Application of tageteoside and difacitinib or ponatinib in blocking esophageal squamous cell carcinoma progression

Abstract

The invention provides application of combination of tageteoside and difacitinib or ponatinib to blocking of esophageal squamous cell carcinoma progression. The tageteoside is combined with an FAK inhibitor defacitinib or an Src family kinase inhibitor ponatinib, so that the metastasis of esophageal squamous carcinoma, especially lymphatic metastasis, can be inhibited, the growth of primary focus and metastatic focus esophageal squamous carcinoma cells can be inhibited, and a synergistic anti-cancer effect is achieved.

Description

Technical Field

[0001] The present invention relates to the field of medicines, and in particular to the use of marigold glycosides in combination with FAK inhibitor defatinib or Src family kinase inhibitor ponatinib to block the progression of esophageal squamous cell carcinoma. Background Art

[0002] Esophageal squamous cell carcinoma (ESCC) is one of the most common solid tumors in my country. Due to its complex pathological mechanism, high malignancy and difficulty in molecular classification, targeted drugs have not been able to achieve effective anti-cancer effects in the treatment of ESCC.

[0003] Clinical specimen studies have shown that phosphorylated FAK tyrosine 576 / 577, Src kinase family members Src tyrosine Y419, Fyn tyrosine Y420 and Lyn tyrosine Y397 are highly expressed in esophageal squamous cell carcinoma tissues, and are closely correlated with tumor malignant progression and negatively correlated with patient survival. In the past few years, clinical trials have been conducted on FAK and Src family kinase inhibitors, but in the treatment of solid tumors, these FAK and Src family kinase inhibitors still cannot be used as single drugs for anticancer. This suggests whether the combination of certain small molecule compounds can effectively enhance the anticancer effect of targeted drugs.

[0004] There have been many studies on the use of small molecule monomer compounds to enhance the sensitivity of anticancer drugs, including targeted drugs, radiochemotherapeutic drugs and immunotherapy. Quercetagitrin is obtained from African marigolds and has the following structural formula:

[0005]

[0006] It has anti-inflammatory and antioxidant biological activities and is widely used in cardiovascular and cerebrovascular diseases and other diseases related to inflammation and oxidation. In recent years, it has also been reported that marigold glycosides have anti-cancer effects by inhibiting the growth of tumor cells. However, whether marigold glycosides can enhance the FAK inhibitor defatinib and the Src family kinase inhibitor ponatinib and play an anti-esophageal squamous cell carcinoma effect still needs further study. Summary of the invention

[0007] The present invention finds that marigold glycoside can significantly enhance the anti-esophageal squamous cell carcinoma effect of FAK inhibitor Defactinib and Src family kinase inhibitor Ponatinib, and has a synergistic anti-cancer effect.

[0008] FAK inhibitor defatinib and Src family kinase inhibitor ponatinib can treat tumor (including esophageal squamous cell carcinoma) progression. However, these targeted drugs are not effective when used alone as a single drug in clinical applications. The reason is closely related to the complexity of the downstream signal network. The present invention found through immunohistochemical experiments that in situ esophageal squamous cell carcinoma tissue, FAK tyrosine 576 / 577, Src kinase family member Src tyrosine Y419, Fyn tyrosine Y420 and Lyn tyrosine Y397 were positively correlated with downstream proliferation-related molecules, including MCM3, MCM4, MCM5, MCM6, MCM7, CDC45, CDK7 and CDK9. In esophageal squamous cell carcinoma tissue with lymphatic metastasis, FAK tyrosine 576 / 577, Src kinase family member Src tyrosine Y419, Fyn tyrosine Y420 and Lyn tyrosine Y397 were positively correlated with downstream invasion-promoting and stemness-related markers, such as SDC2, ITGA5, GALK1, AXL, Myc, Sox2 and OCT4. The present invention constructed a nude mouse popliteal lymph node tumor cell metastasis model to observe the effect of marigold glycosides enhancing the anti-progression effect of FAK inhibitor defatinib and Src family kinase inhibitor ponatinib on esophageal squamous cell carcinoma. The results showed that marigold glycosides could significantly enhance the lymphatic metastasis of esophageal squamous cell carcinoma cell lines KYSE410 and KYSE510 in the mouse model inhibited by defatinib and ponatinib, and inhibit the expression of MCM3, MCM4, MCM5, MCM6, MCM7, CDC45, CDK7, CDK9, proliferation marker Ki67, and lymphangiogenesis marker LYVE1 in carcinoma in situ tissues. Marigold glycosides could significantly enhance the expression of SDC2, ITGA5, GALK1, AXL and the transcriptional activity of Myc, Sox2 and OCT4 in metastatic tumors in the mouse model inhibited by defatinib and ponatinib.

[0009] To this end, in a first aspect, the present invention provides a pharmaceutical composition comprising marigold glycoside or a pharmaceutically acceptable salt, prodrug, metabolite thereof and a FAK inhibitor or a Src family kinase inhibitor.

[0010] In addition, the present invention also provides a kit, which comprises marigold glycoside or a pharmaceutically acceptable salt, prodrug, metabolite thereof and a FAK inhibitor or a Src family kinase inhibitor.

[0011] In one embodiment, the weight ratio of marigold glycoside or a pharmaceutically acceptable salt, prodrug, metabolite thereof to the FAK inhibitor or Src family kinase inhibitor is 0.1-10:1, preferably 1-5:1, and more preferably 2-3:1.

[0012] Preferably, the weight ratio of marigold glycoside or its pharmaceutically acceptable salt, prodrug, metabolite to the FAK inhibitor is 3-9:3, preferably 4-7:3, and more preferably 5:3.

[0013] Preferably, the weight ratio of marigold glycoside or its pharmaceutically acceptable salt, prodrug, metabolite to the Src family kinase inhibitor is 1-4:1, preferably 2-3:1, and more preferably 2.5:1.

[0014] In one embodiment, the FAK inhibitor includes defatinib or a pharmaceutically acceptable salt thereof; the Src family kinase inhibitor includes ponatinib or a pharmaceutically acceptable salt thereof.

[0015] In one embodiment, the ratio of tagetoid glycoside or a pharmaceutically acceptable salt, prodrug, metabolite thereof to defatinib or a pharmaceutically acceptable salt thereof or ponatinib or a pharmaceutically acceptable salt thereof, based on the mass of tagetoid glycoside and defatinib or ponatinib, is 0.1-10:1.

[0016] Preferably, the ratio of tagetoid glycoside or a pharmaceutically acceptable salt, prodrug, metabolite thereof to defatinib or a pharmaceutically acceptable salt thereof or ponatinib or a pharmaceutically acceptable salt thereof, based on the mass of tagetoid glycoside and defatinib or ponatinib, is 1-5:1.

[0017] In a preferred embodiment, the ratio of marigold glycoside or a pharmaceutically acceptable salt, prodrug, metabolite thereof to defatinib or a pharmaceutically acceptable salt thereof, based on the mass of marigold glycoside and defatinib, is 3-9:3, preferably 4-7:3, and more preferably 5:3.

[0018] In a preferred embodiment, the ratio of marigold glycoside or its pharmaceutically acceptable salt, prodrug, metabolite to ponatinib or its pharmaceutically acceptable salt, based on the mass of marigold glycoside and ponatinib, is 1-4:1, preferably 2-3:1, and more preferably 2.5:1.

[0019] In a second aspect, the present invention also provides the use of the pharmaceutical composition in preparing a drug for blocking the progression of esophageal squamous cell carcinoma.

[0020] In a third aspect, the present invention also provides the use of marigold glycoside or a pharmaceutically acceptable salt, prodrug, or metabolite thereof in enhancing the effect of FAK inhibitors or Src family kinase inhibitors in blocking the progression of esophageal squamous cell carcinoma.

[0021] In a fourth aspect, the present invention also provides the use of marigold glycoside or a pharmaceutically acceptable salt, prodrug, or metabolite thereof in the preparation of a drug for enhancing the effect of FAK inhibitors or Src family kinase inhibitors in blocking the progression of esophageal squamous cell carcinoma.

[0022] In one embodiment, blocking the progression of esophageal squamous cell carcinoma includes inhibiting the metastasis of esophageal squamous cell carcinoma cells, inhibiting the growth of primary esophageal squamous cell carcinoma cells, and inhibiting the growth of metastatic esophageal squamous cell carcinoma cells.

[0023] In a preferred embodiment, the metastasis is lymphatic metastasis.

[0024] In a preferred embodiment, the esophageal squamous cell carcinoma cells include esophageal squamous cell carcinoma cell line KYSE410, esophageal squamous cell carcinoma cell line KYSE450, and esophageal squamous cell carcinoma cell line KYSE510, and more preferably esophageal squamous cell carcinoma cell line KYSE410 and esophageal squamous cell carcinoma cell line KYSE510.

[0025] In the present invention, the pharmaceutically acceptable salt of tagetoid glycoside includes a salt formed by tagetoid glycoside and a pharmaceutically acceptable acid or base.

[0026] In the present invention, the prodrug refers to a derivative that can directly or indirectly provide tagetoid glycoside after being administered to a patient, such as an ester of tagetoid glycoside.

[0027] In the present invention, the metabolite refers to a pharmaceutically acceptable metabolic derivative form of marigold glycoside or a pharmaceutically acceptable salt thereof.

[0028] Preferably, the medicine or pharmaceutical composition of the present invention further comprises a pharmaceutically acceptable adjuvant.

[0029] Preferably, the marigold glycoside or its pharmaceutically acceptable salt, prodrug, metabolite and FAK inhibitor or Src family kinase inhibitor in the pharmaceutical composition of the present invention can be administered in the same or different pharmaceutical preparations. The pharmaceutical dosage forms of marigold glycoside or its pharmaceutically acceptable salt, prodrug, metabolite and FAK inhibitor or Src family kinase inhibitor can be the same or different. Marigold glycoside or its pharmaceutically acceptable salt, prodrug, metabolite and FAK inhibitor or Src family kinase inhibitor can be administered simultaneously or sequentially.

[0030] In the medical uses described above, the administration time, number of administrations and frequency of administration of marigold glycoside or its pharmaceutically acceptable salts, prodrugs, metabolites and FAK inhibitors or Src family kinase inhibitors need to be determined according to the specific diagnosis results of the disease, which is within the technical scope mastered by those skilled in the art.

[0031] For example, when a treatment regimen for mice or rats is applied to humans, the effective doses of all drugs for humans can be converted by the effective doses of the drugs for mice or rats, which is also easy to achieve for ordinary technicians in this field.

[0032] As pharmaceutically acceptable adjuvants, any adjuvant known to be suitable for preparing a specific drug or pharmaceutical composition can be used. Examples include, but are not limited to, solvents, excipients, dispersants, emulsifiers, solubilizers, gel formers, ointment bases, antioxidants, preservatives, stabilizers, carriers, fillers, bonding agents, thickeners, complexing agents, disintegrants, buffers, penetration enhancers, polymerizers, lubricants, coating agents, propellants, tonicity regulators, surfactants, colorants, flavoring agents, sweeteners and dyes. Particularly adjuvants suitable for the type of desired formulation and desired mode of administration are used.

[0033] The medicine or pharmaceutical composition of the present invention can be one of tablets, powders, granules, pills, capsules, solutions, eye drops, emulsions, suspensions, ointments, oils, pastes, foams, sprays, injections, skin patches, suppositories, liposome preparations, microparticle preparations, and microcapsule preparations.

[0034] The medicament or pharmaceutical composition of the present invention is suitable for oral, rectal, nasal, topical (including transdermal, buccal and sublingual), vaginal, parenteral (including subcutaneous, intramuscular, intravenous and intradermal) or pulmonary administration.

[0035] The medicament or pharmaceutical composition of the present invention can be produced in a manner known to those skilled in the art, for example, by dissolving, mixing, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

[0036] In the medicine or pharmaceutical composition of the present invention, the amount of marigold glycoside or its pharmaceutically acceptable salt, prodrug, metabolite, defatinib and ponatinib can account for 0.01% to 50% of the total weight, preferably 0.1% to 10%, more preferably 0.5% to 5%, and most preferably 1% to 2%.

[0037] The therapeutic amount can be determined empirically and will vary with the pathology being treated, the weight of the subject being treated, and the efficacy and toxicity of the agent. Similarly, those skilled in the art can easily determine the appropriate dosage formulation and the method of administering the agent. For example, for adult patients, the pharmaceutical composition of the present invention can be administered orally or parenterally in an amount of 0.001 mg to 500 mg as a daily dose, once a day or divided into several times.

[0038] Beneficial effects:

[0039] The present invention provides the use of marigold glycosides in combination with FAK inhibitor defatinib or Src family kinase inhibitor ponatinib to block the progression of esophageal squamous cell carcinoma. Experimental results show that marigold glycosides can inhibit the proliferation and invasion of esophageal squamous cell carcinoma cells, and its combination with FAK inhibitor defatinib or Src family kinase inhibitor ponatinib can inhibit the metastasis of esophageal squamous cell carcinoma, especially lymphatic metastasis, and can inhibit the growth of esophageal squamous cell carcinoma cells in primary and metastatic foci, and has a synergistic anti-cancer effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is a schematic diagram of the results of the marigold glycosides in Example 1 that inhibit the proliferation and invasion of the esophageal squamous cell carcinoma cell line KYSE510 in a dose-dependent manner.

[0041] Figure 2 This is a schematic diagram showing the results that marigold glycoside (Quer) in Example 2 is more effective than quercetin in inhibiting the invasion of esophageal squamous cell carcinoma cell line KYSE30.

[0042] Figure 3 It is a schematic diagram of the results of positive correlation expression between FAK tyrosine 576 / 577, Src kinase family member Src tyrosine Y419, Fyn tyrosine Y420 and Lyn tyrosine Y397 and downstream proliferation-related molecules such as MCM3, MCM4, MCM5, MCM6, MCM7, CDC45 and CDK7, CDK9 in Example 3.

[0043] Figure 4 This is a schematic diagram of the results of positive correlation between the expression of FAK tyrosine 576 / 577, Src kinase family members Src tyrosine Y419, Fyn tyrosine Y420 and Lyn tyrosine Y397 in the lymphatic metastasis of esophageal squamous cell carcinoma in Example 4 and downstream pro-invasion and stemness-related markers such as SDC2, ITGA5, GALK1, AXL, Myc, Sox2 and OCT4.

[0044] Figure 5 This is a schematic diagram of the results showing that marigold glycosides in Example 5 significantly enhance the anti-progression effects of defatinib and ponatinib on esophageal squamous cell carcinoma.

[0045] Figure 6 This is a schematic diagram showing the results that marigold glycosides in Example 6 significantly enhance the inhibitory effects of defatinib and ponatinib on the expression of MCMs / CDC45 molecules in primary esophageal squamous cell carcinoma tissues.

[0046] Figure 7 This is a schematic diagram of the results showing that marigold glycosides in Example 7 significantly enhance the inhibitory effects of defatinib and ponatinib on the expression of proliferation markers CDK7, CDK9 and Ki67 and lymphangiogenesis marker LYVE1 in primary esophageal squamous cell carcinoma tissues.

[0047] Figure 8 This is a schematic diagram of the results that marigold glycosides in Example 8 significantly enhance the inhibitory effects of defatinib and ponatinib on the expression of SDC2, ITGA5, GALK1, and AXL molecules in metastatic esophageal squamous cell carcinoma tissues.

[0048] Fig. 9 This is a schematic diagram showing the results that marigold glycosides in Example 9 significantly enhance the inhibitory effects of defatinib and ponatinib on the transcriptional activities of Myc, Sox2 and OCT4 in metastatic esophageal squamous cell carcinoma tissues.

[0049] In the above figures, * represents P < 0.05; ** represents P < 0.01; *** represents P < 0.001; ns represents no statistical difference. DETAILED DESCRIPTION

[0050] The preferred embodiments of the invention are described in detail below. The examples are provided to better illustrate the content of the invention, but the content of the invention is not limited to the examples. Non-essential improvements and adjustments to the implementation scheme according to the content of the invention still fall within the scope of the invention.

[0051] The experimental methods in the following examples are all conventional methods unless otherwise specified. If no specific techniques or conditions are specified in the examples, they are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions.

[0052] Example 1: Marigold glycoside inhibits the growth and invasion of esophageal squamous cell carcinoma cell KYSE510

[0053] 1. Cell Proliferation Assay

[0054] KYSE510 cells were cultured at 3 × 10 3 The cells were inoculated at a density of 100 cells / well in a 96-well plate, and marigold glycoside (concentrations: 1, 2.5, 5, 10, 25, 50, 100 μM) was added after the cells adhered to the wall. After 72 hours, 10% MTS solution was prepared with RPMI 1640 medium, the cultured cells were taken out, the upper medium was discarded, and the prepared MTS was added and incubated for 2 hours, and the absorbance value was measured at 490 nm by an ELISA reader.

[0055] 2. Cell invasion ability detection

[0056] The KYSE510 cell line was cultured in serum-free medium for 24 hours. Using a transwell chamber with an 8 μm pore size, 100 μL of matrix gel was added to the upper chamber of the chamber and incubated at 37°C for 1 hour to solidify the matrix gel. The prepared KYSE510 cell line was inoculated in the upper chamber of the transwell chamber, and 800 μL of RPMI 1640 medium containing 20% ​​fetal bovine serum was added to the lower chamber, and marigold glycoside (concentration: 10, 25, 50 μM) was added. The transwell chamber was placed in an incubator and incubated for 24 hours. After taking it out, the inner wall of the upper chamber was wiped clean with a cotton swab, and the invading cells in the lower chamber were separated by dissociation solution and then cell dye was added, and the invasion cell rate was calculated by an ELISA instrument.

[0057] Compare with Figure 1 , marigold glycosides inhibited the proliferation and invasion of esophageal squamous cell carcinoma cell line KYSE510 in a dose-dependent manner.

[0058] Example 2: Marigold glycosides are more effective than quercetin in inhibiting the invasion of esophageal squamous cell carcinoma cells

[0059] Cell invasion ability assay

[0060] The KYSE30 cell line was cultured in serum-free medium for 24 hours. Using a transwell with an 8 μm pore size, 100 μL of matrix gel was added to the upper chamber of the chamber and incubated at 37°C for 1 hour to solidify the matrix gel. The prepared KYSE30 cell line was inoculated in the upper chamber of the transwell, and 800 μL of RPMI 1640 medium containing 20% ​​fetal bovine serum was added to the lower chamber, and marigold glycosides or quercetin (concentration of 25 μM) were added. The transwell was placed in an incubator and incubated for 24 hours. After removal, the inner wall of the upper chamber was wiped clean with a cotton swab, and the invasive cells in the lower chamber were separated by dissociation solution and then cell dye was added, and the invasion cell rate was calculated by an ELISA instrument.

[0061] Compare with Figure 2 The results showed that marigold glycosides were more effective than quercetin in inhibiting the invasion of esophageal squamous cell carcinoma cell line KYSE30.

[0062] Example 3: Detection of the expression of FAK tyrosine 576 / 577, Src kinase family member Src tyrosine Y419, Fyn tyrosine Y420 and Lyn tyrosine Y397 and downstream proliferation-related molecules in primary esophageal squamous cell carcinoma

[0063] 1. Immunohistochemistry

[0064] The pathological sections of 18 primary esophageal squamous cell carcinoma tissues were routinely dewaxed and antigen repaired, and then incubated with FAK tyrosine 576 / 577, Src kinase family members Src tyrosine Y419, Fyn tyrosine Y420 and Lyn tyrosine Y397, and MCM3, MCM4, MCM5, MCM6, MCM7, CDC45, CDK7, CDK9 antibodies for color development. The expression intensity of the corresponding proteins in local tissues was observed by immunohistochemistry.

[0065] 2. According to the results of immunohistochemistry, FAK tyrosine 576 / 577, Src kinase family member Src tyrosine Y419, Fyn tyrosine Y420 and Lyn tyrosine Y397 were positively correlated with downstream proliferation-related molecules.

[0066] The immunohistochemical results were calculated by multiplying the staining intensity (staining intensity was differentiated according to four levels: 0, 1, 2, and 3) by the proportion of positive cells in the local tissue (staining area was differentiated according to five levels: 0, 1, 2, 3, and 4). A product greater than 3 indicated high expression (positive), and a product less than or equal to 3 indicated low expression (negative). Pearson correlation analysis was used to observe the expression correlation between FAK tyrosine 576 / 577, Src kinase family member Src tyrosine Y419, Fyn tyrosine Y420, and Lyn tyrosine Y397 and downstream proliferation-related molecules.

[0067] Compare with Figure 3 , FAK tyrosine 576 / 577, Src kinase family member Src tyrosine Y419, Fyn tyrosine Y420 and Lyn tyrosine Y397 were positively correlated with downstream proliferation-related molecules such as MCM3, MCM4, MCM5, MCM6, MCM7, CDC45, CDK7 and CDK9.

[0068] Example 4: Detection of the expression of FAK tyrosine 576 / 577, Src kinase family member Src tyrosine Y419, Fyn tyrosine Y420 and Lyn tyrosine Y397 and downstream invasion-promoting and stemness-related markers in esophageal squamous cell carcinoma with lymphatic metastasis

[0069] 1. Immunohistochemistry

[0070] Pathological sections of 20 cases of esophageal squamous cell carcinoma tissues with lymph node metastasis were routinely dewaxed and antigen repaired, and then incubated with FAK tyrosine 576 / 577, Src kinase family members Src tyrosine Y419, Fyn tyrosine Y420 and Lyn tyrosine Y397, and SDC2, ITGA5, GALK1, AXL, Myc, Sox2 and OCT4 antibodies for color development. The expression intensity of the corresponding proteins in local tissues was observed by immunohistochemistry.

[0071] 2. According to the results of immunohistochemistry, FAK tyrosine 576 / 577, Src kinase family member Src tyrosine Y419, Fyn tyrosine Y420 and Lyn tyrosine Y397 were positively correlated with downstream invasion-promoting and stemness-related molecules.

[0072] Immunohistochemistry results were calculated based on the staining intensity (staining intensity was differentiated according to four levels: 0, 1, 2, and 3) multiplied by the proportion of positive cells in the local tissue (staining area was differentiated according to five levels: 0, 1, 2, 3, and 4). A product greater than 3 indicated high expression (positive), and a product less than or equal to 3 indicated low expression (negative). Pearson correlation analysis was used to observe the expression correlation between FAK tyrosine 576 / 577, Src kinase family member Src tyrosine Y419, Fyn tyrosine Y420, and Lyn tyrosine Y397 and downstream invasion-promoting and stemness-related molecules.

[0073] Compare with Figure 4 In the lymphatic metastasis of esophageal squamous cell carcinoma, FAK tyrosine 576 / 577, Src kinase family members Src tyrosine Y419, Fyn tyrosine Y420 and Lyn tyrosine Y397 were positively correlated with the downstream invasion and stemness-related markers, such as SDC2, ITGA5, GALK1, AXL, Myc, Sox2 and OCT4.

[0074] Example 5: Marigold glycosides combined with FAK inhibitor defatinib and Src family kinase inhibitor ponatinib inhibit lymphatic metastasis of esophageal squamous cell carcinoma cells

[0075] Animal model validates that marigold glycosides combined with FAK inhibitor defatinib and Src family kinase inhibitor ponatinib inhibit lymphatic metastasis of esophageal squamous cell carcinoma cells

[0076] KYSE410 and KYSE510 cells were inoculated into the footpads of nude mice (4-week-old female BALB / c-nu nude mice (average body weight 14-15 g), purchased from Vital River Company). After about 1 week, marigold glycosides (25 mg / kg / day), defatinib (15 mg / kg / day) or ponatinib (10 mg / kg / day) were administered orally, either alone or in combination, for 5 weeks. The popliteal lymph nodes were collected, and the volume was calculated as follows: Volume = Long diameter × Transverse diameter 2 ×0.5.

[0077] Compare with Figure 5 The results showed that marigold glycosides significantly enhanced the anti-progression effects of defatinib and ponatinib on esophageal squamous cell carcinoma.

[0078] Example 6: Marigold glycosides combined with FAK inhibitor defatinib and Src family kinase inhibitor ponatinib inhibit the expression of MCMs / CDC45 molecules in primary esophageal squamous cell carcinoma cells

[0079] Animal model validates that marigold glycosides combined with FAK inhibitor defatinib and Src family kinase inhibitor ponatinib inhibit lymphatic metastasis of esophageal squamous cell carcinoma cells

[0080] KYSE410 and KYSE510 cells were inoculated into the footpads of nude mice (4-week-old female BALB / c-nu nude mice (average weight 14-15 g), purchased from Vital River). After about 1 week, marigold glycosides (25 mg / kg / day), defatinib (15 mg / kg / day) or ponatinib (10 mg / kg / day) were administered orally, either alone or in combination, for 5 weeks. Primary esophageal squamous cell carcinoma tissues were collected and prepared into paraffin sections, and MCM3, MCM4, MCM5, MCM6, MCM7, and CDC45 were observed by immunohistochemistry.

[0081] Compare with Figure 6 The results showed that marigold glycosides significantly enhanced the inhibitory effect of defatinib and ponatinib on the expression of MCMs / CDC45 molecules in primary esophageal squamous cell carcinoma tissues.

[0082] Example 7: Marigold glycosides combined with FAK inhibitor defatinib and Src family kinase inhibitor ponatinib inhibit the expression of CDK7, CDK9, Ki67 and YVE1 in primary esophageal squamous cell carcinoma cells

[0083] KYSE410 and KYSE510 cells were inoculated into the footpads of nude mice (4-week-old female BALB / c-nu nude mice (average body weight 14-15 g), purchased from Vital River). After about 1 week, marigold glycosides (25 mg / kg / day), defatinib (15 mg / kg / day) or ponatinib (10 mg / kg / day) were administered orally, either alone or in combination, for 5 weeks. Primary esophageal squamous cell carcinoma tissues were collected and prepared into protein lysate. The expression levels of proliferation markers CDK7, CDK9 and Ki67 and lymphangiogenesis marker LYVE1 in local tumor tissues were observed by high-throughput enzyme-linked immunosorbent assay (ELISA).

[0084] Compare with Figure 7 The results showed that marigold glycosides significantly enhanced the inhibitory effect of defatinib and ponatinib on the expression of proliferation markers CDK7, CDK9 and Ki67 and lymphangiogenesis marker LYVE1 in primary esophageal squamous cell carcinoma tissues.

[0085] Example 8: Marigold glycosides combined with FAK inhibitor defatinib and Src family kinase inhibitor ponatinib inhibit the expression of SDC2, ITGA5, GALK1, and AXL molecules in esophageal squamous cell carcinoma cells with lymphatic metastasis

[0086] Animal model verified that marigold glycosides combined with FAK inhibitor defatinib and Src family kinase inhibitor ponatinib inhibited the expression of SDC2, ITGA5, GALK1 and AXL molecules in esophageal squamous cell carcinoma cells with lymphatic metastasis

[0087] KYSE410 and KYSE510 cells were inoculated into the footpads of nude mice. After about 1 week, marigold glycosides (25 mg / kg / day), defatinib (15 mg / kg / day) or ponatinib (10 mg / kg / day) were administered orally, either alone or in combination, for 5 weeks. Metastatic esophageal squamous cell carcinoma tissues were collected and prepared into paraffin sections, and the expression of SDC2, ITGA5, GALK1, and AXL was observed by immunohistochemistry.

[0088] Compare with Figure 8 The results showed that marigold glycosides significantly enhanced the inhibitory effect of defatinib and ponatinib on the expression of SDC2, ITGA5, GALK1, and AXL molecules in metastatic esophageal squamous cell carcinoma tissues.

[0089] Example 9: Marigold glycosides combined with FAK inhibitor defatinib and Src family kinase inhibitor ponatinib inhibit the transcriptional activity of Myc, Sox2 and OCT4 molecules in esophageal squamous cell carcinoma cells with lymphatic metastasis

[0090] Animal model verified that marigold glycosides combined with FAK inhibitor defatinib and Src family kinase inhibitor ponatinib inhibited the transcriptional activity of Myc, Sox2 and OCT4 molecules in esophageal squamous cell carcinoma cells with lymphatic metastasis

[0091] KYSE410 and KYSE510 cells were inoculated into the footpads of nude mice (4-week-old female BALB / c-nu nude mice (average weight 14-15 grams), purchased from Vital River Company). After about 1 week, marigold glycosides (25 mg / kg / day), defatinib (15 mg / kg / day) or ponatinib (10 mg / kg / day) were administered orally, either alone or in combination, for 5 weeks. The nuclear proteins were extracted after the metastatic esophageal squamous cell carcinoma tissues were collected, and the transcriptional activity of Myc, Sox2 and OCT4 molecules was observed by transcriptional activity experiments.

[0092] Compare with Fig. 9The results showed that marigold glycosides significantly enhanced the inhibitory effects of defatinib and ponatinib on the transcriptional activity of Myc, Sox2 and OCT4 in metastatic esophageal squamous cell carcinoma tissues.

[0093] Embodiment 10:

[0094] Marigold glycosides were encapsulated in liposomes with defatinib and ponatinib (mass ratio = 5:3 and mass ratio = 5:2, respectively) to prepare liposome drugs for treating esophageal squamous cell carcinoma.

[0095] Embodiment 11:

[0096] After marigold glycosides were mixed with defatinib and ponatinib in proportions (mass ratio = 5:3 and mass ratio = 5:2, respectively), excipients were added in the required proportions of the preparation to prepare tablets.

[0097] Embodiment 12:

[0098] The marigold glycoside is mixed with difatinib and ponatinib in proportions (mass ratio = 5:3 and mass ratio = 5:2, respectively), dissolved in water, finely filtered, and sterilized by filling to prepare an injection.

[0099] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. A pharmaceutical composition comprising marigold glycoside or a pharmaceutically acceptable salt, prodrug, metabolite thereof and a FAK inhibitor or a Src family kinase inhibitor.

2. The pharmaceutical composition according to claim 1, characterized in that The weight ratio of marigold glycoside or its pharmaceutically acceptable salt, prodrug, metabolite to FAK inhibitor or Src family kinase inhibitor is 0.1-10:

1.

3. The pharmaceutical composition according to claim 1, characterized in that The FAK inhibitor includes defatinib or a pharmaceutically acceptable salt thereof; the Src family kinase inhibitor includes ponatinib or a pharmaceutically acceptable salt thereof.

4. The pharmaceutical composition according to claim 3, characterized in that The ratio of marigold glycoside or its pharmaceutically acceptable salt, prodrug, metabolite to defatinib or its pharmaceutically acceptable salt or ponatinib or its pharmaceutically acceptable salt is 0.1-10:1 based on the mass of marigold glycoside and defatinib or ponatinib.

5. A kit comprising marigold glycoside or a pharmaceutically acceptable salt, prodrug, metabolite thereof and a FAK inhibitor or a Src family kinase inhibitor.

6. Use of the pharmaceutical composition according to any one of claims 1 to 4 in the preparation of a drug for blocking the progression of esophageal squamous cell carcinoma.

7. Use of marigold glycoside or its pharmaceutically acceptable salt, prodrug, metabolite in enhancing the effect of FAK inhibitors or Src family kinase inhibitors in blocking the progression of esophageal squamous cell carcinoma.

8. Use of marigold glycoside or a pharmaceutically acceptable salt, prodrug or metabolite thereof in the preparation of a drug for enhancing the effect of FAK inhibitors or Src family kinase inhibitors in blocking the progression of esophageal squamous cell carcinoma.

9. The use according to any one of claims 6 to 8, characterized in that: The blocking of the progression of esophageal squamous cell carcinoma includes inhibiting the metastasis of esophageal squamous cell carcinoma cells, inhibiting the growth of esophageal squamous cell carcinoma cells in primary foci, and inhibiting the growth of esophageal squamous cell carcinoma cells in metastatic foci.

10. The use according to claim 9, characterized in that: The metastasis refers to lymphatic metastasis.

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