Compound for inhibiting osteosarcoma cell invasion and metastasis capability and application

Abstract

The invention relates to a compound for inhibiting osteosarcoma cell invasion and metastasis capability and application of the compound, in particular to application of a PAC channel inhibitor to preparation of a composition or a preparation, and the composition or the preparation is used for preventing and / or treating osteosarcoma. It is found for the first time that PAC channel inhibitors (such as Evans blue (EB), trypan blue (TB), aniline blue (AB), Coomassie brilliant blue (CBB), Cicago sky blue 6B (CSB), direct blue 71 (DB-71) and thiazole yellow (TY)) effectively prevent and / or treat osteosarcoma by inhibiting the activity of PAC channels.

Description

Technical Field

[0001] The present invention relates to the field of biopharmaceuticals, and in particular to a compound capable of inhibiting the invasion and metastasis of osteosarcoma cells and an application thereof. Background Art

[0002] Osteosarcoma is a malignant bone tumor characterized by the direct formation of bone or bone-like tissue from sarcoma cells. It is the most common primary malignant bone tumor. Studies have found that the proton-activated chloride channel (PAC) is highly expressed in malignant osteosarcoma cells, and that PAC expression is positively correlated with the malignancy grade of osteosarcoma, suggesting that PAC is a potential therapeutic target for malignant osteosarcoma.

[0003] Although broad-spectrum chloride channel inhibitors such as DIDS and NFA and PIP2 can inhibit the activity of PAC channels, their poor specificity makes them unsuitable for drug development targeting PAC channels. Therefore, there is an urgent need to discover highly specific inhibitors of PAC.

[0004] Therefore, there is an urgent need in the art to develop a highly specific PAC inhibitor that can effectively treat osteosarcoma. Summary of the Invention

[0005] The purpose of the present invention is to provide a highly specific inhibitor of PAC for effectively treating osteosarcoma.

[0006] A first aspect of the present invention provides a use of a PAC channel inhibitor for preparing a composition or preparation for preventing and / or treating osteosarcoma.

[0007] In another preferred embodiment, the osteosarcoma is malignant osteosarcoma.

[0008] In another preferred embodiment, the composition or preparation prevents and / or treats osteosarcoma by inhibiting PAC channel activity.

[0009] In another preferred embodiment, the PAC channel inhibitors include Evans blue (EB), Trypan blue (TB), Aniline blue (AB), Coomassie brilliant blue (CBB), Chicago sky blue 6B (CSB), Direct blue 71 (DB-71) and Thiazol yellow G (TY).

[0010] In another preferred embodiment, the PAC channel inhibitor is chemically synthesized.

[0011] In another preferred embodiment, the PAC channel inhibitor selectively inhibits PAC channels, and does not inhibit or substantially does not inhibit other ion channels (such as TMEM16A, ClC-2, TRPV1, TRPV2, TRPV3, TRPV4, TRPA1, TRPM8, Kv4.2, Nav1.4, Nav1.5, Nav1.7 and hERG channels).

[0012] In another preferred embodiment, in the composition or preparation, the concentration of the PAC channel inhibitor is 1-100 μM, preferably 1-30 μM, and more preferably 1-10 μM.

[0013] In another preferred embodiment, the composition or preparation is further used for one or more purposes selected from the following groups:

[0014] (i) Inhibit the metastatic ability of osteosarcoma cells;

[0015] (ii) Inhibit the invasive ability of osteosarcoma cells.

[0016] In another preferred embodiment, the composition or preparation further comprises other drugs that can prevent and / or treat osteosarcoma.

[0017] In another preferred embodiment, other drugs that can prevent and / or treat osteosarcoma include doxorubicin, cisplatin, methotrexate, and ifosfamide.

[0018] In another preferred embodiment, the composition comprises a pharmaceutical composition.

[0019] In another preferred embodiment, the pharmaceutical composition contains (a) a PAC channel inhibitor and (b) a pharmaceutically acceptable carrier.

[0020] In another preferred embodiment, the component (a) accounts for 0.1-99.9 wt %, preferably 10-99.9 wt %, and more preferably 70%-99.9 wt % of the total weight of the pharmaceutical composition.

[0021] In another preferred embodiment, the component (a) accounts for 60.0%-99.5wt% of the total weight of the pharmaceutical composition, preferably 70.0-99.5wt%, more preferably 80.0%-99.5wt%.

[0022] In another preferred embodiment, the pharmaceutical composition is in liquid, solid, or semisolid form.

[0023] In another preferred embodiment, the dosage form of the pharmaceutical composition includes tablets, granules, capsules, oral solutions, or injections.

[0024] In another preferred embodiment, the composition is an oral preparation.

[0025] In another preferred embodiment, the composition (eg, pharmaceutical composition) is administered to a mammal by the following means: oral administration, intravenous injection, or local injection.

[0026] In another preferred embodiment, the mammal includes a mammal suffering from osteosarcoma.

[0027] In another preferred embodiment, the mammal includes a human or a non-human mammal.

[0028] In another preferred embodiment, the non-human mammals include rodents, such as mice and rats.

[0029] A second aspect of the present invention provides a pharmaceutical composition for preventing and / or treating osteosarcoma, comprising:

[0030] (a1) a first pharmaceutical composition, comprising (a) a first active ingredient, wherein the first active ingredient is a PAC channel inhibitor; and

[0031] (a2) an optional second pharmaceutical composition, wherein the second pharmaceutical composition comprises (b) a second active ingredient, wherein the second active ingredient is another drug capable of preventing and / or treating osteosarcoma;

[0032] (b) a pharmaceutically acceptable carrier.

[0033] In another preferred embodiment, the first pharmaceutical composition and the second pharmaceutical composition are different pharmaceutical compositions, or the same pharmaceutical composition.

[0034] In another preferred embodiment, the PAC channel inhibitors include Evans blue (EB), trypan blue (TB), aniline blue (AB), Coomassie brilliant blue (CBB), Chicago sky blue 6B (CSB), direct blue 71 (DB-71) and thiazole yellow (TY).

[0035] In another preferred embodiment, other drugs that can prevent and / or treat osteosarcoma include doxorubicin, cisplatin, methotrexate, and ifosfamide.

[0036] In another preferred embodiment, the weight ratio of the first active ingredient to the second active ingredient is 1:100 to 100:1, preferably 1:10 to 10:1.

[0037] In another preferred embodiment, in the pharmaceutical composition, the content of component (a1) is 1%-99%, preferably 10%-90%, and more preferably 30%-70%.

[0038] In another preferred embodiment, in the pharmaceutical composition, the components (a1) and (a2) account for 0.01-99.99 wt%, preferably 0.1-90 wt%, and more preferably 1-80 wt% of the total weight of the product combination. (Please review)

[0039] In another preferred embodiment, in the pharmaceutical composition, the concentration of the component (a1) is 1-100 μM, preferably 1-30 μM, more preferably 1-10 μM.

[0040] In another preferred embodiment, the dosage form of the pharmaceutical composition includes an injection form and an oral dosage form.

[0041] In another preferred embodiment, the oral dosage forms include tablets, capsules, films, and granules.

[0042] In another preferred embodiment, the dosage form of the pharmaceutical composition includes a sustained-release dosage form and a non-sustained-release dosage form.

[0043] In another preferred embodiment, the weight ratio of component (a1) to component (a2) is 1:100 to 100:1, preferably 1:10 to 10:1.

[0044] A third aspect of the present invention provides a medicine kit comprising:

[0045] (a1) a first container, and a PAC channel inhibitor or a drug containing a PAC channel inhibitor in the first container;

[0046] (a2) Optionally, a second container, and other drugs for preventing and / or treating osteosarcoma, or drugs containing other drugs for preventing and / or treating osteosarcoma, located in the second container.

[0047] In another preferred embodiment, the concentration of the PAC channel inhibitor in the first container is 1-100 μM, preferably 1-30 μM, and more preferably 1-10 μM.

[0048] In another preferred embodiment, the first container and the second container are the same or different containers.

[0049] In another preferred embodiment, the drug in the first container is a single-ingredient preparation containing a PAC channel inhibitor.

[0050] In another preferred embodiment, the medicine in the second container is a single-ingredient preparation containing other medicines for preventing and / or treating osteosarcoma.

[0051] In another preferred embodiment, the dosage form of the drug is an oral dosage form or an injection dosage form.

[0052] In another preferred embodiment, the kit further comprises instructions.

[0053] In another preferred embodiment, the description records one or more instructions selected from the following group:

[0054] (a) A method for using a PAC channel inhibitor to prevent and / or treat osteosarcoma;

[0055] (b) A method for preventing and / or treating osteosarcoma by combining a PAC channel inhibitor with other drugs for preventing and / or treating osteosarcoma.

[0056] A fourth aspect of the present invention provides a method for (i) inhibiting the metastatic ability of osteosarcoma cells; and / or (ii) inhibiting the invasive ability of osteosarcoma cells, comprising:

[0057] Osteosarcoma cells are cultured in the presence of a PAC channel inhibitor or the pharmaceutical composition according to the second aspect of the present invention, thereby (i) inhibiting the metastatic ability of osteosarcoma cells; and / or (ii) inhibiting the invasive ability of osteosarcoma cells.

[0058] In another preferred embodiment, the concentration of the PAC channel inhibitor is 1-100 μM, preferably 1-30 μM, more preferably 1-10 μM.

[0059] A fifth aspect of the present invention provides a method for preventing and / or treating osteosarcoma, comprising:

[0060] Administer the PAC channel inhibitor, or the pharmaceutical composition according to the second aspect of the present invention, or the pharmaceutical kit according to the third aspect of the present invention to a subject in need thereof.

[0061] In another preferred embodiment, the subject includes a human or non-human mammal suffering from osteosarcoma.

[0062] In another preferred embodiment, the non-human mammals include rodents and primates, preferably mice, rats, rabbits, and monkeys.

[0063] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] The following drawings are used to illustrate specific embodiments of the present invention and are not used to limit the scope of the present invention defined by the claims.

[0065] Figure 1 The chemical structural formula of the compound Evans Blue (EB) is shown.

[0066] Figure 2It was shown that EB inhibited the current of PAC channels in a dose-dependent manner.

[0067] Figure 3 The chemical structural formula of the compound Trypan Blue (TB) is shown.

[0068] Figure 4 It was shown that TB inhibited the current of PAC channels in a dose-dependent manner.

[0069] Figure 5 The chemical structural formula of the compound aniline blue (AB) is shown.

[0070] Figure 6 It was shown that AB inhibited the current of PAC channels in a dose-dependent manner.

[0071] Figure 7 The chemical structural formula of the compound Coomassie Brilliant Blue (CBB) is shown.

[0072] Figure 8 It was shown that CBB inhibited the current of PAC channels in a dose-dependent manner.

[0073] Figure 9 The chemical structural formula of the compound Chicago Sky Blue 6B (CSB) is shown.

[0074] Figure 10 It was shown that CSB inhibited the current of PAC channels in a dose-dependent manner.

[0075] Figure 11 The chemical structural formula of the compound Direct Blue 71 (DB-71) is shown.

[0076] Figure 12 It was shown that DB-71 inhibited the current of PAC channels in a dose-dependent manner.

[0077] Figure 13 The chemical structural formula of the compound Thiazole Yellow (TY) is shown.

[0078] Figure 14 It was shown that TY inhibited the current of PAC channels in a dose-dependent manner.

[0079] Figure 15 Shown are the concentration-effect curves of EB, TB, AB, CBB, CSB, DB-71, and TY for inhibition of PAC channel currents, fitted with the Hill equation.

[0080] Figure 16 It was shown that 3 μM EB had no inhibitory effect on TMEM16A channels.

[0081] Figure 17 It was shown that 3 μM EB had no inhibitory effect on ClC-2 channels.

[0082] Figure 18 It was shown that 3 μM EB had no inhibitory effect on TRPV1 channels.

[0083] Figure 19 It was shown that 3 μM EB had no inhibitory effect on TRPV2 channels.

[0084] Figure 20 It was shown that 3 μM EB had no inhibitory effect on TRPV3 channels.

[0085] Figure 21 It was shown that 3 μM EB had no inhibitory effect on TRPV4 channels.

[0086] Figure 22 It was shown that 3 μM EB had no inhibitory effect on TRPA1 channels.

[0087] Figure 23 It was shown that 3 μM EB had no inhibitory effect on TRPM8 channels.

[0088] Figure 24 It was shown that 3 μM EB had no inhibitory effect on Kv4.2 channels.

[0089] Figure 25 It was shown that 3 μM EB had no inhibitory effect on Nav1.4 channels.

[0090] Figure 26 It was shown that 3 μM EB had no inhibitory effect on Nav1.5 channels.

[0091] Figure 27 It was shown that 3 μM EB had no inhibitory effect on Nav1.7 channels.

[0092] Figure 28 It was shown that 3 μM EB had no inhibitory effect on hERG channels.

[0093] Figure 29 Statistical graph showing that 3 μM EB has no inhibitory effect on other ion channels.

[0094] Figure 30 It was shown that in the scratch test of the malignant osteosarcoma cell line MG-63 cells, both PAC knockdown and EB treatment could inhibit the metastatic ability of MG-63 cells.

[0095] Figure 31 Statistical graph of the scratch area of ​​MG-63 cells after PAC knockdown and EB treatment.

[0096] Figure 32 It was shown that in the scratch test of the malignant osteosarcoma cell line U2OS cells, both PAC knockdown and EB treatment inhibited the metastatic ability of U2OS cells.

[0097] Figure 33 Statistical graph of the scratch area of ​​U2OS cells after PAC knockdown and EB treatment.

[0098] Figure 34 It was shown that in the transwell invasion assay of the malignant osteosarcoma cell line MG-63 cells, both PAC knockdown and EB treatment inhibited the invasive ability of MG-63 cells.

[0099] Figure 35 Statistical graph showing the number of MG-63 cells that passed through the chamber after PAC knockdown and EB treatment.

[0100] Figure 36 It was shown that in the transwell invasion assay of the malignant osteosarcoma cell line U2OS cells, both PAC knockdown and EB treatment inhibited the invasive ability of U2OS cells.

[0101] Figure 37 Statistical graph showing the number of U2OS cells crossing the chamber after PAC knockdown and EB treatment. DETAILED DESCRIPTION

[0102] After extensive and in-depth research, the inventors unexpectedly discovered that PAC channel inhibitors (such as Evans Blue (EB), Trypan Blue (TB), Aniline Blue (AB), Coomassie Brilliant Blue (CBB), Chicago Sky Blue 6B (CSB), Direct Blue 71 (DB-71) and Thiazole Yellow (TY)) effectively prevent and / or treat osteosarcoma by inhibiting PAC channel activity. In addition, the present invention first discovered that PAC channel inhibitors (such as Evans Blue (EB), Trypan Blue (TB), Aniline Blue (AB), Coomassie Brilliant Blue (CBB), Chicago Sky Blue 6B (CSB), Direct Blue 71 (DB-71) and Thiazole Yellow (TY)) have high selectivity for PAC channels and do not inhibit or substantially do not inhibit other ion channels. On this basis, the inventors completed the present invention.

[0103] osteosarcoma

[0104] Osteosarcoma is a malignant bone tumor characterized by the direct formation of bone or bone-like tissue from sarcoma cells. It is the most common primary malignant bone tumor. Studies have found that the proton-activated chloride channel (PAC) is highly expressed in malignant osteosarcoma cells, and that PAC expression is positively correlated with the malignancy grade of osteosarcoma, suggesting that PAC is a potential therapeutic target for malignant osteosarcoma.

[0105] PAC channel inhibitors

[0106] PAC channels are proton-activated chloride channels, and their inhibitors include those that bind to PAC channels in the presence of protons to inhibit the PAC channel current caused by protons; and those that bind to PAC channels under neutral conditions to prevent protons from activating the channel. Currently reported inhibitors of PAC channels include PIP2 and other broad-spectrum chloride channel inhibitors such as DIDS and NPPB. In a preferred embodiment, PAC channel inhibitors include Evans blue (EB), trypan blue (TB), aniline blue (AB), Coomassie brilliant blue (CBB), Chicago sky blue 6B (CSB), direct blue 71 (DB-71), and thiazole yellow (TY).

[0107] The structural formula of Evans Blue (EB) is Figure 1 shown.

[0108] Evans blue, a blue crystalline powder, is a commonly used azo dye preparation. It has a high affinity with plasma albumin in the blood. Therefore, it is often used as a tracer in neuroscience research to observe the integrity of the blood-brain barrier (BBB) ​​and to measure blood volume. The structural formula of trypan blue (TB) is as follows: Figure 3 shown.

[0109] Trypan blue is commonly used as a cell viability dye to detect the integrity of cell membranes and whether cells are alive. Living cells will not be stained blue, while cells that have lost their activity or have incomplete cell membranes have increased permeability of the cell membrane and can be stained blue by trypan blue. It is generally believed that the loss of cell membrane integrity means that the cell is dead. Therefore, trypan blue staining is one of the most commonly used staining methods for dead cell identification in tissue and cell culture. The structural formula of aniline blue (AB) is as follows: Figure 5 shown.

[0110] Aniline blue is a commonly used biological stain used to stain nerve tissue, cells, and connective tissue. Aniline blue is red in acidic environments and blue in alkaline environments, so it can be used as an acid-base indicator. Aniline blue can also be used to stain plant callose. The structural formula of Coomassie Brilliant Blue (CBB) is as follows: Figure 7 shown.

[0111] Coomassie Brilliant Blue is an anionic dye that can be used for protein analysis in solution. It can be used to determine microgram-level protein content, with a protein concentration range of 0 to 1,000 μg / mL. It is a commonly used method for rapid determination of trace amounts of protein. The structural formula of Chicago Sky Blue 6B (CSB) is as follows: Figure 9 shown.

[0112] Chicago Sky Blue 6B is commonly used as a dye and indicator. In the field of neuroscience, it is generally used as a counterstain for background autofluorescence in fluorescence and immunofluorescence histochemistry; in chemical laboratories, it can be used as an acid-base indicator to detect the acidity and alkalinity of solutions. It can also be used to dye materials such as fibers, plastics, and paints. The structural formula of Direct Blue 71 (DB-71) is as follows: Figure 11 shown.

[0113] Direct Blue 71 is a triazo dye used for staining membrane-immobilized antibodies, proteins, and adsorption studies. Direct Blue 71 is also used to stain tissue sections fixed on PVDF for in situ proteome analysis, and can be used to stain electroporated membranes and brain sections to reveal their structure; it is also used to stain cellulose fibers such as cotton, linen, and viscose. The structural formula of Thiazole Yellow (TY) is as follows: Figure 13 shown.

[0114] Thiazole Yellow G is a pH-sensitive chromogenic ionophore that can be used as a stain and fluorescent indicator for the photocatalytic conversion of azo dyes. Its pH range is 12.0-13.0. It is used for the determination of boron and magnesium, as well as in biochemistry and histochemistry.

[0115] Compound pharmaceutical composition and medicine kit

[0116] The present invention provides a compound pharmaceutical composition comprising the active ingredients (a) a PAC channel inhibitor; optionally (b) a drug for preventing and / or treating osteosarcoma; and (c) a pharmaceutically acceptable carrier. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, powders, and combinations thereof. The pharmaceutical formulation should be compatible with the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, using physiological saline or an aqueous solution containing glucose and other adjuvants by conventional methods. Pharmaceutical compositions such as tablets and capsules can be prepared by conventional methods. Pharmaceutical compositions such as injections, solutions, tablets, and capsules are preferably manufactured under sterile conditions. The pharmaceutical combination of the present invention can also be prepared as a powder for aerosol inhalation. A preferred dosage form is an injectable preparation. In addition, the pharmaceutical composition of the present invention can also be used in conjunction with other therapeutic agents.

[0117] The present invention also provides a medicine kit for preventing and / or treating osteosarcoma, the medicine kit comprising:

[0118] (a1) a first container, and a PAC channel inhibitor or a drug containing a PAC channel inhibitor in the first container;

[0119] (b1) an optional second container, and other drugs for preventing and / or treating osteosarcoma, or a drug containing other drugs for preventing and / or treating osteosarcoma, located in the second container.

[0120] The pharmaceutical composition and kit of the present invention are suitable for preventing and / or treating osteosarcoma.

[0121] The preparation of the present invention can be taken three times a day to once every ten days, or taken once every ten days in a sustained-release manner. The preferred mode is to take it once a day because it is convenient for the patient to adhere to it, thereby significantly improving the patient's compliance with the medication.

[0122] When taking the drug, the total daily dose generally used in most cases should be lower than (or equal to or slightly greater than) the usual daily dose of each single drug in a few cases. Of course, the effective dose of the active ingredient used may vary depending on the mode of administration and the severity of the disease to be treated.

[0123] Treatment

[0124] The present invention also provides a method for preventing and / or treating osteosarcoma using the above-mentioned active ingredients of the present invention or corresponding drugs, which comprises administering to a mammal an effective amount of the active ingredient (a) a PAC channel inhibitor; and optionally (b) other drugs for preventing and / or treating osteosarcoma (e.g., doxorubicin, cisplatin, methotrexate, ifosfamide), or administering a pharmaceutical composition containing the active ingredient (a) and the optional active ingredient (b).

[0125] When the active ingredient of the present invention is used for the above-mentioned purposes, it can be mixed with one or more pharmaceutically acceptable carriers or excipients, such as solvents, diluents, etc., and can be administered orally in the form of tablets, pills, capsules, dispersible powders, granules or suspensions (containing, for example, about 0.05-5% suspending agents), syrups (containing, for example, about 10-50% sugar), and elixirs (containing about 20-50% ethanol), or parenterally in the form of sterile injectable solutions or suspensions (containing about 0.05-5% suspending agents in an isotonic medium). For example, these pharmaceutical preparations can contain about 0.01-99%, more preferably about 0.1%-90% by weight of the active ingredient mixed with a carrier.

[0126] The active ingredient or pharmaceutical composition of the present invention can be administered by conventional routes, including but not limited to: intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, oral, intratumoral or topical administration. Preferred routes of administration include oral administration, intramuscular administration or intravenous administration.

[0127] From the standpoint of ease of administration, preferred pharmaceutical compositions are liquid compositions, especially injections.

[0128] The main advantages of the present invention include:

[0129] (1) The present invention first discovered that PAC channel inhibitors (such as Evans blue (EB), trypan blue (TB), aniline blue (AB), Coomassie brilliant blue (CBB), Chicago sky blue 6B (CSB), direct blue 71 (DB-71) and thiazol yellow (TY)) effectively prevent and / or treat osteosarcoma by inhibiting PAC channel activity. In addition, the present invention first discovered that PAC channel inhibitors (such as Evans blue (EB), trypan blue (TB), aniline blue (AB), Coomassie brilliant blue (CBB), Chicago sky blue 6B (CSB), direct blue 71 (DB-71) and thiazol yellow (TY)) have high selectivity for PAC channels and do not inhibit or substantially inhibit other ion channels.

[0130] (2) The present invention first discovered that the inhibitory activity against PAC channels is good, showing significant inhibition at nM concentrations;

[0131] (3) The present invention first discovered that it has almost no inhibitory effect on other ion channels and has good selectivity;

[0132] (4) The present invention discovered for the first time that it has a significant inhibitory effect on the metastasis and invasion ability of malignant osteosarcoma cells.

[0133] The present invention will be further described below with reference to specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, for which specific conditions are not specified, were generally performed under conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise specified, the materials and reagents used in the examples are all commercially available products.

[0134] The test drugs used in Example 1 include: Evans blue (EB), trypan blue (TB), aniline blue (AB), Coomassie brilliant blue (CBB), Chicago sky blue 6B (CSB), direct blue 71 (DB-71) and thiazole yellow (TY), all of which are commercially available drugs.

[0135] The test drugs used in Example 1 were used in multiple doses, the concentrations used for EB were 0.3 μM, 1 μM, 3 μM, and 10 μM; the concentrations used for TB, AB, CBB, CSB, and DB-71 were 1 μM, 3 μM, 10 μM, 30 μM, and 100 μM; and the concentrations used for TY were 0.01 μM, 0.1 μM, 1 μM, 3 μM, 10 μM, and 30 μM, all of which were dissolved in double distilled water.

[0136] The test drug used in Examples 2 and 3 was Evans blue (EB), and the concentrations of EB used were 0.3 μM, 3 μM, and 10 μM;

[0137] Data Analysis

[0138] All data are expressed as mean ± standard error (SEM). Statistical analysis was performed using GraphPad Prism 8.0 software. Comparisons between two groups were performed using a two-tailed unpaired t-test; comparisons between multiple groups were performed using a one-way analysis of variance; and comparisons between multiple groups using a two-way analysis of variance. ns, no significant difference; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

[0139] Example 1 Electrophysiological Recording

[0140] Patch-clamp recordings were performed using a HEKA EPC10 amplifier and PatchMaster software. Borosilicate glass electrodes were pulled using a P-97 electrode puller to a resistance of 3–8 MΩ. The extracellular solution contained 145 mM NaCl, 2 mM KCl, 2 mM MgCl₂, 1.5 mM CaCl₂, 10 mM HEPES, and 10 mM glucose (adjusted to pH 7.2–7.4 with NaOH); the electrode solution contained 135 mM CsCl, 1 mM MgCl₂, 10 mM HEPES, and 5 mM EGTA (adjusted to pH 7.2–7.4 with CsOH). The HEPES in the extracellular solution was replaced with MES at pH 4.5. Whole-cell recordings were voltage-clamped at ±80 mV, and the current sampling rate was 10 kHz. A gravity perfusion system with a freely rotatable perfusion tube was used for perfusion of peptides and ligands. After the cell membrane was permeabilized using the electrodes, the electrodes lifted the cells off the bottom of the culture dish and placed them in front of the perfusion tube for subsequent perfusion. All experiments were performed at room temperature.

[0141] The results showed that EB, TB, AB, CBB, CSB, DB-71 and TY all showed inhibitory effects on PAC channels and inhibited proton-induced PAC currents in a dose-dependent manner ( Figures 1-14 ), and the half inhibition concentration IC50 were 0.62±0.05μM, 8.28±1.98μM, 3.23±0.15μM, 18.69±1.32μM, 5.70±3.44μM, 2.18±0.32μM, 4.97±0.54μM ( Figure 15 ).

[0142] Electrophysiological recordings were then used to investigate the ion channel selectivity of EB. HEK293T cells were transfected with TMEM16A, ClC-2, TRPV1, TRPV2, TRPV3, TRPV4, TRPA1, TRPM8, Kv4.2, Nav1.4, Nav1.5, Nav1.7, and hERG channels to determine their inhibitory effects. Figures 16-28 The results showed that EB had little ability to inhibit the current of these channels ( Figure 29 ), indicating that EB is a highly selective inhibitor of PAC channels.

[0143] Example 2 Scratch test

[0144] The migration ability of two cancer cell lines was observed by scratch test. PAC knockdown (PAC-KD) cell lines (MG-63 and U2OS cell lines, purchased from the Chinese Academy of Sciences Cell Bank) were constructed using the CRISPR / Cas9 system and verified by Western blotting. Cells were plated at 3×10 cells per well. 5 Cells were seeded at a density of 100 cells / well in a 6-well plate. When the cells reached approximately 90% confluency, they were scratched with a sterile pipette tip and washed twice with phosphate-buffered saline (PBS). The culture medium was then replaced with DMEM supplemented with 1% fetal bovine serum. Images were captured using a microscope at 0, 24, and 48 hours, and the scratch area was measured using Fiji software.

[0145] The metastatic ability of osteosarcoma cells was determined by measuring the scratch area. The larger the scratch area, the weaker the metastatic ability of osteosarcoma cells. The results showed that in MG-63 cells (purchased from the cell bank of the Chinese Academy of Sciences), the metastatic ability of MG-63 cells with PAC knockdown was significantly reduced, while EB could inhibit the metastatic ability of normal MG-63 cells in a dose-dependent manner. Among them, 0.3μM EB had a weak effect, while 1μM EB and 10μM EB significantly inhibited the metastatic ability of normal MG-63 cells ( Figure 30 and Figure 31 , two-way ANOVA, **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. )

[0146] Similar effects were also observed in another malignant osteosarcoma cell line, U2OS cells (purchased from the Cell Bank of the Chinese Academy of Sciences): the metastatic ability of U2OS cells with PAC knockdown was significantly reduced, while EB could dose-dependently inhibit the metastatic ability of normal MG-63 cells, among which 0.3 μM EB had almost no effect, while 1 μM EB and 10 μM EB significantly inhibited the metastatic ability of normal U2OS cells ( Figure 34 and Figure 35 , Two-way ANOVA, ns, no significant difference; ****, p < 0.0001. )

[0147] Example 3 Transwell invasion assay

[0148] MG-63 cells (3×10 5 cells / 200 μl) or U2OS cells (2×10 4 Cells (100 μl / 200 μl) were resuspended in culture medium and added to the upper chamber of a Transwell plated with Matrigel. 600 μl of culture medium (purchased from Gibco) containing 20% ​​fetal bovine serum (FBS) was added to the lower chamber. After 48 hours of culture in EB or DMEM, the cells were fixed with 4% paraformaldehyde for 1 hour, washed three times with PBS, and stained with 0.1% crystal violet solution for 20 minutes. Finally, the chamber was rinsed with PBS. Cell images were captured using a microscope, and cell counts were performed using Fiji software.

[0149] The invasive ability of osteosarcoma cells was determined by measuring the number of cells passing through the chamber. The more cells that passed through the chamber, the stronger the invasive ability of osteosarcoma cells. The results showed that in MG-63 cells, the number of PAC-knockdown MG-63 cells passing through the chamber was much smaller than that of normal MG-63 cells, indicating that their invasive ability was significantly reduced. EB can inhibit the invasive ability of normal MG-63 cells in a dose-dependent manner. Among them, 0.3μM EB had a weak effect, while 1μM EB and 10μM EB significantly inhibited the invasive ability of normal MG-63 cells ( Figure 32 and Figure 33 , one-way ANOVA, *, p < 0.05; ****, p < 0.0001. ).

[0150] Similarly, in U2OS cells, the number of PAC-knockdown U2OS cells that passed through the chamber was much smaller than that of normal U2OS cells, indicating that their invasive ability was significantly reduced. EB can dose-dependently inhibit the invasive ability of normal U2OS cells. Among them, the inhibitory effect of 0.3μM EB almost disappeared, while 1μM EB and 10μM EB significantly inhibited the invasive ability of normal MG-63 cells ( Figure 36 and Figure 37 , one-way ANOVA, ns, no significant difference; ***, p < 0.001; ****, p < 0.0001).

[0151] In summary, EB showed a potent inhibitory effect on PAC channels in cell experiments, and dose-dependently inhibited the invasion and metastasis of malignant osteosarcoma cells, indicating that it is a potential prodrug of anticancer drugs.

[0152] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A use of a PAC channel inhibitor, characterized in that: Used for preparing a composition or preparation for preventing and / or treating osteosarcoma.

2. The use according to claim 1, characterized in that The osteosarcoma is malignant osteosarcoma.

3. The use according to claim 1, characterized in that The composition or preparation prevents and / or treats osteosarcoma by inhibiting the activity of the PAC channel.

4. The use according to claim 1, wherein The PAC channel inhibitors include Evans blue (EB), trypan blue (TB), aniline blue (AB), Coomassie brilliant blue (CBB), Chicago sky blue 6B (CSB), direct blue 71 (DB-71) and thiazolyl yellow (TY).

5. The use according to claim 1, characterized in that The PAC channel inhibitor selectively inhibits PAC channels, and does not inhibit or substantially does not inhibit other ion channels (such as TMEM16A, ClC-2, TRPV1, TRPV2, TRPV3, TRPV4, TRPA1, TRPM8, Kv4.2, Nav1.4, Nav1.5, Nav1.7 and hERG channels).

6. The use according to claim 1, wherein In the composition or preparation, the concentration of the PAC channel inhibitor is 1-100 μM, preferably 1-30 μM, and more preferably 1-10 μM.

7. The use according to claim 1, characterized in that The composition or preparation is also used for one or more uses selected from the following group: (i) Inhibit the metastatic ability of osteosarcoma cells; (ii) Inhibit the invasive ability of osteosarcoma cells.

8. A pharmaceutical composition for preventing and / or treating osteosarcoma, characterized in that: include: (a1) a first pharmaceutical composition, comprising (a) a first active ingredient, wherein the first active ingredient is a PAC channel inhibitor; and (a2) an optional second pharmaceutical composition, wherein the second pharmaceutical composition comprises (b) a second active ingredient, wherein the second active ingredient is another drug capable of preventing and / or treating osteosarcoma; (b) a pharmaceutically acceptable carrier.

9. A medicine box, characterized in that: include: (a1) a first container, and a PAC channel inhibitor or a drug containing a PAC channel inhibitor in the first container; (a2) Optionally, a second container, and other drugs for preventing and / or treating osteosarcoma, or drugs containing other drugs for preventing and / or treating osteosarcoma, located in the second container.

10. A method for (i) inhibiting the metastatic ability of osteosarcoma cells; and / or (ii) inhibiting the invasive ability of osteosarcoma cells, characterized in that: include: Culturing osteosarcoma cells in the presence of a PAC channel inhibitor or the pharmaceutical composition of claim 8, thereby (i) inhibiting the metastatic ability of osteosarcoma cells; and / or (ii) inhibiting the invasive ability of osteosarcoma cells.

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