An anti-esophageal squamous cell carcinoma drug composition

Abstract

This invention provides a pharmaceutical composition for treating esophageal squamous cell carcinoma, comprising zafniform chloride and a FAK inhibitor such as VS4718. The pharmaceutical composition of this invention exhibits synergistic effects in inhibiting the growth and invasion of esophageal squamous cell carcinoma cells, and also synergistically inhibits the metabolism and growth of these cells. The combination of zafniform chloride and VS4718 achieves a synergistic anti-esophageal squamous cell carcinoma effect, which is beneficial for improving anti-tumor efficacy, reducing drug dosage, and enhancing safety.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical compositions, and more specifically, to a pharmaceutical composition for treating esophageal squamous cell carcinoma. Background Technology

[0002] Esophageal squamous cell carcinoma (ESCC) is a serious cancer that threatens human life worldwide, and it is also one of the most common and specific cancers in China. Despite continuous improvements in diagnostic and treatment methods, and significant improvements in the survival rate and quality of life for ESCC patients, the prognosis remains challenging. Exploring and discovering new diagnostic biomarkers and therapeutic drugs is crucial for targeting ESCC.

[0003] Tyrosine kinase signaling pathways are key signaling axes promoting signaling pathways in tumor cells (including ESCC), mediating various malignant phenotypes of ESCC through multiple downstream signaling steps. Among these, the signaling axis centered on focal adhesion kinase (FAK) is particularly crucial in inducing ESCC progression, serving as an important molecular hub. Several small-molecule inhibitors targeting FAK activity have been developed, but their antitumor effects require further investigation.

[0004] Small molecule monomeric compounds from traditional Chinese medicine (TCM) can inhibit multiple signaling pathways, their key targets, and downstream effector molecules, synergistically suppressing the development of malignant phenotypes in tumor progression and reducing the toxicity of anticancer drugs. Therefore, research on TCM monomeric compounds assisting tyrosine kinase-targeted drugs in the treatment of ESCC has become a hot topic in current tumor pharmacology research.

[0005] Nitidine chloride is a quaternary ammonium alkaloid extracted from the roots of the Rutaceae plant *Zanthoxylum nitidum*. It is an active alkaloid compound with anti-inflammatory and antioxidant biological effects, and its structure is as follows:

[0006]

[0007] Zanthoxylum chloride possesses a wide range of biological activities, playing important roles in anti-inflammation, analgesia, antibacterial activity, and immune system regulation. In recent years, there have also been reports that zanthoxylum chloride can inhibit various malignant phenotypes in solid tumor cell lines, such as growth, invasion, and metastasis, thus exhibiting an anticancer effect. Summary of the Invention

[0008] This invention has discovered that zeaxanthin chloride can enhance the anti-progression effect of FAK inhibitors on esophageal squamous cell carcinoma. When zeaxanthin chloride is used in combination with FAK inhibitors, the resulting drug composition can have a synergistic anti-esophageal squamous cell carcinoma effect.

[0009] In one aspect, the present invention provides a pharmaceutical composition comprising zeatin chloride or a pharmaceutically acceptable salt thereof and a FAK inhibitor.

[0010] In another aspect, the present invention also provides a kit comprising nifedipine chloride or a pharmaceutically acceptable salt thereof and a FAK inhibitor.

[0011] In one embodiment, the mass ratio of zebufenozide chloride or its pharmaceutically acceptable salt to the FAK inhibitor in the pharmaceutical composition or the kit is 1-15:1, preferably 1.5-10:1, more preferably 2-8:1, and most preferably 2:1 or 6-7:1.

[0012] In one embodiment, the FAK inhibitor is VS4718 or a pharmaceutically acceptable salt thereof.

[0013] In this invention, the CAS registry number of VS4718 is 1061353-68-1, and its chemical name is 2-[[2-(2-methoxy-4-morpholin-4-ylanilino)-5-(trifluoromethyl)pyridin-4-yl]

[0014] [amino]-N-methylbenzamide (2-[[2-(2-methoxy-4-morpholino-4-ylanilino)-5-(trifluoromethyl)pyridinyl-4-yl]amino]-N-methylbenzamide), its structural formula is:

[0015] .

[0016] Preferably, the mass ratio of cinnamon chloride or its pharmaceutically acceptable salt to VS4718 or its pharmaceutically acceptable salt, based on the mass ratio of cinnamon chloride to VS4718, is 1-15:1, preferably 1.5-10:1, more preferably 2-8:1, and most preferably 2:1, or 6-7:1.

[0017] In another aspect, the present invention also provides the use of the pharmaceutical composition of the present invention in the preparation of a medicament for use against esophageal squamous cell carcinoma.

[0018] In another aspect, the present invention also provides 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.

[0019] In this invention, the anti-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 glucose metabolism of esophageal squamous cell carcinoma cell lines, and inhibiting the amino acid metabolism of esophageal squamous cell carcinoma cell lines.

[0020] In one embodiment, 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, more preferably esophageal squamous cell carcinoma cells KYSE510.

[0021] In this invention, the term "pharmaceutically acceptable salt" includes, but is not limited to, pharmaceutically acceptable acid addition salts, such as salts of inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, metaphosphoric acid, nitric acid, and sulfuric acid, and salts of organic acids such as acetic acid, ethanesulfonic acid, benzenesulfonic acid, benzoic acid, citric acid, fumaric acid, tartaric acid, glycolic acid, hydroxyethanesulfonic acid, lactic acid, lactobionic acid, maleic acid, malic acid, mesylic acid, succinic acid, p-toluenesulfonic acid, and tartaric acid.

[0022] In one embodiment, the pharmaceutical composition or medicament of the present invention further comprises, as well as pharmaceutically acceptable excipients.

[0023] The pharmaceutically acceptable excipients include at least one of the following: carrier, excipient, diluent, lubricant, emulsifier, preservative, sweetener, suspension stabilizer, or flavoring.

[0024] The pharmaceutical compositions or drugs of the present invention can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, or via implantable reservoirs, preferably orally or by injection. The term parenterally as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-articular, intrasynovial, intrasternal, intrathecal, intrawound, and intracranial injection or infusion techniques.

[0025] According to the use of the present invention, the above-described pharmaceutical composition or medicine can be formulated into an oral dosage form. 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 enables the pharmaceutical composition or medicine to be formulated into tablets, capsules, powders, granules, crystals, solutions, suspensions, soups, syrups, elixirs, and chewable tablets, etc. Suitable carriers include excipients, such as fillers like sugars, including lactose, sucrose, mannose, and / or sorbitol; cellulose preparations, such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone (PVP). If desired, a disintegrant such as cross-linked polyvinylpyrrolidone, agar, or alginate or its salts, such as sodium alginate, can be added.

[0026] Alternatively, the pharmaceutical compositions or drugs of the present invention are preferably formulated into dosage forms suitable for inhalation or blowing, intranasal, transdermal or topical administration, said dosage forms being selected from nasal sprays, nasal drops, suspensions, gels, ointments, creams, emulsions and powders.

[0027] For inhalation administration, suitable propellants, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gases, can be used to conveniently administer the pharmaceutical composition or drug according to the invention as an aerosol via pressurized packaging or a nebulizer. When using pressurized gas, a valve can be provided to determine the dosage unit for administration in the prescribed amount. The formulation of the capsules and gelatin sheets within the inhalation device comprises a powder mixture of the compound and a suitable powder base, such as lactose or starch.

[0028] The nasal delivery system used in this invention can take various forms, including aqueous formulations, non-aqueous formulations, and combinations thereof. Aqueous formulations include, for example, hydrogels, aqueous suspensions, liposome aqueous dispersions, aqueous emulsions, aqueous microemulsions, and combinations thereof. Non-aqueous formulations include, for example, non-hydrogels, non-aqueous suspensions, liposome non-aqueous dispersions, non-aqueous emulsions, non-aqueous microemulsions, and combinations thereof. Various forms of nasal delivery systems may include buffers to maintain pH, pharmaceutically acceptable thickeners, and wetting agents. The pH of the buffer can be selected to optimize the absorption of the therapeutic agent through the nasal mucosa.

[0029] Injectable formulations (e.g., sterile injectable aqueous or oily suspensions) can be formulated using appropriate dispersants or wetting agents and suspending agents according to known techniques. Sterile injectable formulations can also be sterile injectable solutions, suspensions, or emulsions in non-toxic, parenteral-acceptable diluents or solvents, such as solutions in 1,3-butanediol. Additionally, sterile, non-volatile oils are commonly used as solvents or suspension media. For this purpose, any mild, non-volatile oil can be used, including synthetic monoglycerides or diglycerides.

[0030] Dosage forms for topical or transdermal administration of the pharmaceutical compositions or drugs of the present invention include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalers, or patches. Under sterile conditions, the active ingredient is mixed with a pharmaceutically acceptable carrier and any desired preservatives or buffer solutions. Ophthalmic preparations, ear drops, eye ointments, powders, and solutions are also included within the scope of the present invention.

[0031] The pharmaceutical composition or drug can be readily available in unit dose form and can be prepared by any method well known in the pharmaceutical field. These methods all involve combining the therapeutic agent with a carrier constituting one or more excipients. Typically, the composition is prepared by uniformly and tightly binding the therapeutic agent with a liquid carrier, a fine solid carrier, or both. The product is then shaped as needed.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] Beneficial effects:

[0037] 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:

[0039] 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.

[0040] 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.

[0041] Figure 3 This is a schematic diagram showing the results of the synergistic inhibition of glucose metabolism in esophageal squamous cell carcinoma cell lines by phenylephrine chloride and VS4718 in Example 3.

[0042] Figure 4 This is a schematic diagram showing the results of the synergistic inhibition of amino acid metabolism in esophageal squamous cell carcinoma cell lines by phenylephrine chloride and VS4718 in Example 4.

[0043] Figure 5 This is a schematic diagram showing the results of Example 5, where chlorhexidine chloride enhances the inhibition of esophageal squamous cell carcinoma cell growth in a tumor-bearing mouse model by VS4718.

[0044] In the above figures, This represents P < 0.01; This represents P < 0.001; This means P < 0.0001. Detailed Implementation

[0045] The following will describe preferred embodiments of the invention in detail. These embodiments are provided to better illustrate the invention and are not intended to limit the invention to these examples. Non-essential improvements and adjustments to the embodiments based on the invention's description still fall within the scope of the invention.

[0046] Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Where specific techniques or conditions are not specified in the embodiments, they should be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Detailed implementation method:

[0048] Example 1: Zanthoxylum nitidate chloride enhances the growth-inhibiting effect of VS4718 on esophageal squamous cell carcinoma cell lines.

[0049] 1. Cell Culture

[0050] The human esophageal squamous cell carcinoma line KYSE510 was placed in RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. The incubation conditions were 37 ℃ and 5% CO2.

[0051] 2. Cell growth capacity assay (MTS method)

[0052] The KYSE510 cell line (culture density 3.5 × 10⁶) was used. 3 After seeding (1 sample per well) into a 96-well plate, add 5 μM nitrate chloride, 0.5 μM VS4718, or a combination thereof. After 72 hours, prepare a 10% MTS solution with RPMI 1640 medium, add it to the 96-well plate, incubate for 1 hour, and measure the absorbance at 490 nm using a microplate reader.

[0053] 3. Cell growth capacity assay (soft agar colony formation assay)

[0054] After preparing the lower agarose layer in a 96-well plate, the plate was incubated at 4°C for 20 minutes. Then, an upper agarose layer containing the KYSE510 cell line was prepared. After completing the preparation of both agarose layers, a combination of 5 μM nitrile chloride and 0.5 μM VS4718 was added to the upper layer. After incubation for 10 days, the chromogenic working solution was added, and the plate was incubated at 37°C for 4 hours. The absorbance was measured at 450 nm using a microplate reader.

[0055] Comparison Appendix Figure 1 The results showed that nitrile chloride and VS4718 had a synergistic effect in inhibiting the growth of esophageal squamous cell carcinoma cell lines.

[0056] The Jin Zhengjun Q-value method was used to evaluate the synergistic effect of the combination of thorn chloride and VS4718 in inhibiting tumor growth. The Q-value was calculated using the following formula: Q = E a+b / (E a +E b -E a ×E b E a+b It is the inhibition rate of the drug combination, E a and E b Q represents the inhibition rate of drug therapy when administered alone. Q < 0.85 indicates antagonistic effect, 0.85 ≤ Q < 1.15 indicates additive effect, and Q ≥ 1.15 indicates synergistic effect.

[0057] Calculations showed that the Q value for the growth inhibition effect mediated by the combined use of chlorpyrifos chloride and VS4718 was 1.52.

[0058] The Q value for the non-anchoring-dependent growth inhibition effect mediated by the combination of chlorpyrifos and VS4718 was 1.56.

[0059] Example 2: Zanthoxylum nitidate chloride enhances the inhibitory effect of VS4718 on the invasion of esophageal squamous cell carcinoma cell lines.

[0060] Cell invasion ability detection:

[0061] The anti-ESCC cell invasion ability of VS4718 sensitized with zafyne chloride was observed using a Tranwell system (8 μm pore size). 100 µL of matrix gel was added to the upper chamber of the transom chamber and incubated at 37°C for 1 hour to allow the matrix gel to solidify. Prepared KYSE510 cell lines were seeded into the upper chamber of the transom chamber. 1 mL of RPMI 1640 medium containing 20% ​​fetal bovine serum was added to the lower chamber, along with zafyne chloride (5 μM), VS4718 (0.5 μM), or a combination thereof. The transom chamber was then incubated for 24 hours. After incubation, uninvaded KYSE510 cells in the upper chamber were wiped clean with a cotton swab. Invaded KYSE510 cells in the lower chamber were separated using a dissociation solution. The separated cells were stained with dye, and the invasion rate was calculated using a microplate reader.

[0062] Comparison Appendix Figure 2 The results showed that nitrile chloride and VS4718 had a synergistic effect in inhibiting the invasion of esophageal squamous cell carcinoma cell lines.

[0063] Calculations showed that the Q value for the invasion inhibition effect mediated by the combined use of chlorpyrifos chloride and VS4718 was 1.43.

[0064] Example 3: Zanthoxylum nitidate chloride enhances the inhibitory effect of VS4718 on glucose metabolism in esophageal squamous cell carcinoma cell lines.

[0065] KYSE510 cell lines were seeded into 6-well plates. After cell adhesion, 5 μM nitrate chloride, 0.5 μM VS4718, or a combination thereof were added. After 24 hours, the conditioned medium was collected. Using a lactate assay kit (Nanjing Jiancheng Bioengineering Institute Co., Ltd., catalog number: A019-3-1), the conditioned medium under different treatments was incubated with different reagents according to the reagent instructions, and the absorbance was measured using a microplate reader to determine the lactate content. Similarly, using a glucose assay kit (Beyotime Biotechnology Research Institute Co., Ltd., catalog number: S0201S), the conditioned medium under different treatments was incubated with different reagents according to the reagent instructions, and the absorbance was measured using a microplate reader to determine the glucose content.

[0066] Comparison Appendix Figure 3 The results showed that nitrile chloride and VS4718 had a synergistic effect in inhibiting glucose metabolism in esophageal squamous cell carcinoma cell lines.

[0067] Calculations showed that the Q value for lactate inhibition mediated by the combined use of phenylephrine chloride and VS4718 was 1.28. The Q value for glucose inhibition mediated by the combined use of phenylephrine chloride and VS4718 was 1.3.

[0068] Example 4: Enhancement of amino acid metabolism inhibition of VS4718 by nephrite chloride in esophageal squamous cell carcinoma cell lines

[0069] KYSE510 cell lines were seeded in 6-well plates. After cell attachment, 5 μM nitrate chloride, 0.5 μM VS4718, or a combination thereof were added. After 24 hours, cell lysates were collected. Using a glutamine assay kit (Abcam, catalog number: ab197011), cell lysates under different treatment conditions were incubated with different reagents according to the kit instructions. The absorbance was then measured using a microplate reader to determine the glutamine content in the cells.

[0070] Comparison Appendix Figure 4 The results showed that nitrile chloride and VS4718 had a synergistic effect in inhibiting amino acid metabolism in esophageal squamous cell carcinoma cell lines.

[0071] Calculations showed that the Q value for the amino acid inhibition effect mediated by the combined use of chlorhexidine chloride and VS4718 was 1.52.

[0072] Example 5: Zanthoxylum nitidate chloride enhances the anti-tumor growth effect of VS4718 on esophageal squamous cell carcinoma cell line KYSE510 tumor-bearing mice.

[0073] KYSE510 cells were injected subcutaneously into the upper limb of nude mice (4-week-old female BALB / c-nu nude mice (average weight 14-15 grams), purchased from Vital River Pharmaceuticals), until the tumor grew to approximately 100 mm. 3 Afterwards, KYSE510 tumor-bearing mice were randomly divided into four groups: a control group, a styrax chloride group, a VS4718 group, and a styrax chloride plus VS4718 group. The styrax chloride group (20 mg / kg) received intraperitoneal injection once daily, the VS4718 group (10 mg / kg) received gavage once daily, and the styrax chloride plus VS4718 group received VS4718 (10 mg / kg, once daily) by gavage in addition to styrax chloride (20 mg / kg, intraperitoneal injection, once daily), for a total of 3 weeks. The control group received the same volume of physiological saline by gavage daily for 3 weeks. The tumor volume was calculated using the formula: Volume = Tumor long diameter × Tumor transverse diameter. 2 ×0.5.

[0074] Comparison Appendix Figure 5 The results showed that acetamiprid chloride and VS4718 had a synergistic effect in inhibiting the growth of esophageal squamous cell carcinoma cells in a tumor-bearing mouse model.

[0075] Calculations showed that the Q value for the tumor growth inhibition effect mediated by the combination of styrax chloride and VS4718 was 1.2.

[0076] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not intended 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 to it in form and detail without departing from the scope defined by the claims of the present invention.

Claims

1. A pharmaceutical composition comprising nitidine chloride or a pharmaceutically acceptable salt thereof and a FAK inhibitor, the FAK inhibitor being VS4718 or a pharmaceutically acceptable salt thereof, the mass ratio of nitidine chloride or a pharmaceutically acceptable salt thereof to VS4718 or a pharmaceutically acceptable salt thereof being 1-15:1, based on the mass ratio of nitidine chloride to VS4718. 2.A kit comprising nitidine chloride or a pharmaceutically acceptable salt thereof and a FAK inhibitor, the FAK inhibitor being VS4718 or a pharmaceutically acceptable salt thereof, the mass ratio of nitidine chloride or a pharmaceutically acceptable salt thereof to VS4718 or a pharmaceutically acceptable salt thereof being 1-15:1, based on the mass ratio of nitidine chloride to VS4718. 3.The use of the pharmaceutical composition according to claim 1 in the preparation of a medicament for resisting esophageal squamous cell carcinoma.

4. Use according to claim 3, characterized in that, The resisting esophageal squamous cell carcinoma comprises 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 lines, and inhibiting the amino acid metabolism of esophageal squamous cell lines.

5. Use according to claim 4, characterized in that, The esophageal squamous cell carcinoma cells comprise esophageal squamous cell KYSE410, esophageal squamous cell KYSE450, and esophageal squamous cell KYSE510.

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