An anti-esophageal squamous cell carcinoma pharmaceutical composition containing tagetesin and its application

Abstract

The present invention provides an anti-esophageal squamous cell carcinoma pharmaceutical composition comprising tagetal glycoside and its application. In the present invention, tagetal glycoside and cisplatin have a synergistic anti-cancer effect. The pharmaceutical composition of tagetal glycoside and cisplatin has an enhanced anti-esophageal squamous cell carcinoma effect and can effectively prevent and treat esophageal squamous cell carcinoma.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to an anti-esophageal squamous cell carcinoma pharmaceutical composition comprising marigold glycoside and an application thereof. Background Art

[0002] Esophageal cancer is a common malignant tumor and a digestive system disease. The pathological types of esophageal cancer are mainly divided into esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (Adenocarcinoma of Esophagus), and the most common type in my country is squamous cell carcinoma. At present, the main treatments for esophageal squamous cell carcinoma include surgical resection, radiotherapy and chemotherapy. However, esophageal squamous cell carcinoma has the characteristics of invasive growth, and patients often develop resistance to long-term chemotherapy and radiotherapy drugs. At the same time, some patients have difficulty tolerating the adverse reactions and toxic side effects of chemotherapy and radiotherapy due to their poor physical condition. Therefore, the prognosis of esophageal squamous cell carcinoma is usually poor, and the patient's survival time is short.

[0003] Platinum compounds are the first-line treatment for esophageal squamous cell carcinoma, but they are prone to drug resistance and tumor recurrence after treatment. Several studies have reported on the use of small molecule monomer compounds to enhance the sensitivity of anticancer drugs, including targeted drugs, chemotherapeutic drugs, and immunotherapy. Quercetagitrin is obtained from African marigolds and has the following structural formula:

[0004]

[0005] Tagetes glycosides possess anti-inflammatory and antioxidant properties and are widely used in cardiovascular and cerebrovascular diseases, as well as other diseases associated with inflammation and oxidation. In recent years, there have also been reports that tagetes glycosides may have anti-cancer effects by inhibiting tumor cell growth. However, whether tagetes glycosides enhance the anti-cancer effects of cisplatin remains to be further investigated. Summary of the Invention

[0006] The present invention found that in the treatment of esophageal squamous cell carcinoma, marigold glycoside can significantly enhance the anti-cancer effect of cisplatin, achieving a synergistic anti-cancer effect.

[0007] To this end, the present invention provides a use of marigold glycoside or a pharmaceutically acceptable salt, prodrug, or metabolite thereof in enhancing the anti-esophageal squamous cell carcinoma effect of cisplatin.

[0008] The present invention also provides a use of marigold glycoside or a pharmaceutically acceptable salt, prodrug, or metabolite thereof in the preparation of a drug that enhances the anti-esophageal squamous cell carcinoma effect of cisplatin.

[0009] In another aspect, the present invention provides a pharmaceutical composition comprising tagetal glycoside or a pharmaceutically acceptable salt, prodrug, metabolite thereof and cisplatin.

[0010] The present invention also provides a kit comprising tagetal glycoside or a pharmaceutically acceptable salt, prodrug, metabolite thereof and cisplatin.

[0011] In one embodiment, the ratio of tagetal glycoside or a pharmaceutically acceptable salt, prodrug, or metabolite thereof to cisplatin is 0.8-60:1, based on the mass of tagetal glycoside and cisplatin.

[0012] Preferably, the ratio of marigold glycoside or its pharmaceutically acceptable salt, prodrug, metabolite to cisplatin, based on the mass of marigold glycoside and cisplatin, is 0.8:1, 2:1, 4.1:1, 8.2:1, 10:1, 20:1, 30:1, 35:1, 40:1, 50:1, 60:1, preferably, it is 2-40:1, or 4.1-35:1, most preferably, it is 4.1-8.2:1, or 35:1.

[0013] In another aspect, the present invention also provides use of the pharmaceutical composition in preparing drugs for treating esophageal squamous cell carcinoma.

[0014] In the present invention, the anti-esophageal squamous cell carcinoma agent includes at least one of inhibiting the growth of esophageal squamous cell carcinoma cells, inhibiting the invasion of esophageal squamous cell carcinoma cells, and inducing apoptosis of esophageal squamous cell carcinoma cells.

[0015] Also preferably, 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 KYSE450.

[0016] In the present invention, the pharmaceutically acceptable salt of tagetal glycoside includes a salt formed by tagetal glycoside and a pharmaceutically acceptable acid or base.

[0017] In the present invention, the prodrug refers to a derivative that can directly or indirectly provide tagetin after being administered to a patient, such as an ester of tagetin.

[0018] In the present invention, the metabolite refers to a pharmaceutically acceptable metabolic derivative of tagetal glycoside or a pharmaceutically acceptable salt thereof.

[0019] Preferably, the medicine or pharmaceutical composition of the present invention further comprises a pharmaceutically acceptable adjuvant.

[0020] Preferably, the tagetal glycoside or its pharmaceutically acceptable salt, prodrug, metabolite and cisplatin in the pharmaceutical composition of the present invention can be administered in the same or different pharmaceutical formulations. The pharmaceutical dosage forms of tagetal glycoside or its pharmaceutically acceptable salt, prodrug, metabolite and cisplatin can be the same or different. Tadal glycoside or its pharmaceutically acceptable salt, prodrug, metabolite and cisplatin can be administered simultaneously or sequentially.

[0021] 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 cisplatin, etc., 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.

[0022] 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.

[0023] As pharmaceutically acceptable adjuvants, any adjuvant known to be suitable for preparing a particular medicament 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, binding agents, thickeners, complexing agents, disintegrants, buffers, penetration enhancers, polymerizing agents, lubricants, coating agents, propellants, tonicity regulators, surfactants, colorants, flavorings, sweeteners, and dyes. In particular, adjuvants of a type suitable for the desired formulation and desired route of administration are used.

[0024] 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.

[0025] 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.

[0026] The medicaments or pharmaceutical compositions 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.

[0027] In the medicine or pharmaceutical composition of the present invention, the amount of marigold glycoside or its pharmaceutically acceptable salt, prodrug, metabolite 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%.

[0028] 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, one skilled in the art can readily determine the appropriate dosage formulation and method of administering the agent. For example, for adult patients, the pharmaceutical composition of the present invention can be administered orally or parenterally in a daily dosage of 0.001 mg to 500 mg, once a day or divided into several doses.

[0029] Beneficial effects:

[0030] The present invention provides a pharmaceutical composition for treating esophageal squamous cell carcinoma containing tagetagelin and its use. Experimental results show that tagetagelin and cisplatin have a synergistic anticancer effect. Tagetagelin can enhance cisplatin's inhibitory effect on the growth and invasion of esophageal squamous cell carcinoma cells, enhance cisplatin's effect in promoting apoptosis of esophageal squamous cell carcinoma cells, and strengthen cisplatin's anti-tumor growth effect and anti-tumor invasion-promoting effect in esophageal squamous cell carcinoma-bearing mice. Therefore, the pharmaceutical composition of tagetagelin and cisplatin has enhanced anti-esophageal squamous cell carcinoma activity and can effectively prevent and treat esophageal squamous cell carcinoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a schematic diagram showing the results that marigold glycosides in Example 1 can enhance the growth inhibition of esophageal squamous cell carcinoma cell lines by cisplatin.

[0032] Figure 2 This is a schematic diagram showing the results that marigold glycosides in Example 2 can enhance the effect of cisplatin on the invasion of esophageal squamous cell carcinoma cell lines.

[0033] Figure 3 This is a schematic diagram of the results of Example 3 in which marigold glycoside doses enhance cisplatin-induced apoptosis in multiple esophageal squamous cell carcinoma cell lines and upregulation of cleaved PARP expression.

[0034] Figure 4 This is a schematic diagram of the results of Example 4 showing that marigold glycoside enhances the effect of cisplatin in inhibiting the growth of KYSE450 tumors and the expression of Ki67, CD31 and LYVE1 in a xenogeneic tumor-bearing mouse model.

[0035] Figure 5 This is a schematic diagram of the results of Example 5 in which marigold glycoside enhances cisplatin's ability to inhibit the lymphatic metastasis of KYSE450 tumors in a xenogeneic tumor-bearing mouse model.

[0036] In the above figures, * represents P < 0.05; ** represents P < 0.01; *** represents P < 0.001. DETAILED DESCRIPTION

[0037] The following describes a preferred embodiment of the invention in detail. The examples are provided to better illustrate the invention and are not intended to limit the invention to the examples. Non-essential improvements and adjustments to the embodiments based on the invention remain within the scope of the invention.

[0038] 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 were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions.

[0039] Example 1: Tagetes erecta enhances the growth inhibitory effect of cisplatin on esophageal squamous cell carcinoma cell lines

[0040] 1. Cell Culture

[0041] Human esophageal squamous cell carcinoma cell line KYSE450 was cultured in RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin in an incubator at 37°C with 5% CO2.

[0042] 2. Cell Growth Ability Determination

[0043] The KYSE450 cell line was inoculated into a 96-well plate. After the cells adhered to the wall, different concentrations of cisplatin were added to the cisplatin group. In the embodiment, different concentrations of cisplatin and marigold glycoside (25 μM) were added. After 72 hours, 10% MTS solution was prepared with RPMI 1640 culture medium. The cultured cells were taken out, the upper culture medium was discarded, and the prepared MTS was added and incubated for 2 hours. The absorbance value was measured at 490 nm using a microplate reader. The growth inhibition rate of different concentrations of cisplatin was compared with that of the control group and input into GraphPadPrism software, and IC was analyzed using the "Dose-response inhibition" model. 50 The fitting value.

[0044] Compare with Figure 1 , the results showed that marigold glycosides could enhance the growth inhibition of esophageal squamous cell carcinoma cell lines by cisplatin.

[0045] Example 2: Tagetes erecta enhances the inhibitory effect of cisplatin on the invasion of esophageal squamous cell carcinoma cell lines

[0046] Cell invasion ability assay

[0047] The KYSE450 cell line was cultured in serum-free medium for 24 hours. Using a transwell chamber with an 8 μm pore size, 100 μL of matrigel was added to the upper chamber of the chamber and incubated at 37°C for 1 hour to allow the matrigel to solidify. The prepared KYSE450 cell line was inoculated into 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. Cisplatin (10 μM), marigold glycoside (25 μM) and cisplatin (10 μM) were added. The transwell chamber 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. The invading cells in the lower chamber were separated by dissociation solution and cell dye was added. The invasion cell rate was calculated by microplate reader.

[0048] Compare with Figure 2 , the results showed that marigold glycosides could enhance the cisplatin-inhibitory effect on the invasion of esophageal squamous cell carcinoma cell lines.

[0049] Example 3: Tagetes erecta enhances the apoptosis-promoting effect of cisplatin on esophageal squamous cell carcinoma cell lines

[0050] 1. Determination of Cell Apoptosis Index

[0051] The KYSE450 cell line was inoculated in RPMI 1640 medium containing 10% fetal bovine serum. After the cells adhered, marigold glycoside (25 μM) and cisplatin (5 μM) were added alone or in combination. After incubation for 24 hours, the supernatant was collected by centrifugation. The supernatant was added to the wells of the 96-well plate apoptosis detection kit, sealed with a blocking membrane, and incubated at room temperature for 1 hour. The sample was removed, the 96-well plate was repeatedly washed, and the substrate solution (100 μL / well) was added. After shaking for 15 minutes, the 96-well plate was tapped and the absorbance value was measured at 405 nm.

[0052] 2. Detection of key apoptosis proteins in esophageal squamous cell carcinoma cell lines

[0053] The KYSE450 cell line was inoculated in RPMI 1640 medium containing 10% fetal bovine serum. After the cells adhered, marigold glycoside (25μM) and cisplatin (5μM) were added alone or in combination. After incubation for 24 hours, the cells were collected together with the supernatant, centrifuged at 4°C, 12000rpm for 10 minutes, and the precipitate was collected. RIPA lysis buffer was added and lysed on ice for 1 hour, with shaking every 10 minutes. After lysis, the supernatant was collected after centrifugation at 4°C, 12000rpm for 20 minutes to obtain the protein lysate. The enzyme-linked immunosorbent assay (ELISA) method was further used to observe the expression of cleaved poly (ADP-ribose polymerase, PARP) in high throughput. PARP is a key apoptosis protein, and the expression of cleaved PARP can clearly measure the degree of cell apoptosis. Samples (100 μL / well) were sequentially added to a 96-well cleaved PARP ELISA plate and incubated at 37°C. After incubation, the 96-well plate was repeatedly washed. The cleaved PARP antibody was added to the 96-well plate and incubated at room temperature for 1 hour, then repeatedly washed and discarded. The reaction substrate was added to each well and incubated at room temperature in the dark. The reaction stop solution was then added and the absorbance was measured at 450 nm using a microplate reader.

[0054] Compare with Figure 3 The results showed that marigold glycosides enhanced cisplatin-induced apoptosis of esophageal squamous cell carcinoma cells and upregulation of cleavage-type PARP expression.

[0055] Example 4: Tagetes erecta enhances the anti-tumor growth effect of cisplatin on esophageal squamous cell carcinoma cell line KYSE450-bearing mice

[0056] KYSE450 cells were injected into the subcutaneous tissue of the upper limbs of nude mice (4-week-old female BALB / c-nu nude mice (average weight 14-15 g), purchased from Vital River Company) until the tumor grew to about 100 mm. 3 After about 3 weeks, the KYSE450 tumor-bearing mice were randomly divided into four groups: control group, marigold glycoside group, cisplatin group and marigold glycoside plus cisplatin group. The marigold glycoside group (25 mg / kg) was administered orally once a day, the cisplatin group (5 mg / kg) was intraperitoneally injected with cisplatin once a week, and the cisplatin group and marigold glycoside were administered orally with marigold glycoside (25 mg / kg) and intraperitoneally with cisplatin (5 mg / kg, once a week) daily for a total of 3 weeks. The control group was given the same dose of normal saline by gavage every day, and after 3 consecutive weeks of administration, the tumor volume was measured. The formula for calculating tumor volume is: Volume = Tumor Long Diameter × Tumor Transverse Diameter 2× 0.5. The expression of proliferation marker Ki67, angiogenesis marker CD31, and lymphangiogenesis marker LYVE1 in KYSE450 tissues was observed by quantitative ELISA.

[0057] Compare with Figure 4 The results showed that marigold glycosides enhanced the inhibitory effect of cisplatin on the growth of KYSE450 tumors in a xenograft-bearing mouse model and the induction of Ki67, CD31, and LYVE1 expression in KYSE450 tissues.

[0058] The Jin Zhengjun Q value method was used to evaluate the synergistic effect of the combination of marigold glycosides and cisplatin 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 is the inhibition rate of the drug combination, E a and E b is the inhibition rate of drug treatment when administered alone. Q < 0.85 indicates antagonism, 0.85 ≤ Q < 1.15 indicates additive effect, and Q ≥ 1.15 indicates synergism.

[0059] The calculated Q value for tumor volume was 1.20; for Ki67, the Q value was 1.33; for CD31, the Q value was 1.17; and for LYVE1, the Q value was 1.75.

[0060] Example 5: Tagetes erecta enhances the anti-tumor invasion-promoting effect of cisplatin on esophageal squamous cell carcinoma cell line KYSE450-bearing mice

[0061] KYSE450 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). Approximately one week later, marigold glycosides (25 mg / kg / day) and / or cisplatin (5 mg / kg / week) were administered via gavage. The experiment lasted for 5 weeks. Popliteal lymph nodes were collected, and lymphatic volume was calculated. The volume calculation formula is: Volume = Long diameter × Transverse diameter 2 ×0.5.

[0062] Compare with Figure 5 The results showed that marigold glycosides enhanced the effect of cisplatin in inhibiting the lymphatic metastasis of KYSE450 tumors in a xenograft-bearing mouse model.

[0063] Similarly, the Jin Zhengjun Q value judgment method was used to evaluate the synergistic effect of the combination of marigold glycosides and cisplatin in inhibiting tumor lymphatic metastasis, and the Q value was 1.36.

[0064] 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 limiting. 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 tagetoid glycoside or a pharmaceutically acceptable salt thereof and cisplatin for use in the preparation of a drug for treating esophageal squamous cell carcinoma, wherein the active ingredients of the pharmaceutical composition are composed of tagetoid glycoside or a pharmaceutically acceptable salt thereof and cisplatin, and the ratio of tagetoid glycoside or a pharmaceutically acceptable salt thereof to cisplatin, calculated on the weight of tagetoid glycoside and cisplatin, is 35:1; wherein, The structure of the marigold glycoside is: 。

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