Application of Ginkgo biloba extract enhancing VS4718 or ponatinib in the progression of esophageal squamous cell carcinoma

Abstract

This invention provides the application of ginkgo biloba extract enhancing VS4718 or ponatinib in the anti-progression of esophageal squamous cell carcinoma. This invention discovers that ginkgo biloba extract can enhance the inhibitory effects of VS4718 or ponatinib on the growth and invasion of esophageal squamous cell carcinoma cells; ginkgo biloba extract sensitizes the inhibitory effect of VS4718 or ponatinib on the glycolipid metabolism of tumor cells; and the combined use of ginkgo biloba extract with VS4718 or ponatinib produces a synergistic anti-cancer effect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of pharmaceutical compositions, in particular, the present application relates to the application of ginkgetin in enhancing the anti-ESCC effect of VS4718 or ponatinib. BACKGROUND

[0002] Esophageal squamous cell carcinoma (ESCC) is a serious solid tumor that endangers human health worldwide, and is one of the cancer types with high incidence in China. With the improvement of diagnosis and treatment level, the survival rate and survival status of ESCC patients have been improved, but the prognosis is still not ideal. It is crucial to find new diagnostic markers and treatment strategies in targeted ESCC.

[0003] Tyrosine kinases can regulate various malignant phenotypes of ESCC by mediating the transmission of signal pathways. Among them, the focal adhesion kinase (FAK) / Src signaling axis is an important signaling pathway that promotes the progression of ESCC. Several small molecule inhibitors targeting the FAK / Src axis have been developed, but their anticancer effects still need further study.

[0004] Traditional Chinese medicine small molecule monomer compounds can inhibit the progression of tumor cells through multiple pathways and multiple targets, while reducing drug toxicity. Therefore, the research on traditional Chinese medicine assisted targeted drugs for anti-ESCC has become the focus of current oncology research.

[0005] Ginkgetin is a biflavone from Ginkgo biloba, which is a flavonoid compound with anti-inflammatory, antioxidant and other biological effects. Its structure is as follows:

[0006]

[0007] Ginkgetin has a wide range of biological activities, such as anti-inflammatory, antioxidant, prevention of cardiovascular and cerebrovascular diseases, etc. In recent years, it has also been reported that ginkgetin can inhibit various malignant phenotypes of solid tumor cell lines, such as growth, invasion and metastasis, and has anticancer effect. However, there is no research on the enhancement of ginkgetin on the anticancer effect of FAK inhibitors and Src family kinase inhibitors. SUMMARY

[0008] The present application found that ginkgetin has the effect of enhancing the anti-ESCC effect of FAK inhibitors and Src family kinase inhibitors. When ginkgetin is used in combination with FAK inhibitors or Src family kinase inhibitors, the drug combination can have a synergistic anticancer effect.

[0009] In a first aspect, the present application provides a pharmaceutical composition comprising ginkgetin or a pharmaceutically acceptable salt thereof and a FAK inhibitor or a Src family kinase inhibitor.

[0010] In a second aspect, the present application also provides a kit comprising ginkgetin or a pharmaceutically acceptable salt thereof and a FAK inhibitor or a Src family kinase inhibitor.

[0011] Preferably, the molar ratio of ginkgetin or a pharmaceutically acceptable salt thereof to the FAK inhibitor or the Src family kinase inhibitor is 1-30:1, preferably 3-8:1, more preferably 5:1.

[0012] Preferably, the FAK inhibitor comprises VS4718 or a pharmaceutically acceptable salt thereof; and the Src family kinase inhibitor comprises ponatinib or a pharmaceutically acceptable salt thereof.

[0013] In the present application, the CAS registry number of 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; and the CAS registry number of ponatinib is 943319-70-8, and the chemical name is 3-(2-imidazo[1,2-b]pyridazin-3-ylethynyl)-4-methyl-N-[4-[(4-methyl-1-piperazinyl)methyl]-3-(trifluoromethyl)phenyl]benzamide.

[0014] Preferably, the molar ratio of ginkgetin or a pharmaceutically acceptable salt thereof to VS4718 or a pharmaceutically acceptable salt thereof is 0.5-15:1, preferably 1-6:1, more preferably 2:1; and the molar ratio of ginkgetin or a pharmaceutically acceptable salt thereof to ponatinib or a pharmaceutically acceptable salt thereof is 0.5-15:1, preferably 1-6:1, more preferably 2:1.

[0015] In a third aspect, the present application also provides use of the pharmaceutical composition in the preparation of a drug for resisting progression of esophageal squamous cell carcinoma.

[0016] In a fourth aspect, the present invention provides the use of ginkgo biloba flavonoids or pharmaceutically acceptable salts thereof in enhancing the progression of esophageal squamous cell carcinoma against FAK inhibitors or Src family kinase inhibitors.

[0017] In a fifth aspect, the present invention also provides the use of ginkgo biloba flavonoids or pharmaceutically acceptable salts thereof in the preparation of a medicament for enhancing the anti-progression effect of FAK inhibitors or Src family kinase inhibitors on esophageal squamous cell carcinoma.

[0018] In this invention, the anti-esophageal squamous cell carcinoma progression includes inhibiting the growth of esophageal squamous cell carcinoma cells, inhibiting the invasion of metastatic esophageal squamous cell carcinoma cells, inhibiting the glucose metabolism of esophageal squamous cell carcinoma cell lines, and inhibiting the fatty acid metabolism of esophageal squamous cell carcinoma cell lines.

[0019] In a preferred embodiment, the esophageal squamous cell carcinoma cells include esophageal squamous cell carcinoma cells KYSE410, esophageal squamous cell carcinoma cell line KYSE450, and esophageal squamous cell carcinoma cells KYSE510, more preferably esophageal squamous cell carcinoma cells KYSE450.

[0020] In this invention, the term "pharmaceutically acceptable salt" refers to a salt that retains the bioavailability and properties of a given compound and is not biologically or otherwise undesirable. Pharmaceutically acceptable salts include pharmaceutically acceptable base addition salts and pharmaceutically acceptable acid addition salts. Pharmaceutically acceptable base addition salts can be prepared from inorganic and organic bases. Salts derived from inorganic bases include (by way of example only) sodium, potassium, lithium, ammonium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, such as alkylamines, dialkylamines, trialkylamines, substituted alkylamines, di(substituted alkyl)amines, tri(substituted alkyl)amines, alkenylamines, dienylamines, trienylamines, substituted alkenylamines, di(substituted alkenyl)amines, tri(substituted alkenyl)amines, monocyclic alkylamines, dicyclic alkylamines or tricyclic alkylamines, monoarylamines, diarylamines or triarylamines or mixed amines, etc. Specific examples of suitable amines include (by way of example only) isopropylamine, trimethylamine, diethylamine, tri(isopropyl)amine, tri(n-propyl)amine, ethanolamine, 2-dimethylaminoethanol, piperazine, piperidine, morpholine, N-ethylpiperidine, etc. Pharmaceutically acceptable acid addition salts can be prepared from inorganic and organic acids. Inorganic acids from which salts can be derived include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc. Organic acids from which salts can be derived include acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, etc.

[0021] In one embodiment, the medicament or pharmaceutical composition of the present invention further comprises a pharmaceutically acceptable carrier or excipient.

[0022] The term "pharmaceutically acceptable carrier or excipient" refers to a non-toxic, inert solid, semi-solid, or liquid filler, diluent, capsule-forming material, or any type of pharmaceutical adjuvant. Some examples of materials that can serve as pharmaceutically acceptable carriers are sugars, such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth gum; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; ethylene glycols, such as propylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffers, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol and phosphate buffers; and other non-toxic, compatible lubricants, such as sodium dodecyl sulfate and magnesium stearate, as well as colorants, release agents, coating agents, sweeteners, flavorings, and aromatizers, etc.

[0023] 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.

[0024] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is mixed with at least one inert, pharmaceutically acceptable excipient or carrier (e.g., sodium citrate or calcium hydrogen phosphate) and / or: a) a filler or swelling agent, such as starch, lactose, sucrose, glucose, mannitol, and silicate; b) a binder, such as carboxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and gum arabic; c) a wetting agent, such as glycerin; d) a disintegrant, such as agar, calcium carbonate, potato or cassava starch, alginate, certain silicates, and sodium carbonate; e) a solution retardant, such as paraffin; f) an absorption enhancer, such as quaternary ammonium compounds; g) a wetting agent, such as cetyl alcohol and glyceryl monostearate; h) an absorbent, such as clay and bentonite; and i) a lubricant, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium dodecyl sulfate, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may also contain buffers.

[0025] Oral liquid dosage forms include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active compound, the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizers, and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butanediol, dimethylformamide; fatty acid esters of oils (particularly cottonseed, peanut, corn, germ, olive, castor, and sesame oils), glycerin, tetrahydrofurfuryl alcohol, polyethylene glycol, and sorbitol, and mixtures thereof. Besides inert diluents, oral compositions may also include adjuvants such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, and aromatizers.

[0026] 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.

[0027] 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.

[0028] In one embodiment, ginkgo biloba extract or its pharmaceutically acceptable salt in the pharmaceutical composition, kit, or medicament of the present invention, along with a FAK inhibitor or a Src family kinase inhibitor, can be administered in the same or different pharmaceutical formulations. The dosage forms of ginkgo biloba extract or its pharmaceutically acceptable salt, and the FAK inhibitor or Src family kinase inhibitor, can be the same or different. Ginkgo biloba extract or its pharmaceutically acceptable salt, along with the FAK inhibitor or Src family kinase inhibitor, can be administered simultaneously or sequentially.

[0029] In the pharmaceutical uses described herein, the timing, frequency, and duration of administration of ginkgo biloba extract or its pharmaceutically acceptable salts and FAK inhibitors or Src family kinase inhibitors, etc., 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.

[0030] For example, when a treatment regimen for mice or rats is applied to humans, the effective dose of all drugs for humans can be converted to the effective dose of the same drug for mice or rats, which is easily achievable by those skilled in the art.

[0031] The pharmaceutical 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, preparing a sugar coating, grinding, emulsifying, forming capsules, embedding, or lyophilizing processes.

[0032] Beneficial effects:

[0033] This invention provides the application of ginkgo biloba extract in enhancing the effects of VS4718 and ponatinib on the progression of esophageal squamous cell carcinoma (ESCC). Evaluation using the MTS assay and soft agar colony formation assay showed that ginkgo biloba extract enhanced the growth inhibition of ESCC cell line KYSE450 mediated by VS4718 and ponatinib. Evaluation using the Transwell assay showed that ginkgo biloba extract enhanced the inhibitory effect of VS4718 and ponatinib on the invasion of ESCC cells. Ginkgo biloba extract significantly enhanced the metabolism of VS4718 and ponatinib in the ESCC cell line KYSE450, including reducing the levels of lactate, glucose, and fatty acids in ESCC cells. The combined use of ginkgo biloba extract with VS4718 and ponatinib achieved a synergistic anti-ESCC effect, which is beneficial for improving anti-tumor efficacy and thus enhancing the anti-cancer activity of FAK inhibitors and Src family kinase inhibitors. Attached Figure Description Attached image description:

[0035] Figure 1 This is a schematic diagram illustrating the results of Ginkgo biloba flavonoids in Example 1 enhancing the inhibition of esophageal squamous cell carcinoma cell lines by VS4718 or ponatinib.

[0036] Figure 2 This is a schematic diagram illustrating the effect of Ginkgo biloba flavonoids in Example 2 on enhancing the inhibition of esophageal squamous cell carcinoma cell line invasion by VS4718 or ponatinib.

[0037] Figure 3 This is a schematic diagram illustrating the results of Ginkgo biloba extract enhancing the inhibition of glucose metabolism in VS4718 or ponatinib in esophageal squamous cell carcinoma cell lines in Example 3.

[0038] Figure 4 This is a schematic diagram illustrating the results of Ginkgo biloba extract enhancing fatty acid metabolism in VS4718 or ponatinib in Example 4.

[0039] In the above figures, *** represents P<0.001. Detailed Implementation

[0040] 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.

[0041] 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:

[0043] Example 1: Ginkgo biloba extract enhances the growth-inhibiting effect of VS4718 or ponatinib on esophageal squamous cell carcinoma cell lines.

[0044] 1. Cell Culture

[0045] The human esophageal squamous cell carcinoma line KYSE450 was placed in RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. The incubator conditions were 37°C and 5% CO2.

[0046] 2. Cell growth capacity assay (MTS method)

[0047] The KYSE450 cell line (culture density 3×10⁶) was used. 3 After seeding (number of cells / well) into 96-well plates, add Ginkgo biloba extract (1 μM), VS4718 (0.5 μM), ponatinib (0.5 μM), or a combination thereof. After 72 hours, prepare 10% MTS solution with RPMI 1640 medium, add to the 96-well plates, incubate for 2 hours, and measure the absorbance at 490 nm using a microplate reader.

[0048] 3. Cell growth capacity assay (soft agar colony formation assay)

[0049] 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 KYSE450 cell line was prepared. After completing the preparation of both the upper and lower agarose layers, a combination of ginkgo biloba extract (1 μM) and either VS4718 (0.5 μM) or ponatinib (0.5 μM) was added to the upper layer. After incubation for 8 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.

[0050] Comparison Appendix Figure 1 The results showed that Ginkgo biloba flavonoids could enhance the inhibition of esophageal squamous cell carcinoma cell lines by VS4718 or ponatinib.

[0051] The Jin Zhengjun Q-value method was used to evaluate the synergistic effect of the combination of Ginkgo biloba extract and VS4718 or ponatinib 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.

[0052] Calculations showed that the Q value for the growth inhibition effect mediated by Ginkgo biloba extract combined with VS4718 was 1.25; and the Q value for the growth inhibition effect mediated by Ginkgo biloba extract combined with ponatinib was 1.22.

[0053] The Q value for the non-fixation-dependent growth inhibition effect mediated by Ginkgo biloba extract combined with VS4718 was 1.31; the Q value for the non-fixation-dependent growth inhibition effect mediated by Ginkgo biloba extract combined with ponatinib was 1.25.

[0054] Example 2: Ginkgo biloba extract enhances the inhibitory effect of VS4718 or ponatinib on the invasion of esophageal squamous cell carcinoma cell lines.

[0055] Cell invasion ability detection:

[0056] The anti-ESCC cell invasion ability of VS4718 cells sensitized by Ginkgo biloba extract and ponatinib was observed using a Tranwell system (8 μm pore size). 100 μL of matrix gel was added to the upper chamber of the transposition chamber and incubated at 37°C for 1 hour to allow the matrix gel to solidify. Prepared KYSE450 cell lines were seeded into the upper chamber of the transposition chamber. 800 μL of RPMI 1640 medium containing 20% ​​fetal bovine serum was added to the lower chamber, along with Ginkgo biloba extract (1 μM), VS4718 (0.5 μM), ponatinib (0.5 μM), or a combination thereof. The transposition chamber was then incubated for 24 hours. After removal, the uninvaded KYSE450 cells in the upper chamber were wiped clean with cotton swabs. Invaded KYSE450 cells in the lower chamber were separated using dissociation buffer. The separated cells were stained with dye, and the invasion rate was calculated using a microplate reader.

[0057] Comparison Appendix Figure 2 The results showed that Ginkgo biloba flavonoids could enhance the inhibition of esophageal squamous cell carcinoma cell lines by VS4718 or ponatinib.

[0058] Calculations showed that the Q value for the invasion inhibition effect mediated by Ginkgo biloba extract combined with VS4718 was 1.3; and the Q value for the invasion inhibition effect mediated by Ginkgo biloba extract combined with ponatinib was 1.27.

[0059] Example 3: Ginkgo biloba extract enhances the inhibitory effect of VS4718 or ponatinib on glucose metabolism in esophageal squamous cell carcinoma cell lines.

[0060] KYSE450 cell lines were seeded in 6-well plates. After cell adhesion, ginkgo biloba extract (1 μM), VS4718 (0.5 μM), ponatinib (0.5 μM), or combinations thereof were added. After 24 hours, the conditioned medium was collected. Using a lactate assay kit (Nanjing Jiancheng Bioengineering Research 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.

[0061] Comparison Appendix Figure 3 The results showed that Ginkgo biloba flavonoids could enhance the inhibition of glucose metabolism in esophageal squamous cell carcinoma cell lines by VS4718 or ponatinib.

[0062] Calculations showed that the Q value for lactate inhibition mediated by Ginkgo biloba extract combined with VS4718 was 1.32; the Q value for lactate inhibition mediated by Ginkgo biloba extract combined with ponatinib was 1.34. The Q value for glucose inhibition mediated by Ginkgo biloba extract combined with VS4718 was 1.31; the Q value for glucose inhibition mediated by Ginkgo biloba extract combined with ponatinib was 1.36.

[0063] Example 4: Ginkgo biloba extract enhances the inhibitory effect of VS4718 or ponatinib on fatty acid metabolism in esophageal squamous cell carcinoma cell lines.

[0064] KYSE450 cell lines were seeded in 6-well plates. After cell attachment, ginkgo biloba extract (1 μM), VS4718 (0.5 μM), ponatinib (0.5 μM), or a combination thereof were added. After 24 hours, the conditioned medium was collected. Using a fatty acid assay kit (Nanjing Jiancheng Bioengineering Research Institute Co., Ltd., catalog number: A042-2-1), the conditioned medium under different treatments was incubated with different reagents according to the reagent instructions. The absorbance was then measured using a microplate reader to determine the fatty acid content in the conditioned medium.

[0065] Comparison Appendix Figure 4The results showed that Ginkgo biloba flavonoids could enhance the inhibition of fatty acid metabolism in esophageal squamous cell carcinoma cell lines by VS4718 or ponatinib.

[0066] Calculations showed that the Q value for fatty acid inhibition mediated by VS4718 in combination with ginkgo biloba extract was 1.26; and the Q value for fatty acid inhibition mediated by ponatinib in combination with ginkgo biloba extract was 1.22.

[0067] 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 ginkgetin or a pharmaceutically acceptable salt thereof and ponatinib or a pharmaceutically acceptable salt thereof, the molar ratio of ginkgetin or a pharmaceutically acceptable salt thereof to ponatinib or a pharmaceutically acceptable salt thereof being 2:

1. 2.A kit comprising ginkgetin or a pharmaceutically acceptable salt thereof and ponatinib or a pharmaceutically acceptable salt thereof, the molar ratio of ginkgetin or a pharmaceutically acceptable salt thereof to ponatinib or a pharmaceutically acceptable salt thereof being 2:

1. 3.The use of the pharmaceutical composition according to claim 1 in the preparation of a drug for resisting the progression of esophageal squamous cell carcinoma.

4. Use according to claim 3, characterized in that, The resisting the progression of esophageal squamous cell carcinoma comprises inhibiting the growth of esophageal squamous cell carcinoma cells, inhibiting the invasion of metastatic esophageal squamous cell carcinoma cells, inhibiting the sugar metabolism of esophageal squamous cell carcinoma cell lines, and inhibiting the fatty acid metabolism of esophageal squamous cell carcinoma cell lines.

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

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