Application of medicine in treatment of chordoma of different molecular types

Abstract

The invention relates to the technical field of targeted intervention of chordoma, in particular to application of a medicine to treatment of chordoma of different molecular types. The invention provides application of anlotinib, an oxidative phosphorylation inhibitor and desumab in preparation of products for preventing and / or treating chordoma. The chordoma comprises interstitial epithelial transformed chordoma, mesenchymal derived chordoma and bone microenvironment dominant chordoma. The invention also provides a pharmaceutical composition, which comprises an effective dose of anlotinib, and / or an oxidative phosphorylation inhibitor, and / or desumab. Under the condition that current treatment selection is limited, proliferation, clone formation and tumor growth of tumor cells can be remarkably inhibited by applying the medicine to specific molecular subtype chordoma, and compared with existing treatment medicine, the medicine has a better treatment effect, has good safety and shows potential clinical application value.

Description

Technical Field

[0001] The present application relates to the technical field of targeted intervention for chordomas, and in particular to the use of drugs in treating chordomas of different molecular types. Background Art

[0002] Chordoma is a rare malignant tumor that originates from residual cells of the notochord that have not degenerated during embryonic development, and mainly occurs at the fusion site of the axial bones of the spine and skull base. Chordoma is locally invasive and usually has low to moderate malignancy. Although surgical resection is the main treatment for chordoma, because chordoma is often located in areas with complex anatomical structures, it is difficult to completely remove tumor tissue through surgical resection, resulting in a high recurrence rate. In addition, chordoma shows significant tolerance to traditional chemotherapy and radiotherapy, which makes the prognosis of patients generally poor.

[0003] Molecular pathological studies of chordomas have revealed their significant heterogeneity, and chordomas have been further divided into different molecular subtypes. Chordomas of different subtypes differ in molecular mechanisms, resulting in different sensitivities to drugs. Therefore, chordomas of different molecular subtypes often require different drugs for treatment. Existing drugs are not effective in chordomas of specific molecular subtypes, and some drugs that are effective in other types of chordomas often fail to produce the expected therapeutic effects in these types. At the same time, research also needs to gain a deeper understanding of the key regulatory factors and microenvironmental changes in different processes in order to develop more precise and personalized treatment plans and improve the treatment effects of specific types of chordomas. Summary of the invention

[0004] Based on the defects of the above-mentioned prior art, the present application develops new drugs and treatment strategies to target the molecular characteristics and signaling pathways unique to mesenchymal epithelial transformation chordoma, mesenchymal chordoma, and bone microenvironment-dominated chordoma, break its drug resistance mechanism, and reactivate sensitivity to treatment. The present application found that anlotinib has a significant inhibitory effect in mesenchymal epithelial transformation chordoma, especially inhibiting mesenchymal epithelial transformation chordoma cell proliferation and tumor growth, and based on this discovery, studied the mechanism of anlotinib in the treatment of mesenchymal epithelial transformation chordoma. The present application also found and confirmed that S-Gboxin can effectively inhibit the growth of mesenchymal chordoma, and its mechanism is mainly through inhibiting the activity of mitochondrial complex V, blocking the oxidative phosphorylation pathway, reducing the generation of ATP in tumor cells, and then inhibiting the proliferation and survival of tumor cells. The present application also found that denosumab has the effect of inhibiting bone microenvironment-dominated chordoma.

[0005] In a first aspect, the present application provides use of anlotinib or a pharmaceutically acceptable salt thereof in preparing a product for preventing and / or treating chordoma.

[0006] The product comprises an effective dose of anlotinib or a pharmaceutically acceptable salt thereof.

[0007] Preferably, the effective dose of anlotinib or a pharmaceutically acceptable salt thereof is greater than or equal to 3 mg / kg body weight.

[0008] More preferably, the effective dose of anlotinib or a pharmaceutically acceptable salt thereof is 3 to 6 mg / kg body weight.

[0009] The chordoma includes mesenchymal-epithelial transformation chordoma.

[0010] The second aspect of the present application provides the use of an oxidative phosphorylation inhibitor in the preparation of a product for preventing and / or treating chordoma.

[0011] The product comprises an effective amount of an oxidative phosphorylation inhibitor.

[0012] Preferably, the oxidative phosphorylation inhibitor comprises S-Gboxin or a pharmaceutically acceptable salt thereof.

[0013] More preferably, the effective dose of the oxidative phosphorylation inhibitor is greater than or equal to 10 mg / kg body weight.

[0014] The chordoma includes mesenchymal chordoma.

[0015] The third aspect of the present application provides the use of denosumab in preparing a product for preventing and / or treating chordoma.

[0016] The product comprises an effective dose of denosumab.

[0017] Preferably, the effective dose of denosumab is greater than or equal to 2 mg / kg body weight.

[0018] The chordoma includes bone microenvironment-dominant chordoma.

[0019] The fourth aspect of the present application provides a pharmaceutical composition comprising an effective dose of any one or more of the following:

[0020] 1) Anlotinib or a pharmaceutically acceptable salt thereof;

[0021] 2) Oxidative phosphorylation inhibitors;

[0022] 3) Desumebambo.

[0023] The beneficial effects of this application are:

[0024] This application provides a new treatment plan for the clinical development of chordomas with different molecular classifications. By using specific drugs for specific molecular classifications, it has better targeting and achieves better treatment effects.

[0025] This application unexpectedly discovered that anlotinib has a significant effect in treating mesenchymal epithelial transformation chordoma. Under the current limited treatment options, the use of anlotinib at 0.5 μM can significantly inhibit tumor cell proliferation and cloning, and at 3 mg / kg can significantly inhibit tumor growth. It has better therapeutic effects than existing therapeutic drugs and has good safety, showing potential clinical application value. This application not only deepens the understanding of the mechanism of action of anlotinib, but also provides theoretical support and practical basis for the precise treatment of mesenchymal epithelial transformation chordoma.

[0026] Based on the study of the molecular pathological characteristics of chordoma in this application, it was found that there is significant heterogeneity within mesenchymal chordoma, and its cells show a high dependence on the oxidative phosphorylation pathway. Tumor growth can be effectively inhibited by intervening in this pathway. This application discovered and confirmed for the first time that S-Gboxin can effectively inhibit the growth of this type of chordoma, and the dose can inhibit chordoma when it is greater than or equal to 10 mg / kg body weight, and there is no toxic reaction. The therapeutic effect of S-Gboxin on mesenchymal chordoma is better than other existing therapeutic drugs. This application clarifies the mechanism of action of S-Gboxin, and therefore this application provides a new, effective and targeted treatment strategy for the treatment of mesenchymal chordoma.

[0027] This application creatively discovered for the first time that denosumab can inhibit the generation and activity of osteoclasts, reduce bone destruction, and improve patient prognosis; the therapeutic effect is significant, and only 2 mg / kg body weight of denosumab can effectively inhibit the tumor growth of bone microenvironment-dominated chordoma, slow tumor progression, and reduce tumor volume. In addition, denosumab treatment in PDX did not cause obvious toxic side effects and showed good safety. It provides a new targeted intervention strategy for the treatment of bone microenvironment-dominated chordoma, has broad clinical application prospects, not only fills the gap in existing treatment methods, but also provides important theoretical and practical support for the future development of anti-tumor drugs based on the RANKL pathway. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 A is the IC50 curve of anlotinib in the chordoma cell line UCH2 in Example 1; Figure 1 BD is a schematic diagram of anlotinib inhibiting the proliferation and clone formation of chordoma cell line UCH2 in Example 1.

[0029] Figure 2 This is a schematic diagram showing that anlotinib inhibits the expression of RTK (FGFR, PDGFR and VEGFR) in the chordoma cell line UCH2 in Example 2.

[0030] Figure 3AD is a schematic diagram of anlotinib inhibiting the tumor volume and weight of mesenchymal-epithelial transition chordoma PDX in Example 3; Figure 3 E is a schematic diagram of the body weight changes of mice bearing mesenchymal-epithelial transition-type chordoma PDX in Example 3 in which anlotinib inhibits the growth of chordoma PDX.

[0031] Figure 4 AC is a schematic diagram of the inhibition of tumor volume and weight of mesenchymal-epithelial transition chordoma CDX by anlotinib in Example 4; Figure 4 D is a schematic diagram of the body weight changes of mice bearing mesenchymal-epithelial transition-type chordoma CDX in Example 4 in which anlotinib inhibits the effect.

[0032] Figure 5 A is the IC50 curve of S-Gboxin in the chordoma cell line CH22 in Example 5 of the present application; Figure 5 BD is a schematic diagram of S-Gboxin inhibiting the proliferation and clone formation of chordoma cell line CH22 in Example 5 of the present application.

[0033] Figure 6 AB is a schematic diagram of S-Gboxin reducing the oxygen consumption rate (OCR) of chordoma cell line CH22 in Example 6; Figure 6 CD is a schematic diagram of S-Gboxin reducing the extracellular acidification rate (ECAR) of the chordoma cell line CH22 in Example 6.

[0034] Figure 7 AC is a schematic diagram showing that S-Gboxin inhibits the tumor volume and weight of mesenchymal chordoma PDX in Example 7; Figure 7 D is a schematic diagram of the effect of S-Gboxin on the weight changes of mice bearing mesenchymal chordoma PDX in Example 7.

[0035] Figure 8 AC is a schematic diagram showing that S-Gboxin inhibits the tumor volume of mesenchymal chordoma CDX in Example 8; Figure 8 D is a schematic diagram of the body weight changes of mice bearing mesenchymal chordoma CDX inhibited by S-Gboxin in Example 8 of the present application.

[0036] Fig. 9 AC is a schematic diagram of the inhibition of tumor volume and weight of bone microenvironment-dominant chordoma PDX by denosumab in Example 9. DETAILED DESCRIPTION

[0037] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0038] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing the specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0039] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

[0040] In this application, prevention refers to taking measures to prevent the occurrence of a disease or reduce the risk of a disease, and can be divided into primary prevention, which is taking measures before the disease occurs in order to prevent the occurrence of the disease; secondary prevention, which aims to detect and treat the disease in its early stages to prevent the development and worsening of the disease; and tertiary prevention, which is taking measures after the disease has occurred to prevent further worsening of the disease or the development of complications.

[0041] Treatment refers to taking steps after a disease occurs to relieve symptoms, cure a disease, or manage a condition in order to restore health or improve quality of life. Treatment can include the use of drugs to treat a disease.

[0042] The chordoma is a rare malignant tumor derived from the residual cells of the notochord that have not degenerated during embryonic development, and mainly occurs at the fusion site of the axial bones of the spine and the base of the skull. Chordoma is locally invasive and usually low to moderately malignant. Although surgical resection is the main method for treating chordoma, since chordoma is often located in a part with complex anatomical structure, it is difficult to completely remove tumor tissue by surgical resection, resulting in a high recurrence rate. In addition, chordoma shows significant tolerance to traditional chemotherapy and radiotherapy, which generally makes the patient's prognosis poor. Chordoma includes but is not limited to classic chordoma, chondroid chordoma, dedifferentiated chordoma, ossifying chordoma, clear cell chordoma, and neurochordoma.

[0043] Molecular pathological studies of chordoma have revealed its significant heterogeneity, and chordoma has been further divided into different molecular subtypes. The research of this application found that chordoma has multiple molecular subtypes, including mesenchymal epithelial transformation chordoma, mesenchymal chordoma, and bone microenvironment-dominated chordoma. Among them, the bone microenvironment-dominated type shows a specific protein expression spectrum related to bone destruction, showing the characteristics of bone microenvironment cells including osteoclasts, osteoblasts, mast cells, neutrophils, NK cells, macrophages, T cells and B cells; the mesenchymal type shows the characteristics of mesenchymal cells and mitochondrial oxidative phosphorylation, and its characteristic proteins are closely related to energy metabolism; the mesenchymal epithelial transformation type shows the characteristics of enrichment of epithelial characteristics (mesenchymal epithelial transformation, epithelial cell proliferation), decomposition of extracellular matrix and response to tyrosine kinase (PDGF).

[0044] Most of the existing drugs target classic chordoma targets, such as Brachyury protein, mTOR, and EGFR, but these targets may lose their original expression or activity in mesenchymal-epithelial-transition chordoma, resulting in a significant decrease in drug efficacy. In addition, mesenchymal-epithelial-transition chordoma cells have stronger drug resistance and survival advantages, which further limits the effectiveness of existing therapies, making the efficacy of many drugs that are effective in other types of chordomas unsatisfactory in mesenchymal-epithelial-transition chordoma.

[0045] On the other hand, the reprogramming of gene expression and signaling pathways brought about by the mesenchymal-epithelial transition process increases the difficulty of developing targeted drugs. Traditional targeted drugs are difficult to work in mesenchymal-epithelial transition chordomas because these tumor cells may activate alternative survival pathways during the mesenchymal-epithelial transition process, such as Wnt, TGF-β, and Notch signaling pathways, which are closely related to the proliferation, survival, and metastasis of tumor cells, but the targeting effects of existing drugs on these pathways are relatively limited. In addition, the microenvironment of mesenchymal-epithelial transition chordomas will also undergo significant changes, such as increased secretion of immunosuppressive molecules and profibrotic factors, which allows tumor cells to more effectively evade the surveillance of the immune system and counteract the effects of tumor suppressors during treatment.

[0046] Compared with other chordoma subtypes, mesenchymal chordomas usually show worse clinical prognosis and treatment effects. The difficulty in treating mesenchymal chordomas lies in their unique and complex biological characteristics. First, this type of chordomas shows high heterogeneity. The diversity of tumor cells and microenvironment increases the difficulty of treatment, making it difficult to fully cover with a single treatment. In addition, the high invasiveness of the tumor makes it easy to invade important structures such as surrounding nerves, blood vessels and bones, making surgical resection difficult and increasing the risk of tumor residue and recurrence. Mesenchymal chordomas are highly resistant to radiotherapy and chemotherapy, partly because they have strong DNA repair ability and immunosuppressive microenvironment, which reduces the effectiveness of traditional treatment. Due to the incomplete understanding of molecular mechanisms, the treatment effects are mixed. In addition, due to the lack of effective prognostic evaluation markers, the selectivity of individualized treatment is limited, and it is difficult to formulate a highly targeted treatment plan. The high recurrence and metastasis rates further increase the complexity of treatment, making long-term management a major challenge. The multifactorial resistance and complex pathological characteristics of mesenchymal chordomas make it face significant difficulties in diagnosis, treatment and management. Therefore, new treatment options need to be developed.

[0047] Bone microenvironment-dominated chordomas also face unique challenges in treatment and medication, mainly due to the complex interaction between the tumor and the bone microenvironment. The bone microenvironment provides the tumor with a wealth of growth factors, intercellular communication signals, and structural support, allowing tumor cells to survive and proliferate in it, and acquire stronger invasive capabilities and drug resistance. Existing treatment options mostly focus on targeting specific molecules or signaling pathways in the chordoma cells themselves, such as Brachyury protein, mTOR, and EGFR. Although they have certain effects in other types of chordomas, they perform poorly in bone microenvironment-dominated chordomas. This situation occurs in part because these drugs fail to effectively penetrate and regulate the complex cellular and matrix components in the bone microenvironment, thereby failing to inhibit the interaction between tumor cells and the microenvironment.

[0048] In addition, osteoblasts, osteoclasts, and bone marrow stromal cells in the bone microenvironment can secrete a variety of cytokines, such as TGF-β, RANKL, and osteoprotegerin (OPG), which not only play an important role in maintaining bone homeostasis, but also provide growth-promoting and anti-apoptotic signals for tumor cells. This dynamic microenvironment forms a "shelter" with chordoma cells that is conducive to tumor growth and spread, making many drugs that are effective in in vitro tests ineffective during in vivo treatment due to the barrier effect of the microenvironment. The protective effect of this bone microenvironment allows tumor cells to evade the surveillance of the immune system and become resistant to drug treatment.

[0049] Although existing drugs have shown some efficacy in other types of chordomas, they are less effective in bone microenvironment-dominated chordomas, partly because these drugs are difficult to target regulatory factors and signaling pathways unique to the bone microenvironment. Therefore, a single therapeutic strategy that targets tumor cells themselves is often not enough to effectively curb this type of chordoma.

[0050] In the present application, the product includes but is not limited to drugs, combinations of drugs, pharmaceutical compositions, reagents, and kits.

[0051] In this application, an effective dose refers to a dose of a drug, treatment or other intervention measure that achieves the desired therapeutic effect without causing unacceptable side effects or toxicity, and the effective dose is expressed as the weight of the drug administered per kilogram of body weight to each individual.

[0052] The present application found that anlotinib has a significant inhibitory effect in mesenchymal epithelial transformation chordoma, and further explored the mechanism of anlotinib in the treatment of mesenchymal epithelial transformation chordoma. The present application found that anlotinib effectively inhibits multiple RTK signaling pathways such as FGFR, PDGFR, and VEGFR, thereby inhibiting the proliferation and angiogenesis of tumor cells, and significantly inhibiting the growth of mesenchymal epithelial transformation chordoma. Specifically, the research of the present invention shows that anlotinib can significantly reduce the expression levels of FGFR, PDGFR, and VEGFR in the mesenchymal epithelial transformation chordoma cell line UCH2, thereby inhibiting the proliferation and cloning ability of the cell line. In addition, the present invention further confirmed the inhibitory effect of anlotinib on mesenchymal epithelial transformation chordoma in vivo by CDX and PDX. In the CDX model, anlotinib was administered orally at a dose of 3 mg / kg and 6 mg / kg, which significantly inhibited tumor growth; in the PDX model, anlotinib was administered orally at a dose of 6 mg / kg, which significantly inhibited tumor volume growth without causing significant weight loss, indicating that anlotinib has good safety while inhibiting tumor growth. In addition, the present application found that anlotinib has a more significant therapeutic effect on mesenchymal epithelial transformation chordoma than other drugs.

[0053] The present application first provides the use of anlotinib or a pharmaceutically acceptable salt thereof in preparing a product for preventing and / or treating chordoma.

[0054] Anlotinib, CAS No. 1058156-90-3, molecular formula C 23 H 22 FN3O3, molecular weight is 407.44, its structural formula is as follows:

[0055]

[0056] The pharmaceutically acceptable salt refers to the salt of anlotinib, which is prepared by anlotinib of the present application and a relatively non-toxic acid or base. When the anlotinib of the present application contains a relatively acidic functional group, a base addition salt can be obtained by contacting the neutral form of the compound of the present application with a sufficient amount of base in a pure solution or a suitable inert solvent. Pharmaceutically acceptable bases include salts prepared from inorganic bases and organic bases, and the salts of the inorganic bases include aluminum salts, ammonium salts, calcium salts, copper salts, iron salts, ferrous salts, lithium salts, magnesium salts, manganese salts, manganous salts, potassium salts, sodium salts, zinc salts, etc. The salts of the organic non-toxic bases include salts of primary amines, secondary amines and tertiary amines, including substituted amines and cyclic amines. For example: N, N'-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, aminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucosamine, glucosamine, histidine, hydroxocobalamin, isopropylamine, lysine, methylglucosamine, morpholine, piperazine, piperidine, guanidine, polyamine resin, procaine, purine, theobromine, triethylamine, trimethylamine, tripropylamine, etc. When the compounds of the present application contain relatively basic functional groups, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of acid in a pure solution or a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts, such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, hydrogen sulfate, hydroiodic acid, phosphorous acid, etc.; and organic acid salts, such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid and methanesulfonic acid and the like. For example, the salt of anlotinib in the present application can be anlotinib hydrochloride.

[0057] In a specific embodiment of the present application, the chordoma is a mesenchymal-epithelial transition chordoma. The mesenchymal-epithelial transition chordoma is significantly difficult to treat, mainly due to its unique biological characteristics and high heterogeneity. Mesenchymal-epithelial transition chordoma undergoes an epithelial-mesenchymal transition process, which enables tumor cells to acquire stronger migration and invasiveness, resulting in different molecular phenotypes and activation patterns of signaling pathways during growth.

[0058] The epithelial-mesenchymal transition (EMT) is a biological process involving the transformation of cells from an epithelial cell phenotype to a mesenchymal cell phenotype. In cancer, the EMT process is closely related to tumor invasion and metastasis, and tumor cells acquire the following characteristics through EMT: Reduced intercellular adhesion: making it easier for tumor cells to detach from the primary tumor; Enhanced migration and invasion: enabling tumor cells to pass through the basement membrane and enter surrounding tissues or blood vessels; Improved chemotherapy resistance: the mesenchymal cell phenotype may be related to the resistance of tumor cells to certain chemotherapy drugs; Participation in the formation of the tumor microenvironment: affecting the interaction between the tumor and surrounding normal cells.

[0059] The product has any one or more of the following effects:

[0060] 1) Inhibit RTK-related signaling pathways;

[0061] 2) inhibiting the growth and / or proliferation of chordoma cells;

[0062] 3) Inhibit angiogenesis in chordoma cells;

[0063] 4) Reduce the colony formation of chordoma cells;

[0064] 5) Inhibit the growth of chordoma;

[0065] 5) Prevent and / or treat chordoma.

[0066] In a specific embodiment of the present application, the chordoma can inhibit RTK-related signaling pathways, which refer to a series of signal transduction pathways mediated by receptor tyrosine kinases (RTKs). RTKs are cell surface receptors that can respond to specific extracellular signaling molecules (ligands), such as growth factors, cytokines, and neurotrophic factors, thereby activating the intracellular signal transduction network. The activation process of RTKs usually includes the following steps: Ligand binding: specific extracellular ligands, such as epidermal growth factor (EGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), etc., bind to the extracellular domain of RTKs; Receptor dimerization: after ligand binding, RTKs dimerize, that is, the two receptor molecules are close to each other; Autophosphorylation: the dimerized RTKs activate their intrinsic tyrosine kinase activity, resulting in mutual phosphorylation (autophosphorylation) between receptor molecules; Downstream signal transduction: phosphorylated RTKs act as a signal platform to recruit and activate a variety of downstream signaling molecules, such as RAS, PI3K, AKT, STAT, etc., thereby triggering a cascade reaction. Several typical examples of RTK-related signaling pathways include: EGFR signaling pathway, PDGFR signaling pathway, FGFR signaling pathway, VEGFR signaling pathway, MET signaling pathway, INSR signaling pathway.

[0067] In a specific embodiment of the present application, the RTK-related signaling pathway includes any one or more of the FGFR signaling pathway, the PDGFR signaling pathway, and the VEGFR signaling pathway.

[0068] The inhibiting of the growth and / or proliferation of chordoma cells refers to taking measures to inhibit the growth rate and increase in the number of chordoma cells.

[0069] In a specific embodiment of the present application, compared with the blank control without anlotinib, when anlotinib was administered to the subject, the growth rate of the mesenchymal-epithelial-transition chordoma cell line UCH2 was reduced, in particular, anlotinib significantly inhibited the proliferation of the mesenchymal-epithelial-transition chordoma cell line UCH2 in a time-dependent manner at a dose of 0.5 μM.

[0070] The inhibition of angiogenesis of chordoma cells refers to limiting the growth and spread of chordoma by inhibiting the process of tumor angiogenesis.

[0071] The inhibiting of chordoma cell clone formation refers to reducing the chordoma cell clone formation, which refers to reducing the ability of a single chordoma cell to develop into a cell population (clone).

[0072] In a specific embodiment of the present application, the mesenchymal-epithelial transition chordoma cells are UCH2 cells.

[0073] In a specific embodiment of the present application, when anlotinib is administered to a subject, the cloning ability of the mesenchymal-epithelial transition chordoma cell line UCH2 is reduced compared to a blank control to which anlotinib is not administered.

[0074] The inhibition of chordoma growth refers to taking measures to slow down or stop the volume growth of chordoma and the proliferation of tumor cells.

[0075] In a specific embodiment of the present application, when anlotinib is administered to a subject, the weight and volume of the mesenchymal-epithelial transition chordoma in vivo are reduced compared to a blank control to which anlotinib is not administered.

[0076] The product comprises an effective dose of anlotinib or a pharmaceutically acceptable salt thereof.

[0077] In a specific embodiment of the present application, the effective dose of anlotinib or a pharmaceutically acceptable salt thereof is greater than or equal to 3 mg / kg body weight.

[0078] In a specific embodiment of the present application, the effective dose of anlotinib or a pharmaceutically acceptable salt thereof is 3 to 6 mg / kg body weight. When 3 mg / kg body weight of anlotinib is administered to a subject, it can inhibit tumor growth without toxic side effects.

[0079] In a specific embodiment of the present application, the product may include, in addition to anlotinib, a pharmaceutically acceptable excipient or carrier.

[0080] In drug preparations, pharmaceutically acceptable excipients refer to those auxiliary materials that do not affect the efficacy and safety of the main ingredients of the drug and are non-toxic in themselves. Examples of these excipients include: sterile water or saline, which serves as a solvent or diluent for the drug; stabilizers, which are used to maintain the stability of the drug and prevent decomposition; excipients, which help form a specific form of the drug, such as tablets, capsules, etc.; antioxidants, such as ascorbic acid, which prevent the drug from oxidizing; buffers: such as phosphates, citric acid, etc., which are used to maintain the pH value of the drug; preservatives, which prevent the drug from being contaminated by microorganisms during storage; surfactants, such as polyethylene glycol (PEG), Tween, etc., which are used to increase the solubility or stability of the drug; chelating agents, such as EDTA, which are used to bind to metal ions to prevent drug degradation; binders, which are used to fix drug components together. In addition, the pharmaceutical preparation may also contain: low molecular weight polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; amino acids such as glycine, glutamic acid, asparagine, arginine and lysine; sugars or carbohydrates such as polysaccharides and monosaccharides; sugar alcohols such as mannitol or sorbitol. When preparing aqueous solutions for injection, the following may also be used: physiological saline; isotonic solutions containing glucose or other auxiliary drugs; solubilizers such as alcohols (ethanol), polyols (such as propylene glycol, PEG) and non-ionic surfactants (such as Tween 80, HCO-50). The types and proportions of these excipients or carriers can be adjusted according to the type and requirements of the final pharmaceutical preparation.

[0081] In a specific embodiment of the present application, in the product, the anlotinib or a pharmaceutically acceptable salt thereof can be a single active ingredient, or it can be combined with one or more other active ingredients that have a therapeutic effect on chordoma to form a combined preparation, and the efficacy and safety of each component do not conflict. Other active ingredients can be various other drugs that can be used to treat chordoma, such as chemotherapy drugs (such as cisplatin, doxorubicin, irinotecan), drugs targeting specific molecular targets of tumor cells (such as Herceptin, pazopanib), attacking tumor cells by activating or enhancing the patient's own immune system (such as pembrolizumab, nivolumab), bone protective agents (such as denosumab), and radioactive drugs (such as radium-223). The content of each component in the combined preparation is usually a safe and effective amount, which can be adjusted based on actual usage (such as patient weight, application type, disease condition, severity).

[0082] The product can be administered to mammals, such as humans; primates, such as monkeys and other primates; livestock, such as sheep, cattle, horses, donkeys, and pigs; pets, such as dogs and cats; laboratory animals, such as mice, rabbits, rats, guinea pigs, and hamsters; captured wild animals, such as foxes and deer; in a preferred embodiment of the present application, the product is administered to humans or mice.

[0083] The product can also be used in combination with other methods for treating chordoma, which can be used simultaneously or in a sequential order. Other methods for treating chordoma include but are not limited to surgical resection, radiotherapy, chemotherapy, targeted therapy, immunotherapy, and supportive care.

[0084] On the other hand, through in-depth research on mesenchymal chordoma, the present application discovered and confirmed for the first time that S-Gboxin can effectively inhibit the growth of this type of chordoma. The present application found that its mechanism is mainly through inhibiting the activity of mitochondrial complex V, blocking the oxidative phosphorylation pathway, reducing the generation of ATP in tumor cells, and thereby inhibiting the proliferation and survival of mesenchymal chordoma tumor cells.

[0085] In this application, S-Gboxin, as a new type of oxidative phosphorylation inhibitor, involves the following aspects:

[0086] In vitro studies: Studies on the mesenchymal chordoma cell line CH22 have shown that S-Gboxin can significantly reduce the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of the cell line, thereby inhibiting its oxidative phosphorylation activity, thereby hindering the cell's energy production, and inhibiting cell proliferation and clone formation.

[0087] In vivo studies: S-Gboxin showed significant anti-tumor activity in mesenchymal chordoma patient-derived xenograft models (PDX) and cell line-derived xenograft models (CDX). Specifically, S-Gboxin was able to significantly inhibit tumor growth of mesenchymal chordoma in PDX and CDX models, and no obvious toxicity was observed in the models.

[0088] On the other hand, the present application provides use of an oxidative phosphorylation inhibitor in the preparation of a product for preventing and / or treating chordoma.

[0089] The oxidative phosphorylation inhibitor refers to a compound that can inhibit the oxidative phosphorylation process of cells, including inhibitors targeting different complexes, such as complex I inhibitors (such as rotenone or anoroxone), complex II inhibitors (such as malathion), complex III inhibitors (such as antimycin A), complex IV inhibitors (such as cyanide), and ATP synthase inhibitors (such as oligomycin).

[0090] In a specific embodiment of the present application, the chordoma includes mesenchymal chordoma.

[0091] The study of this application found that the mesenchymal chordoma refers to chordoma cells originating from the mesenchyme, and mesenchymal chordoma usually shows worse clinical prognosis and treatment effect. The difficulty in treating mesenchymal chordoma lies in its unique and complex biological characteristics. First of all, this type of chordoma shows a high degree of heterogeneity. The diversity of tumor cells and microenvironment increases the difficulty of treatment, making it difficult for a single treatment to cover it comprehensively. Mesenchymal chordoma is highly invasive and tends to invade surrounding key structures such as nerves, blood vessels, and bones, which makes it complicated to completely remove the tumor through surgery and increases the possibility of postoperative tumor residue and recurrence. This molecular classification of chordomas is resistant to radiotherapy and chemotherapy, in part because they have effective DNA repair mechanisms and can create an immunosuppressive environment, which reduces the effectiveness of traditional treatments.

[0092] In a specific embodiment of the present application, the product has one or more of the following effects:

[0093] 1) Reduce the oxygen consumption rate of chordoma cells;

[0094] 2) reduce the extracellular acidification rate of chordoma cells;

[0095] 3) inhibiting the growth and / or proliferation of chordoma cells;

[0096] 4) Inhibit the clone formation of chordoma cells;

[0097] 5) Inhibit the growth of chordoma.

[0098] In a specific embodiment of the present application, the mesenchymal chordoma chordoma cells may be CH22 cells.

[0099] The oxygen consumption rate refers to the rate at which tumor cells consume oxygen within a certain period of time. In a specific embodiment of the present application, after the administration of an oxidative phosphorylation inhibitor, the oxygen consumption rate of mesenchymal chordoma chordoma cells is reduced compared to the blank control.

[0100] The extracellular acidification rate refers to the rate at which cells release acidic substances (such as lactic acid) into the culture medium or the extracellular environment within a certain period of time. In a specific embodiment of the present application, after the administration of an oxidative phosphorylation inhibitor, the extracellular acidification rate of mesenchymal chordoma chordoma cells is reduced compared with the blank control.

[0101] The growth and proliferation of the chordoma cells refers to the increase in volume and number of chordoma cells under in vivo or in vitro culture conditions. In a specific embodiment of the present application, after the administration of an oxidative phosphorylation inhibitor, the growth and proliferation of mesenchymal chordoma cells are inhibited compared to the blank control.

[0102] The clonal formation of chordoma cells refers to the process in which a single chordoma cell or a small group of chordoma cells proliferates and forms a visible cell colony under in vitro culture conditions. In a specific embodiment of the present application, when an oxidative phosphorylation inhibitor is administered, the clonal formation of mesenchymal chordoma cells is inhibited compared to the blank control.

[0103] The growth of the chordoma refers to the process of proliferation and expansion of chordoma cells in vivo or in vitro, including cell proliferation, invasion, metastasis and interaction with surrounding tissues. In a specific embodiment of the present application, when an oxidative phosphorylation inhibitor is administered, the growth of mesenchymal chordoma is inhibited compared to a blank control, for example, including the volume and weight of the tumor.

[0104] In a specific embodiment of the present application, the oxidative phosphorylation inhibitor includes S-Gboxin or a pharmaceutically acceptable salt thereof.

[0105] The molecular formula of the S-Gboxin is C27H32F3IN2O2, the molecular weight is 600.45, the IC50 is 470nM, the CAS number is 2101317-21-7, and the structural formula is as follows:

[0106]

[0107] The pharmaceutically acceptable salt refers to a salt of S-Gboxin, which is prepared from the S-Gboxin of the present application and a relatively non-toxic acid or base. Examples of pharmaceutically acceptable salts are as described above.

[0108] In a specific embodiment of the present application, the S-Gboxin can be synthesized by itself, or is a ready-made commercial product that can be purchased.

[0109] In a specific embodiment of the present application, when an oxidative phosphorylation inhibitor is used to reduce the oxygen consumption rate and extracellular acidification rate of mesenchymal chordoma chordoma tumor cells, or inhibit the growth, proliferation and clone formation of tumor cells, the effective dose of the oxidative phosphorylation inhibitor is greater than or equal to 1 μM, for example, it can be 1 μM, 2 μM, 3 μM, 4 μM, >4 μM. When the dose of S-Gboxin is 1 μM, S-Gboxin inhibits the proliferation and clone formation of the chordoma cell line CH22 in a time-dependent manner.

[0110] In a specific embodiment of the present application, when an oxidative phosphorylation inhibitor is used to inhibit the growth of mesenchymal chordoma, prevent or treat chordoma, the effective dose of the oxidative phosphorylation inhibitor is greater than or equal to 10 mg / kg body weight. For example, when the dose of S-Gboxin is 10 mg / kg, it significantly inhibits the growth of mesenchymal chordoma and has no cytotoxicity.

[0111] In a specific embodiment of the present application, the product may include, in addition to the oxidative phosphorylation inhibitor, a pharmaceutically acceptable excipient or carrier. Examples of the pharmaceutically acceptable excipient or carrier are as described above.

[0112] In a specific embodiment of the present application, in the product, the oxidative phosphorylation inhibitor can be a single active ingredient, or it can be combined with one or more other active ingredients that have a therapeutic effect on chordoma to form a combined preparation, and the efficacy and safety of each component do not conflict. Other active ingredients can be various other drugs that can be used to treat chordoma, such as chemotherapy drugs (such as cisplatin, doxorubicin, irinotecan), drugs targeting specific molecular targets of tumor cells (such as Herceptin, pazopanib), attacking tumor cells by activating or enhancing the patient's own immune system (such as pembrolizumab, nivolumab, bone protective agents (such as denosumab), radioactive drugs (such as radium-223). The content of each component in the combined preparation is usually a safe and effective amount, which can be adjusted based on actual usage (such as patient weight, application type, disease condition, severity).

[0113] The administration object of the product is preferably a mammal, such as but not limited to humans, primates, livestock (such as sheep, cattle, horses, donkeys, pigs), pets (such as dogs, cats), laboratory test animals (such as mice, rabbits, rats, guinea pigs, hamsters) or captured wild animals (such as foxes, deer). Preferably, the subject is a primate, and more preferably, the subject is a human.

[0114] The product can also be used in combination with other methods for treating chordoma, which can be used simultaneously or in a sequential order. Other methods for treating chordoma include but are not limited to surgical resection, radiotherapy, chemotherapy, targeted therapy, immunotherapy, and supportive care.

[0115] On the other hand, this application discovered and confirmed for the first time the significant inhibitory effect of denosumab in bone microenvironment-dominated chordoma, and conducted in vivo experiments using a patient-derived chordoma xenograft model (PDX), and found the therapeutic effect of denosumab in this type of chordoma. The experimental results showed that denosumab can significantly inhibit the growth of bone microenvironment-dominated chordoma tumors, reduce tumor volume, and effectively slow down tumor-related bone destruction. In addition, denosumab showed good safety in in vivo experiments, and there was no significant decrease in the weight of mice, further confirming the clinical application potential of the drug.

[0116] Therefore, the present application also provides the use of denosumab in preparing a product for preventing and / or treating chordoma.

[0117] In a specific embodiment of the present application, the chordoma includes a bone microenvironment-dominant chordoma.

[0118] The present study found that the bone microenvironment-dominant chordoma exhibits significant bone destruction characteristics. Tumor cells of this subtype interact closely with the bone microenvironment, activating the generation and activity of osteoclasts, leading to severe bone destruction and promoting the aggressive growth of the tumor. Osteoclasts are activated through the RANK / RANKL signaling pathway, and RANKL (receptor activating factor ligand) is a key regulator of osteoclast generation and activity.

[0119] Denosumab is a fully human IgG2 monoclonal antibody, which can be a commercial product obtained by conventional means and is widely used in osteoporosis. The present application found that the monoclonal antibody Denosumab targeting RANKL can inhibit the generation and activity of osteoclasts and reduce bone destruction by blocking the binding of RANKL to its receptor RANK. Thereby effectively inhibiting the growth of bone microenvironment-dominant chordomas, slowing bone destruction, and significantly inhibiting the progression of bone microenvironment-dominant chordomas.

[0120] In a specific embodiment of the present application, the product has one or more of the following effects:

[0121] 1) Blocking the RANKL pathway;

[0122] 2) Inhibit the activity of osteoclasts;

[0123] 3) Inhibit the formation of osteoclasts;

[0124] 4) Reduce bone destruction;

[0125] 5) Inhibit the growth of chordoma;

[0126] 6) Prevent and / or treat chordoma.

[0127] The blocking RANKL pathway refers to inhibiting the function of nuclear factor κB receptor activator ligand (RANKL) by drugs or other intervention means, thereby reducing the generation and activity of osteoclasts and inhibiting the process of bone resorption. RANKL is an important factor in bone metabolism. It promotes the differentiation and maturation of osteoclasts by binding to its receptor RANK, leading to increased bone resorption. In diseases such as osteoporosis, rheumatoid arthritis, and giant cell tumor of bone, the RANKL pathway is often overactivated, leading to bone destruction and loss. The main signal components involved include RANKL itself, its receptor RANK (Receptor Activator of Nuclear Factor-κB), and osteoprotegerin (OPG, Osteoprotegerin).

[0128] The inhibition of osteoclastogenesis refers to the process of preventing the differentiation, maturation and activity of osteoclasts (OCs) through drugs or other interventions, thereby reducing bone resorption. Osteoclasts are cells specifically responsible for bone resorption. They dissolve bone minerals and organic matrix by secreting acidic substances and enzymes, leading to bone loss.

[0129] The inhibition of osteoclast activity refers to reducing the function of osteoclasts (OCs) through certain drugs or biological factors, thereby reducing their absorption and destruction of bone. Osteoclasts are cells specifically responsible for bone absorption, which dissolve bone minerals and organic matrix by secreting acidic substances and enzymes, leading to bone loss. Inhibiting the activity of osteoclasts helps reduce bone loss.

[0130] The inhibition of chordoma growth refers to controlling or slowing down the growth and expansion of chordoma cells by surgery, radiotherapy, chemotherapy, targeted therapy or immunotherapy. In a specific embodiment of the present application, after administration of denosumab, the growth of the bone microenvironment-dominated type is inhibited compared to the blank control, for example, including the volume and weight of the tumor.

[0131] In a specific embodiment of the present application, the product comprises an effective dose of denosumab.

[0132] In a specific embodiment of the present application, the effective dose of denosumab is greater than or equal to 2 mg / kg body weight. For example, when a dose of 2 mg / kg body weight of denosumab is administered to a subject, the tumor volume of bone microenvironment-dominant chordoma PDX is significantly inhibited, and the tumor weight is also significantly reduced; and no significant weight loss is caused to the mice.

[0133] In a specific embodiment of the present application, the product may include, in addition to denosumab, a pharmaceutically acceptable excipient or carrier. Examples of the pharmaceutically acceptable excipient or carrier are as described above.

[0134] In a specific embodiment of the present application, in the product, the denosumab can be a single active ingredient, or it can be combined with one or more other active ingredients that have a therapeutic effect on chordoma to form a combined preparation, and the efficacy and safety of each component do not conflict. Other active ingredients can be various other drugs that can be used to treat chordoma, such as chemotherapy drugs (such as cisplatin, doxorubicin, irinotecan), drugs targeting specific molecular targets of tumor cells (such as Herceptin, pazopanib), attacking tumor cells by activating or enhancing the patient's own immune system (such as pembrolizumab, nivolumab, bone protective agents (such as denosumab), radioactive drugs (such as radium-223). The content of each component in the combined preparation is generally a safe and effective amount, which can be adjusted based on actual usage (such as patient weight, application type, disease condition, severity).

[0135] The administration object of the product is preferably a mammal, such as but not limited to humans, primates, livestock (such as sheep, cattle, horses, donkeys, pigs), pets (such as dogs, cats), laboratory test animals (such as mice, rabbits, rats, guinea pigs, hamsters) or captured wild animals (such as foxes, deer). Preferably, the subject is a primate, and more preferably, the subject is a human.

[0136] The product can also be used in combination with other methods for treating chordoma, which can be used simultaneously or in a sequential order. Other methods for treating chordoma include but are not limited to surgical resection, radiotherapy, chemotherapy, targeted therapy, immunotherapy, and supportive care.

[0137] The present application also provides a pharmaceutical composition comprising an effective dose of any one or more of the following:

[0138] 1) Anlotinib or a pharmaceutically acceptable salt thereof;

[0139] 2) Oxidative phosphorylation inhibitors;

[0140] 3) Desumebambo.

[0141] In a specific embodiment of the present application, the pharmaceutical composition may include an effective dose of anlotinib or a pharmaceutically acceptable salt thereof alone, and the anlotinib or a pharmaceutically acceptable salt thereof is as described above.

[0142] In a specific embodiment of the present application, when the pharmaceutical composition is used to inhibit the growth of mesenchymal-epithelial transformation chordoma, prevent and treat mesenchymal-epithelial transformation chordoma, the effective dose of anlotinib or a pharmaceutically acceptable salt thereof is greater than or equal to 3 mg / kg body weight.

[0143] In a preferred embodiment of the present application, the effective dose of anlotinib or a pharmaceutically acceptable salt thereof is 3 to 6 mg / kg body weight.

[0144] In addition, when the pharmaceutical composition is used to inhibit the proliferation and clone formation of mesenchymal-epithelial transition chordoma cells, the effective dose of anlotinib or a pharmaceutically acceptable salt thereof is greater than or equal to 0.5 μM, for example, 0.5, 1 and 5 μM.

[0145] In a specific embodiment of the present application, the pharmaceutical composition may include an effective dose of an oxidative phosphorylation inhibitor alone.

[0146] In a specific embodiment of the present application, the oxidative phosphorylation inhibitor includes S-Gboxin or a pharmaceutically acceptable salt thereof, and the S-Gboxin or a pharmaceutically acceptable salt thereof is as described above.

[0147] In a specific embodiment of the present application, when the pharmaceutical composition is used to inhibit the growth, prevent or treat mesenchymal chordoma, the effective dose of the oxidative phosphorylation inhibitor is greater than or equal to 10 mg / kg body weight.

[0148] In a preferred embodiment of the present application, the effective dose of the oxidative phosphorylation inhibitor is 10 mg / kg body weight.

[0149] In addition, when the pharmaceutical composition is used to reduce the oxygen consumption rate and extracellular acidification rate of mesenchymal chordoma tumor cells, or inhibit the growth, proliferation and clone formation of mesenchymal chordoma tumor cells, the effective dose of the oxidative phosphorylation inhibitor is greater than or equal to 1 μM, for example, it can be 1, 2 and 4 μM.

[0150] In a specific embodiment of the present application, the pharmaceutical composition may alone include an effective dose of denosumab, and the denosumab is as described above.

[0151] In a specific embodiment of the present application, when the pharmaceutical composition is used to inhibit the growth, prevent or treat bone microenvironment-dominant chordoma, the effective dose of denosumab is greater than or equal to 2 mg / kg body weight.

[0152] In a preferred embodiment of the present application, the effective dose of denosumab is 2 mg / kg body weight.

[0153] In the present application, the pharmaceutical composition may include anlotinib or a pharmaceutically acceptable salt thereof alone; or an oxidative phosphorylation inhibitor alone; or denosumab alone; or any combination of any two of the above; or a combination of the three above.

[0154] In the pharmaceutical composition, the above-mentioned ingredients can be a single active ingredient, or they can be combined with one or more other active ingredients that have therapeutic effects on chordoma to form a combined preparation, and there is no conflict between the efficacy and safety of the components. The other active ingredients that have therapeutic effects on chordoma are as described above.

[0155] The pharmaceutical composition may further include a pharmaceutically acceptable excipient or carrier, and the pharmaceutically acceptable excipient or carrier is as described above.

[0156] The subject of administration of the pharmaceutical composition can be mammals, such as humans; primates, such as monkeys and other primates; livestock, such as sheep, cattle, horses, donkeys, pigs; pets, such as dogs and cats; laboratory animals, such as mice, rabbits, rats, guinea pigs, hamsters; captured wild animals, such as foxes, deer, etc.; in a preferred embodiment of the present application, the subject of administration of the pharmaceutical composition is humans or mice.

[0157] The pharmaceutical composition can also be used in combination with other methods for treating chordoma, and the other methods for treating chordoma are as described above.

[0158] The present application also provides a method for preventing and / or treating mesenchymal epithelial transformation chordoma, which is to administer an effective dose of anlotinib or a pharmaceutically acceptable salt thereof, or a product containing an effective dose of anlotinib, or a pharmaceutical composition containing an effective dose of anlotinib to a subject. The method can also be used in combination with other methods for treating chordoma or active ingredients or drugs that have a therapeutic effect on chordoma.

[0159] The method also includes regularly monitoring the tumor volume to evaluate the tumor inhibitory effect of anlotinib, or evaluating the inhibitory effect of anlotinib on mesenchymal-epithelial transformation chordoma by measuring the proliferation rate, clone formation ability and activity of RTK signaling pathway of tumor cells.

[0160] The present application also provides a method for preventing and / or treating mesenchymal chordoma, which comprises administering to a subject an effective dose of an oxidative phosphorylation inhibitor, or a product containing an effective dose of an oxidative phosphorylation inhibitor, or a pharmaceutical composition containing an effective dose of an oxidative phosphorylation inhibitor. The method can also be used in combination with other methods for treating chordoma or active ingredients or drugs that have a therapeutic effect on chordoma.

[0161] The method also includes regularly monitoring tumor volume to evaluate the tumor inhibitory effect of oxidative phosphorylation inhibitors, or evaluating the effect of S-Gboxin on tumor cell oxidative phosphorylation by measuring oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), and evaluating the inhibitory effect of anlotinib on mesenchymal-epithelial transition chordoma by detecting the proliferation rate and clone formation ability of tumor cells.

[0162] The present application also provides a method for preventing and / or treating bone microenvironment-dominant chordoma, which comprises administering to a subject an effective dose of denosumab, or the above-mentioned product containing an effective dose of denosumab, or the above-mentioned pharmaceutical composition containing an effective dose of denosumab. The method can also be used in combination with other methods for treating chordoma or active ingredients or drugs that have a therapeutic effect on chordoma.

[0163] The method also includes evaluating the inhibitory effect of denosumab on tumor growth by regularly measuring tumor volume and observing bone destruction.

[0164] In the above method, the object can be a mammal, such as a human; a primate, such as a monkey and other primates; livestock, such as sheep, cattle, horses, donkeys, pigs; pets, such as dogs and cats; laboratory animals, such as mice, rabbits, rats, guinea pigs, hamsters; captured wild animals, such as foxes, deer, etc.; in a preferred embodiment of the present application, the object is a human or a mouse.

[0165] The present application is further illustrated by the following examples, but the scope of the present application is not limited thereby. Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and conventional techniques in related fields in the field of the present technology. These technologies have been fully described in the existing literature. Unless otherwise specified, the instruments, materials, and reagents of this application are all obtained through conventional channels. Animal experiments were conducted in accordance with the guidelines of the institutional animal welfare and ethics committee. Chordoma tumor tissue was provided by Shanghai First People's Hospital, and the patients were informed and in compliance with ethical regulations.

[0166] Example 1 Anlotinib inhibits proliferation and clone formation of mesenchymal-epithelial transition chordoma cell line UCH2

[0167] 1. Materials and Methods

[0168] IC50 experiment: UCH2 cells (derived from ATCC, based on proteomic sequencing in molecular typing, belong to mesenchymal epithelial transition chordoma) were inoculated in 96-well plates, with 4×10 3 After the cells adhered to the wall, anlotinib (purchased from Selleckchem) with a concentration gradient was added, and the cells were cultured in the medium containing anlotinib for 24 hours. After 24 hours of culture, the ATP level in the cells was measured using an ATP detection kit (purchased from Promega).

[0169] MTT assay: UCH2 cells were exposed to 0 μM, 0.5 μM, 1 μM, and 5 μM concentrations of anlotinib (purchased from Selleckchem) at the indicated time points. The proliferation ability of cells was evaluated by MTT assay. UCH2 cells were seeded in 96-well plates (4 × 10 3 Cells were harvested every 24 hours and incubated with MTT (purchased from Sigma-Aldrich) for 4 hours. The reaction was terminated by adding dimethyl sulfoxide (DMSO) solution and incubating for 20 minutes, and then the absorbance of the sample was measured at 490 nm.

[0170] Cloning experiment: UCH2 cells were seeded in 6-well plates at a density of 1000 cells per well, and 0 μM, 0.5 μM, 1 μM and 5 μM concentrations of anlotinib (purchased from Selleckchem) were added. After culturing the cells for 2 weeks, they were fixed with 4% paraformaldehyde, stained with crystal violet (purchased from Sigma-Aldrich), and then rinsed three times with phosphate-buffered saline (PBS). Then, the stained cells were dissolved with 95% ethanol, diluted tenfold, and incubated for 10 minutes. Finally, the absorbance of the samples was measured at 570 nm and the cells were counted.

[0171] 2. Results

[0172] The experimental results are as follows Figure 1 As shown, Figure 1 A shows that the IC50 of anlotinib against the mesenchymal-epithelial transition chordoma cell line UCH2 is 0.7324 μM. Figure 1 BD's MTT assay and clone formation assay confirmed that anlotinib significantly inhibited the proliferation and clone formation of mesenchymal epithelial transition chordoma cell line UCH2 in a time-dependent manner at doses of 0.5, 1, and 5 μM, indicating that anlotinib can significantly inhibit the growth of mesenchymal epithelial transition chordoma cell line UCH2.

[0173] Example 2 Anlotinib inhibits the expression of RTK (FGFR, PDGFR and VEGFR) in the mesenchymal-epithelial transition chordoma cell line UCH2

[0174] 1. Materials and Methods

[0175] UCH2 cells were seeded in 6-well plates at a density of 10,000 cells per well. After the cells adhered, 0 μM and 2 μM concentrations of anlotinib (Selleckchem) were added, and the cells were cultured in a medium containing anlotinib for 24 h. The UCH2 cell line was lysed with ice-cold RIPA lysis buffer (Sigma-Aldrich) for 30 minutes, centrifuged at 12,000 g for 15 minutes at 4 ° C, and the supernatant was collected. The protein concentration was determined using a BCA protein quantification kit (Thermo). 20 μg of protein was separated on a 10% SDS-PAGE gel. The electrophoresis conditions were 80V constant voltage to run the sample to the boundary between the concentrated gel and the separation gel. After entering the separation gel, it was run under 120V constant voltage until the bromophenol blue indicator reached the bottom of the separation gel. Then 200mA constant current transfer was performed for 2 hours. The membrane was blocked with 5% skim milk powder at room temperature for 1 hour. Then, the cells were incubated overnight at 4°C with antibodies to VEGF Receptor 1+VEGF Receptor 2 (purchased from Abcam, AB36844), PDGFR alpha+PDGFR beta (purchased from Abcam, AB32570), FGF Receptor 1 (purchased from CST, #9740), and beta Actin (purchased from Diagbio, db7283). The cells were incubated at room temperature for 1 hour with a 1:10000 dilution of HRP-labeled anti-rabbit secondary antibody (purchased from Proteintech). Chemiluminescence detection was performed using ECL development reagent (purchased from Thermo) according to the manufacturer's instructions.

[0176] 2. Results

[0177] The experimental results are as follows Figure 2 As shown, anlotinib significantly inhibited the protein expression of RTK (FGFR, PDGFR and VEGFR) in the mesenchymal-epithelial-transition chordoma cell line UCH2, indicating that anlotinib significantly reduced the expression of receptor tyrosine kinases in mesenchymal-epithelial-transition chordoma.

[0178] Example 3 Anlotinib inhibits the growth of mesenchymal-epithelial transition chordoma PDX

[0179] 1. Materials and Methods

[0180] The mesenchymal-epithelial transition chordoma tumor tissue was cut into several 2-3 mm 3 The tumor fragments were manipulated and washed on ice, and then implanted subcutaneously into the left abdomen of male nude mice (5 weeks old, 18-20 g, Shanghai Slake Laboratory Animal Co., Ltd., China). When the tumor volume reached 500 mm 3 When the tumor is removed, it is transplanted into another nude mouse. This process needs to be repeated twice.

[0181] Nude mice were randomly assigned to different experimental groups. The experimental group was treated with 6 mg / kg of anlotinib by gavage, and the control group was treated with an equal volume of PBS by gavage. The mice were treated for 16 days, and their body weight was monitored every 3 days for signs of toxicity. Under general anesthesia, the tumor size of tibial chordoma was evaluated by micro-MRI and analyzed by two independent researchers using ITK-SNAP 3.6 software. In addition, the tumor volume of the PDX model was monitored after tumor resection until the experimental endpoint. The tumor volume was estimated using the following formula: V = ab 2 / 2 (V represents volume; a represents length; b represents width). Then, the mice were anesthetized and killed by cervical dislocation.

[0182] 2. Results

[0183] The experimental results are as follows Figure 3 As shown, Figure 3 AD shows that in the PDX model of mesenchymal-epithelial transition chordoma, 6 mg / kg of S anlotinib significantly inhibited the growth of chordoma; Figure 3 E shows that in the PDX model of mesenchymal-epithelial transition chordoma, 6 mg / kg of anlotinib did not cause significant weight loss, indicating that anlotinib significantly inhibited the growth of mesenchymal-epithelial transition chordoma PDX.

[0184] Example 4 Anlotinib inhibits the growth of mesenchymal-epithelial transition chordoma CDX

[0185] The chordoma cell line UCH2 was injected into the left tibia of male nude mice (5 weeks old, 18-20 g, Shanghai Slake Laboratory Animal Co., Ltd., China) at 5×106 cells per dose suspended in 60 μL RPMI-1640 medium.

[0186] Nude mice were randomly assigned to different experimental groups. The experimental groups were treated with 3 mg / kg and 6 mg / kg of anlotinib by gavage, and the control group was treated with an equal volume of PBS by gavage. The mice were treated for 16 days, and their body weight was monitored every 3 days for signs of toxicity. Under general anesthesia, the tumor size of tibial chordoma was assessed by micro-MRI and analyzed by two independent researchers using ITK-SNAP 3.6 software. In addition, the tumor volume of the PDX model was monitored after tumor resection until the experimental endpoint. The tumor volume was estimated using the following formula: V = ab 2 / 2 (V represents volume; a represents length; b represents width). Then, the mice were anesthetized and killed by cervical dislocation.

[0187] 2. Results

[0188] The experimental results are as follows Figure 4 As shown, Figure 4AC illustrates that in the tibial xenograft model of UCH2 cells, anlotinib significantly inhibited the growth of chordoma at doses of 3 mg / kg and 6 mg / kg; Figure 4 D shows that in the tibial xenograft model of UCH2 cells, the doses of 3 mg / kg and 6 mg / kg of anlotinib did not cause a significant decrease in the body weight of nude mice, indicating that anlotinib significantly inhibited the growth of mesenchymal-epithelial transition chordoma CDX.

[0189] Example 5 S-Gboxin inhibits the proliferation and colony formation of chordoma cell line CH22

[0190] 1. Materials and Methods

[0191] 1.1 IC50 experiment

[0192] CH22 cells (derived from the Chordoma Foundation and classified as mesenchymal chordoma based on proteomic sequencing in molecular typing) were seeded in 96-well plates, with 4 × 10 3 After the cells adhered to the wall, S-Gboxin (purchased from Selleckchem) with a concentration gradient was added, and the cells were cultured in the medium containing S-Gboxin for 24 hours. After 24 hours of culture, the ATP level in the cells was measured using an ATP detection kit (purchased from Promega).

[0193] 1.2 MTT assay

[0194] The cells were exposed to 0 μM, 1 μM, 2 μM and 4 μM concentrations of S-Gboxin (purchased from Selleckchem) at the indicated time points (1d, 2d, 3d, 4d, 5d). The proliferation ability of the cells was evaluated by MTT assay. CH22 cells were seeded in 96-well plates (4 × 10 3 Cells were harvested every 24 hours and incubated with MTT (purchased from Sigma-Aldrich) for 4 hours. The reaction was terminated by adding dimethyl sulfoxide (DMSO) solution and incubating for 20 minutes, and then the absorbance of the sample was measured at 490 nm.

[0195] 1.3 Clone formation experiment

[0196] CH22 cells were seeded in 6-well plates at a density of 1000 cells per well, and 0 μM, 1 μM, 2 μM and 4 μM concentrations of S-Gboxin (purchased from Selleckchem) were added. After the cells were cultured for 2 weeks, they were fixed with 4% paraformaldehyde, stained with crystal violet (purchased from Sigma-Aldrich), and then rinsed three times with phosphate buffered saline (PBS). Then, the stained cells were dissolved with 95% ethanol, diluted tenfold, and incubated for 10 minutes. Finally, the absorbance of the sample was measured at 570 nm and the cell count was performed.

[0197] 2. Results

[0198] like Figure 5 As shown in A, the IC50 of S-Gboxin against the chordoma cell line CH22 is 3.37 μM; Figure 5 As shown in B-5D, the MTT experiment and clone formation experiment confirmed that S-Gboxin significantly inhibited the proliferation and clone formation of the chordoma cell line CH22 in a time-dependent manner at doses of 1, 2 and 4 μM, indicating that S-Gboxin can significantly inhibit the growth of the chordoma cell line CH22 and can exert an inhibitory effect at a dose of 1 μM.

[0199] Example 6 S-Gboxin reduces the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of chordoma cell line CH22

[0200] 1. Materials and Methods

[0201] 1.1 Extracellular acidification rate (ECAR) experiment

[0202] Glycolytic rate determination was performed using a Seahorse XFe24 analyzer (Agilent Technologies). The sensor cartridge used for measurement was hydrated overnight in a 37°C CO2-free incubator before the experiment. CH22 cells were cultured at 2 × 10 4Cells were seeded at a density of 100 μM in 24-well plates and pretreated with different concentrations of S-Gboxin (0 μM, 1 μM, and 4 μM) for 12 h. After 12 h, the medium was replaced and the cell culture plates were placed in a CO2-free incubator at 37 °C for 1 h. For the glycolytic rate assay, 10 mM glucose, 1 μM oligomycin, and 50 mM 2-deoxy-D-glucose (2DG) were injected sequentially. Subsequently, the culture plates were loaded into the Seahorse XF analyzer together with the sensor cartridge. The analyzer automatically mixed the cells, waited for temperature and pH to stabilize, and then measured the extracellular acidification rate (ECAR) at specified time intervals. At the end of the experiment, the ECAR data were collected and analyzed using the SeahorseWave software analysis tool. To eliminate the influence of differences in cell density, the results were normalized according to the number of cells or protein content per well.

[0203] 1.2 Oxygen consumption rate (OCR) experiment

[0204] Mito stress tests were performed using a Seahorse XFe24 analyzer (Agilent Technologies). The sensor cartridge used for the measurements was hydrated overnight in a 37°C CO2-free incubator prior to the experiment. CH22 cells were cultured at 2 × 10 4 Cells were seeded at a density of 100 μM in 24-well plates and pretreated with different concentrations of S-Gboxin (0 μM, 1 μM, and 4 μM) for 12 h. After 12 h, the medium was replaced and the cell culture plates were placed in a CO2-free incubator at 37 °C for 1 h. In the Mito stress test, 1.5 μM oligomycin, 0.5 μM FCCP, and 0.5 μM rotenone + antimycin A were injected sequentially. Subsequently, the culture plates were loaded into the Seahorse XF analyzer together with the sensor cartridge. The analyzer automatically mixed the cells, waited for the temperature and pH to stabilize, and then measured the oxygen consumption rate (OCR) at the specified time intervals. At the end of the experiment, the OCR data were collected and analyzed using the Seahorse Wave software analysis tool. To eliminate the influence of differences in cell density, the results were normalized according to the number of cells or protein content per well.

[0205] 2. Results

[0206] like Figure 6 As shown, Figure 6 AB shows that the oxygen consumption rate (OCR) of CH22 was significantly inhibited under the action of 1 and 4 μM S-Gboxin. Figure 6CD showed that the extracellular acidification rate (ECAR) of CH22 was significantly inhibited under the action of 1 and 4 μM S-Gboxin. S-Gboxin significantly reduced the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of the chordoma cell line CH22, indicating that S-Gboxin can significantly inhibit the oxidative phosphorylation of the chordoma cell line CH22.

[0207] Example 7S-Gboxin inhibits the growth of mesenchymal chordoma PDX

[0208] 1. Materials and Methods

[0209] Mesenchymal chordoma tumor tissue was cut into several 2-3 mm 3 The tumor fragments were manipulated and washed on ice, and then implanted subcutaneously into the left abdomen of male nude mice (5 weeks old, 18-20 g, Shanghai Slake Laboratory Animal Co., Ltd., China). When the tumor volume reached 500 mm 3 When the tumor is removed, it is transplanted into another nude mouse. This process needs to be repeated twice.

[0210] Nude mice were randomly assigned to different experimental groups, and the experimental groups were treated with 10 mg / kg of S-Gboxin intraperitoneally, and the control groups were treated with an equal volume of PBS intraperitoneally. The mice were treated for 10 days, and their body weight was monitored every 3 days for signs of toxicity. Under general anesthesia, the tumor size of tibial chordoma was assessed by micro-MRI and analyzed by two independent researchers using ITK-SNAP 3.6 software. In addition, the tumor volume of the PDX model was monitored after tumor resection until the experimental endpoint. Tumor volume was estimated using the following formula: V = ab 2 / 2 (V represents volume; a represents length; b represents width). Then, the mice were anesthetized and killed by cervical dislocation.

[0211] 2. Results

[0212] like Figure 7 As shown, Figure 7 AC shows that compared with the control group, the tumor volume of mesenchymal chordoma PDX was significantly inhibited and the tumor weight was significantly reduced under the intraperitoneal injection of 10 mg / kg S-Gboxin; Figure 7 D shows that there was no significant change in the body weight of mice after intraperitoneal injection of 10 mg / kg S-Gboxin. In the PDX model of mesenchymal chordoma, 10 mg / kg S-Gboxin significantly inhibited the growth of chordoma and did not cause significant weight loss, indicating that S-Gboxin significantly inhibited the growth of mesenchymal chordoma PDX.

[0213] Example 8 S-Gboxin inhibits the growth of mesenchymal chordoma CDX

[0214] 1. Materials and Methods

[0215] Chordoma cell line CH22 was dosed at 5×10 6 The cells were suspended in 60 μL RPMI-1640 medium and injected into the left tibia of male nude mice (5 weeks old, 18-20 g, Shanghai Slake Laboratory Animal Co., Ltd., China).

[0216] Nude mice were randomly assigned to different experimental groups, and the experimental groups were treated with 10 mg / kg of S-Gboxin intraperitoneally, and the control groups were treated with an equal volume of PBS intraperitoneally. The mice were treated for 10 days, and their body weight was monitored every 3 days for signs of toxicity. Under general anesthesia, the tumor size of tibial chordoma was assessed by micro-MRI and analyzed by two independent researchers using ITK-SNAP 3.6 software. In addition, the tumor volume of the PDX model was monitored after tumor resection until the experimental endpoint. Tumor volume was estimated using the following formula: V = ab 2 / 2 (V represents volume; a represents length; b represents width). Then, the mice were anesthetized and killed by cervical dislocation.

[0217] 2. Results

[0218] like Figure 8 As shown, Figure 8 AC shows that compared with the control group, the tumor volume of mesenchymal chordoma PDX was significantly inhibited after intraperitoneal injection of 10 mg / kg S-Gboxin; Figure 8 D shows that there was no significant change in the body weight of mice after intraperitoneal injection of 10 mg / kg S-Gboxin. In the tibial xenograft model of CH22 cells, S-Gboxin significantly inhibited the growth of chordoma at a dose of 10 mg / kg, and the body weight of nude mice did not decrease significantly, indicating that S-Gboxin significantly inhibited the growth of mesenchymal chordoma CDX.

[0219] Example 9 Desumetab inhibits the growth of bone microenvironment-dominant chordoma PDX;

[0220] 1. Materials and Methods

[0221] Bone microenvironment dominant chordoma tumor tissue was cut into several 2-3 mm 3 The tumor fragments were manipulated and washed on ice, and then implanted subcutaneously into the left abdomen of male nude mice (5 weeks old, 18-20 g, Shanghai Slake Laboratory Animal Co., Ltd., China). When the tumor volume reached 500 mm 3When the tumor is removed, it is transplanted into another nude mouse. This process needs to be repeated twice.

[0222] Nude mice were randomly assigned to different experimental groups. The experimental group was treated with 2 mg / kg of denosumab intraperitoneally, and the control group was treated with an equal volume of PBS intraperitoneally. The mice were treated for 34 days, and their body weight was monitored every 3 days for signs of toxicity. The tumor size of tibial chordoma was assessed by micro-MRI under general anesthesia and analyzed by two independent researchers using ITK-SNAP 3.6 software. In addition, the tumor volume of the PDX model was monitored after tumor resection until the experimental endpoint. The tumor volume was estimated using the following formula: V = ab 2 / 2 (V represents volume; a represents length; b represents width). Then, the mice were anesthetized and killed by cervical dislocation.

[0223] 2. Results

[0224] Fig. 9 AC shows that compared with the control group, the tumor volume of bone microenvironment-dominant chordoma PDX was significantly inhibited and the tumor weight was significantly reduced under intraperitoneal injection of 2 mg / kg denosumab; and it did not cause significant weight loss in mice; indicating that denosumab significantly inhibits the growth of bone microenvironment-dominant chordoma PDX.

[0225] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

Claims

1. Use of anlotinib or a pharmaceutically acceptable salt thereof in the preparation of a product for preventing and / or treating chordoma.

2. The use according to claim 1, characterized in that The product has any one or more of the following effects: 1) Inhibit RTK-related signaling pathways; 2) inhibiting the growth and / or proliferation of chordoma cells; 3) Inhibit angiogenesis in chordoma cells; 4) Inhibit the clone formation of chordoma cells; 5) Inhibit the growth of chordoma; 6) Prevention and / or treatment of chordoma; Preferably, the RTK-related signaling pathway includes any one or more of the FGFR signaling pathway, the PDGFR signaling pathway, and the VEGFR signaling pathway.

3. The use according to claim 1, characterized in that The product contains an effective dose of anlotinib or a pharmaceutically acceptable salt thereof; preferably, the effective dose of anlotinib or a pharmaceutically acceptable salt thereof is greater than or equal to 3 mg / kg body weight; more preferably, the effective dose of anlotinib or a pharmaceutically acceptable salt thereof is 3 to 6 mg / kg body weight.

4. The use according to claim 1, characterized in that The chordoma includes mesenchymal-epithelial transformation chordoma.

5. Use of an oxidative phosphorylation inhibitor in the preparation of a product for preventing and / or treating chordoma.

6. The use according to claim 5, characterized in that The product has one or more of the following effects: 1) Reduce the oxygen consumption rate of chordoma cells; 2) reduce the extracellular acidification rate of chordoma cells; 3) inhibiting the growth and / or proliferation of chordoma cells; 4) Inhibit the clone formation of chordoma cells; 5) Inhibit the growth of chordoma; 6) Prevent and / or treat chordoma.

7. The use according to claim 5, characterized in that The product contains an effective dose of an oxidative phosphorylation inhibitor; preferably, the oxidative phosphorylation inhibitor includes S-Gboxin or a pharmaceutically acceptable salt thereof; more preferably, the effective dose of the oxidative phosphorylation inhibitor is greater than or equal to 10 mg / kg body weight.

8. The use according to claim 5, characterized in that The chordoma includes mesenchymal chordoma.

9. Use of denosumab in the preparation of a product for preventing and / or treating chordoma.

10. The use according to claim 9, characterized in that The product has one or more of the following effects: 1) Blocking the RANKL pathway; 2) Inhibit the formation of osteoclasts; 3) Inhibit the activity of osteoclasts; 4) Reduce bone destruction; 5) Inhibit the growth of chordoma; 6) Prevent and / or treat chordoma.

11. The use according to claim 9, characterized in that The product contains an effective dose of denosumab; preferably, the effective dose of denosumab is greater than or equal to 2 mg / kg body weight.

12. The use according to claim 9, characterized in that The chordoma includes bone microenvironment-dominant chordoma.

13. A pharmaceutical composition comprising an effective dose of any one or more of the following: 1) Anlotinib or a pharmaceutically acceptable salt thereof; 2) Oxidative phosphorylation inhibitors; 3) Desumebambo.

14. The pharmaceutical composition according to claim 13, characterized in that The pharmaceutical composition may further include one or more of the following features: 1) The effective dose of anlotinib or a pharmaceutically acceptable salt thereof is greater than or equal to 3 mg / kg body weight; preferably, the effective dose of anlotinib or a pharmaceutically acceptable salt thereof is 3 to 6 mg / kg body weight; 2) The oxidative phosphorylation inhibitor includes S-Gboxin or a pharmaceutically acceptable salt thereof; the effective dose of the oxidative phosphorylation inhibitor is greater than or equal to 10 mg / kg body weight; 3) The effective dose of denosumab is greater than or equal to 2 mg / kg body weight.

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