Anti-esophageal squamous cell carcinoma pharmaceutical composition containing tageteoside and application thereof

Abstract

The invention provides an esophageal squamous cell carcinoma resisting pharmaceutical composition containing tageteoside and application of the esophageal squamous cell carcinoma resisting pharmaceutical composition. According to the pharmaceutical composition disclosed by the invention, the tageteoside and the cis-platinum have a synergistic anti-cancer effect, and the pharmaceutical composition of the tageteoside and the cis-platinum has an enhanced esophageal squamous cell carcinoma resisting effect and can be used for effectively preventing and treating the esophageal squamous cell carcinoma.

Description

Technical Field

[0001] The present invention relates to the field of medicines, and in particular to an anti-esophageal squamous cell carcinoma pharmaceutical composition comprising marigold glycoside and 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 radiotherapy and chemotherapy due to their poor physical constitution. Therefore, the prognosis of esophageal squamous cell carcinoma is usually poor, and the patient's survival time is short.

[0003] The first-line drug for clinical treatment of esophageal squamous cell carcinoma is platinum compounds, but they are very easy to develop drug resistance and tumor recurrence after treatment. There have been many studies on 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 structural formula:

[0004]

[0005] Tagetes glycosides have anti-inflammatory and antioxidant biological activities and are widely used in cardiovascular and cerebrovascular diseases and other diseases related to inflammation and oxidation. In recent years, it has also been reported that marigold glycosides have anti-cancer effects by inhibiting the growth of tumor cells. However, whether marigold glycosides can enhance the anti-cancer effect of cisplatin remains to be further studied. Summary of the invention

[0006] The present invention finds 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 an application of marigold glycoside or a pharmaceutically acceptable salt, prodrug, or metabolite thereof in the preparation of a drug for enhancing the anti-esophageal squamous cell carcinoma effect of cisplatin.

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

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

[0011] In one embodiment, the ratio of tagetoid glycoside or a pharmaceutically acceptable salt, prodrug, metabolite thereof to cisplatin is 0.8-60:1, based on the mass of tagetoid 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 comprises 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, 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 tagetoid glycoside includes a salt formed by tagetoid 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 tagetoid glycoside after being administered to a patient, such as an ester of tagetoid glycoside.

[0018] In the present invention, the metabolite refers to a pharmaceutically acceptable metabolic derivative form of marigold 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 marigold 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 preparations. The pharmaceutical dosage forms of marigold glycoside or its pharmaceutically acceptable salt, prodrug, metabolite and cisplatin can be the same or different. Marigold 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 specific drug or pharmaceutical composition can be used. Examples include, but are not limited to, solvents, excipients, dispersants, emulsifiers, solubilizers, gel formers, ointment bases, antioxidants, preservatives, stabilizers, carriers, fillers, bonding agents, thickeners, complexing agents, disintegrants, buffers, penetration enhancers, polymerizers, lubricants, coating agents, propellants, tonicity regulators, surfactants, colorants, flavoring agents, sweeteners and dyes. Particularly adjuvants suitable for the type of desired formulation and desired mode of administration are used.

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

[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, those skilled in the art can easily determine the appropriate dosage formulation and the method of administering the agent. For example, for adult patients, the pharmaceutical composition of the present invention can be administered orally or parenterally in an amount of 0.001 mg to 500 mg as a daily dose, once a day or divided into several times.

[0029] Beneficial effects:

[0030] The present invention provides an anti-esophageal squamous cell carcinoma pharmaceutical composition comprising tagetoid glycosides and its application. Experimental results show that tagetoid glycosides and cisplatin have a synergistic anti-cancer effect, tagetoid glycosides can enhance the inhibitory effect of cisplatin on the growth and invasion of esophageal squamous cell carcinoma cells, enhance the effect of cisplatin in promoting the apoptosis of esophageal squamous cell carcinoma cells, and enhance the anti-tumor growth effect and anti-tumor invasion promoting effect of cisplatin on esophageal squamous cell carcinoma tumor-bearing mice. Therefore, the pharmaceutical composition of tagetoid glycosides and cisplatin has an enhanced anti-esophageal squamous cell carcinoma effect and can effectively prevent and treat esophageal squamous cell carcinoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a schematic diagram showing the results that marigold glycosides of Example 1 can enhance the inhibitory effect of cisplatin on the growth of esophageal squamous cell carcinoma cell lines.

[0033] Figure 2 This is a schematic diagram of the results showing that marigold glycosides in Example 2 can enhance the effect of cisplatin on the invasion of esophageal squamous cell carcinoma cell lines.

[0034] Figure 3 This is a schematic diagram of the results of marigold glycoside dose enhancement in Example 3 to induce apoptosis of multiple esophageal squamous cell carcinoma cell lines and up-regulation of cleaved PARP expression.

[0035] Figure 4 This is a schematic diagram of the results of Example 4 showing that marigold glycoside enhances the effect of cisplatin on inhibiting the growth of KYSE450 tumors and the expression of Ki67, CD31 and LYVE1 in a xenogeneic tumor-bearing mouse model.

[0036] Figure 5 This is a schematic diagram of the results of Example 5 in which marigold glycosides enhance cisplatin's inhibition of lymphatic metastasis of KYSE450 tumors in a xenogeneic tumor-bearing mouse model.

[0037] In the above figures, * represents P < 0.05; ** represents P < 0.01; *** represents P < 0.001. DETAILED DESCRIPTION

[0038] The preferred embodiments of the invention are described in detail below. The examples are provided to better illustrate the content of the invention, but the content of the invention is not limited to the examples. Non-essential improvements and adjustments to the implementation scheme according to the content of the invention still fall within the scope of the invention.

[0039] The experimental methods in the following examples are all conventional methods unless otherwise specified. If no specific techniques or conditions are specified in the examples, they are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions.

[0041] Example 1: Marigold glycosides enhance the growth inhibitory effect of cisplatin on esophageal squamous cell carcinoma cell lines

[0042] 1. Cell Culture

[0043] 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.

[0044] 2. Cell Growth Ability Determination

[0045] The KYSE450 cell line was inoculated in a 96-well plate. After the cells adhered to the wall, different concentrations of cisplatin were added to the cisplatin group. In the example, different concentrations of cisplatin and marigold glycoside (25 μM) were added. After 72 hours, a 10% MTS solution was prepared with RPMI 1640 medium, the cultured cells were taken out, the upper medium was discarded, and the prepared MTS was added and incubated for 2 hours. The absorbance value was measured at 490 nm using an enzyme reader. The growth inhibition rate of different concentrations of cisplatin compared to the control group was input into the GraphPadPrism software, and the IC was analyzed using the "Dose-response inhibition" model. 50 The value of the fit.

[0046] Compare with Figure 1 , the results showed that marigold glycosides could enhance the inhibitory effect of cisplatin on the growth of esophageal squamous cell carcinoma cell lines.

[0047] Example 2: Marigold glycosides enhance the inhibitory effect of cisplatin on the invasion of esophageal squamous cell carcinoma cell lines

[0048] Cell invasion ability assay

[0049] 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 matrix gel was added to the upper chamber of the chamber, and incubated at 37°C for 1 hour to solidify the matrix gel. The prepared KYSE450 cell line was inoculated in the upper chamber of the transwell chamber, and 800 μL of RPMI 1640 medium containing 20% ​​fetal bovine serum was added to the lower chamber, and 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 taking it out, the inner wall of the upper chamber was wiped clean with a cotton swab, and the invading cells in the lower chamber were separated by dissociation solution and then cell dye was added, and the invasion cell rate was calculated by an enzyme plate reader.

[0050] Compare with Figure 2 , the results showed that marigold glycosides could enhance the cisplatin-inhibitory invasion of esophageal squamous cell carcinoma cell lines.

[0051] Example 3: Marigold glycosides enhance the apoptosis-promoting effect of cisplatin on esophageal squamous cell carcinoma cell lines

[0052] 1. Determination of cell apoptosis index

[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 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, incubated at room temperature for 1 hour, and the sample was removed. After repeatedly washing the 96-well plate, the substrate solution (100μL / well) was added, and after shaking for 15 minutes, the 96-well plate was tapped and the absorbance value was measured at 405nm.

[0054] 2. Detection of key apoptosis proteins in esophageal squamous cell carcinoma cell lines

[0055] 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, and the precipitate was collected after centrifugation at 4°C and 12000rpm for 10 minutes. RIPA lysis buffer was added and lysed on ice for 1 hour, with shaking every 10 minutes. After the lysis was completed, the supernatant was collected after centrifugation at 4°C and 12000rpm for 20 minutes to obtain the protein lysate. The cleaved poly ADP-ribose polymerase (PARP) was further observed by high-throughput enzyme-linked immunosorbent assay (ELISA). PARP is a key protein for apoptosis, and the expression of cleaved PARP can clearly measure the degree of cell apoptosis. Add samples (100 μL / well) to the 96-well plate of sheared PARP ELISA in sequence and incubate at 37°C. After incubation, wash the 96-well plate repeatedly. Add the sheared PARP antibody to the 96-well plate, incubate at room temperature for 1 hour, wash repeatedly and discard. Add the reaction substrate to each well, incubate at room temperature in the dark, add the reaction stop solution, and measure the absorbance value at 450nm using an ELISA reader.

[0056] Compare with Figure 3 The results showed that marigold glycosides enhanced cisplatin-induced apoptosis of esophageal squamous cell carcinoma cells and upregulation of cleaved PARP expression.

[0057] Example 4: Marigold glycosides enhance the anti-tumor growth effect of cisplatin on esophageal squamous cell carcinoma cell line KYSE450 tumor-bearing mice

[0058] 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 body 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: a control group, a marigold glycoside group, a cisplatin group, and a marigold glycoside plus cisplatin group. The marigold glycoside group (25 mg / kg) was gavaged 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 gavaged with marigold glycoside (25 mg / kg) and intraperitoneally injected with cisplatin (5 mg / kg, once a week) daily for a total of 3 weeks. The control group was gavaged with the same dose of normal saline daily, and the tumor volume was measured after 3 consecutive weeks of administration. 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.

[0059] 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 the expression of Ki67, CD31, and LYVE1 in KYSE450 tissues.

[0060] 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 growth. The Q value was calculated using the following formula: Q = E a+b / (E a +E b -E a ×E b ). a+b is the inhibition rate of the drug combination, E a and E b It 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 synergistic effect.

[0061] After calculation, the 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.

[0062] Example 5: Marigold glycosides enhance the anti-tumor invasion promoting effect of cisplatin on esophageal squamous cell carcinoma cell line KYSE450 tumor-bearing mice

[0063] 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). After about 1 week, marigold glycosides (25 mg / kg / day) and / or cisplatin (5 mg / kg / week) were administered orally. The experiment lasted for 5 weeks. The popliteal lymph nodes were collected and the lymph node volume was calculated. The volume calculation formula is: Volume = Long diameter × Transverse diameter 2 ×0.5.

[0064] Compare with Figure 5 The results showed that marigold glycosides enhanced the inhibition of cisplatin on the lymphatic metastasis of KYSE450 tumors in a xenograft-bearing mouse model.

[0065] Similarly, Jin Zhengjun's 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.

[0066] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. Use of marigold glycoside or its pharmaceutically acceptable salt, prodrug, metabolite in enhancing the anti-esophageal squamous cell carcinoma effect of cisplatin.

2. Use of marigold glycoside or its pharmaceutically acceptable salt, prodrug, metabolite in the preparation of a drug for enhancing the anti-esophageal squamous cell carcinoma effect of cisplatin.

3. A pharmaceutical composition comprising marigold glycoside or a pharmaceutically acceptable salt, prodrug, metabolite thereof and cisplatin.

4. The pharmaceutical composition according to claim 3, characterized in that The ratio of marigold glycoside or its pharmaceutically acceptable salt, prodrug, metabolite to cisplatin is 0.8-60:1 based on the mass of marigold glycoside and cisplatin.

5. The pharmaceutical composition according to claim 3, characterized in that The ratio of marigold glycoside or its pharmaceutically acceptable salt, prodrug, metabolite to cisplatin is 2-40:1 based on the mass of marigold glycoside and cisplatin.

6. The pharmaceutical composition according to claim 3, characterized in that The ratio of marigold glycoside or its pharmaceutically acceptable salt, prodrug, metabolite to cisplatin is 4.1-8.2:1 based on the mass of marigold glycoside and cisplatin.

7. The pharmaceutical composition according to claim 3, characterized in that The ratio of marigold glycoside or its pharmaceutically acceptable salt, prodrug, metabolite to cisplatin is 35:1, based on the mass of marigold glycoside and cisplatin.

8. A kit comprising tagetoid glycoside or a pharmaceutically acceptable salt, prodrug, metabolite thereof and cisplatin.

9. Use of the pharmaceutical composition according to any one of claims 3 to 7 in the preparation of drugs for treating esophageal squamous cell carcinoma.

10. The use according to claim 9, characterized in that: The anti-esophageal squamous cell carcinoma agent comprises 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.

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