Pharmaceutical composition for resisting esophageal squamous carcinoma

Abstract

The invention provides a pharmaceutical composition for resisting esophageal squamous carcinoma. The pharmaceutical composition comprises nitidine chloride and an FAK inhibitor such as VS4718. The pharmaceutical composition disclosed by the invention has the effects of synergistically inhibiting growth of esophageal squamous carcinoma cells, synergistically inhibiting invasion of esophageal squamous carcinoma cells, synergistically inhibiting metabolism of esophageal squamous carcinoma cells and synergistically inhibiting growth of esophageal squamous carcinoma cells. The nitidine chloride is combined with the VS4718 to obtain a synergistic esophageal squamous cell carcinoma resisting effect, so that the anti-tumor effect is improved, the medicine dosage is reduced, and the safety is improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of pharmaceutical compositions, in particular, the present application relates to an anti-esophageal squamous cell carcinoma pharmaceutical composition. BACKGROUND

[0002] Esophageal squamous cell carcinoma (ESCC) is a tumor that seriously threatens human life worldwide, and is one of the cancer types with high incidence and specificity in China. Although the level of diagnosis and treatment is constantly improving, the survival rate and quality of life of ESCC patients have been greatly improved, but the prognosis of patients is still not optimistic. It is particularly important to explore and discover new diagnostic markers and therapeutic drugs for targeting ESCC.

[0003] The tyrosine kinase signaling pathway is a key signal axis that promotes the signaling pathway in tumor cells, including ESCC, which can mediate the generation of multiple malignant phenotypes of ESCC through multiple downstream signal links. Among them, the signal axis centered on focal adhesion kinase (FAK) is particularly critical in inducing the progression of ESCC and is an important molecular hub. Several small molecule inhibitors targeting FAK activity have been developed, but their anti-tumor effects still need further study.

[0004] Traditional Chinese medicine small molecule monomer compounds can inhibit multiple signaling pathways and their key target points and downstream effector molecules, synergistically inhibit the generation of malignant phenotypes in tumor progression, and reduce the toxicity of anticancer drugs. Therefore, the research on traditional Chinese medicine monomer compounds assisting tyrosine kinase targeted drugs in resisting ESCC has become a hot spot in current tumor pharmacology research.

[0005] Nitidine chloride is a quaternary ammonium alkaloid extracted from the roots of the plant Picrasma quassioides, which is an active biological alkaloid compound with anti-inflammatory, antioxidant and other biological effects. Its structure is as follows: Nitidine chloride has a wide range of biological activities, such as anti-inflammatory, analgesic, antibacterial and important role in regulating the immune system. In recent years, it has also been reported that nitidine chloride can inhibit various malignant phenotypes of solid tumor cell lines, such as growth, invasion and metastasis, and has an anticancer effect. SUMMARY

[0006] The present application found that nitidine chloride can enhance the effect of FAK inhibitors in resisting the progression of esophageal squamous cell carcinoma. When nitidine chloride is used in combination with FAK inhibitors, the resulting pharmaceutical composition can have a synergistic effect in resisting esophageal squamous cell carcinoma.

[0007] In one aspect, the present application provides a pharmaceutical composition comprising plumbagin or a pharmaceutically acceptable salt thereof and a FAK inhibitor.

[0008] In another aspect, the present application also provides a kit comprising plumbagin or a pharmaceutically acceptable salt thereof and a FAK inhibitor.

[0009] In one embodiment, in the pharmaceutical composition or the kit, the mass ratio of plumbagin or a pharmaceutically acceptable salt thereof to the FAK inhibitor, based on the mass ratio of plumbagin to the FAK inhibitor, is 1-15:1, preferably 1.5-10:1, more preferably 2-8:1, preferably 2:1, or 6-7:1.

[0010] In one embodiment, the FAK inhibitor is VS4718 or a pharmaceutically acceptable salt thereof.

[0011] In the present application, the CAS registration number of the VS4718 is 1061353-68-1, and the chemical name is 2-[[2-(2-methoxy-4-morpholin-4-ylanilino)-5-(trifluoromethyl)pyridin-4-yl]amino]-N-methylbenzamide (2-[[2-(2-methoxy-4-morpholin-4-yl anilino)-5-(trifluoromethyl)pyridin-4-yl]amino]-N-methylbenzamide), and the structural formula is: .

[0012] Preferably, the mass ratio of plumbagin or a pharmaceutically acceptable salt thereof to VS4718 or a pharmaceutically acceptable salt thereof, based on the mass ratio of plumbagin to VS4718, is 1-15:1, preferably 1.5-10:1, more preferably 2-8:1, preferably 2:1, or 6-7:1.

[0013] In yet another aspect, the present application also provides the use of the pharmaceutical composition of the present application in the preparation of a medicament for resisting esophageal squamous cell carcinoma.

[0014] In still another aspect, the present application also provides the use of plumbagin or a pharmaceutically acceptable salt thereof in the preparation of a medicament for enhancing the effect of a FAK inhibitor on resisting esophageal squamous cell carcinoma.

[0015] In the present application, the resisting esophageal squamous cell carcinoma includes inhibiting the growth of esophageal squamous cell carcinoma cells, inhibiting the invasion of esophageal squamous cell carcinoma cells, inhibiting the sugar metabolism of esophageal squamous cell carcinoma cell lines, and inhibiting the amino acid metabolism of esophageal squamous cell carcinoma cell lines. ​

[0016] In one embodiment, the esophageal squamous cell carcinoma cell comprises esophageal squamous cell carcinoma cell KYSE410, esophageal squamous cell carcinoma cell KYSE450, esophageal squamous cell carcinoma cell KYSE510, more preferably esophageal squamous cell carcinoma cell KYSE510.

[0017] In the present application, the term "pharmaceutically acceptable salt" includes, but is not limited to, pharmaceutically acceptable acid addition salts of the compounds of the present application with inorganic acids such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric, and sulfuric acids or with organic acids such as acetic, ethanesulfonic, benzenesulfonic, benzoic, citric, fumaric, gluconic, glycolic, isethionic, lactic, lactobionic, maleic, malic, methanesulfonic, succinic, p-toluenesulfonic, and tartaric acids.

[0018] In one embodiment, the pharmaceutical composition or medicament of the present application further comprises at least one pharmaceutically acceptable excipient.

[0019] The pharmaceutically acceptable excipient includes at least one of a carrier, an excipient, a diluent, a lubricant, an emulsifier, a preservative, a sweetener, a suspension stabilizer, or a flavoring agent.

[0020] The pharmaceutical composition or medicament of the present application can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir, preferably by oral administration or by injection. The term parenteral as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques.

[0021] According to the use of the present application, the above pharmaceutical composition or medicament can be formulated into a dosage form for oral administration. For example, it can be formulated by combining the active compound with a pharmaceutically acceptable carrier well known to those skilled in the art. Such a carrier allows the pharmaceutical composition or medicament to be formulated into tablets, capsules, powders, granules, crystals, solutions, suspensions, soups, syrups, elixirs, and chewable agents, etc. Suitable carriers include excipients such as fillers, including sugars such as lactose, sucrose, mannitol, and / or sorbitol; cellulose preparations such as corn starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl cellulose, sodium hydroxymethyl cellulose, and / or polyvinylpyrrolidone (PVP). If necessary, a disintegrant such as cross-linked polyvinylpyrrolidone, agar, or alginic acid or its salts such as sodium alginate can be added.

[0022] Alternatively, the pharmaceutical composition or medicament of the present application is preferably formulated into a dosage form suitable for inhalation or insufflation, intranasal, transdermal or local administration, selected from the group consisting of nasal sprays, nose drops, suspensions, gels, ointments, creams, lotions, and powders.

[0023] For administration by inhalation, the pharmaceutical compositions or drugs for use according to the present application are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichloromonofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator can be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

[0024] Nasal delivery systems useful in the present application can take a variety of forms including aqueous formulations, non-aqueous formulations, and combinations thereof. Aqueous formulations include, for example, hydrogels, hydro-suspensions, liposomal aqueous dispersions, aqueous emulsions, aqueous microemulsions, and combinations thereof. Non-aqueous formulations include, for example, non-hydrogels, non-hydro-suspensions, liposomal non-aqueous dispersions, non-aqueous emulsions, non-aqueous microemulsions, and combinations thereof. Nasal delivery systems in various forms can include buffers to maintain pH, pharmaceutically acceptable thickening and wetting agents. The pH of the buffer can be selected to optimize the absorption of the therapeutic agent through the nasal mucosa.

[0025] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions, can be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono- or diglycerides.

[0026] Dosage forms for topical or transdermal administration of a pharmaceutical composition or drug of this application include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, or patches. In some embodiments, the active component is admixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers as can be required. Ophthalmic formulation, eardrops, eye drops, eye ointments, powders and solutions are also contemplated as being within the scope of this application.

[0027] The pharmaceutical compositions or drugs can be conveniently presented in unit dosage form and can be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the therapeutic agent(s) with the carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing into association the therapeutic agent(s) with liquid carriers, fine solid carriers, or both, and then, if necessary, shaping the product.

[0028] In one embodiment, the pharmaceutical composition, kit, or medicament of the present invention, containing zebufenozide chloride or a pharmaceutically acceptable salt thereof, and a FAK inhibitor, can be administered in the same or different pharmaceutical formulations. The pharmaceutical dosage forms of zebufenozide chloride or a pharmaceutically acceptable salt thereof and the FAK inhibitor can be the same or different. Zebufenozide chloride or a pharmaceutically acceptable salt thereof and the FAK inhibitor can be administered simultaneously or sequentially.

[0029] The effective dosage of the pharmaceutical composition of the present invention can be determined according to age, weight, sex, method of administration, health condition, and severity of illness. For example, the dosage for an adult weighing 70 kg is 0.1-1000 mg / day, preferably 1-500 mg / day. Such administration can be done once or multiple times a day, as determined by a physician or pharmacist.

[0030] In the pharmaceutical applications described herein, the timing, frequency, and duration of administration of the pharmaceutical composition or drug of this invention need to be determined based on the specific diagnostic results of the condition, which is within the technical scope of those skilled in the art.

[0031] The pharmaceutical compositions or drugs of the present invention can be produced in a manner known to those skilled in the art, for example by dissolving, mixing, granulating, preparing a sugar coating, grinding, emulsifying, forming capsules, embedding, or lyophilizing processes.

[0032] Beneficial effects: This invention provides an anti-esophageal squamous cell carcinoma drug composition comprising pekinensis chloride and VS4718. Evaluations using the MTS assay and soft agar colony formation assay showed that pekinensis chloride and VS4718 synergistically inhibited the growth of esophageal squamous cell carcinoma cells. Evaluation using the Transwell assay showed that pekinensis chloride and VS4718 synergistically inhibited the invasion of esophageal squamous cell carcinoma cells. Furthermore, pekinensis chloride and VS4718 synergistically inhibited the metabolism of esophageal squamous cell carcinoma cells, including reducing the levels of lactate, glucose, and glutamine in these cells. Animal studies demonstrated that pekinensis chloride and VS4718 synergistically inhibited the growth of esophageal squamous cell carcinoma. The combination of pekinensis chloride and VS4718 achieved a synergistic anti-esophageal squamous cell carcinoma effect, which is beneficial for improving anti-tumor efficacy and reducing drug dosage. Attached Figure Description Attached image description: Figure 1 This is a schematic diagram showing the results of the synergistic inhibition of esophageal squamous cell carcinoma cell line growth by phenylephrine chloride and VS4718 in Example 1.

[0034] Figure 2 This is a schematic diagram showing the results of the synergistic inhibition of esophageal squamous cell carcinoma cell line invasion by phenylephrine chloride and VS4718 in Example 2.

[0035] Figure 3 Figure 3 shows the results of the synergistic inhibition of glucose metabolism of an esophageal squamous cell carcinoma cell line by nitidine chloride and VS4718 of Example 3.

[0036] Figure 4 Figure 4 shows the results of the synergistic inhibition of amino acid metabolism of an esophageal squamous cell carcinoma cell line by nitidine chloride and VS4718 of Example 4.

[0037] Figure 5 Figure 5 shows the results of the synergistic inhibition of the growth of an esophageal squamous cell carcinoma cell line in a tumor-bearing mouse model by nitidine chloride and VS4718 of Example 5.

[0038] In the above figures, represents P < 0.01; represents P < 0.001; represents P < 0.0001. DETAILED DESCRIPTION

[0039] The preferred examples of the present application will be described in detail below. The examples are provided to better illustrate the present application and should not be construed to limit the present application to only the examples. Non-essential improvements and modifications to the embodiments according to the present application are still within the scope of the present application.

[0040] The experimental methods in the following examples are routine methods unless otherwise specified. When a specific technique or condition is not specified in the examples, the technique or condition described in the literature in the art or according to the product manual is used. DETAILED DESCRIPTION Example 1: Nitidine chloride enhances the growth inhibition of an esophageal squamous cell carcinoma cell line by VS4718 1. Cell culture The human esophageal squamous cell carcinoma cell line KYSE510 was maintained in RPMI1640 medium containing 10% fetal bovine serum, 1% penicillin streptomycin. The incubator condition was 37 °C with 5% CO2.

[0042] 2. Cell growth capacity assay (MTS method) KYSE510 cell line (3.5 x 10 3 After the KYSE510 cell line was seeded in a 96-well plate, nitidine chloride (5 μM), VS4718 (0.5 μM), or a combination thereof was added. After 72 hours, 10% MTS solution was prepared with RPMI1640 medium and added to the 96-well plate and incubated for 1 hour. The absorbance value was measured at 490 nm using a microplate reader.

[0043] 3. Cell growth capacity assay (soft agar colony formation experiment) After the preparation of the lower agarose in 96-well plates, the plates were placed at 4℃ for 20 minutes. The upper agarose containing KYSE510 cell line was prepared. After the preparation of the upper and lower agarose gel was completed, the combination of nitidine chloride (5 μM) and VS4718 (0.5 μM) was added to the upper layer. After 10 days of incubation, the color developing solution was added, and the plates were incubated at 37℃ for 4 hours. The absorbance value was measured at 450 nm using a microplate reader.

[0044] Control Figure 1 The results showed that nitidine chloride and VS4718 had a synergistic effect on inhibiting the growth of esophageal squamous cell carcinoma cell lines.

[0045] The Jin Zhengjun Q value judgment method was used to evaluate the synergistic effect of nitidine chloride and VS4718 on tumor growth inhibition. The Q value was calculated as follows: Q = E a+b / (E a +E b -E a ×E b ). E a+b is the inhibition rate of drug combination, E a and E b is the inhibition rate of drug treatment alone. Q < 0.85 indicates antagonism, 0.85 ≤ Q < 1.15 indicates additive effect, and Q ≥ 1.15 indicates synergistic effect.

[0046] The calculated Q value for the growth inhibition effect mediated by nitidine chloride combined with VS4718 was 1.52.

[0047] The Q value for the anchorage-independent growth inhibition effect mediated by nitidine chloride combined with VS4718 was 1.56.

[0048] Example 2: Nitidine chloride enhances the inhibitory effect of VS4718 on the invasion of esophageal squamous cell carcinoma cell lines Cell invasion ability detection: The invasion ability of VS4718 against ESCC cells was observed by Tranwell system (8 μm pore size) with the sensitization of nitidine chloride. 100 μL Matrigel was added to the upper chamber of the transwell chamber and incubated at 37℃ for 1 hour to solidify the Matrigel. The prepared KYSE510 cell line was inoculated in the upper chamber of the transwell chamber, 1 mL of RPMI 1640 culture medium containing 20% fetal bovine serum was added to the lower chamber, and nitidine chloride (5 μM), VS4718 (0.5 μM) or their combination was added, and the transwell chamber was incubated in an incubator for 24 hours. After taking out, the upper chamber was wiped with a cotton swab to remove the uninvaded KYSE510 cells, and the invaded KYSE510 cells in the lower chamber were separated by dissociation solution. The separated cells were added with dye, and the invasion rate of the cells was calculated by an enzyme-labeled instrument after staining.

[0049] Control Figure 2 The results showed that nitidine chloride and VS4718 had a synergistic effect on inhibiting the invasion of esophageal squamous cell carcinoma cell lines.

[0050] According to the calculation, the Q value of the invasion inhibition effect mediated by nitidine chloride combined with VS4718 was 1.43.

[0051] Example 3: Nitidine chloride enhances the inhibition of glucose metabolism of VS4718 on esophageal squamous cell carcinoma cell lines The KYSE510 cell line was inoculated in a 6-well plate, and after the cells adhered, nitidine chloride (5 μM), VS4718 (0.5 μM) or their combination was added. After 24 hours, the conditioned medium was collected, and the lactic acid detection kit (Nanjing Jiancheng Biological Engineering Research Co., Ltd., Catalog No.: A019-3-1) was used to incubate the conditioned medium under different treatment conditions with different reagents according to the instructions, and then the absorbance was displayed by an enzyme-labeled instrument to determine the lactic acid content. The glucose detection kit (Bi Yun Tian Biological Technology Research Co., Ltd., Catalog No.: S0201S) was used to incubate the conditioned medium under different treatment conditions with different reagents according to the instructions, and then the absorbance was displayed by an enzyme-labeled instrument to determine the glucose content.

[0052] Control Figure 3 The results showed that nitidine chloride and VS4718 had a synergistic effect on inhibiting the glucose metabolism of esophageal squamous cell carcinoma cell lines.

[0053] According to the calculation, the Q value of the lactic acid inhibition effect mediated by nitidine chloride combined with VS4718 was 1.28, and the Q value of the glucose inhibition effect mediated by nitidine chloride combined with VS4718 was 1.3.

[0054] Example 4: Nitidine chloride enhances the inhibition of amino acid metabolism of VS4718 on esophageal squamous cell carcinoma cell lines KYSE510 cell line was inoculated in 6-well plates, and after the cells adhered, nitidine chloride (5 μM), VS4718 (0.5 μM) or their combination was added. After 24 hours, the cell lysate was collected, and the glutamine content in the cell lysate under different treatments was determined by glutamine detection kit (Abeam, ab197011) according to the instructions of the reagent. After incubation with different reagents, the absorbance was displayed by the enzyme-labeled instrument.

[0055] Control Figure 4 The results showed that nitidine chloride and VS4718 had a synergistic effect on inhibiting the amino acid metabolism of esophageal squamous cell carcinoma cell lines.

[0056] According to the calculation, the Q value of the amino acid inhibition effect mediated by nitidine chloride combined with VS4718 was 1.52.

[0057] Example 5: Nitidine chloride enhances the anti-tumor growth effect of VS4718 on esophageal squamous cell carcinoma cell line KYSE510 tumor-bearing mice KYSE510 cells were injected into the upper limbs of nude mice (4-week-old female BALB / c-nu nude mice (average body weight 14-15 g), purchased from Vantian Lihua Company) subcutaneously, and when the tumor grew to about 100 mm 3 left and right, the KYSE510 tumor-bearing mice were randomly divided into four groups: control group, nitidine chloride group, VS4718 group and nitidine chloride plus VS4718 group. The nitidine chloride group (20 mg / kg) was administered intraperitoneally once a day, the VS4718 group (10 mg / kg) was administered orally once a day, and the nitidine chloride plus VS4718 group was administered orally with VS4718 (10 mg / kg) on the basis of nitidine chloride (20 mg / kg, administered intraperitoneally once a day), and the administration was continued for 3 weeks. The control group was administered with the same dose of normal saline orally every day for 3 weeks. The formula for calculating the tumor volume was: volume = tumor length x tumor width 2 × 0.5.

[0058] Control Figure 5 The results showed that nitidine chloride and VS4718 had a synergistic effect on inhibiting the growth of esophageal squamous cell carcinoma cells in tumor-bearing mouse models.

[0059] According to the calculation, the Q value of the tumor growth inhibition effect mediated by nitidine chloride combined with VS4718 was 1.2.

[0060] Finally, it should be noted that the above preferred embodiments are merely intended to illustrate the technical solutions of the present application, not to limit the present application. Even though the present application has been described in detail by the above preferred embodiments, those skilled in the art should understand that various modifications can be made in form and details thereof without departing from the scope of the present application as defined by the appended claims.

Claims

1. A pharmaceutical composition comprising nifedipine chloride or a pharmaceutically acceptable salt thereof and a FAK inhibitor.

2. The pharmaceutical composition according to claim 1, characterized in that, The mass ratio of zeylan chloride or its pharmaceutically acceptable salt to FAK inhibitor is 1-15:

1.

3. The pharmaceutical composition according to claim 1, characterized in that, The FAK inhibitor is VS4718 or a pharmaceutically acceptable salt thereof.

4. The pharmaceutical composition according to claim 3, characterized in that, The mass ratio of alkaloid chloride or a pharmaceutically acceptable salt thereof to VS4718 or a pharmaceutically acceptable salt thereof, based on the mass ratio of alkaloid chloride to VS4718, is 1-15:

1.

5. A kit comprising nifedipine chloride or a pharmaceutically acceptable salt thereof and a FAK inhibitor.

6. The reagent kit according to claim 5, characterized in that, The FAK inhibitor is VS4718 or a pharmaceutically acceptable salt thereof.

7. Use of the pharmaceutical composition according to any one of claims 1-4 in the preparation of a medicament for use against esophageal squamous cell carcinoma.

8. The use of neem chloride or a pharmaceutically acceptable salt thereof in the preparation of a medicament for enhancing the efficacy of FAK inhibitors against esophageal squamous cell carcinoma.

9. The application according to claim 7 or 8, characterized in that, The anti-esophageal squamous cell carcinoma method includes inhibiting the growth of esophageal squamous cell carcinoma cells, inhibiting the invasion of esophageal squamous cell carcinoma cells, inhibiting the glucose metabolism of esophageal squamous cell carcinoma cell lines, and inhibiting the amino acid metabolism of esophageal squamous cell carcinoma cell lines.

10. The application according to claim 9, characterized in that, The esophageal squamous cell carcinoma cells include esophageal squamous cell carcinoma cells KYSE410, esophageal squamous cell carcinoma cells KYSE450, and esophageal squamous cell carcinoma cells KYSE510.

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