Targeted therapy of SMO inhibitor Sonidegib for bone metastasis of luminal breast cancer

Abstract

The present invention provides a targeted treatment method for luminal breast cancer bone metastasis using the SMO inhibitor Sonidegib. Specifically, the present invention provides a new use of the SMO inhibitor Sonidegib for treating luminal breast cancer bone metastasis. The present invention has found through experiments that the SMO targeted inhibitor Sonidegib can reduce the differentiation of osteoblasts around bone metastases, can effectively inhibit the bone metastasis of breast cancer cells, and provides a new treatment method for clinical breast cancer bone metastasis patients.

Description

Technical Field

[0001] The present invention relates to the field of medicine and biology, and in particular to the use of the SMO inhibitor Sonidegib in targeted treatment of luminal breast cancer bone metastasis. Background Art

[0002] Breast cancer is the most common female tumor in the world, and distant metastasis is the main cause of death in breast cancer patients. The main metastatic sites of breast cancer include bones, lungs, brain, and liver, among which bones are the most common metastatic target organs. More than 70% of patients with advanced breast cancer will have bone metastasis, and bone metastasis is also a major cause of death in patients. An important clinical phenomenon is that the metastasis of different subtypes of breast cancer has obvious organ preference. Breast cancer can be divided into different subtypes such as luminal, HER2-positive, and triple-negative. Among them, luminal is the main subtype, accounting for about 70% of all breast cancers. Although luminal breast cancer generally has a good prognosis and the risk of metastasis and recurrence to organs such as the lungs, liver, and brain is lower than other subtypes, the risk of bone metastasis is higher than other subtypes. Bone metastasis mainly occurs in luminal subtype breast cancer, but the reason for this is not clear, which has hindered the clinical targeted treatment of bone metastasis. It can be said that bone metastasis is one of the most important problems in the treatment of luminal breast cancer.

[0003] At present, the treatment of breast cancer bone metastasis is mainly aimed at inhibiting the activity of osteoclasts, and the commonly used drugs are bisphosphonates and denosumab. However, clinical data show that these treatments can only slow down the progression of bone metastasis and cannot effectively improve the overall survival of patients. In addition, the research and development of these drugs was mainly carried out in the cell animal model of triple-negative breast cancer. Therefore, it is urgent to develop a new treatment for luminal breast cancer bone metastasis to provide a basis for clinical treatment.

[0004] Therefore, there is a need in the art to develop an effective method for treating bone metastasis of luminal breast cancer. Summary of the invention

[0005] The purpose of the present invention is to provide a method for effectively treating luminal breast cancer bone metastasis.

[0006] In a first aspect of the present invention, there is provided a use of a SMO inhibitor for preparing a composition or a preparation for preventing and / or treating cancer metastasis.

[0007] In another preferred embodiment, the SMO inhibitor is a small molecule compound.

[0008] In another preferred embodiment, the SMO inhibitor is Sonidegib or a pharmaceutically acceptable salt thereof.

[0009] In another preferred embodiment, the cancer is a cancer in which proteins related to the Hedgehog signaling pathway are overexpressed.

[0010] In another preferred embodiment, the Hedgehog signaling pathway related proteins include: SCUBE2, Hedgehog ligand, SMO, or a combination thereof.

[0011] In another preferred embodiment, the overexpression refers to that the ratio of the expression level (F1) of the Hedgehog signaling pathway related protein to the expression level (F0) under physiological conditions (ie, F1 / F0) is ≥2, preferably ≥3, and more preferably ≥5.

[0012] In another preferred embodiment, the Hedgehog ligand is a ligand that specifically binds to the receptor PTCH1.

[0013] In another preferred embodiment, the Hedgehog ligands include: SHH, IHH and DHH.

[0014] In another preferred embodiment, the cancer is breast cancer.

[0015] In another preferred embodiment, the breast cancer is selected from the following group: luminal breast cancer, HER2-positive breast cancer, triple-negative breast cancer, or a combination thereof.

[0016] In another preferred embodiment, the breast cancer is luminal breast cancer.

[0017] In another preferred embodiment, the breast cancer metastasis is selected from the group consisting of breast cancer bone metastasis, breast cancer lung metastasis, breast cancer liver metastasis, breast cancer brain metastasis, breast cancer lymph node metastasis, or a combination thereof.

[0018] In another preferred embodiment, the breast cancer metastasis is bone metastasis of breast cancer.

[0019] In another preferred embodiment, the transfer is transfer mediated by Hedgehog signaling.

[0020] In another preferred embodiment, the composition or preparation is used to prevent and / or treat Hedgehog signaling-mediated luminal breast cancer bone metastasis.

[0021] In another preferred embodiment, the composition or preparation is used to prepare a drug for one or more of the following uses:

[0022] (i) Inhibit osteogenic differentiation of cells surrounding cancer metastases;

[0023] (ii) inhibiting the immune escape of breast cancer bone metastasis cells;

[0024] (iii) inhibiting bone metastasis of breast cancer cells;

[0025] (iv) Prolong the survival of breast cancer patients.

[0026] In another preferred embodiment, the cancer cells are breast cancer cells.

[0027] In another preferred embodiment, the breast cancer cells are MCF7 cells.

[0028] In another preferred embodiment, the immune escape is immune escape mediated by Hedgehog signaling.

[0029] In another preferred embodiment, the preparation is an oral preparation or a non-oral preparation.

[0030] In another preferred embodiment, the preparation is selected from the following group: injection, inhalation, tincture, powder, granule, capsule, oral solution, tablet, pill, suspension, emulsion, lozenge, or pill.

[0031] In another preferred embodiment, the preparation is administered orally or by injection.

[0032] In another preferred embodiment, the preparation further comprises other drugs for preventing and / or treating breast cancer metastasis.

[0033] In another preferred embodiment, the subject to which the preparation is administered is a human or a non-human mammal (such as a rodent).

[0034] In a second aspect of the present invention, a pharmaceutical composition is provided, comprising:

[0035] (A1) a therapeutically effective amount of a first active ingredient, wherein the first active ingredient is a SMO inhibitor;

[0036] (A2) a therapeutically effective amount of a second active ingredient selected from:

[0037] (Z1) Ingredients for preventing and / or treating cancer;

[0038] (Z2) an ingredient for preventing and / or treating cancer metastasis; or

[0039] (Z3) a combination of Z1 and Z2;

[0040] (B) a pharmaceutically acceptable carrier or excipient.

[0041] In another preferred embodiment, the first active ingredient and the second active ingredient are different.

[0042] In another preferred embodiment, the SMO inhibitor is Sonidegib or a pharmaceutically acceptable salt thereof.

[0043] In another preferred embodiment, the cancer is breast cancer.

[0044] In another preferred embodiment, the ingredients for preventing and / or treating cancer include: anti-estrogen drugs, targeted therapeutic agents, or chemotherapeutic agents.

[0045] In another preferred embodiment, the anti-estrogen drug includes: tamoxifen, ovarian function inhibitors, aromatase inhibitors or a combination thereof.

[0046] In another preferred embodiment, the chemotherapeutic agent includes: anthracyclines (doxorubicin, epirubicin, daunorubicin and aclatocycline), taxanes (paclitaxel, liposome paclitaxel, albumin paclitaxel and docetaxel) or a combination thereof.

[0047] In another preferred embodiment, the targeted therapeutic agent includes: trastuzumab, pertuzumab, or a combination thereof.

[0048] In another preferred embodiment, the targeted therapeutic agent further comprises a SCUBE2 neutralizing antibody, a Hedgehog ligand neutralizing antibody, other Hedgehog signal inhibitors, or a combination thereof.

[0049] In another preferred embodiment, the Hedgehog ligand neutralizing antibody is a SHH neutralizing antibody.

[0050] In another preferred embodiment, the breast cancer is luminal breast cancer.

[0051] In another preferred embodiment, the breast cancer metastasis is bone metastasis of breast cancer.

[0052] In another preferred embodiment, the mass percentage of the first active ingredient to the total weight of the drug is 0.1-99%.

[0053] In the third aspect of the present invention, there is provided a use of the pharmaceutical composition according to the second aspect of the present invention, wherein the pharmaceutical composition is used to prepare:

[0054] (a) drugs for the prevention and / or treatment of breast cancer;

[0055] (b) Drugs for preventing and / or treating breast cancer metastasis.

[0056] In a fourth aspect of the present invention, there is provided an in vitro non-therapeutic method for inhibiting osteogenic differentiation of mesenchymal stem cells (MSCs), comprising the steps of:

[0057] Mesenchymal stem cells are cultured in the presence of a SMO inhibitor, thereby inhibiting the osteogenic differentiation of the mesenchymal stem cells.

[0058] In another preferred embodiment, the SMO inhibitor is Sonidegib or a pharmaceutically acceptable salt thereof.

[0059] In another preferred embodiment, the osteogenic differentiation of MSCs is induced by cancer cells that overexpress proteins related to the Hedgehog signaling pathway.

[0060] In another preferred embodiment, in the step, cancer cells overexpressing Hedgehog signaling pathway-related proteins and mesenchymal stem cells are co-cultured in the presence of a SMO inhibitor, thereby inhibiting the osteogenic differentiation of the mesenchymal stem cells.

[0061] In another preferred embodiment, the cancer cells are breast cancer cells.

[0062] In a fifth aspect of the present invention, a method for treating and / or preventing breast cancer bone metastasis is provided, comprising the steps of:

[0063] A therapeutically effective amount of a SMO inhibitor or a pharmaceutical composition containing a SMO inhibitor is administered to a subject in need thereof, thereby treating and / or preventing breast cancer bone metastasis.

[0064] In another preferred embodiment, the treatment and / or prevention is selected from the following group:

[0065] (i) Inhibit osteogenic differentiation of cells surrounding breast cancer metastases;

[0066] (ii) inhibiting the immune escape of breast cancer bone metastasis cells;

[0067] (iii) inhibiting bone metastasis of breast cancer cells;

[0068] (iv) Any combination of the above i to iii.

[0069] 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 specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] 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.

[0071] Figure 1 (a) shows the expression level of SCUBE2 in in situ tumors of patients with different subtypes of breast cancer.

[0072] Figure 1 (b) shows the bone-free survival rate of patients in the high SCUBE2 group and the low SCUBE2 group.

[0073] Figure 1 (c) shows the SCUBE2 levels in the serum of breast cancer patients in different metastasis groups.

[0074] Figure 1 (d) shows the optical signals of bone metastasis in mice after iliac artery injection of SCUBE2 knockdown and control breast cancer cells.

[0075] Figure 1 (e) shows the ability of SCUBE2 knockdown and control breast cancer cells to induce osteogenic differentiation. The figure shows the osteoblast calcification area stained with Alizarin red, and the right figure shows the osteoblast staining area statistics.

[0076] Figure 1 (f) shows the optical signals of bone metastasis in the hind limbs of mice after iliac artery injection of Scube2-overexpressing and control breast cancer cells.

[0077] Figure 1 (g) shows the ability of Scube2 overexpression and control breast cancer cells to induce osteogenic differentiation. The figure shows the osteoblast calcification area stained with Alizarin red, and the right figure shows the osteoblast staining area statistics.

[0078] Figure 1 (h) shows the distribution of ALP-positive osteoblasts around tumor cells after iliac artery injection of SCUBE2 knockdown or Scube2 overexpressing breast cancer cells. The right figure shows the percentage of osteoblasts in the tumor microenvironment. *, P<0.05; **, P<0.01; ***, P<0.001; NS, no significant difference.

[0079] Figure 2 (a) shows the SHH content in the conditioned culture medium of breast cancer cells in the SCUBE2 knockdown or overexpression group and the control group.

[0080] Figure 2 (b) shows the activation of Hh reporter gene in MSC induced by breast cancer cell conditioned medium in SCUBE2 knockdown or overexpression group and control group.

[0081] Figure 2 (c) shows the osteogenic differentiation level of MSCs with SMO knockdown induced by the conditioned medium of breast cancer cells in the Scube2 overexpression group and the control group. The figure shows the osteoblast calcification area stained with Alizarin red, and the right figure shows the osteoblast staining area statistics.

[0082] Figure 2 (d) shows the optical signal of bone metastasis in the hind limbs of mice after iliac artery injection of mixed SMO knockdown MSCs, Scube2 overexpressing and control breast cancer cells.

[0083] Figure 2(e) shows the distribution of ALP-positive osteoblasts around tumor cells after sectioning the leg bones of mice. The right figure shows the percentage of osteoblasts in the microenvironment cells around the tumor. *, P<0.05; **, P<0.01; ***, P<0.001; NS, no significant difference.

[0084] Figure 3 The inhibitory effect of Sonidegib treatment on bone metastasis was shown.

[0085] Figure 3 (a) shows the effect of Sonidegib treatment on the expression of downstream target genes of Hedgehog signaling in MSCs.

[0086] Figure 3 (b) shows the effect of Sonidegib treatment on osteogenic differentiation.

[0087] Figure 3 (cd) Show the tumor size (c) and bioluminescent signal intensity of the hind limb leg bone (d) formed after orthotopic injection of mouse breast cancer cell line into the fat pad and the effect of Sonidegib treatment.

[0088] Figure 3 (ef) shows the bioluminescence intensity of bone metastasis (e) and the overall survival rate of mice (f) after left ventricular injection of mouse breast cancer cell lines and the effect of Sonidegib treatment. *, P < 0.05; **, P < 0.01; ***, P < 0.001; NS, no significant difference.

[0089] Figure 4 The SCUBE2 action model is shown. (1) Luminal breast cancer cells highly express SCUBE2 protein, which promotes tumor cells to release SHH protein that adheres to the cell membrane. (2) SHH released by breast cancer cells activates the Hedgehog signaling pathway of osteoblast precursor cells and promotes osteogenic differentiation. (3) Hedgehog signaling-activated osteoblasts release COL1 that binds to the immune cell surface receptor LAIR1, inhibiting immune cell activation. DETAILED DESCRIPTION

[0090] After extensive and in-depth research, the inventors unexpectedly discovered for the first time a new use of the SMO inhibitor Sonidegib in the treatment of luminal breast cancer bone metastasis. The inventors found through experiments that the SMO targeted inhibitor Sonidegib unexpectedly can reduce the differentiation of osteoblasts around bone metastases and inhibit the occurrence of bone metastases. At the same time, Sonidegib blocks the activation of Hedgehog signals, which can not only inhibit breast cancer bone metastasis, but also has the effect of reducing immune escape and enhancing the killing effect of immune cells on bone metastases. On this basis, the present invention was completed.

[0091] Experiments have shown that Sonidegib can inhibit osteogenic differentiation of cells and effectively inhibit bone metastasis of breast cancer cells. It also has the effect of reducing the immune escape of bone metastases, providing a new treatment for clinical breast cancer bone metastasis patients.

[0092] the term

[0093] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0094] As used herein, the terms "comprise", "include", and "contain" are used interchangeably and include not only closed definitions, but also semi-closed and open definitions. In other words, the terms include "consisting of", "consisting essentially of".

[0095] As used herein, the term "pharmaceutically acceptable carrier" refers to a substance that is suitable for use in humans and / or animals without excessive adverse side effects (such as toxicity, irritation and allergic response), ie, has a reasonable benefit / risk ratio.

[0096] As used herein, the term "therapeutically effective amount" refers to an amount that produces a function or activity in humans and / or animals and is acceptable to humans and / or animals. It should be understood by those skilled in the art that the "therapeutically effective amount" may vary depending on the form of the pharmaceutical composition, the route of administration, the adjuvants of the drug used, the severity of the disease, and the combination with other drugs.

[0097] Hedgehog Signaling

[0098] Hedgehog signaling was first discovered in Drosophila and is a highly conserved signaling pathway that plays an important role in embryonic development, tissue homeostasis, and tumorigenesis. The activation of the classical Hedgehog signaling pathway depends on the interaction between the corresponding ligand and receptor. In the absence of Hedgehog ligands (including SHH, IHH, and DHH), the receptor PTCH1 corresponding to the Hedgehog ligand inhibits the activation of Hedgehog signaling by inhibiting the activity of SMO. Once the Hedgehog ligand binds to PTCH1, the inhibition of SMO is released, thereby transmitting the activation signal to the downstream transcription factor GLI, and the overall Hedgehog signal is activated, which further plays an important role in the occurrence and development of tumors. Osteoblasts activated by Hedgehog signaling can release COL1, bind to the immune cell surface receptor LAIR1, inhibit the activation of immune cells, thereby inhibiting the killing of tumors by immune cells, achieving immune escape of breast cancer bone metastasis, and promoting the occurrence of bone metastasis.

[0099] In the present invention, an activation pathway of the Hedgehog signaling pathway is examined using luminal breast cancer as an example. The experiments of the present invention show that the Hedgehog signaling pathway is regulated by the SCUBE2 protein expressed by luminal breast cancer cells. Luminal breast cancer cells highly express the SCUBE2 protein, which promotes tumor cells to release the Hedgehog ligand (SHH protein) adhered to the cell membrane. SHH released by breast cancer cells releases the inhibition of SMO by binding to the corresponding receptor PTCH1, and transmits an activation signal to the downstream transcription factor GLI, thereby activating the Hedgehog signaling pathway of osteoblast precursor cells and promoting osteogenic differentiation.

[0100] In tumors with abnormally elevated secretion of SCUBE2 protein and Hedgehog ligand, SMO is ultimately used to transmit signals to activate the Hedgehog signaling pathway and promote osteogenic differentiation. Therefore, the present invention finds that SMO inhibitors can be used to prevent and / or inhibit tumor metastasis caused by abnormally elevated secretion of SCUBE2 protein and Hedgehog ligand.

[0101] SMO inhibitors

[0102] SMO protein refers to the seven-transmembrane transduction protein Smoothened, which is an important signal transduction protein of the Hedgehog pathway. In an embodiment of the present invention, the SMO protein is preferably a human SMO protein, and its Uniprot ID is Q99835.

[0103] As used herein, the term "SMO inhibitor" refers to any inhibitor that can directly or indirectly reduce the activity of the SMO protein, for example, by binding to the SMO protein, thereby inhibiting the activation of the downstream transcription factor GLI by the SMO protein.

[0104] In the present invention, the SMO inhibitor is preferably Sonidegib and a pharmaceutically acceptable salt thereof.

[0105] Sonidegib is an effective and selective SMO antagonist. Its molecular formula is C26H26F3N3O3 and its CAS number is 956697-53-3. Currently, the SMO inhibitor Sonidegib has been approved by the FDA for the clinical treatment of basal cell tumors. However, there is still a lack of application of Sonidegib for tumor metastasis, especially in the treatment of bone metastasis of luminal breast cancer.

[0106] The present invention unexpectedly discovered for the first time that Sonidegib, a targeted inhibitor of SMO, can reduce the differentiation of osteoblasts around bone metastases and inhibit the occurrence of bone metastases. At the same time, Sonidegib blocks the activation of Hedgehog signals, which can not only inhibit breast cancer bone metastasis, but also reduce immune escape and enhance the killing effect of immune cells on bone metastasis cells. Therefore, the present invention discovered that Sonidegib can be used to prepare drugs for inhibiting breast cancer bone metastasis.

[0107] Cancer bone metastasis

[0108] As used herein, "bone metastasis" refers to a disease in which certain malignant tumors originating from non-bone tissues metastasize to bone tissues through the blood circulation, thereby causing fractures, pain, and other fatal complications. Bone metastasis mainly occurs in breast cancer patients.

[0109] In a preferred embodiment of the present invention, SMO inhibitors are used to prepare drugs for treating breast cancer bone metastasis. The breast cancer described in the present invention includes different subtypes of breast cancer, such as luminal breast cancer, HER2-positive breast cancer and triple-negative breast cancer. Among them, bone metastasis mainly occurs in luminal subtype breast cancer. Breast cancer mainly achieves bone metastasis by promoting the differentiation of mesenchymal stem cells (MSC) into osteoblasts.

[0110] Preferably, the bone metastasis is bone metastasis mediated by Hedgehog signaling.

[0111] The present invention has discovered one of the mechanisms of breast cancer bone metastasis, and the mechanism model is as follows Figure 4 Specifically, the present invention first discovered that:

[0112] (1) SCUBE2 is highly expressed in luminal breast cancer cells, which promotes the release of SHH into the extracellular space, explaining why high SCUBE2 levels were detected in the serum of patients with luminal breast cancer and suggesting that SCUBE2 could be a new target for the detection of breast cancer bone metastasis;

[0113] (2) Osteoblast precursor cells (such as MSC cells) activated by SHH released by breast cancer cells undergo osteogenic differentiation, helping breast cancer cells survive in the bone microenvironment;

[0114] (3) Hedgehog signaling activates osteoblasts to release COL1, which binds to the inhibitory receptor LAIR1 on the surface of immune cells, inhibiting immune activation, allowing tumor cells to escape immune killing and reducing tumor cell apoptosis.

[0115] Pharmaceutical composition

[0116] The present invention also provides a pharmaceutical composition, comprising:

[0117] (A1) a therapeutically effective amount of a first active ingredient, wherein the first active ingredient is a SMO inhibitor;

[0118] (A2) a therapeutically effective amount of a second active ingredient selected from:

[0119] (Z1) Ingredients for preventing and / or treating cancer;

[0120] (Z2) an ingredient for preventing and / or treating cancer metastasis; or

[0121] (Z3) a combination of Z1 and Z2;

[0122] (B) a pharmaceutically acceptable carrier or excipient.

[0123] The pharmaceutical composition provided by the present invention preferably contains 0.1-99 wt % of the first active ingredient, and the remainder is the second active ingredient, a pharmaceutically acceptable carrier, a diluent or a solution or a saline solution.

[0124] The first active ingredient of the present invention can be used directly to treat or prevent cancer metastasis. In addition, it can also be used in combination with other therapeutic agents, i.e., the second active ingredient.

[0125] The second active ingredient can be any pharmaceutical ingredient capable of preventing and / or treating cancer or cancer metastasis, including but not limited to chemotherapeutic agents, endocrine therapeutic agents, targeted therapeutic agents, and the like.

[0126] When necessary, one or more pharmaceutically acceptable carriers may be added to the drug of the present invention, including conventional diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants, etc. in the pharmaceutical field.

[0127] The compounds and pharmaceutical compositions provided by the present invention may be in various forms, such as tablets, injections, capsules, powders, syrups, solutions, suspensions and aerosols, and may be present in suitable solid or liquid carriers or diluents and in suitable sterile apparatus for injection or instillation.

[0128] The various dosage forms of the pharmaceutical composition of the present invention can be prepared according to conventional preparation methods in the pharmaceutical field. The unit dosage of the preparation formula generally contains 0.05-1000 mg of the active compound of the present invention, preferably, the unit dosage of the preparation formula contains 1 mg-500 mg of the active compound of the present invention.

[0129] The pharmaceutical composition of the present invention can be used clinically in mammals, including humans and animals, and can be administered through the mouth, nose, skin, lungs or gastrointestinal tract. Oral administration is the most preferred. The most preferred daily dose is 0.01-400 mg / kg body weight, taken at one time, or 0.01-200 mg / kg body weight taken in divided doses. Regardless of the method of administration, the optimal dose for an individual should be determined based on the specific treatment. Usually, a small dose is started and the dose is gradually increased until the most suitable dose is found.

[0130] The drugs or inhibitors of the present invention can be administered in various ways, for example, by injection, spraying, nasal drops, eye drops, penetration, absorption, physical or chemical mediated methods, such as introduction into the body into muscle, intradermal, subcutaneous, intravenous, mucosal tissue; or mixed or encapsulated in other substances and introduced into the body.

[0131] Typically, the active ingredient of the present invention or the pharmaceutical composition containing it can be administered in a unit dosage form, and the administration route can be enteral or parenteral, such as oral, intravenous, intramuscular, subcutaneous, nasal, oral mucosa, eyes, lungs and respiratory tract, skin, vagina, rectum, etc.

[0132] The dosage form can be a liquid dosage form, a solid dosage form or a semisolid dosage form. Liquid dosage forms can be solutions (including true solutions and colloidal solutions), emulsions (including O / W types, W / O types and multiple emulsions), suspensions, injections (including water injections, powder injections and infusions), eye drops, nasal drops, lotions and liniments, etc.; solid dosage forms can be tablets (including ordinary tablets, enteric-coated tablets, lozenges, dispersible tablets, chewable tablets, effervescent tablets, orally disintegrating tablets), capsules (including hard capsules, soft capsules, enteric-coated capsules), granules, powders, micropills, dropping pills, suppositories, films, patches, aerosols (powders), sprays, etc.; semisolid dosage forms can be ointments, gels, pastes, etc.

[0133] The active ingredients of the present invention can be made into common preparations, sustained-release preparations, controlled-release preparations, targeted preparations and various microparticle drug delivery systems.

[0134] In order to prepare the active ingredients of the present invention into tablets, various excipients known in the art can be widely used, including diluents, binders, wetting agents, disintegrants, lubricants, and glidants. The diluent may be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.; the wetting agent may be water, ethanol, isopropanol, etc.; the adhesive may be starch slurry, dextrin, syrup, honey, glucose solution, microcrystalline cellulose, acacia slurry, gelatin slurry, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, ethyl cellulose, acrylic resin, carbomer, polyvinyl pyrrolidone, polyethylene glycol, etc.; the disintegrant may be dry starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, cross-linked polyvinyl pyrrolidone, cross-linked sodium carboxymethyl cellulose, sodium carboxymethyl starch, sodium bicarbonate and citric acid, polyoxyethylene sorbitan fatty acid ester, sodium dodecyl sulfate, etc.; the lubricant and glidant may be talc, silicon dioxide, stearate, tartaric acid, liquid paraffin, polyethylene glycol, etc.

[0135] The tablets can be further made into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer tablets and multi-layer tablets.

[0136] In order to prepare the dosing unit into a capsule, the active ingredient of the present invention can be mixed with a diluent and a glidant, and the mixture can be directly placed in a hard capsule or a soft capsule. The active ingredient can also be first made into granules or pellets with a diluent, a binder, and a disintegrant, and then placed in a hard capsule or a soft capsule. The diluents, binders, wetting agents, disintegrants, and glidants used to prepare the tablets of the present invention can also be used to prepare the capsules of the present invention.

[0137] In order to prepare the active ingredient of the present invention into an injection, water, ethanol, isopropanol, propylene glycol or a mixture thereof can be used as a solvent and an appropriate amount of a solubilizer, a cosolvent, a pH adjuster, and an osmotic pressure regulator commonly used in the art can be added. The solubilizer or cosolvent can be poloxamer, lecithin, hydroxypropyl-β-cyclodextrin, etc.; the pH adjuster can be phosphate, acetate, hydrochloric acid, sodium hydroxide, etc.; the osmotic pressure regulator can be sodium chloride, mannitol, glucose, phosphate, acetate, etc. If a lyophilized powder injection is prepared, mannitol, glucose, etc. can also be added as a support agent.

[0138] Furthermore, if necessary, colorants, preservatives, perfumes, flavoring agents or other additives may be added to the pharmaceutical preparations.

[0139] The active ingredient or composition of the present invention can be taken alone or in combination with other therapeutic drugs or symptomatic drugs.

[0140] In a preferred embodiment of the present invention, a SMO inhibitor can be used in combination with a SCUBE2 neutralizing antibody, a Hedgehog ligand neutralizing antibody, or other Hedgehog signaling inhibitors to enhance the effect of preventing tumor bone metastasis.

[0141] When the active ingredient of the present invention has a synergistic effect with other therapeutic drugs, its dosage should be adjusted according to the actual situation.

[0142] The main advantages of the present invention include:

[0143] (1) For the first time, it was discovered that the SCUBE2 protein highly expressed in luminal breast cancer cells can promote the release of SHH into the extracellular space of tumor cells and affect the bone metastasis of breast cancer. Therefore, it was proposed for the first time that SCUBE2 can be used as a new detection target for predicting breast cancer bone metastasis.

[0144] (2) For the first time, it was discovered that SHH released by breast cancer cells can activate osteoblast precursor cells to differentiate into osteoblasts, thus helping breast cancer cells survive in the bone microenvironment.

[0145] (3) For the first time, they discovered that osteoblasts activated by Hedgehog signaling can release COL1, which binds to the inhibitory receptor LAIR1 on the surface of immune cells, inhibiting immune activation, allowing tumor cells to escape immune killing, and reducing tumor cell apoptosis.

[0146] (4) For the first time, it was discovered that the SMO inhibitor Sonidegib can effectively alleviate the progression of breast cancer bone metastasis.

[0147] The present invention will be further described below in conjunction with 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 where specific conditions are not specified are usually 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 under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

[0148] Example 1 Relationship between SCUBE2 and bone metastasis of luminal breast cancer

[0149] In this example, by analyzing clinical sequencing data, it was found that compared with several other subtypes, patients with luminal breast cancer expressed high levels of SCUBE2 ( Figure 1 a). Bone metastasis survival analysis found that the bone metastasis-free survival rate of patients in the high-expression SCUBE2 group was significantly lower than that in the low-expression SCUBE2 group, and the incidence of bone metastasis was significantly increased ( Figure 1b). The serum of patients was collected for enzyme-linked immunosorbent assay (ELISA) and it was found that the serum SCUBE2 protein level of breast cancer patients in the bone metastasis group was significantly higher than that in the control group without metastasis ( Figure 1 c).

[0150] The results of this example show that SCUBE2 is closely related to bone metastasis of luminal breast cancer.

[0151] Example 2 Role of SCUBE2 in Bone Metastasis of Breast Cancer

[0152] As an important component of the bone microenvironment, osteoblasts often play an important role in bone metastasis. In this example, the effect of SCUBE2 on osteoblast differentiation and its role in breast cancer bone metastasis were detected.

[0153] First, SCUBE2 was knocked down in the luciferase-labeled breast cancer cell line MCF7, and then the cells were injected into immunodeficient mice through the iliac artery, and the light signal corresponding to the metastatic load was observed by injection of luciferase substrate. In addition, Scube2 was overexpressed in the luciferase-labeled mouse cell line Py8119, and injected into immune-normal mice through the iliac artery to observe the effect of overexpressed SCUBE2 on breast cancer metastasis.

[0154] Results: Figure 1 As shown in the figure, pLKO.1, pLVX, and DMEM are all control groups. The optical signal of tumor cells showed that knocking down SCUBE2 can significantly inhibit the bone metastasis of breast cancer ( Figure 1 d). Furthermore, knockdown of SCUBE2 significantly inhibited the ability of breast cancer cells to induce mesenchymal stem cells (MSCs) to differentiate into osteoblasts ( Figure 1 e). Overexpression of Scube2 can significantly enhance bone metastasis of breast cancer ( Figure 1 f), and the ability of tumor cells to induce MSC osteogenic differentiation in vitro was significantly enhanced ( Figure 1 g). In addition, the aggregation degree of ALP-positive osteoblasts around tumor cells in mouse leg bone slices changed with the expression of SCUBE2, and the proportion of osteoblasts around tumor cells in the SCUBE2 high expression group increased significantly ( Figure 1 h).

[0155] This example illustrates that SCUBE2 promotes the occurrence of bone metastasis by regulating the osteogenic microenvironment.

[0156] Example 3 Effect of SCUBE2 on Hedgehog signaling pathway

[0157] 3.1 Effect of SCUBE2 on Shh release

[0158] SCUBE2 is a key protein in the Hedgehog signaling pathway. It participates in the cleavage and release of Hedgehog signaling pathway ligands attached to the cell membrane, maintains their solubility outside the cell, and activates Hedgehog signals in target cells. Our mechanistic studies have found that SCUBE2 can promote tumor cells to release the Hedgehog signaling pathway ligand SHH and increase the extracellular SHH content.

[0159] In this example, the effect of SCUBE2 on SHH release was detected by knocking down or overexpressing SCUBE2 and detecting the SHH content in the culture medium.

[0160] Results: Compared with the control pLKO.1 group, the SHH content released into the culture supernatant of breast cancer cells MCF7 after SCUBE2 knockdown was significantly decreased, while compared with the control pLVX group, the SHH content released into the culture supernatant of breast cancer cells MCF7 after SCUBE2 overexpression was significantly increased ( Figure 2 a).

[0161] 3.2 Effect of SCUBE2 on Hedgehog signaling activation

[0162] After SHH binds to its receptor PTCH1, it can release the inhibition of SMO activity, thereby transmitting the activation signal to the downstream transcription factor GLI. Therefore, in this example, the activation of the GLI reporter system was used to detect the effect of SCUBE2 on the activation of Hedgehog signaling.

[0163] Results: Compared with the blank control DMEM, the supernatant of MCF7 cells in the control pLKO.1 group could significantly activate the GLI reporter system, while the activation of the GLI system by the supernatant of MCF7 cells was significantly inhibited after knockdown of SCUBE2. Similarly, compared with the supernatant of MDA-MB-231 breast cancer cells in the control pLVX group, the activation of the GLI system was significantly enhanced after overexpression of SCUBE2 ( Figure 2 b).

[0164] This example shows that SCUBE2 can affect breast cancer cells to release SHH to the extracellular space, and further affect the activation of Hedgehog signaling in surrounding cells.

[0165] Example 4 Effect of knockdown of SMO in MSCs on breast cancer bone metastasis

[0166] The Hedgehog signaling pathway plays an important role in promoting osteoblast differentiation. Osteoblasts, as bone microenvironment cells, are important for the survival, colonization and growth of bone metastatic cells.

[0167] In this example, the effect of knocking down SMO, a key factor in the Hedgehog signaling pathway, on the differentiation of MSC into osteoblasts was detected in MSCs. The MSC cells with knocked down SMO were mixed with tumor cells overexpressing Scube2 and injected into mice to observe the bone metastasis of breast cancer in mice. The proportion of osteoblasts around bone metastases was observed by immunofluorescence staining of mouse leg bones.

[0168] Results: Knockdown of SMO in MSCs inhibited the differentiation of MSCs into osteoblasts ( Figure 2 c). In addition, knocking down SMO to inhibit the Hedgehog signaling pathway can inhibit the formation of breast cancer bone metastasis ( Figure 2 d), and significantly reduced the distribution ratio of osteoblasts around bone metastases ( Figure 2 e).

[0169] This example shows that knocking down SMO in MSCs can effectively inhibit breast cancer bone metastasis.

[0170] Example 5 Effect of Sonidegib on Osteogenic Differentiation of MSC Cells

[0171] In this example, MSCs were treated with Sonidegib, a specific inhibitor of SMO, and the expression of Hedgehog signal downstream target genes Runx2, Osterix, Alpl, Cd1a1, Gli1, and Ptch1 in MSCs was detected. The osteogenic differentiation of MSCs was detected by alkaline phosphatase activity.

[0172] Results: Sonidegib could significantly inhibit the activation of Hedgehog signaling pathway in MSCs ( Figure 3 a), and the alkaline phosphatase activity in the Sonidegib-treated group was significantly lower than that in the control group, proving that Sonidegib can inhibit the osteogenic differentiation of MSCs ( Figure 3 b).

[0173] Example 6 Effect of Sonidegib on Breast Cancer Metastasis in Mice

[0174] The SMO inhibitor Sonidegib was used in mice, and the size of tumors formed after orthotopic injection of mouse breast cancer cell lines into the fat pad was measured to detect whether Sonidegib could significantly reduce the metastasis of breast cancer cells from the orthotopic site to the bone tissue.

[0175] Results: Drug treatment had little effect on the growth of in situ tumors ( Figure 3 c). However, the bone bioluminescence signal of mice in the Sonidegib treatment group was significantly reduced ( Figure 3d). In the left ventricular injection bone metastasis model, Sonidegib treatment also significantly inhibited bone metastasis signals ( Figure 3 e) and prolonged the overall survival of mice ( Figure 3 f).

[0176] This example demonstrates that Sonidegib significantly inhibits bone metastasis of tumor cells and prolongs the overall survival of mice, but Sonidegib has no significant effect on the size of the in situ tumor.

[0177] discuss

[0178] In summary, the present invention discovered for the first time that targeted inhibitors of SMO, a key factor in Hedgehog signaling in microenvironmental cells (such as Sonidegib), can effectively reduce the differentiation of osteoblasts around breast cancer bone metastases and inhibit the occurrence of breast cancer bone metastases.

[0179] The Hedgehog signaling pathway plays an important role in the occurrence and development of tumors, especially in the bone metastasis of tumors. Therapeutic methods targeting Hedgehog include, for example, Hedgehog ligand inhibitors, Hedgehog transduction factor SMO inhibitors, and Hedgehog transcription factor GLI inhibitors.

[0180] Through the embodiments of the present invention, an important activation pathway of Hedgehog signaling and the effect of Hedgehog signaling on breast cancer bone metastasis were verified. Specifically, it was found that luminal breast cancer cells promote the release of SHH protein on the cell membrane by highly expressing SCUBE2. SHH protein releases the inhibition of PTCH1 on SMO protein activity by binding to its corresponding receptor PTCH1, thereby releasing the activation signal to the downstream transcription factor GLI, thereby activating the Hedgehog signaling in osteoblasts, in addition to causing tumor bone metastasis.

[0181] In addition, since osteoblasts can release COL1 after being activated by Hedgehog signals, it binds to the immune inhibitory receptor LAIR1 on the surface of immune cells, inhibiting the activation of immune cells, thereby inhibiting the killing of tumors by immune cells, and achieving immune escape of breast cancer bone metastasis. Therefore, the present invention can not only inhibit breast cancer bone metastasis by blocking Hedgehog signal activation through Sonidegib, but also has the effect of alleviating immune escape, providing a new treatment method for clinical breast cancer bone metastasis patients.

[0182] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A use of a SMO inhibitor, characterized in that: Used for preparing a composition or preparation, wherein the composition or preparation is used for treating bone metastasis of luminal breast cancer, and the SMO inhibitor is Sonidegib or a pharmaceutically acceptable salt thereof.

2. A pharmaceutical composition for preparing a drug for treating bone metastasis of luminal breast cancer, characterized in that: The pharmaceutical composition comprises: (A1) a therapeutically effective amount of a first active ingredient, wherein the first active ingredient is Sonidegib or a pharmaceutically acceptable salt thereof; (A2) a therapeutically effective amount of a second active ingredient selected from: (Z1) Ingredients for the prevention and / or treatment of cancer; (Z2) compositions for preventing and / or treating cancer metastasis; and (B) a pharmaceutically acceptable carrier.

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