Use of long-acting β2AR agonists in the preparation of cancer therapeutic agents

Abstract

The present invention discloses the use of a long-acting β2AR agonist or a pharmaceutically acceptable salt thereof in the preparation of a cancer treatment drug. The long-acting β2AR agonist of the present invention uses the β2AR receptor on the cancer cell membrane as a single functional molecular target, and can inhibit the self-renewal ability of cancer stem cells, thereby inhibiting the development and progression of cancer, and can be used clinically in the treatment of cancer. Furthermore, long-acting β2AR agonists can improve the success rate of drug discovery in clinical translation by redeveloping existing drugs.

Description

[Technical Field]

[0001] The present invention relates to the field of small molecule drugs, and specifically to the use of long-acting β2AR agonists in the preparation of cancer therapeutic drugs. [Background technology]

[0002] Cancer is the second leading cause of death in humans after cardiovascular and cerebrovascular diseases, which are the leading causes of death. In 2020, 19.3 million people were diagnosed with cancer worldwide, and 10 million people died from cancer, accounting for approximately one-sixth of the total deaths that year.

[0003] Surgical resection began to be formally used in the treatment of cancer in the mid-19th century. The discovery of radioactive elements later laid the foundation for cancer radiation therapy. In 1949, the first chemotherapy drug, nitrogen mustard, was approved by the U.S. FDA for the treatment of Hodgkin's lymphoma. Since then, surgery, radiation therapy, and chemotherapy have gradually become the three main approaches to cancer treatment. The discovery of the double helix structure of DNA in 1953 ushered in the era of molecular biology. In recent decades, as researchers have delved deeper into cancer at the molecular level and applied it to further transformation, molecular targeted therapy and immunotherapy have become new treatments. While surgery, radiation therapy, chemotherapy, molecular targeted therapy, and immunotherapy have significantly improved the five-year survival rate and prognosis for cancer patients, the five-year survival rate for most cancers, including lung cancer, liver cancer, pancreatic cancer, and gastric cancer, remains below 30%. Therefore, the development of new drugs with effective therapeutic effects is urgently needed.

[0004] Small molecule targeted drugs have the advantages of being easy to synthesize, inexpensive, rapidly absorbed, and easy to store. Imatinib is the first small molecule targeted drug used clinically, and targets BCR, a fusion protein formed by recombination of a proto-oncogene. -It specifically inhibits the activation of ABL tyrosine kinase and induces apoptosis in cancer cells. Clinical data show that patients treated with imatinib as a first-line treatment for chronic myeloid leukemia had a high overall 10-year survival rate of 83.3%. By the end of 2020, 89 small molecule targeted anticancer drugs had been approved by the US FDA and the China National Medical Products Administration. These include gefitinib, an inhibitor targeting epidermal growth factor receptor tyrosine kinase (EGFR-TK), everolimus, an inhibitor targeting mTOR, and vemurafenib, an inhibitor targeting BRAF, all of which are typical clinical drugs in cancer treatment.

[0005] However, in the case of small molecule targeted drugs, phenomena such as unstable response, drug resistance, and recurrence remain the main problems currently faced by clinical cancer patients after administration.In recent years, many studies have demonstrated that cancer tissues are heterogeneous, and even within the same cancer tissue, there exist cancer cells with completely different genotypes, phenotypes, and functions.

[0006] In 1937, Furth and Kanh discovered that single mouse cancer cells could form new tumors when transplanted into recipient mice. In 1960, Pierce discovered highly tumorigenic cells in malignant teratomas, and these cells could further differentiate into various non-tumorigenic cells. In the early 1990s, with the development of hematopoietic stem cell research and flow cytometry technology, Dick et al. demonstrated the CD34 expression of CD34 in a mouse transplant model of acute myeloid leukemia (AML). + CD38 - He discovered that only cancer cells from this group could form transplantable tumors, and that the frequency of the cells of origin of transplantable tumors was one in a million. This group of cells was defined as cancer stem cells in AML, and the concept of "cancer stem cells" was clearly proposed for the first time. In 2003, Clarke first applied the concept of "cancer stem cells" and xenotransplantation experimental methods to solid tumors of the breast, and found that CD44 + CD24 -In the following 20 years, the study of cancer stem cells has continued to deepen and has been demonstrated in various types of cancer.

[0007] The results of a large number of animal experiments have further demonstrated that cancer stem cells, although accounting for a relatively small proportion of the total cancer cell population, have the ability to self-renew and differentiate into multiple cells, and are the main causes of cancer initiation, growth, drug resistance, immune evasion, recurrence and metastasis. This has led to the hypothesis that targeted inhibition of cancer stem cells is the most ideal means of clinical cancer treatment.

[0008] However, currently, there is no single molecular targeted drug in clinical practice that can specifically target cancer stem cells and further treat cancer. Therefore, finding a specific cancer stem cell target and developing a single molecular targeted drug that specifically targets cancer stem cells, has strong efficacy, is highly safe, and is inexpensive, which may significantly extend and improve the survival of cancer patients. Summary of the Invention

[0009] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. For this purpose, the present invention provides a use of a long-acting β2AR agonist or a pharmaceutically acceptable salt thereof in preparing a cancer treatment drug, wherein the long-acting β2AR agonist can target and inhibit cancer stem cells for cancer treatment.

[0010] According to one aspect of the present invention, there is provided use of a long-acting β2AR agonist or a pharmaceutically acceptable salt thereof in the preparation of a cancer therapeutic agent.

[0011] According to a specific embodiment of the present invention, the specific long-acting β2AR agonist has at least the following beneficial effects: the surface receptor β2AR on the cancer cell membrane is the sole functional molecular target, and by targeting and inhibiting the self-renewal ability of cancer stem cells, it exerts the function of inhibiting the development and progression of cancer, and can be clinically used in cancer treatment. Furthermore, long-acting β2AR agonists can improve the success rate of drug discovery in clinical translation by redeveloping existing drugs.

[0012] In some embodiments of the present invention, the long-acting β2AR agonist includes all clinically approved long-acting specific β2 adrenergic receptor agonists.

[0013] In some embodiments of the present invention, the long-acting β2AR agonist is a specific β2 adrenergic receptor agonist with a duration of efficacy of 12 hours or more.

[0014] In some embodiments of the invention, the long-acting β2AR agonist comprises at least one of Arformoterol, Bambuterol, Clenbuterol, Formoterol, Indacaterol, Olodaterol, Salmeterol, Tulobuterol, and Vilanterol.

[0015] In some preferred embodiments of the invention, the long-acting β2AR agonist comprises at least one of Arformoterol Tartrate, Bambuterol Hydrochloride, Clenbuterol Hydrochloride, Formoterol Hemifumarate, Indacaterol Maleate, Olodaterol Hydrochloride, Salmeterol Xinafoate, Tulobuterol Hydrochloride, and Vilanterol Trifenate.

[0016] In some embodiments of the invention, the cancer comprises at least one of breast cancer, colorectal cancer, lung cancer, head and neck cancer, esophageal cancer, bladder cancer, melanoma, hematological malignancies, prostate cancer, liver cancer, ovarian cancer, kidney cancer, gastric cancer, pancreatic cancer, brain cancer, osteosarcoma, and cervical cancer.

[0017] In some embodiments of the present invention, the cancer therapeutic agent is a drug that targets and inhibits cancer stem cells.

[0018] In some preferred embodiments of the present invention, the cancer stem cell-targeted inhibition is targeted binding to the extracellular domain of the cancer stem cell β2AR receptor.

[0019] In some embodiments of the present invention, the dosage form of the drug comprises an aerosol, a spray, an emulsion, a suspension, an enema, a paste, a plaster, a gel, a lotion, a suppository, a patch, a film, a capsule, a tablet, a pill, a powder, a granule, a tincture, an injection, a syrup, or a solution.

[0020] In some embodiments of the present invention, the route of administration of the drug includes oral administration, sublingual administration, inhalation administration, mucosal administration, intravenous injection, arterial injection, intramuscular injection, intradermal injection, subcutaneous injection, intraperitoneal injection, rectal administration, vaginal administration, transdermal administration, or topical administration. As used herein, the drug of the present invention can be administered by any suitable route, such as oral administration by capsule, injection administration by injection solution, topical administration by plaster or lotion, rectal administration by suppository, or transdermal administration by patch delivery system.

[0021] In some embodiments of the present invention, the pharmaceutically acceptable salt is a salt formed between the long-acting β2AR agonist and an acid or a base, wherein the acid comprises an inorganic acid or an organic acid, wherein the inorganic acid is selected from hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, phosphoric acid, sulfuric acid, and perchloric acid, wherein the organic acid comprises acetic acid, oxalic acid, malic acid, maleic acid, lactic acid, pyruvic acid, methanesulfonic acid, ethanesulfonic acid, paratoluenesulfonic acid, salicylic acid, tartaric acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, succinic acid, fumaric acid, phenylacetic acid, triphenylacetic acid, mandelic acid, gluconic acid, glutamic acid, isethionic acid, mucic acid, pamoic acid, pantothenic acid, sinafenic acid, or malonic acid, and wherein the base is an inorganic base comprising an alkali metal cation, an alkaline earth metal cation, or an ammonium cation salt.

[0022] In some embodiments of the invention, the medicament further comprises a pharmaceutically acceptable carrier.

[0023] As used herein, the term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or carrier, such as a liquid or solid filler, stabilizer, dispersant, suspending agent, diluent, excipient, thickener, solvent, or encapsulating material, that is involved in the transport or delivery of a useful compound of the present invention within or into a patient's body so that it can perform its desired function. Typically, such constructs can be delivered to an organ or part of the body, or transported from one organ or part of the body to another. Some examples of materials that can be used as pharmaceutically acceptable carriers include sugars such as lactose, glucose, and sucrose, starches such as corn starch and potato starch, cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and acetylcellulose, tragacanth gum powder, malt, gelatin, talc, excipients such as cocoa butter and suppository wax, oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil, glycols such as propylene glycol, polyols such as glycerol, sorbitol, mannitol, and polyethylene glycol, esters such as ethyl oleate and ethyl laurate, agar, buffers such as magnesium hydroxide, aluminum hydroxide, surfactants, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethanol, phosphate buffer, and other non-toxic compatible substances used in drug formulations. As used herein, "pharmaceutically acceptable carrier" further includes any and all coatings, antibacterial, antifungal, and absorption delaying agents, etc., that are compatible with the activity of the compounds of the invention and are physiologically acceptable to the patient. Supplementary active compounds can also be incorporated into the compositions. [Brief explanation of the drawings]

[0024] In the following the invention will be further explained with reference to the figures and examples. The description of the figures is given next. [Figure 1] FIG. 1 is a diagram showing the mechanism of action of a long-acting β2AR agonist against cancer in an example of the present invention. [Figure 2]FIG. 1 is a schematic diagram showing the expression status of β2AR in various cancer tissues according to an embodiment of the present invention. [Figure 3] 1 is a flowchart of an animal experiment in Example 1 of the present invention. [Figure 4] FIG. 1 is a diagram showing the results of an animal experiment on the inhibition of breast cancer orthotopic tumor growth by salmeterol xinafoate in an example of the present invention. [Figure 5] FIG. 1 is a diagram showing the results of an animal experiment on colorectal cancer tumor inhibition by salmeterol xinafoate in an example of the present invention. [Figure 6] FIG. 1 is a diagram showing the results of an animal experiment on lung cancer tumor inhibition by salmeterol xinafoate in an example of the present invention. [Figure 7] FIG. 1 is a diagram showing the results of an animal experiment on head and neck cancer tumor inhibition by salmeterol xinafoate in an example of the present invention. [Figure 8] FIG. 1 is a diagram showing the results of an animal experiment on esophageal cancer tumor inhibition by salmeterol xinafoate in an example of the present invention. [Figure 9] FIG. 1 is a diagram showing the results of an animal experiment on bladder cancer tumor inhibition by salmeterol xinafoate in an example of the present invention. [Figure 10] FIG. 1 is a diagram showing the results of an animal experiment on melanoma tumor inhibition by salmeterol xinafoate in an example of the present invention. [Figure 11] FIG. 1 is a diagram showing experimental results of the inhibition of breast cancer cell sphere formation by salmeterol xinafoate in an example of the present invention. [Figure 12] FIG. 1 is a diagram showing experimental results of inhibition of colorectal cancer cell sphere formation by salmeterol xinafoate in an example of the present invention. [Figure 13] FIG. 1 is a diagram showing experimental results of the inhibition of lung cancer cell sphere formation by salmeterol xinafoate in an example of the present invention. [Figure 14] FIG. 1 is a diagram showing experimental results of the inhibition of head and neck cancer cell sphere formation by salmeterol xinafoate in an example of the present invention. [Figure 15] FIG. 1 is a diagram showing experimental results of the inhibition of esophageal cancer cell sphere formation by salmeterol xinafoate in an example of the present invention. [Figure 16] FIG. 1 is a diagram showing experimental results of the inhibition of bladder cancer cell sphere formation by salmeterol xinafoate in an example of the present invention. [Figure 17] FIG. 1 is a diagram showing experimental results of the inhibition of melanoma cell sphere formation by salmeterol xinafoate in an example of the present invention. [Figure 18] FIG. 1 shows experimental results of the inhibition of blood tumor cell sphere formation by salmeterol xinafoate in an example of the present invention. [Figure 19] FIG. 1 is a diagram showing experimental results of the inhibition of prostate cancer cell sphere formation by salmeterol xinafoate in an example of the present invention. [Figure 20] FIG. 1 is a diagram showing experimental results of inhibition of hepatic cancer cell sphere formation by salmeterol xinafoate in an example of the present invention. [Figure 21] FIG. 1 is a diagram showing experimental results of the inhibition of ovarian cancer cell sphere formation by salmeterol xinafoate in an example of the present invention. [Figure 22] FIG. 1 is a diagram showing experimental results of inhibition of renal cancer cell sphere formation by salmeterol xinafoate in an example of the present invention. [Figure 23] FIG. 1 is a diagram showing experimental results of inhibition of gastric cancer cell sphere formation by salmeterol xinafoate in an example of the present invention. [Figure 24] FIG. 1 is a diagram showing experimental results of inhibition of pancreatic cancer cell sphere formation by salmeterol xinafoate in an example of the present invention. [Figure 25] FIG. 1 is a diagram showing experimental results of inhibition of brain cancer cell sphere formation by salmeterol xinafoate in an example of the present invention. [Figure 26] FIG. 1 is a diagram showing experimental results of inhibition of osteosarcoma cell sphere formation by salmeterol xinafoate in an example of the present invention. [Figure 27] FIG. 1 is a diagram showing experimental results of the inhibition of cervical cancer cell sphere formation by salmeterol xinafoate in an example of the present invention. [Figure 28]FIG. 1 shows experimental results of the inhibition of sphere formation of various cancer cells by alfomoterol tartrate in an example of the present invention. [Figure 29] FIG. 1 shows experimental results of the inhibition of sphere formation of various cancer cells by bambuterol hydrochloride in an example of the present invention. [Figure 30] This is a diagram showing the experimental results of the inhibition of sphere formation of various cancer cells by clenbuterol hydrochloride in an example of the present invention. [Figure 31] FIG. 1 shows experimental results of the inhibition of sphere formation of various cancer cells by formoterol fumarate in an example of the present invention. [Figure 32] FIG. 1 shows experimental results of the inhibition of sphere formation of various cancer cells by indacaterol maleate in an example of the present invention. [Figure 33] FIG. 1 shows experimental results of the inhibition of sphere formation of various cancer cells by olodaterol hydrochloride in an example of the present invention. [Figure 34] FIG. 1 shows experimental results of the inhibition of sphere formation of various cancer cells by tulobuterol hydrochloride in an example of the present invention. [Figure 35] FIG. 1 shows experimental results of the inhibition of sphere formation of various cancer cells by vilanterol triphenylacetate in an example of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0025] In order to fully understand the objectives, features, and advantages of the present invention, the concept and technical effects of the present invention will be clearly and completely explained below through examples. It should be understood that the examples described are only a portion of the examples of the present invention, and are not all examples. Other examples obtained by those skilled in the art based on the examples of the present invention without requiring creative effort are all within the scope of the present invention. Unless otherwise specified, all test methods used in the examples are conventional methods. Unless otherwise specified, all materials, reagents, etc. used are commercially available reagents and materials.

[0026] In the present example, we developed molecular targeted drugs targeting a single functional molecular target specifically expressed on the surface of cancer stem cells, and discovered that a long-acting β2AR agonist can target and inhibit cancer stem cells and further treat cancer. The mechanism of action is shown in Figure 1.

[0027] The beta2-adrenoceptor (β2AR) is the earliest discovered and most extensively studied member of the G protein-coupled receptor superfamily. β2AR receptors are expressed on the cell membrane and have a typical seven-transmembrane structure. Long-acting β2AR agonists are commonly used as bronchodilators to treat asthma and chronic obstructive pulmonary disease. Long-acting β2AR agonists have a long duration of action, lasting for 12 hours or more. The long-acting β2AR agonists of the present invention include all clinically approved long-acting specific β2-adrenoceptor agonists. There are nine long-acting β2AR agonists used in the examples of the present invention, including Arformoterol Tartrate, Bambuterol Hydrochloride, Clenbuterol Hydrochloride, Formoterol Hemifumarate, Indacaterol Maleate, Olodaterol Hydrochloride, Salmeterol Xinafoate, Tulobuterol Hydrochloride, and Vilanterol Trifenate, all of which are small molecule drugs that specifically target β2AR.

[0028] Analysis of the genome expression spectrum of clinical cancer patients demonstrated that β2AR is expressed in various cancer tissues (Figure 2). Examples of the present invention have discovered and demonstrated that the use of any specific long-acting β2AR agonist alone can inhibit the function of cancer stem cells and further treat cancer. That is, β2AR can be used as a single functional molecular target specific to cancer stem cells, and specific long-acting β2AR agonists are small molecule targeted drugs against this target.

[0029] The mRNA expression of ADRB2 (β2AR) in various cancer tissues (data from The Cancer Genome Atlas database) is as follows: ACC (adrenal carcinoma), BLCA (bladder cancer), BRCA (breast cancer), CESC (cervical cancer), CHOL (cholangiocarcinoma), COAD (colon cancer), DLBC (large B-cell lymphoma), ESCA (esophageal cancer), GBM (glioma), HNSC (head and neck cancer), KICH (chromophilic renal cell carcinoma), KIRC (renal clear cell carcinoma), KIRP (renal papillary cell carcinoma), LAML (myeloid leukemia), LGG (low-grade glioma), LIHC (liver cancer), L UAD (lung adenocarcinoma), LUSC (lung squamous cell carcinoma), MESO (mesothelioma), OV (ovarian cancer), PAAD (pancreatic cancer), PCPG (pheochromocytoma and paraganglioma), PRAD (prostate cancer), READ (rectal cancer), SARC (sarcoma), SKCM (melanoma), STAD (gastric cancer), TGCT (testicular cancer), THCA (thyroid cancer), THYM (thymic carcinoma), UCEC (endometrial cancer), UCS (uterine carcinosarcoma), UVM (uveal melanoma).

[0030] Salmeterol xinafoate is inexpensive, easily available, and is the long-acting β2AR agonist that has been used earliest in clinical practice and has the largest market share. Therefore, in the examples of the present invention, animal experiments were first carried out using salmeterol xinafoate as an example.

[0031] Example 1: Cancer treatment effect of salmeterol xinafoate 1.1 Animal studies of tumor inhibition by salmeterol xinafoate The experimental methods and procedures were as follows. Cancer cells were treated in vitro with salmeterol xinafoate (SX) for 3 to 7 days, followed by subcutaneous or orthotopic tumor formation experiments in mice. The flow of the animal experiment is shown in Figure 3. As a result of this experiment, salmeterol xinafoate was found to have inhibitory effects on the development and progression of breast cancer, colorectal cancer, lung cancer, head and neck cancer, esophageal cancer, bladder cancer, and melanoma (Figures 4 to 10).

[0032] 1.1.1 Inhibition of orthotopic breast cancer tumor initiation and progression by salmeterol xinafoate MDA-MB-231 breast cancer cell line was treated with 10 μM salmeterol xinafoate (SX) for 7 days, and then 1 × 10 SX cells were injected into the orthotopic mammary gland sites on both sides of the body of control and salmeterol xinafoate-treated mice (five mice per group). 4 In tumorigenesis experiments, 8 orthotopic tumors were formed at 10 injection sites in the orthotopic mammary gland of 5 mice in the control group, whereas only 6 orthotopic tumors were formed at 10 injection sites in the orthotopic mammary gland of 5 mice in the salmeterol xinafoate-treated group.

[0033] The experimental results, shown in Figure 4, show tumor growth curves and representative tumor photographs. Two-way ANOVA analysis was used with Fisher's LSD test as a post-hoc test to analyze statistical differences between tumor growth curves (****: P<0.0001). The experimental results demonstrated that salmeterol xinafoate has therapeutic effects on breast cancer and inhibits the development and progression of orthotopic breast cancer tumors.

[0034] 1.1.2 Inhibition of the development and progression of subcutaneous colorectal cancer tumors by salmeterol xinafoate CT-26 colorectal cancer cell line was treated with 10 μM salmeterol xinafoate (SX) for 7 days, and then 1 × 10 5In a tumor formation experiment, 10 tumors were formed at 10 injection sites in the control group (five mice), while 8 tumors were formed at 10 injection sites in the salmeterol xinafoate-treated group (five mice).

[0035] Figure 5 shows the experimental results, including tumor growth curves and representative tumor photographs. Two-way ANOVA analysis was used with Fisher's LSD test as a post-hoc test to analyze statistical differences between tumor growth curves (****: P<0.0001). The experimental results demonstrated that salmeterol xinafoate has therapeutic effects on colorectal cancer and inhibits the development and progression of subcutaneous colorectal cancer tumors.

[0036] 1.1.3 Inhibition of the development and progression of subcutaneous lung cancer tumors by salmeterol xinafoate In Figure 6A, H460 lung cancer cell line was treated with 10 μM salmeterol xinafoate (SX) for 7 days, and then 3 × 10 mice were injected subcutaneously into both sides of the back of the control group (7 mice) and the salmeterol xinafoate-treated group (5 mice). 5 In a tumor formation experiment, 7 mice in the control group developed 11 tumors out of 14 injection sites, whereas 5 mice in the salmeterol xinafoate-treated group did not develop tumors out of 10 injection sites.

[0037] In Figure 6B, LLC lung cancer cell line was treated with 10 μM salmeterol xinafoate (SX) for 7 days, and then 1 × 10 mice were injected subcutaneously into both sides of the back of the control group (8 mice) and the salmeterol xinafoate-treated group (5 mice). 5 Tumor formation experiments were performed using cells injected into each group. Eight mice in the control group developed tumors at all 16 injection sites, whereas five mice in the salmeterol xinafoate-treated group did not develop tumors at any of the 10 injection sites.

[0038] Figure 6 shows the experimental results, including tumor growth curves and representative tumor photographs. Two-way ANOVA analysis was used with Fisher's LSD test as a post-hoc test to analyze statistical differences between tumor growth curves (****: P<0.0001). The experimental results demonstrated that salmeterol xinafoate has therapeutic effects on lung cancer and inhibits the development and progression of subcutaneous lung cancer tumors.

[0039] 1.1.4 Salmeterol xinafoate inhibits the development and progression of subcutaneous tumors in head and neck cancer patients CAL-27 head and neck cancer cell line was treated with 10 μM salmeterol xinafoate (SX) for 3 days, and then 1 × 10 6 The cells were injected into each mouse and tumor formation experiments were carried out.

[0040] The experimental results, shown in Figure 7, show the tumor growth curves. Two-way ANOVA analysis was used with Fisher's LSD test as a post-hoc test to analyze statistical differences between the tumor growth curves (****: P<0.0001). The experimental results demonstrated that salmeterol xinafoate has a therapeutic effect on head and neck cancer and inhibits the development and progression of subcutaneous head and neck cancer tumors.

[0041] 1.1.5 Salmeterol xinafoate inhibits the development and progression of subcutaneous esophageal cancer tumors KYSE-150 esophageal cancer cell line was treated with 10 μM salmeterol xinafoate (SX) for 3 days, and then 1 × 10 6 The cells were injected into each mouse and tumor formation experiments were carried out.

[0042] The experimental results, shown in Figure 8, show the tumor growth curves. Two-way ANOVA analysis was used, with Fisher's LSD test as a post-hoc test, to analyze statistical differences between the tumor growth curves (****: P<0.0001). The experimental results demonstrated that salmeterol xinafoate has a therapeutic effect on esophageal cancer and inhibits the development and progression of subcutaneous esophageal cancer tumors.

[0043] 1.1.6 Salmeterol xinafoate inhibits the development and progression of subcutaneous bladder cancer tumors RT4 bladder cancer cell line was treated with 10 μM salmeterol xinafoate (SX) for 3 days, and then 1 × 10 6 The cells were injected into each mouse and tumor formation experiments were carried out.

[0044] Figure 9 shows the experimental results, showing the tumor growth curves. Two-way ANOVA analysis was used with Fisher's LSD test as a post-hoc test to analyze statistical differences between the tumor growth curves (****: P<0.0001). The experimental results demonstrated that salmeterol xinafoate has a therapeutic effect on bladder cancer and inhibits the development and progression of subcutaneous bladder cancer tumors.

[0045] 1.1.7 Inhibition of melanoma orthotopic tumor initiation and progression by salmeterol xinafoate B16F10 melanoma cell line was treated with 10 μM salmeterol xinafoate (SX) for 3 days, and then 1 × 10 6 mice were injected subcutaneously into both sides of the back of the control and salmeterol xinafoate-treated mice (4 mice per group). 6 Cells were injected into each mouse and tumor formation experiments were performed. Tumors formed at all eight injection sites in the four control mice. Tumors also formed at all eight injection sites in the four salmeterol xinafoate-treated mice.

[0046] The experimental results, shown in Figure 10, show tumor growth curves and representative tumor photographs. Two-way ANOVA analysis was used with Fisher's LSD test as a post-hoc test to analyze statistical differences between tumor growth curves (****: P<0.0001). The experimental results demonstrated that salmeterol xinafoate has therapeutic effects on melanoma and inhibits the development and progression of orthotopic melanoma tumors.

[0047] The above in vivo tumor formation experiments demonstrated that salmeterol xinafoate, a specific long-acting β2AR agonist, has therapeutic effects on various cancers, and can not only inhibit the development of cancer but also significantly inhibit cancer growth.

[0048] 1.2 Inhibition of tumorsphere formation by salmeterol xinafoate Numerous studies have proven that cancer stem cells have the ability to self-renew, and determine the occurrence and progression of cancer.Tumorsphere formation experiment, which is the gold standard for detecting the self-renewal ability of cancer stem cells in vitro, can detect that a single cancer stem cell continuously self-renews in conditioned medium and forms a multicellular tumorsphere.This in vitro experiment has been frequently used to detect the responsiveness of clinical patients to anti-cancer drugs, and further to guide the in vivo administration of clinical patients.

[0049] Similarly, through tumorsphere formation experiments, this example also demonstrated that salmeterol xinafoate could inhibit the self-renewal ability of cancer stem cells of the above seven types of cancer (FIGS. 11 to 17).

[0050] 1.2.1 Salmeterol xinafoate inhibits the sphere-forming ability of breast cancer cells Tumorsphere formation experiments were performed on breast cancer cell lines MDA-MB-231 (A), 4T1 (B), 168FARN (C), 67NR (D), and 4TO7 (E) under the culture conditions listed in Table 1. Simultaneously, salmeterol xinafoate (SX) treatment was performed at concentrations of 1 μM, 3 μM, or 10 μM. After 7 days, tumorspheres were counted and photographed in both the control and salmeterol xinafoate-treated groups.

[0051] [Table 1]

[0052] The experimental results in Figure 11 show the number of tumor spheres and representative tumor sphere photographs from at least three independent experiments. One-way ANOVA analysis was used with Fisher's LSD test as a post-hoc test to analyze statistical differences in the number of tumor spheres between groups (*: P<0.05; **: P<0.01; ***: P<0.001; ****: P<0.0001). The experimental results demonstrated that salmeterol xinafoate concentration-dependently inhibited the self-renewal ability of breast cancer stem cells.

[0053] 1.2.2 Inhibition of colorectal cancer cell sphere-forming ability by salmeterol xinafoate Tumorsphere formation experiments were performed on colorectal cancer cell lines CT-26 (A), SW620 (B), HCT-15 (C), and DLD-1 (D) under the culture conditions shown in Table 2. Simultaneously, salmeterol xinafoate (SX) treatment was performed at concentrations of 1 μM, 3 μM, or 10 μM, respectively. After 7 days, tumorspheres were counted and photographed in both the control and salmeterol xinafoate-treated groups.

[0054] [Table 2]

[0055] The experimental results in Figure 12 show the number of tumor spheres and representative tumor sphere photographs from three independent experiments. One-way ANOVA analysis was performed with Fisher's LSD test as a post-hoc test to analyze statistical differences in the number of tumor spheres between groups (**: P<0.01; ***: P<0.001; ****: P<0.0001; ns: not significant). The experimental results demonstrated that salmeterol xinafoate inhibited the self-renewal ability of colorectal cancer stem cells in a concentration-dependent manner.

[0056] 1.2.3 Salmeterol xinafoate inhibits the sphere-forming ability of lung cancer cells Tumorsphere formation experiments were performed on lung cancer cell lines LLC (A), H1299 (B), H460 (C), and H446 (D) under the culture conditions listed in Table 3. Simultaneously, salmeterol xinafoate (SX) treatment was performed at concentrations of 1 μM, 3 μM, or 10 μM, respectively. After 7 days, tumorspheres were counted and photographed in both the control and salmeterol xinafoate-treated groups.

[0057] [Table 3]

[0058] The experimental results in Figure 13 show the number of tumor spheres and representative tumor sphere photographs from three independent experiments. One-way ANOVA analysis was performed with Fisher's LSD test as a post-hoc test to analyze the statistical differences in the number of tumor spheres between groups (**: P<0.01; ***: P<0.001; ****: P<0.0001). The experimental results demonstrated that salmeterol xinafoate concentration-dependently inhibited the self-renewal ability of lung cancer stem cells.

[0059] 1.2.4 Salmeterol xinafoate inhibits the sphere-forming ability of head and neck cancer cells Tumorsphere formation experiments were performed on the head and neck cancer cell line CAL-27 under the culture conditions listed in Table 4. At the same time, salmeterol xinafoate (SX) was added at concentrations of 1 μM, 3 μM, or 10 μM. After 7 days, tumorspheres were counted and photographed in both the control and salmeterol xinafoate-treated groups.

[0060] [Table 4]

[0061] The experimental results in Figure 14 show the number of tumor spheres and representative tumor sphere photographs from three independent experiments. One-way ANOVA analysis was used with Fisher's LSD test as a post-hoc test to analyze the statistical difference in the number of tumor spheres between groups (****: P<0.0001). The experimental results demonstrated that salmeterol xinafoate inhibits the self-renewal ability of head and neck cancer stem cells.

[0062] 1.2.5 Salmeterol xinafoate inhibits the sphere-forming ability of esophageal cancer cells Tumorsphere formation experiments were performed on the esophageal cancer cell line KYSE-150 under the culture conditions shown in Table 5. Cells were also treated with 10 μM salmeterol xinafoate (SX). After 7 days, tumorspheres were counted and photographed in both the control and salmeterol xinafoate-treated groups.

[0063] [Table 5]

[0064] The experimental results in Figure 15 show the number of tumor spheres and representative tumor sphere photographs from three independent experiments. One-way ANOVA analysis was used with Fisher's LSD test as a post-hoc test to analyze statistical differences in the number of tumor spheres between groups (*: P<0.05). The experimental results demonstrated that salmeterol xinafoate inhibits the self-renewal ability of esophageal cancer stem cells.

[0065] 1.2.6 Salmeterol xinafoate inhibits the sphere-forming ability of bladder cancer cells A tumorsphere formation experiment was conducted on the bladder cancer cell line RT4 under the culture conditions shown in Table 6. At the same time, salmeterol xinafoate (SX) was added at concentrations of 1 μM, 3 μM, or 10 μM. After 7 days, tumorspheres were counted and photographed in both the control and salmeterol xinafoate-treated groups.

[0066] [Table 6]

[0067] The experimental results in Figure 16 show the number of tumor spheres and representative tumor sphere photographs from three independent experiments. One-way ANOVA analysis was used with Fisher's LSD test as a post-hoc test to analyze the statistical differences in the number of tumor spheres between groups (***: P<0.001; ****: P<0.0001). The experimental results demonstrated that salmeterol xinafoate concentration-dependently inhibited the self-renewal ability of bladder cancer stem cells.

[0068] 1.2.7 Inhibition of melanoma cell sphere-forming ability by salmeterol xinafoate Tumorsphere formation experiments were performed on melanoma cell lines B16F10 (A), A375 (B), and A2058 (C) under the culture conditions listed in Table 7. Simultaneously, salmeterol xinafoate (SX) treatment was performed at concentrations of 1 μM, 3 μM, or 10 μM, respectively. After 7 days, tumorspheres were counted and photographed in both the control and salmeterol xinafoate-treated groups.

[0069] [Table 7]

[0070] The experimental results in Figure 17 show the number of tumor spheres and representative tumor sphere photographs from three independent experiments. One-way ANOVA analysis was used with Fisher's LSD test as a post-hoc test to analyze the statistical differences in the number of tumor spheres between groups (**: P<0.01; ***: P<0.001; ****: P<0.0001). The experimental results demonstrated that salmeterol xinafoate concentration-dependently inhibited the self-renewal ability of melanoma stem cells.

[0071] Tumorsphere formation experiments demonstrated that salmeterol xinafoate could inhibit the self-renewal ability of cancer stem cells, a conclusion consistent with in vivo tumor formation experiments. In other words, in vitro tumorsphere formation experiments not only demonstrated the inhibition of cancer stem cell self-renewal by long-acting β2AR agonists, but also reflected the inhibition of cancer development and progression by long-acting β2AR agonists. Furthermore, the 3R principle for experimental animals was strictly adhered to, and in vitro cell experiments were used instead of in vivo animal experiments whenever possible, provided that experimental conclusions were consistent.

[0072] Therefore, this example demonstrates the therapeutic effect of salmeterol xinafoate on other common cancers (blood tumors, prostate cancer, liver cancer, ovarian cancer, kidney cancer, stomach cancer, pancreatic cancer, brain cancer, osteosarcoma, and cervical cancer) through tumorsphere formation experiments.

[0073] 1.2.8 Salmeterol xinafoate inhibits the sphere-forming ability of hematologic tumor cells Tumorsphere formation experiments were performed on the hematological tumor cell line SU-DHL-8 under the culture conditions listed in Table 8. At the same time, salmeterol xinafoate (SX) was added at concentrations of 1 μM, 3 μM, or 10 μM. After 7 days, tumorspheres were counted and photographed in both the control and salmeterol xinafoate-treated groups.

[0074] [Table 8]

[0075] The experimental results in Figure 18 show the number of tumor spheres and representative tumor sphere photographs from three independent experiments. One-way ANOVA analysis was used with Fisher's LSD test as a post-hoc test to analyze the statistical difference in the number of tumor spheres between groups (****: P<0.0001). The experimental results demonstrated that salmeterol xinafoate concentration-dependently inhibited the self-renewal ability of hematopoietic tumor stem cells.

[0076] 1.2.9 Salmeterol xinafoate inhibits the sphere-forming ability of prostate cancer cells Tumorsphere formation experiments were performed on prostate cancer cell lines PC-3 (A), DU145 (B), and RM-1 (C) under the culture conditions listed in Table 9. Simultaneously, salmeterol xinafoate (SX) treatment was performed at concentrations of 1 μM, 3 μM, or 10 μM, respectively. After 7 days, tumorspheres were counted and photographed in both the control and salmeterol xinafoate-treated groups.

[0077] [Table 9]

[0078] The experimental results in Figure 19 show the number of tumor spheres and representative tumor sphere photographs from three independent experiments. One-way ANOVA analysis was used with Fisher's LSD test as a post-hoc test to analyze the statistical differences in the number of tumor spheres between groups (*: P<0.05; ***: P<0.001; ****: P<0.0001). The experimental results demonstrated that salmeterol xinafoate inhibited the self-renewal ability of prostate cancer stem cells in a concentration-dependent manner.

[0079] 1.2.10 Inhibition of the sphere-forming ability of hepatocellular carcinoma cells by salmeterol xinafoate Tumorsphere formation experiments were performed on the liver cancer cell line Hep G2 under the culture conditions listed in Table 10. At the same time, salmeterol xinafoate (SX) was added at concentrations of 1 μM, 3 μM, or 10 μM. After 7 days, tumorspheres were counted and photographed in both the control and salmeterol xinafoate-treated groups.

[0080] [Table 10]

[0081] The experimental results in Figure 20 show the number of tumor spheres and representative tumor sphere photographs from three independent experiments. One-way ANOVA analysis was used with Fisher's LSD test as a post-hoc test to analyze the statistical differences in the number of tumor spheres between groups (***: P<0.001; ****: P<0.0001). The experimental results demonstrated that salmeterol xinafoate concentration-dependently inhibited the self-renewal ability of liver cancer stem cells.

[0082] 1.2.11 Inhibition of sphere-forming ability of ovarian cancer cells by salmeterol xinafoate Tumorsphere formation experiments were performed on the ovarian cancer cell line SKOV3 under the culture conditions shown in Table 11. At the same time, salmeterol xinafoate (SX) was added at concentrations of 1 μM, 3 μM, or 10 μM. After 7 days, tumorspheres were counted and photographed in both the control and salmeterol xinafoate-treated groups.

[0083] [Table 11]

[0084] The experimental results in Figure 21 show the number of tumor spheres and representative tumor sphere photographs from three independent experiments. One-way ANOVA analysis was used with Fisher's LSD test as a post-hoc test to analyze statistical differences in the number of tumor spheres between groups (*: P<0.05; **: P<0.01). The experimental results demonstrated that salmeterol xinafoate concentration-dependently inhibited the self-renewal ability of ovarian cancer stem cells.

[0085] 1.2.12 Salmeterol xinafoate inhibits the sphere-forming ability of renal cancer cells Tumorsphere formation experiments were performed on renal cancer cell lines G-401 (A) and 786-O (B) under the culture conditions shown in Table 12. Simultaneously, salmeterol xinafoate (SX) treatment was performed at concentrations of 1 μM, 3 μM, or 10 μM, respectively. After 7 days, tumorspheres were counted and photographed in both the control and salmeterol xinafoate-treated groups.

[0086] [Table 12]

[0087] The experimental results in Figure 22 show the number of tumor spheres and representative tumor sphere photographs from three independent experiments. One-way ANOVA analysis was used with Fisher's LSD test as a post-hoc test to analyze the statistical differences in the number of tumor spheres between groups (**: P<0.01; ***: P<0.001; ****: P<0.0001). The experimental results demonstrated that salmeterol xinafoate concentration-dependently inhibited the self-renewal ability of renal cancer stem cells.

[0088] 1.2.13 Salmeterol xinafoate inhibits the sphere-forming ability of gastric cancer cells A tumorsphere formation experiment was conducted on the gastric cancer cell line SNU-1 under the culture conditions shown in Table 13. At the same time, the cells were treated with 10 μM salmeterol xinafoate (SX). After 7 days, tumorspheres were counted and photographed in both the control and salmeterol xinafoate-treated groups.

[0089] [Table 13]

[0090] The experimental results in Figure 23 show the number of tumor spheres and representative tumor sphere photographs from three independent experiments. One-way ANOVA analysis was used with Fisher's LSD test as a post-hoc test to analyze the statistical difference in the number of tumor spheres between groups (***: P<0.001). The experimental results demonstrated that salmeterol xinafoate inhibits the self-renewal ability of gastric cancer stem cells.

[0091] 1.2.14 Salmeterol xinafoate inhibits the sphere-forming ability of pancreatic cancer cells Tumorsphere formation experiments were performed on pancreatic cancer cell lines CFPAC-1 (A) and PANC-1 (B) under the culture conditions shown in Table 14. At the same time, salmeterol xinafoate (SX) was added at concentrations of 1 μM, 3 μM, or 10 μM, respectively. After 7 days of culture, tumorspheres were counted and photographed in both the control and salmeterol xinafoate-treated groups.

[0092] [Table 14]

[0093] The experimental results in Figure 24 show the number of tumor spheres and representative tumor sphere photographs from three independent experiments. One-way ANOVA analysis was performed with Fisher's LSD test as a post-hoc test to analyze statistical differences in the number of tumor spheres between groups (*: P<0.05; **: P<0.01; ***: P<0.001; ****: P<0.0001; ns: not significant). The experimental results demonstrated that salmeterol xinafoate concentration-dependently inhibited the self-renewal ability of pancreatic cancer stem cells.

[0094] 1.2.15 Salmeterol xinafoate inhibits the sphere-forming ability of brain cancer cells Tumorsphere formation experiments were performed on the brain cancer cell line SF126 under the culture conditions shown in Table 15. At the same time, salmeterol xinafoate (SX) was added at concentrations of 1 μM, 3 μM, or 10 μM. After 7 days, tumorspheres were counted and photographed in both the control and salmeterol xinafoate-treated groups.

[0095] [Table 15]

[0096] The experimental results in Figure 25 show the number of tumor spheres and representative tumor sphere photographs from three independent experiments. One-way ANOVA analysis was used with Fisher's LSD test as a post-hoc test to analyze the statistical differences in the number of tumor spheres between groups (**: P<0.01; ****: P<0.0001; ns: not significant). The experimental results demonstrated that salmeterol xinafoate concentration-dependently inhibited the self-renewal ability of brain cancer stem cells.

[0097] 1.2.16 Salmeterol xinafoate inhibits the sphere-forming ability of osteosarcoma cells Tumorsphere formation experiments were performed on osteosarcoma cell line U2OS under the culture conditions shown in Table 16. At the same time, salmeterol xinafoate (SX) was added at concentrations of 1 μM, 3 μM, or 10 μM. After 7 days, tumorspheres were counted and photographed in both the control and salmeterol xinafoate-treated groups.

[0098] [Table 16]

[0099] The experimental results in Figure 26 show the number of tumor spheres and representative tumor sphere photographs from three independent experiments. One-way ANOVA analysis was used with Fisher's LSD test as a post-hoc test to analyze the statistical difference in the number of tumor spheres between groups (**: P<0.01). The experimental results demonstrated that salmeterol xinafoate inhibited the self-renewal ability of osteosarcoma stem cells.

[0100] 1.2.17 Inhibition of the sphere-forming ability of cervical cancer cells by salmeterol xinafoate Tumorsphere formation experiments were performed on the cervical cancer cell line Hela under the culture conditions shown in Table 17. At the same time, salmeterol xinafoate (SX) was added at concentrations of 1 μM, 3 μM, or 10 μM. After 7 days, tumorspheres were counted and photographed in both the control and salmeterol xinafoate-treated groups.

[0101] [Table 17]

[0102] The experimental results in Figure 27 show the number of tumor spheres and representative tumor sphere photographs from three independent experiments. One-way ANOVA analysis was performed with Fisher's LSD test as a post-hoc test to analyze the statistical differences in the number of tumor spheres between groups (**: P<0.01; ***: P<0.001; ****: P<0.0001). The experimental results demonstrated that salmeterol xinafoate concentration-dependently inhibited the self-renewal ability of cervical cancer stem cells.

[0103] Example 2: Cancer treatment effect of alfomoterol tartrate This example demonstrates the ability of alfomoterol tartrate to treat cancer by inhibiting cancer cell sphere formation.

[0104] According to the culture conditions in Tables 1 to 17 of Example 1, breast cancer cell line MDA-MB-231 (A), colorectal cancer cell line SW620 (B), colorectal cancer cell line CT-26 (C), lung cancer cell line H460 (D), lung cancer cell line H446 (E), melanoma cell line B16F10 (F), melanoma cell line A375 (G), head and neck cancer cell line CAL-27 (H), bladder cancer cell line RT4 (I), blood tumor cell line SU-DHL-8 (J), prostate cancer cell line PC-3 (K), liver cancer cell line Hep Tumorsphere formation experiments were performed on G2 (L), renal cancer cell line 786-O (M), gastric cancer cell line SNU-1 (N), pancreatic cancer cell line CFPAC-1 (O), pancreatic cancer cell line PANC-1 (P), brain cancer cell line SF126 (Q), and cervical cancer cell line Hela (R). Simultaneously, cells were treated with alfomoterol tartrate (AT) at concentrations of 1 μM, 3 μM, or 10 μM, respectively. After 7 days, tumorspheres were counted and photographed in the control and alfomoterol tartrate-treated groups.

[0105] The experimental results show the number of tumor spheres and representative tumor sphere photographs from three independent experiments. One-way ANOVA analysis was performed with Fisher's LSD test as a post-hoc test to analyze statistical differences in the number of tumor spheres between groups (*: P<0.05; **: P<0.01; ***: P<0.001; ****: P<0.0001, ns: not significant). The experimental results shown in Figure 28 demonstrate that alfomoterol tartrate has therapeutic effects on various cancers, and that this effect is concentration-dependent. The specific, long-acting β2AR agonist alfomoterol tartrate is effective in treating breast cancer, colorectal cancer, lung cancer, melanoma, head and neck cancer, esophageal cancer, bladder cancer, hematological malignancies, prostate cancer, liver cancer, ovarian cancer, kidney cancer, gastric cancer, pancreatic cancer, brain cancer, osteosarcoma, and cervical cancer.

[0106] Example 3: Cancer treatment effect of bambuterol hydrochloride This example demonstrates the cancer therapeutic potential of bambuterol hydrochloride by inhibiting cancer cell sphere formation.

[0107] According to the culture conditions of Tables 1 to 17 in Example 1, breast cancer cell line MDA-MB-231 (A), colorectal cancer cell line SW620 (B), colorectal cancer cell line CT-26 (C), lung cancer cell line H460 (D), lung cancer cell line H446 (E), melanoma cell line B16F10 (F), melanoma cell line A375 (G), head and neck cancer cell line CAL-27 (H), esophageal cancer cell line KYSE-150 (I), bladder cancer cell line RT4 (J), hematological tumor cell line SU-DHL-8 (K), prostate cancer cell line PC-3 (L), liver cancer cell line Hep Tumorsphere formation experiments were performed on G2 (M), ovarian cancer SKOV3 (N), renal cancer cell line 786-O (O), pancreatic cancer cell line CFPAC-1 (P), pancreatic cancer cell line PANC-1 (Q), brain cancer cell line SF126 (R), osteosarcoma cell line U2OS (S), and cervical cancer cell line Hela (T). Simultaneously, bambuterol hydrochloride (BH) treatment was performed at concentrations of 1 μM, 3 μM, or 10 μM, respectively. After 7 days, tumorspheres were counted and photographed in both the control and bambuterol hydrochloride-treated groups.

[0108] The experimental results show the number of tumor spheres and representative tumor sphere photographs from three independent experiments. One-way ANOVA analysis was performed with Fisher's LSD test as a post-hoc test to analyze the statistical differences in the number of tumor spheres between groups (*: P<0.05; **: P<0.01; ***: P<0.001; ****: P<0.0001). The experimental results shown in Figure 29 demonstrated that bambuterol hydrochloride has therapeutic effects on various cancers, and that its effects are concentration-dependent. Cancer cell sphere formation experiments demonstrated that the specific, long-acting β2AR agonist bambuterol hydrochloride has therapeutic effects on breast cancer, colorectal cancer, lung cancer, melanoma, head and neck cancer, esophageal cancer, bladder cancer, hematological tumors, prostate cancer, liver cancer, ovarian cancer, kidney cancer, gastric cancer, pancreatic cancer, brain cancer, osteosarcoma, and cervical cancer.

[0109] Example 4: Cancer treatment effect of clenbuterol hydrochloride This example demonstrates clenbuterol hydrochloride's ability to treat cancer by inhibiting cancer cell sphere formation.

[0110] Tumorsphere formation experiments were performed on breast cancer cell line MDA-MB-231 (A), colorectal cancer cell line SW620 (B), colorectal cancer cell line CT-26 (C), lung cancer cell line H460 (D), lung cancer cell line H446 (E), melanoma cell line B16F10 (F), melanoma cell line A375 (G), head and neck cancer cell line CAL-27 (H), esophageal cancer cell line KYSE-150 (I), bladder cancer cell line RT4 (J), hematological tumor cell line SU-DHL-8 (K), prostate cancer cell line PC-3 (L), liver cancer cell line Hep G2 (M), kidney cancer cell line 786-O (N), pancreatic cancer cell line CFPAC-1 (O), pancreatic cancer cell line PANC-1 (P), and cervical cancer cell line Hela (Q) under the culture conditions shown in Tables 1 to 17 of Example 1. At the same time, mice were treated with clenbuterol hydrochloride (CH) at concentrations of 1 μM, 3 μM, or 10 μM. After 7 days, tumor spheres were counted and photographed in both the control and clenbuterol hydrochloride-treated groups.

[0111] The experimental results show the number of tumor spheres and representative tumor sphere photographs from three independent experiments. One-way ANOVA analysis was performed with Fisher's LSD test as a post-hoc test to analyze statistical differences in the number of tumor spheres between groups (*: P<0.05; **: P<0.01; ***: P<0.001; ****: P<0.0001). The experiment shown in Figure 30 demonstrated that clenbuterol hydrochloride has therapeutic effects on various cancers, and that this effect is concentration-dependent. Cancer cell sphere formation experiments demonstrated that clenbuterol hydrochloride, a specific, long-acting β2AR agonist, has therapeutic effects on breast cancer, colorectal cancer, lung cancer, melanoma, head and neck cancer, esophageal cancer, bladder cancer, hematological tumors, prostate cancer, liver cancer, ovarian cancer, kidney cancer, gastric cancer, pancreatic cancer, brain cancer, osteosarcoma, and cervical cancer.

[0112] Example 5: Cancer treatment effect of formoterol fumarate This example demonstrates formoterol fumarate's ability to treat cancer by inhibiting cancer cell sphere formation.

[0113] Tumorsphere formation experiments were performed on breast cancer cell line MDA-MB-231 (A), colorectal cancer cell line SW620 (B), colorectal cancer cell line CT-26 (C), lung cancer cell line H460 (D), lung cancer cell line H446 (E), melanoma cell line B16F10 (F), melanoma cell line A375 (G), head and neck cancer cell line CAL-27 (H), esophageal cancer cell line KYSE-150 (I), bladder cancer cell line RT4 (J), hematological tumor cell line SU-DHL-8 (K), prostate cancer cell line PC-3 (L), liver cancer cell line Hep G2 (M), kidney cancer cell line 786-O (N), pancreatic cancer cell line CFPAC-1 (O), pancreatic cancer cell line PANC-1 (P), and cervical cancer cell line Hela (Q) under the culture conditions shown in Tables 1 to 17 of Example 1. At the same time, the mice were treated with formoterol hemifumarate (FH) at concentrations of 1 μM, 3 μM, or 10 μM. After 7 days, tumor spheres were counted and photographed in both the control and formoterol fumarate-treated groups.

[0114] The experimental results show the number of tumor spheres and representative tumor sphere photographs from three independent experiments. One-way ANOVA analysis was performed with Fisher's LSD test as a post-hoc test to analyze statistical differences in the number of tumor spheres between groups (*: P<0.05; **: P<0.01; ***: P<0.001; ****: P<0.0001). The experiment shown in Figure 31 demonstrated that formoterol fumarate has therapeutic effects on various cancers, and that this effect is concentration-dependent. Cancer cell sphere formation experiments demonstrated that formoterol fumarate, a specific, long-acting β2AR agonist, has therapeutic effects on breast cancer, colorectal cancer, lung cancer, melanoma, head and neck cancer, esophageal cancer, bladder cancer, hematological tumors, prostate cancer, liver cancer, ovarian cancer, kidney cancer, gastric cancer, pancreatic cancer, brain cancer, osteosarcoma, and cervical cancer.

[0115] Example 6: Cancer treatment effect of indacaterol maleate This example demonstrates indacaterol maleate's ability to treat cancer by inhibiting cancer cell sphere formation.

[0116] According to the culture conditions of Tables 1 to 17 in Example 1, breast cancer cell line MDA-MB-231 (A), colorectal cancer cell line SW620 (B), colorectal cancer cell line CT-26 (C), lung cancer cell line H460 (D), lung cancer cell line H1299 (E), lung cancer cell line LLC (F), lung cancer cell line H446 (G), melanoma cell line B16F10 (H), melanoma cell line A375 (I), head and neck cancer cell line CAL-27 (J), esophageal cancer cell line KYSE-150 (K), bladder cancer cell line RT4 (L), blood tumor cell line SU-DHL-8 (M), prostate cancer cell line PC-3 (N), liver cancer cell line Hep Tumorsphere formation experiments were performed on G2 (O), renal cancer cell line G-401 (P), gastric cancer cell line SNU-1 (Q), pancreatic cancer cell line CFPAC-1 (R), pancreatic cancer cell line PANC-1 (S), brain cancer cell line SF126 (T), osteosarcoma cell line U2OS (U), and cervical cancer cell line Hela (V). Simultaneously, indacaterol maleate (IM) treatment was performed at concentrations of 1 μM, 3 μM, or 10 μM, respectively. After 7 days, tumorspheres were counted and photographed in the control and indacaterol maleate-treated groups.

[0117] The experimental results show the number of tumor spheres and representative tumor sphere photographs from three independent experiments. One-way ANOVA analysis was performed with Fisher's LSD test as a post-hoc test to analyze statistical differences in the number of tumor spheres between groups (*: P<0.05; **: P<0.01; ***: P<0.001; ****: P<0.0001; ns: not significant). The experimental results shown in Figure 32 demonstrate that indacaterol maleate has therapeutic effects on various cancers, and that this effect is concentration-dependent. Cancer cell sphere formation experiments demonstrated that indacaterol maleate, a specific, long-acting β2AR agonist, has therapeutic effects on breast cancer, colorectal cancer, lung cancer, melanoma, head and neck cancer, esophageal cancer, bladder cancer, hematological tumors, prostate cancer, liver cancer, ovarian cancer, kidney cancer, gastric cancer, pancreatic cancer, brain cancer, osteosarcoma, and cervical cancer.

[0118] Example 7: Cancer treatment effect of olodaterol hydrochloride This example demonstrates the ability of olodaterol hydrochloride to treat cancer by inhibiting cancer cell sphere formation.

[0119] According to the culture conditions of Tables 1 to 17 in Example 1, breast cancer cell line MDA-MB-231 (A), colorectal cancer cell line SW620 (B), colorectal cancer cell line CT-26 (C), lung cancer cell line H460 (D), lung cancer cell line H446 (E), melanoma cell line B16F10 (F), melanoma cell line A375 (G), head and neck cancer cell line CAL-27 (H), esophageal cancer cell line KYSE-150 (I), bladder cancer cell line RT4 (J), hematological tumor cell line SU-DHL-8 (K), prostate cancer cell line PC-3 (L), liver cancer cell line Hep Tumorsphere formation experiments were performed on G2 (M), ovarian cancer cell line SKOV3 (N), renal cancer cell line G-401 (O), pancreatic cancer cell line CFPAC-1 (P), pancreatic cancer cell line PANC-1 (Q), brain cancer cell line SF126 (R), osteosarcoma cell line U2OS (S), and cervical cancer cell line Hela (T). Simultaneously, tumors were treated with olodaterol hydrochloride (OH) at concentrations of 1 μM, 3 μM, or 10 μM, respectively. After 7 days, tumorspheres were counted and photographed in both the control and olodaterol hydrochloride-treated groups.

[0120] The experimental results show the number of tumor spheres and representative tumor sphere photographs from three independent experiments. One-way ANOVA analysis was performed with Fisher's LSD test as a post-hoc test to analyze statistical differences in the number of tumor spheres between groups (*: P<0.05; **: P<0.01; ***: P<0.001; ****: P<0.0001). The experimental results shown in Figure 33 demonstrate that olodaterol hydrochloride has therapeutic effects on various cancers, and that this effect is concentration-dependent. Cancer cell sphere formation experiments demonstrated that olodaterol hydrochloride, a specific, long-acting β2AR agonist, has therapeutic effects on breast cancer, colorectal cancer, lung cancer, melanoma, head and neck cancer, esophageal cancer, bladder cancer, hematological tumors, prostate cancer, liver cancer, ovarian cancer, kidney cancer, gastric cancer, pancreatic cancer, brain cancer, osteosarcoma, and cervical cancer.

[0121] Example 8: Cancer treatment effect of tulobuterol hydrochloride This example demonstrates the cancer therapeutic potential of tulobuterol hydrochloride by inhibiting cancer cell sphere formation.

[0122] According to the culture conditions of Tables 1 to 17 in Example 1, breast cancer cell line MDA-MB-231 (A), colorectal cancer cell line SW620 (B), colorectal cancer cell line CT-26 (C), lung cancer cell line H460 (D), lung cancer cell line H1299 (E), lung cancer cell line LLC (F), lung cancer cell line H446 (G), melanoma cell line B16F10 (H), melanoma cell line A375 (I), head and neck cancer cell line CAL-27 (J), esophageal cancer cell line KYSE-150 (K), bladder cancer cell line RT4 (L), blood tumor cell line SU-DHL-8 (M), prostate cancer cell line PC-3 (N), liver cancer cell line Hep Tumorsphere formation experiments were performed on G2 (O), renal cancer cell line G-401 (P), renal cancer cell line 786-O (Q), gastric cancer cell line SNU-1 (R), pancreatic cancer cell line CFPAC-1 (S), brain cancer cell line SF126 (T), and cervical cancer cell line Hela (U). Tulobuterol hydrochloride (TH) was simultaneously treated at concentrations of 1 μM, 3 μM, or 10 μM, respectively. After 7 days, tumorspheres were counted and photographed in both the control and tulobuterol hydrochloride-treated groups.

[0123] The experimental results show the number of tumor spheres and representative tumor sphere photographs from three independent experiments. One-way ANOVA analysis was performed with Fisher's LSD test as a post-hoc test to analyze statistical differences in the number of tumor spheres between groups (*: P<0.05; **: P<0.01; ***: P<0.001; ****: P<0.0001; ns: not significant). The experimental results shown in Figure 34 demonstrate that tulobuterol hydrochloride has therapeutic effects on various cancers, and that this effect is concentration-dependent. Cancer cell sphere formation experiments demonstrated that tulobuterol hydrochloride, a specific long-acting β2AR agonist, has therapeutic effects on breast cancer, colorectal cancer, lung cancer, melanoma, head and neck cancer, esophageal cancer, bladder cancer, hematological tumors, prostate cancer, liver cancer, ovarian cancer, kidney cancer, gastric cancer, pancreatic cancer, brain cancer, osteosarcoma, and cervical cancer.

[0124] Example 9: Cancer treatment effect of vilanterol triphenylacetate This example demonstrates the ability of vilanterol triphenylacetate to treat cancer by inhibiting cancer cell sphere formation.

[0125] According to the culture conditions of Tables 1 to 17 in Example 1, breast cancer cell line MDA-MB-231 (A), colorectal cancer cell line SW620 (B), colorectal cancer cell line CT-26 (C), lung cancer cell line H460 (D), lung cancer cell line H1299 (E), lung cancer cell line LLC (F), lung cancer cell line H446 (G), melanoma cell line B16F10 (H), melanoma cell line A375 (I), head and neck cancer cell line CAL-27 (J), esophageal cancer cell line KYSE-150 (K), bladder cancer cell line RT4 (L), blood tumor cell line SU-DHL-8 (M), prostate cancer cell line PC-3 (N), liver cancer cell line Hep Tumorsphere formation experiments were performed on G2 (O), renal cancer cell line G-401 (P), renal cancer cell line 786-O (Q), gastric cancer cell line SNU-1 (R), pancreatic cancer cell line CFPAC-1 (S), pancreatic cancer cell line PANC-1 (T), brain cancer cell line SF126 (U), and cervical cancer cell line Hela (V). Simultaneously, vilanterol triphenylacetate (VT) was treated at concentrations of 1 μM, 3 μM, or 10 μM, respectively. After 7 days, tumorspheres were counted and photographed in the control and vilanterol triphenylacetate-treated groups.

[0126] The experimental results show the number of tumor spheres and representative tumor sphere photographs from three independent experiments. One-way ANOVA analysis was performed with Fisher's LSD test as a post-hoc test to analyze statistical differences in the number of tumor spheres between groups (*: P<0.05; **: P<0.01; ***: P<0.001; ****: P<0.0001; ns: not significant). The experimental results shown in Figure 35 demonstrate that vilanterol triphenylacetate has therapeutic effects on various cancers, and that this effect is concentration-dependent. Cancer cell sphere formation experiments demonstrated that vilanterol triphenylacetate, a specific, long-acting β2AR agonist, has therapeutic effects on breast cancer, colorectal cancer, lung cancer, melanoma, head and neck cancer, esophageal cancer, bladder cancer, hematological tumors, prostate cancer, liver cancer, ovarian cancer, kidney cancer, gastric cancer, pancreatic cancer, brain cancer, osteosarcoma, and cervical cancer.

[0127] Taking the experimental results of Examples 1 to 9 together, the examples of the present invention are Arformoterol Tartrate, Bambuterol Hydrochloride, Clenbuterol Hydrochloride, Formoterol Hemifumarate, Indacaterol Maleate, Olodaterol Hydrochloride, Salmeterol Xinafoate, Tulobuterol Hydrochloride, Vilanterol Triphenylacetate, and Nine specific long-acting β2AR agonists, such as β2AR agonist (β2AR agonist), ...

[0128] The following conclusions can be drawn from the experimental results of the above-described embodiments of the present invention. 1. Specific long-acting β2AR agonists can inhibit cancer development and growth. The present invention demonstrates through orthotopic or subcutaneous tumor formation experiments in mice that the use of a specific long-acting β2AR agonist alone at a drug concentration of 10 μM can significantly inhibit the development and progression of cancers such as breast cancer, colorectal cancer, lung cancer, head and neck cancer, esophageal cancer, bladder cancer, and melanoma.

[0129] 2. Specific long-acting β2AR agonists can inhibit the function of cancer stem cells. Cancer stem cells have the ability to self-renew and determine the development and progression of cancer. Tumorsphere formation experiments, the gold standard for detecting the self-renewal ability of cancer stem cells in vitro, can detect single cancer stem cells continuously self-renewing in conditioned medium and forming multicellular tumorspheres.

[0130] Therefore, the present invention has demonstrated through a cancer cell sphere formation experiment that alfomoterol tartrate, bambuterol hydrochloride, clenbuterol hydrochloride, formoterol hemifumarate, indacaterol maleate, olodaterol hydrochloride, salmeterol xinafoate, tulobuterol hydrochloride, vilanterol triphenylacetate, and the like are effective in cancer cell formation. They demonstrated that the single use of any one of nine specific long-acting β2AR agonists, known as β2AR agonists (B2AR agonists), can significantly inhibit the self-renewal ability of cancer stem cells in 17 types of cancer, including breast cancer, colorectal cancer, lung cancer, head and neck cancer, esophageal cancer, bladder cancer, melanoma, blood cancer, prostate cancer, liver cancer, ovarian cancer, kidney cancer, gastric cancer, pancreatic cancer, brain cancer, osteosarcoma, and cervical cancer.

[0131] Therefore, the in vitro tumor sphere formation experiments and the in vivo tumor formation experiments reached a consistent conclusion. Taking the above points 1 and 2 together, it was demonstrated that specific long-acting β2AR agonists can inhibit the development and progression of cancer by targeting and inhibiting the self-renewal ability of cancer stem cells.

[0132] 3. β2AR can be a single functional molecular target specifically expressed on the surface of cancer stem cell membranes, and specific long-acting β2AR agonists are small molecule targeted drugs against this target. β2AR is a member of the G protein-coupled receptor superfamily, has a typical seven-transmembrane structure, and is expressed in various malignant tumors in humans and mice. Specific long-acting β2AR agonists target the surface receptor β2AR on cancer cell membranes as a single functional molecular target, and inhibit the self-renewal ability of cancer stem cells, thereby inhibiting the development and progression of cancer and can be used clinically in cancer treatment.

[0133] Although the present invention has been described above with reference to the drawings, the present invention is not limited to the above-described embodiments, and various modifications may be made within the scope of the knowledge of those skilled in the art without departing from the spirit and scope of the present invention. Furthermore, the embodiments of the present invention and the features of the embodiments may be combined with each other unless they are inconsistent.

Claims

1. long-acting beta ２ Use of an AR agonist or a pharmaceutically acceptable salt thereof in the preparation of a cancer therapeutic agent.

2. The long-acting beta ２ AR agonists are specific β ２ 2. The use according to claim 1, characterized in that it is an adrenoceptor agonist.

3. The long-acting beta ２ 3. The use according to claim 1 or 2, characterized in that the AR agonist comprises at least one of Arfomoterol, Bambuterol, Clenbuterol, Formoterol, Indacaterol, Olodaterol, Salmeterol, Tulobuterol, Vilanterol.

4. The long-acting beta ２ AR agonists include alfomoterol tartrate, bambuterol hydrochloride, clenbuterol hydrochloride, formoterol hemifumarate, indacaterol maleate, olodaterol hydrochloride, salmeterol xinafoate, tulobuterol hydrochloride, and vilanterol triphenylacetate.

4. The use according to claim 3, characterized in that it comprises at least one of the following:

5. 2. The use according to claim 1, characterized in that the cancer comprises at least one of breast cancer, colorectal cancer, lung cancer, head and neck cancer, esophageal cancer, bladder cancer, melanoma, hematological tumors, prostate cancer, liver cancer, ovarian cancer, kidney cancer, gastric cancer, pancreatic cancer, brain cancer, osteosarcoma, and cervical cancer.

6. The use according to claim 1, characterized in that the dosage form of the drug includes an aerosol, a spray, an emulsion, a suspension, an enema, a paste, a plaster, a gel, a lotion, a suppository, a patch, a film, a capsule, a tablet, a pill, a powder, a granule, a tincture, an injection, a syrup, or a solution.

7. 2. The use according to claim 1, characterized in that the route of administration of the drug comprises oral administration, sublingual administration, inhalation administration, mucosal administration, intravenous injection, arterial injection, intramuscular injection, intradermal injection, subcutaneous injection, intraperitoneal injection, rectal administration, vaginal administration, transdermal administration, or topical administration.

8. The pharmaceutically acceptable salts may be used in combination with the long-acting β ２ 2. The use according to claim 1, characterized in that the salt is formed between an AR agonist and an acid or a base, the acid comprising an inorganic acid or an organic acid, the inorganic acid being selected from hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, phosphoric acid, sulfuric acid, and perchloric acid, the organic acid comprising acetic acid, oxalic acid, malic acid, maleic acid, lactic acid, pyruvic acid, methanesulfonic acid, ethanesulfonic acid, paratoluenesulfonic acid, salicylic acid, tartaric acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, succinic acid, fumaric acid, phenylacetic acid, triphenylacetic acid, mandelic acid, gluconic acid, glutamic acid, isethionic acid, mucic acid, pamoic acid, pantothenic acid, sinafenic acid, and malonic acid, and the base is an inorganic base comprising an alkali metal cation, an alkaline earth metal cation, or an ammonium cation salt.

9. The use according to claim 1, characterized in that the medicament further comprises a pharmaceutically acceptable carrier.

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