinations and uses thereof

Abstract

The present disclosure provides a pharmaceutical combination comprising a Ganoderma immunomodulatory protein (GMI), a recombinant thereof, or a fragment thereof; and a Danshensu acid B or a pharmaceutically acceptable salt thereof. The present disclosure further provides uses of the pharmaceutical combination for treating cancer stem cells.

Description

Technical Field

[0001] This disclosure relates to a combination of medicines, and more particularly to a combination of medicines and its use. Background Technology

[0002] The cancer stem cell (CSC) theory is receiving increasing attention in cancer research. Multidrug resistance (MDR) in cancer is thought to be primarily caused by the resilience of CSCs. Therefore, extensive research has explored the potential mechanisms of MDR in these cells.

[0003] Cancer is identified as being driven by a subset of self-renewing cell stem cells (CSCs) that can generate various differentiated cell types that constitute tumors. CSCs can be identified and isolated from tumors in various systems, including the hematopoietic system, as well as breast cancer, lung cancer, prostate cancer, colon cancer, brain cancer, head and neck cancer, and pancreatic cancer.

[0004] Current therapies primarily target most tumor cells to reduce tumor size. However, many cancer cells have limited proliferative potential, so the ability to shrink a tumor depends mainly on the ability to eliminate these cells. Treatments that achieve complete tumor regression may still leave sufficient cancer cells (CSCs) to allow for tumor regrowth. Only therapies specifically targeting CSCs are likely to produce more durable responses and hold the promise of curing metastatic tumors. Summary of the Invention

[0005] This disclosure provides a pharmaceutical combination comprising Ganoderma immunomodulatory protein (GMI), its recombinant form or a fragment thereof; and salvianolic acid B or a pharmaceutically acceptable salt thereof.

[0006] This disclosure further provides a method for inhibiting and / or killing CSCs in an individual in need, comprising administering a combination of drugs to the individual.

[0007] This disclosure also provides a method for treating and / or preventing cancers associated with CSC cancers and / or treating metastatic, difficult-to-treat, chemotherapy- or radiotherapy-resistant, chemotherapy-resistant, or recurrent cancers in an individual after initial treatment in an individual in need, comprising administering a combination of drugs to the individual.

[0008] Example 1: A drug combination or composition or kit comprising: Ganoderma lucidum immunomodulatory protein (GMI), its recombinant form or fragment thereof; and salvianolic acid B or a pharmaceutically acceptable salt thereof.

[0009] Example 2: A drug combination or composition or kit as in Example 1, wherein the GMI, its recombinant or fragment thereof is derived from Ganoderma lucidum, Ganoderma tsugae, Ganoderma microsporum or Ganoderma sinensis.

[0010] Example 3: A drug combination or composition or kit as described in any of Examples 1 and 2, wherein the GMI, its recombinant or fragment thereof is derived from Ganoderma lucidum microspores.

[0011] Example 4: A drug combination or composition or kit as described in any one of Examples 1 to 3, wherein the GMI, its recombinant or fragment thereof comprises the amino acid sequence of SEQ ID NO: 3.

[0012] Example 5: A pharmaceutical combination or composition or kit as described in any one of Examples 1 to 4, wherein the recombinant GMI comprises the amino acid sequence of SEQ ID NO: 4.

[0013] Example 6: A pharmaceutical combination or composition or kit as described in any one of Examples 1 to 5, wherein the fragment of GMI comprises the amino acid sequence of SEQ ID NO: 1 or 2.

[0014] Example 7: A pharmaceutical combination or composition or kit as described in any of Examples 1 to 6, further comprising at least one pharmaceutically acceptable carrier.

[0015] Example 8: A drug combination or composition or kit as described in any of Examples 1 to 7, which is in the form of a kit of parts for combined administration.

[0016] Example 9: A combination or set of drugs, such as any one of Examples 1 to 8, in the form of a combination product or pharmaceutical composition.

[0017] Example 10: A method for inhibiting and / or killing cancer stem cells in an individual in need, comprising administering to the individual a combination or set of drugs as described in any one of Examples 1 to 8.

[0018] Example 11: The method of Example 10, wherein these cancer stem cells are selected from a group composed of pancreatic cancer stem cells and glioblastoma cancer stem cells.

[0019] Example 12: The method of any one of Examples 10 and 11, wherein the cancer stem cells are selected from a group composed of pancreatic cancer stem cells and neuroblastoma cancer stem cells.

[0020] Example 13: The method of any one of Examples 10 to 12, wherein the pancreatic cancer stem cells are pancreatic cancer cells expressing at least one of CD133, CD24, CD44, DCLK1, CXCR4, ESA, Oct4 and ABCB1.

[0021] Example 14: The method of any one of Examples 10 to 13, wherein the glioblastoma cancer stem cells are glioblastoma cancer cells expressing at least one of CD133, CD90, CD44, L1CAM, A2B5 and GPD1.

[0022] Example 15: The method of any one of Examples 10 to 14, wherein the GMI, its recombinant or fragment thereof, and salvianolic acid B or its pharmaceutically acceptable salt are administered simultaneously or independently.

[0023] Example 16: The method of any one of Examples 10 to 15, wherein the GMI, its recombinant or fragment thereof, and salvianolic acid B or its pharmaceutically acceptable salt are administered separately at time intervals.

[0024] Example 17: A method for treating and / or preventing cancer associated with CSC cancer or treating metastatic, difficult-to-treat, chemotherapy- or radiotherapy-resistant, chemotherapy-resistant, or recurrent cancer in an individual after initial treatment in an individual in need, comprising administering to the individual a combination of drugs as described in technical solution 1.

[0025] Example 18: The method of Example 17, wherein the cancer is a metastatic, refractory or recurrent cancer selected from breast cancer, head and neck cancer, lung cancer, ovarian cancer, pancreatic cancer, colorectal cancer, prostate cancer, liver cancer, melanoma, multiple myeloma, brain tumor, sarcoma, neuroblastoma, glioblastoma, leukemia and any of the above cancers.

[0026] Example 19: The method of any one of Examples 17 and 18, wherein the pancreatic cancer comprises pancreatic cancer cells expressing at least one of CD133, CD24, CD44, DCLK1, CXCR4, ESA, Oct4 and ABCB1.

[0027] Example 20: The method of any one of Examples 17 to 19, wherein the glioblastoma cancer comprises glioblastoma cancer cells expressing at least one of CD133, CD90, CD44, L1CAM, A2B5 and GPD1.

[0028] Example 21: The method of any one of Examples 17 to 20, wherein the GMI, its recombinant or fragment thereof, and salvianolic acid B or its pharmaceutically acceptable salt are administered simultaneously or independently.

[0029] Example 22: The method of any one of Examples 17 to 21, wherein the GMI, its recombinant or fragment thereof, and salvianolic acid B or its pharmaceutically acceptable salt are administered separately at time intervals. Attached Figure Description

[0030] Figures 1A to 1C This study demonstrates the cytotoxic effects of GMI and SalB on PBMCs. Figure 1A GMI. Figure 1B SalB. Figure 1C GMI + SalB.

[0031] Figures 2A to 2C This demonstrates the synergistic effect of GMI and SalB on pancreatic cancer stem cells (PCSC). Figure 2A GMI. Figure 2B SalB. Figure 2C GMI + SalB.

[0032] Figures 3A to 3C This study demonstrates the synergistic effect of GMI and SalB on glioblastoma cancer stem cells (GBCSC). Figure 3A GMI. Figure 3B SalB. Figure 3C GMI + SalB. Detailed Implementation

[0033] Unless otherwise defined, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. While any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of this disclosure, preferred methods and materials are described here. All publications referenced herein are incorporated herein by reference.

[0034] In this application, unless otherwise specifically stated, the use of the singular includes the plural, the article “a” or “an” means “at least one”, and the use of “or” means “and / or”.

[0035] The term “preventing” or “prevention” is recognized in this art and, when used in conjunction with symptoms, includes administering a medication to reduce the frequency or severity of an individual’s medical symptoms or to delay their onset relative to an individual who has not received the medication.

[0036] The terms “individual,” “subject,” “host,” and “patient” used interchangeably in this article may refer to mammals, including (but not limited to) rodents (e.g., rats, mice), non-human primates, humans, canines, felines, ungulates (e.g., equines, bovines, sheep, suidae, goats), etc.

[0037] As used herein, the term "effective amount" means the amount by which a particular biological outcome is effectively achieved, such as, but not limited to, the biological outcomes disclosed, described, or illustrated herein. Such outcomes may include, but are not limited to, the treatment of cancer stem cells.

[0038] Cancer stem cells (CSCs) are capable of self-renewal and differentiation, which may be related to chemotherapy resistance. The cancer stem cell theory has attracted widespread attention because cancer stem cells can evade the cytotoxic effects of chemotherapy drugs, potentially explaining chemotherapy failure. Like normal stem cells, cancer stem cells typically remain in the G0 phase of the cell cycle, preventing them from entering the cell cycle. Currently, most chemotherapy drugs used in clinical settings focus on inhibiting the cell cycle and cell proliferation. Therefore, chemotherapy drugs can inhibit the growth of non-stem cancer cells that actively cycle but cannot kill cancer stem cells. After chemotherapy ends, surviving cancer stem cells can regenerate and proliferate, leading to cancer recurrence. Additionally, some literature suggests that chemotherapy drugs can promote subsequent proliferation of cancer stem cells. Current research indicates that known cancer treatments (chemotherapy drugs) or drugs that treat non-stem cancer cells may be insufficient to treat or kill cancer stem cells.

[0039] This disclosure unexpectedly reveals that Ganoderma lucidum immunomodulatory proteins and salvianolic acid B can kill cancer stem cells. Specifically, Ganoderma lucidum immunomodulatory proteins and salvianolic acid B have a synergistic effect on cancer stem cells, such as pancreatic cancer stem cells and glioblastoma cancer stem cells.

[0040] Therefore, this disclosure provides a pharmaceutical combination comprising GMI, its recombinant form or a fragment thereof; and salvianolic acid B or a pharmaceutically acceptable salt thereof.

[0041] Fungal immunomodulatory proteins (FIPs) are a group of proteins found in fungi whose immunomodulatory activities have been extensively studied, including activating immune cells to produce immune-mediated anti-allergic, anti-inflammatory, and anti-tumor effects. GMI is a type of FIP derived from Ganoderma lucidum, Ganoderma tsugae, Ganoderma microsporum, or Ganoderma sinensis.

[0042] The preparation of GMI, its recombinant form, or fragments thereof has been described in U.S. Patent No. 7,601,808. Specifically, in U.S. Patent No. 7,601,808, the Ganoderma lucidum immunomodulatory protein is referred to as GMI, the recombinant form of the Ganoderma lucidum immunomodulatory protein is referred to as reGMI, and the fragments of the Ganoderma lucidum immunomodulatory protein are referred to as SEQ ID NO: 2 and 3.

[0043] In one embodiment, the GMI described herein, its recombinant form, or a fragment thereof comprises the amino acid sequence of SEQ ID NO: 3. In one embodiment, the recombinant form of GMI comprises the amino acid sequence of SEQ ID NO: 4. In one embodiment, a fragment of GMI comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to 2.

[0044] The sequences of SEQ ID NO: 1 to 4 are listed below.

[0045] (SEQ ID NO: 1)

[0046] LAWNVK

[0047] (SEQ ID NO: 2)

[0048] DLGVRPSYAV

[0049] (SEQ ID NO: 3)

[0050] MSDTALIFTLAWNVKQLAFDYTPNWGRGRPSSFIDTVTFPTVLTDKAYTYRVVVSGKDLGVRPSYAVESDGSQKINFLEYNSGYGIADTNTIQVYVIDPDTGNNFIVAQWN

[0051] (SEQ ID NO: 4)

[0052] EEAEFMSDTALIFTLAWNVKQLAFDYTPNWGRGRPSSFIDTVTFPTVLTDKAYTYRVVVSGKDLGVRPSYAVESDGSQKINFLEYNSGYGIADTNTIQVYVIDPDTGNNFIVAQWNYLEQKLISEEDLNSAVDHHHHHH

[0053] Tanshinone is a water-soluble component produced by many species of the genus *Salvia*, primarily extracted from *Salvia miltiorrhiza*. It is a group of polyphenolic acids composed of caffeic acid and tanshinone (tanshinone acid) through various combinations of ester and enol bonds. Tanshinone B (SalB) consists of three molecules of tanshinone and one molecule of caffeic acid. SalB is the most abundant and biologically active member of the hydrophilic components in *Danshen*, containing seven phenolic hydroxyl groups, which have been found to be closely related to redox potential and / or antioxidant activity.

[0054] In some embodiments of this disclosure, the pharmaceutical combination may be in the form of a kit of dispensing portions for combined administration. Alternatively, the pharmaceutical combination may be in the form of a combination product or a pharmaceutical composition.

[0055] In some embodiments of this disclosure, the drug combination is used to treat cancer, particularly to treat cancer stem cells. In some embodiments of this disclosure, the cancer stem cells are pancreatic cancer stem cells and / or glioblastoma cancer stem cells. Pancreatic cancer stem cells can be isolated from pancreatic cancer cells and can express CD133, CD24, CD44, DCLK1, CXCR4, ESA, Oct4, and / or ABCB1. Glioblastoma cancer stem cells can be isolated from glioblastoma cancer cells and can express CD133, CD90, CD44, L1CAM, A2B5, and / or GPD1.

[0056] In some embodiments of this disclosure, the cancer is pancreatic cancer and / or glioblastoma cancer. Pancreatic cancer may include pancreatic cancer cells expressing CD133, CD24, CD44, DCLK1, CXCR4, ESA, Oct4, and / or ABCB1. Glioblastoma cancer may include glioblastoma cancer cells expressing CD133, CD90, CD44, L1CAM, A2B5, and / or GPD1.

[0057] In some embodiments of this disclosure, GMI, its recombinant form or fragment thereof, and salvianolic acid B or its pharmaceutically acceptable salt may be administered simultaneously or independently, or separately at time intervals.

[0058] In some embodiments, the combination of GMI and salvianolic acid B discussed herein can be administered simultaneously as a single composition in a pharmaceutically acceptable carrier, or simultaneously as a single composition each comprising GMI and salvianolic acid B in a pharmaceutically acceptable carrier. In another embodiment, the combination of GMI and salvianolic acid B can be administered sequentially. For example, GMI and salvianolic acid B can be administered sequentially, such as administering GMI first and then salvianolic acid B, or administering salvianolic acid B first and then GMI. Furthermore, if more than one dose of the combination therapy is administered sequentially, the order of administration can be reversed or maintained at each administration point, and sequential administration can be combined with simultaneous administration, or any combination thereof. For example, the first administration of the combination of GMI and salvianolic acid B can be simultaneous, the second administration can be sequentially GMI first and then salvianolic acid B, and the third administration can be sequentially salvianolic acid B first and then GMI, etc. Another representative dosing regimen may involve a first administration, which may be in the order of first administering salvianolic acid B followed by GMI, or first administering GMI followed by salvianolic acid B, and subsequent administrations may be carried out simultaneously.

[0059] The dosage of a pharmaceutical combination suitable for use according to this disclosure can be determined by a person skilled in the art based on this disclosure. The pharmaceutical formulation contains an effective dose (depending on the route of administration of the active agent and pharmacokinetics) of a suitable pharmaceutical carrier and excipient suitable for a specific route of administration (i.e., oral, non-enteral, local, or inhalation) of the formulation. The pharmaceutical combination is incorporated into the pharmaceutical composition through processes such as mixing, dissolving, granulation, tablet coating, emulsification, encapsulation, coating, or lyophilization.

[0060] Each of the two components of the disclosed drug combination can be administered to a patient alone or in combination with a suitable carrier and excipient. The disclosed drug combination can be administered non-enterically, such as by intravenous injection or infusion, intraperitoneal injection, subcutaneous injection, or intramuscular injection. The disclosed drug combination can be administered orally or rectally by appropriate formulation with a carrier and excipient to form tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions, and the like. The disclosed drug combination can be administered topically, such as via a skin patch. The disclosed drug combination can be formulated into a topical cream, skin or mucosal patch, liquid, or gel suitable for topical application to the skin or mucous membrane surface. The disclosed drug combination can be administered into the respiratory tract via an inhaler for local or systemic treatment.

[0061] The information presented in this disclosure, along with recent advances in CSC research, enables this disclosure to provide a range of methods aimed at inhibiting CSCs or specifically treating CSC-containing cancers, as well as for the broader treatment of cancers. Therefore, methods for inhibiting and / or killing CSCs through combinations of this disclosure, or for treating and / or preventing CSC-related cancers using combinations of this disclosure, are also provided.

[0062] Cancers with CSCs are known to be good candidates for this type of treatment, and include (but are not limited to): various types of breast cancer, head and neck cancer, lung cancer, ovarian cancer, pancreatic cancer, colorectal cancer, prostate cancer, liver cancer, melanoma, multiple myeloma, brain tumors, sarcomas, neuroblastoma, glioblastoma, and leukemia. Many metastatic forms of these cancers (such as metastatic breast cancer) also show the presence of CSCs. In one embodiment, the methods described herein can be applied to treat cancers selected from this group. In one embodiment, the methods disclosed herein can be used to treat cancers including (but not limited to) lung cancer, breast cancer, cervical cancer, colorectal cancer, liver cancer, head and neck cancer, pancreatic cancer, gastric cancer, brain cancer, glioblastoma, and prostate cancer.

[0063] Because CSC has been shown to play an important role in tumor formation, cancer metastasis and recurrence, the methods disclosed herein can be applied to the treatment of metastatic cancer that is resistant to chemotherapy or radiotherapy, is itself resistant to chemotherapy, or recurs after initial treatment.

[0064] The following examples are used to illustrate the pharmaceutical combinations and uses of this disclosure, but are not intended to limit the scope of this disclosure.

[0065] Example 1

[0066] 1.1 Culture of human pancreatic cancer stem cells and glioblastoma cancer stem cells

[0067] Pancreatic cancer stem cells (PCSCs) and glioblastoma cancer stem cells (GBCSCs) were purchased from Celprogen (CA, USA). PCSCs and GBCSCs were seeded in 10 cm Petri dishes containing AIM-V medium (Thermo Fisher Scientific, MA, USA) with 10% FBS. When confluence was achieved, cells were isolated with 0.15% trypsin and incubated at 37°C for 6 min. The collected cells were then subcultured at the initial seeding density every 3 days. Cell morphology was photographed using a phase-contrast microscope.

[0068] 1.2 Preparation of PBMCs

[0069] Whole blood was collected into heparin-coated blood collection tubes and centrifuged at 400×g for 20 min at room temperature. After centrifugation, the plasma fraction was removed. The erythrocyte sedimentation rate (ESR) buffy coat and erythrocyte fraction were mixed with an equal volume of PBS and slowly and gently released into a 15 ml conical tube containing 4 ml of ficoll-paque solution without disturbing the ficoll interface. The tubes were centrifuged at 600×g for 20 min at room temperature without braking. The mononuclear cell layer was collected into a new 15 ml conical tube and washed three times with PBS. Finally, the cells were resuspended in cell culture medium and seeded into 24-well plates (2×10⁶ cells / wells). 5 (cells / well).

[0070] 1.3 CFSE staining

[0071] PCSC and GBCSC are set at 1×10 7 Cells were suspended at a concentration of [number] cells / mL in 1× assay buffer. An equal volume of CFSE solution was added to the cell suspension, and the cells were incubated at 37°C for 15 minutes. After incubation, the cells were washed three times with PBS. Finally, the cells were resuspended in cell culture medium and seeded into 24-well plates (1×10⁶ cells / mL). 5 (cells / well).

[0072] 1.4 Cytotoxicity Analysis

[0073] The cytotoxicity of GMI and SalB was assessed using 7-AAD staining. In short, PCSC / GBCSC (1×10⁻⁶) was used. 5 (cells / well) and PBMCs (2×10) 5 Cells (per well) were seeded in 24-well plates and incubated at 37°C for 48 h in the presence of different concentrations of GMI and SalB, followed by 7-AAD staining (Pharmingen, CA, USA). Cells were then analyzed using a flow cytometer (BD Accuracy). TM C6 Plus) analysis of cell death rate.

[0074] 1.5 Statistical Analysis

[0075] Statistical data are presented as mean ± SEM. Student's t-test was used for statistical analysis to compare the two groups, with significance defined as a p-value less than 0.05.

[0076] Example 2

[0077] 2.1 Cytotoxic effects of GMI and salvianolic acid B on PBMCs

[0078] PBMC (2×10) 5Cells were seeded in 24-well plates and treated with different concentrations of GMI and salvianolic acid B for 48 hours. After 7-AAD staining, the cytotoxic effects of GMI and salvianolic acid B were further analyzed by flow cytometry. Figure 1A As shown, there was no significant difference between the control and GMI-treated PBMCs. Although salvianolic acid B slightly induced cell death in PBMCs, the cell death rate was less than 10%. Figure 1B We also found that under GMI and salvianolic acid B treatment, the cell death rate of PBMCs was less than 10%. Figure 1C Based on these results, we confirm that GMI and salvianolic acid B treatment will not cause serious damage to PBMCs.

[0079] Example 3

[0080] 3.1 Synergistic effect of GMI and salvianolic acid B on pancreatic cancer stem cells

[0081] To simulate the tumor microenvironment, we investigated the cytotoxic effects of GMI and salvianolic acid B on PCSCs in the presence of PBMCs. First, PCSCs (1×10⁻⁶) were... 5 (cells) and PBMCs (2×10) 5 PCSCs were seeded in 24-well plates and treated with different concentrations of GMI or salvianolic acid B for 48 hours. After 7-AAD staining, PCSC mortality was analyzed by flow cytometry. Figure 2A As shown, GMI treatment induced cell death in PCSCs. However, tanshinone B treatment had no effect on PCSCs. Figure 2B However, when combined with salvianolic acid B, the sensitivity to GMI in PCSC was significantly improved ( Figure 2C Overall, these findings confirm the enhanced anti-PCSC activity through the synergistic effect of GMI and salvianolic acid B.

[0082] 3.2 Synergistic effect of the disclosed GMI and salvianolic acid B against PCSC cytotoxicity

[0083] Three different effects of various drugs have been proposed, including (1) synergistic effect; (2) additive effect; and (3) antagonistic effect. The combination index (CI), published by Chou TC et al., Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors, Adv Enzyme Regul, Vol. 22, 1984, pp. 27-55, and Liang Zhao et al., Evaluation of Combination Chemotherapy: Integration of Nonlinear Regression, Curve Shift, Isobologram, and Combination Index Analysis, Clinical Cancer Research, Vol. 10, 2004, pp. 7994-8004, can be used to analyze drug-drug interactions. The calculation formula is CI = C A,x / IC x,A + C B,x / IC x,B Among them, C A,x and C B,x IC represents the concentration of drug A and drug B when used in combination to achieve x% drug effect. x,A and IC x,B CI: The concentration of a single drug agent required to achieve x% of drug effect. CI < 1 indicates a synergistic effect when used in combination. CI = 1 indicates an additive effect when used in combination. CI > 1 indicates an antagonistic effect when used in combination.

[0084] In this example, Figures 2A to 2C The results present statistical data on cytotoxicity rates from three independent experiments analyzed by flow cytometry. Figure 2A As shown, the cytotoxicity rates of the control group, the GMI group (20 μg / mL), and the GMI group (100 μg / mL) were 1.17%, 25.4%, and 86.53%, respectively. Figure 2B As shown, the cytotoxicity rates of the salvianolic acid B group (100 μg / mL) and the salvianolic acid B group (200 μg / mL) were 1.67% and 2.0%, respectively. Linear equations for cytotoxicity in the GMI group and the salvianolic acid B group were calculated using these equivalent values. An example is the treatment with GMI and salvianolic acid B (GMI 20 μg / mL + salvianolic acid B 100 μg / mL). Figure 2C The cytotoxicity of 44.9% was placed into the y-values ​​of the GMI cytotoxicity linear equation and the salvianolic acid B cytotoxicity linear equation, respectively, to obtain the IC50. x,GMI =50.79, and IC x,SalB =10818.79. Next, this value is placed in the CI formula, resulting in CI = (20 / 50.79) + (100 / 10818.79) = 0.4. A CI value < 1 indicates that the GMI (20 μg / mL) plus salvianolic acid B (100 μg / mL) group has a synergistic effect on PCSC cytotoxicity. The CI value of the GMI (20 μg / mL) plus salvianolic acid B (200 μg / mL) group was calculated using the same method. Figure 2C The CI value was 0.36. A CI value < 1 indicates that the GMI (20 μg / mL) plus salvianolic acid B (200 μg / mL) group also had a synergistic effect on PCSC cytotoxicity. On the other hand, the CI value of the GMI (100 μg / mL) plus salvianolic acid B (100 μg / mL) group was 0.92; and the CI value of the GMI (100 μg / mL) plus salvianolic acid B (200 μg / mL) group was 0.9. CI values ​​< 1 for both groups indicate that the GMI treatment alone ( Figure 2A ) and treatment with only salvianolic acid B ( Figure 2B Compared to GMI plus salvianolic acid B treatment () Figure 2C It has a synergistic effect on PCSC cytotoxicity.

[0085] Example 4

[0086] 4.1 Synergistic effect of GMI and Tanshinone B on glioblastoma cancer stem cells

[0087] To simulate the tumor microenvironment, we investigated the cytotoxic effects of GMI and salvianolic acid B on GBCSCs in the presence of PBMCs. First, GBCSCs (1×10⁻⁶) were... 5 (cells) and PBMCs (2×10) 5 GBCSCs were seeded in 24-well plates and treated with different concentrations of GMI for 48 hours. After 7-AAD staining, GBCSC mortality was analyzed using flow cytometry. Figure 3A As shown, GMI treatment induced cell death in GBCSCs. Although salvianolic acid B slightly induced cell death in GBCSCs, the cell death rate was less than 10%. Figure 3B However, when combined with salvianolic acid B, the sensitivity to GMI in GBCSCs was significantly improved. Figure 3C Overall, these findings confirm the enhanced anti-GBCSC activity through the synergistic effect of GMI and salvianolic acid B.

[0088] 4.2 Synergistic effect of the disclosed GMI and salvianolic acid B against GBCSC cytotoxicity

[0089] Three different effects of various drugs have been proposed, including (1) synergistic effect; (2) additive effect; and (3) antagonistic effect. The combination index (CI), published by Chou TC et al., Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors, Adv Enzyme Regul, Vol. 22, 1984, pp. 27-55, and Liang Zhao et al., Evaluation of Combination Chemotherapy: Integration of Nonlinear Regression, Curve Shift, Isobologram, and Combination Index Analysis, Clinical Cancer Research, Vol. 10, 2004, pp. 7994-8004, can be used to analyze drug-drug interactions. The calculation formula is CI = C A,x / IC x,A + C B,x / IC x,B Among them, C A,x and C B,x IC represents the concentration of drug A and drug B when used in combination to achieve x% drug effect. x,A and IC x,B CI: The concentration of a single drug agent required to achieve x% of drug effect. CI < 1 indicates a synergistic effect when used in combination. CI = 1 indicates an additive effect when used in combination. CI > 1 indicates an antagonistic effect when used in combination.

[0090] In this example, Figures 3A to 3C The results present statistical data on cytotoxicity rates from three independent experiments analyzed by flow cytometry. Figure 3A As shown, the cytotoxicity rates of the control group, the GMI group (20 μg / mL), and the GMI group (100 μg / mL) were 0.7%, 32%, and 67.8%, respectively. Figure 3BAs shown, the cytotoxicity rates of the salvianolic acid B group (100 μg / mL) and the salvianolic acid B group (200 μg / mL) were 4.9% and 5.67%, respectively. Linear equations for cytotoxicity in the GMI group and the salvianolic acid B group were calculated using these equivalent values. An example is the treatment with GMI and salvianolic acid B (GMI 20 μg / mL + salvianolic acid B 100 μg / mL). Figure 3C The cytotoxicity of 56.2% was placed into the y-values ​​of the GMI cytotoxicity linear equation and the salvianolic acid B cytotoxicity linear equation, respectively, to obtain the IC50 value. x,GMI =77.12, and IC x,SalB =2282.87. Next, this value is placed in the CI formula, resulting in CI = (20 / 77.12) + (100 / 2282.87) = 0.3. A CI value < 1 indicates that the GMI (20 μg / mL) plus salvianolic acid B (100 μg / mL) group has a synergistic effect on GBCSC cytotoxicity. The CI value of the GMI (20 μg / mL) plus salvianolic acid B (200 μg / mL) group was calculated using the same method. Figure 3C The CI value was 0.28. A CI value < 1 indicates that the GMI (20 μg / mL) plus salvianolic acid B (200 μg / mL) group also had a synergistic effect against GBCSC cytotoxicity. On the other hand, the CI value of the GMI (100 μg / mL) plus salvianolic acid B (100 μg / mL) group was 0.83; and the CI value of the GMI (100 μg / mL) plus salvianolic acid B (200 μg / mL) group was 0.82. CI values ​​< 1 for both groups indicate that the GMI treatment alone ( Figure 3A ) and treatment with only salvianolic acid B ( Figure 3B Compared to GMI plus salvianolic acid B treatment () Figure 3C It has a synergistic effect on GBCSC cytotoxicity.

[0091] While this disclosure has been described and illustrated with reference to specific embodiments thereof, such description and illustration are not limiting. Those skilled in the art will understand that various changes and alternatives may be made without departing from the true spirit and scope of this disclosure as defined by the appended claims. Illustrations need not be drawn to scale. Differences may exist between artistic representations in this disclosure and actual installations due to manufacturing processes and tolerances. Other embodiments of this disclosure may exist that are not specifically described. This specification and accompanying drawings should be considered illustrative rather than limiting. Modifications may be made to adapt particular circumstances, materials, compositions, methods, or processes to the objectives, spirit, and scope of this disclosure. All such modifications are intended to be within the scope of the appended claims. Although the methods disclosed herein have been described with reference to specific operations performed in a particular order, it should be understood that these operations may be combined, subdivided, or reordered to form equivalent methods without departing from the teachings of this disclosure. Therefore, unless specifically indicated herein, the order and grouping of operations are not limitations of this disclosure.

Claims

1. A drug combination comprising: Ganoderma immunomodulatory protein (GMI), its recombinant form or fragment thereof; and Salvianolic acid B or its pharmaceutically acceptable salt.

2. The pharmaceutical combination of claim 1, wherein the GMI, its recombinant form or fragment thereof is derived from Ganoderma lucidum, Ganoderma tsugae, Ganoderma microsporum or Ganoderma sinensis.

3. The pharmaceutical combination of claim 1, wherein the GMI, its recombinant form or fragment thereof is derived from Ganoderma lucidum microspores.

4. The pharmaceutical combination of claim 1, wherein the GMI, its recombinant form or fragment thereof comprises the amino acid sequence of SEQ ID NO:

3.

5. The pharmaceutical combination of claim 1, wherein the recombinant GMI comprises the amino acid sequence of SEQ ID NO:

4.

6. The pharmaceutical combination of claim 1, wherein the fragment of GMI comprises the amino acid sequence of SEQ ID NO: 1 or 2.

7. The pharmaceutical combination of claim 1, further comprising at least one pharmaceutically acceptable carrier.

8. The drug combination as claimed in claim 1, wherein it is in the form of a kit of dispensing portions for combined administration.

9. The pharmaceutical combination as described in claim 1, wherein it is in the form of a combination product or a pharmaceutical composition.

10. Use of the pharmaceutical combination of claim 1 in the preparation of a medicament for inhibiting and / or killing cancer stem cells in an individual in need.

11. The use as claimed in claim 10, wherein the cancer stem cells are selected from a group consisting of pancreatic cancer stem cells and glioblastoma cancer stem cells.

12. The use as claimed in claim 11, wherein the pancreatic cancer stem cells are pancreatic cancer cells expressing at least one of CD133, CD24, CD44, DCLK1, CXCR4, ESA, Oct4, and ABCB1.

13. The use as claimed in claim 11, wherein the glioblastoma cancer stem cells are glioblastoma cancer cells expressing at least one of CD133, CD90, CD44, L1CAM, A2B5 and GPD1.

14. The use as described in claim 10, wherein the GMI, its recombinant form or a fragment thereof, and salvianolic acid B or a pharmaceutically acceptable salt thereof are administered simultaneously or independently.

15. The use as claimed in claim 10, wherein the GMI, its recombinant form or a fragment thereof, and salvianolic acid B or a pharmaceutically acceptable salt thereof are administered separately at time intervals.

16. Use of the pharmaceutical combination of claim 1 in the preparation of a medicament for treating and / or preventing cancer associated with CSC cancer or for treating metastatic, difficult-to-treat, chemotherapy- or radiotherapy-resistant, or recurrent cancer in an individual after initial treatment in an individual in need.

17. The use as described in claim 16, wherein the cancer is a metastatic, refractory, or recurrent cancer selected from breast cancer, head and neck cancer, lung cancer, ovarian cancer, pancreatic cancer, colorectal cancer, prostate cancer, liver cancer, melanoma, multiple myeloma, brain tumor, sarcoma, neuroblastoma, glioblastoma, leukemia, and any of the above-mentioned cancers.

18. The use as claimed in claim 17, wherein the pancreatic cancer comprises pancreatic cancer cells expressing at least one of CD133, CD24, CD44, DCLK1, CXCR4, ESA, Oct4, and ABCB1.

19. The use as claimed in claim 17, wherein the glioblastoma cancer comprises glioblastoma cancer cells expressing at least one of CD133, CD90, CD44, L1CAM, A2B5, and GPD1.

20. The use as claimed in claim 16, wherein the GMI, its recombinant form or a fragment thereof, and salvianolic acid B or a pharmaceutically acceptable salt thereof are administered simultaneously or independently.

21. The use as claimed in claim 16, wherein the GMI, its recombinant form or a fragment thereof, and salvianolic acid B or a pharmaceutically acceptable salt thereof are administered separately at time intervals.

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