An anti-tumor pharmaceutical composition and its application

Abstract

The present invention belongs to the field of medical technology, and discloses an anti-tumor pharmaceutical composition and its application, wherein the pharmaceutical composition comprises trozastilboside and cisplatin. The present invention also provides the application of the combined use of trozastilboside and cisplatin in the preparation of a drug for treating tumors, and the application of trozastilboside in the preparation of a drug for enhancing the efficacy of cisplatin and reducing the toxicity of cisplatin. The trozastilboside of the present invention combined with cisplatin shows an obvious synergistic effect in inhibiting lung cancer cell proliferation, and can effectively improve the anti-tumor activity of cisplatin. At the same time, trozastilboside can also alleviate the side effects of sudden weight loss, acute liver and kidney damage, and decreased appetite caused by cisplatin; it has good application prospects in the prevention and treatment of lung cancer.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular, relates to an anti-tumor pharmaceutical composition and application thereof. Background Art

[0002] Cisplatin is a metal platinum complex, a periodic nonspecific anti-tumor drug, and a first-line drug for the treatment of various solid tumors. It has the characteristics of a broad anti-tumor spectrum, strong effects, and definite efficacy. It is widely used in clinical practice, but there are three serious problems: acquired drug resistance, narrow safety range, and obvious tissue or organ toxicity, especially liver and kidney toxicity. These three major problems seriously endanger the quality of life of patients, leading to early termination of treatment and a decrease in the use rate of drugs, which is a thorny problem in the current application of cisplatin. Therefore, it is necessary to develop a combination with cisplatin to synergistically enhance sensitization, improve efficacy, and reduce toxicity.

[0003] The NCCN guidelines include combination therapy in the clinical medication norms from the perspective of enhancing drug effects, improving patients' quality of life, and reducing toxic side effects, and point out that it has an irreplaceable role. Therefore, fully analyzing and summarizing drug characteristics and deeply exploring combination therapy strategies are the key to further improving efficacy and overcoming drug resistance, and have important scientific research value and clinical significance.

[0004] The plant source of taurostilbene ((E)-1-(3,5-dihydroxyphenyl)-2-(3-hydroxy-4-O-β-D-pyranose phenyl)ethylene or 3,5,3',4'-tetrahydroxystilbene-3'-O-β-glucoside) is the rhizome of Rheum officinale Lhasa, and piceatannol is the aglycone of taurostilbene. Studies have shown that taurostilbene has anti-inflammatory, antioxidant, and free radical scavenging activities.

[0005] Chinese patent application number 201010116358.2 discloses the use of trozastilbene in the preparation of preparations for preventing and treating cardiovascular and cerebral ischemic diseases and its preparation method; Chinese patent application number 201210198159.X discloses the use of a stilbene compound in the preparation of drugs for preventing and treating depression; Chinese patent application number 201210202957.5 discloses the use of trozastilbene in the preparation of drugs for improving microcirculatory disorders. Studies in the prior art show that trozastilbene has the activity of preventing and treating ischemic cardiovascular and cerebrovascular diseases, anti-depression and improving microcirculation, but there is no literature report that the combination of trozastilbene and cisplatin can enhance the anti-tumor efficacy of cisplatin and reduce the toxic side effects of cisplatin, so the present invention has important clinical significance.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] The technical problem to be solved by the present invention is to overcome the deficiencies of the prior art and provide an anti-tumor pharmaceutical composition and its application. The combination of the tadalafil glycoside of the present invention and cisplatin shows an obvious synergistic effect in inhibiting lung cancer cell proliferation, and can effectively improve the anti-tumor activity of cisplatin. At the same time, tadalafil glycoside can also alleviate the side effects of cisplatin such as sudden weight loss, acute liver and kidney damage, and decreased appetite; it has good application prospects in the prevention and treatment of lung cancer.

[0008] In order to solve the above technical problems, the basic concept of the technical solution adopted by the present invention is:

[0009] The first object of the present invention is to provide an anti-tumor pharmaceutical composition, which comprises trazastilboside and cisplatin.

[0010] Although cisplatin is a first-line drug for the treatment of various solid tumors, it has a narrow safety range and severe toxic side effects. The inventors found through a large number of experiments that the combination of trazastilbin and cisplatin in vitro has significant synergistic inhibitory activity on the proliferation of lung cancer PC9 cells; in vivo, the combined use can effectively inhibit the proliferation of nude mouse xenograft tumors and significantly reduce the toxicity of cisplatin.

[0011] In a further embodiment, the molar ratio of trazastilboside to cisplatin is 1-100 μM: 0.5-60 μM;

[0012] Preferably, the molar ratio of trazastilboside to cisplatin is 10-100 μM:5-60 μM.

[0013] In a further scheme, the ratios of combined use of quzaqi glycoside and cisplatin include: 100μM quzaqi glycoside and 53μM cisplatin; 33.33μM quzaqi glycoside and 17.67μM cisplatin; 11.11μM quzaqi glycoside and 5.89μM cisplatin; 3.70μM quzaqi glycoside and 1.96μM cisplatin; 1.23μM quzaqi glycoside and 0.65μM cisplatin.

[0014] In a further embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient;

[0015] Preferably, the auxiliary material is selected from one or more of fillers, disintegrants, binders, lubricants, flavoring agents, preservatives, antioxidants, and colorants.

[0016] In a further embodiment, the dosage form of the pharmaceutical composition is selected from injection, tablet, capsule, powder, pill, granule or oral solution.

[0017] In a further embodiment, the pharmaceutical composition is a single compound preparation, which contains both trazosin and cisplatin.

[0018] In a further embodiment, the pharmaceutical composition is divided into two separate preparations of trazalin and cisplatin, and the two separate preparations are administered simultaneously, separately or sequentially.

[0019] In a further embodiment, the dosage of trozalin is 20-1000 mg / person / day.

[0020] In a further embodiment, the tumor comprises lung cancer.

[0021] The second object of the present invention is to provide the use of a combination of trazastilboside and cisplatin in the preparation of a drug for treating tumors.

[0022] In a further embodiment, the tumor comprises lung cancer.

[0023] The third object of the present invention is to provide the use of trozastilbin in the preparation of drugs for enhancing the efficacy of cisplatin and reducing the toxicity of cisplatin.

[0024] After adopting the above technical scheme, the present invention has the following beneficial effects compared with the prior art.

[0025] 1. The present invention discloses for the first time the use of trozastilboside in the preparation of drugs for sensitizing cisplatin. Studies have shown that in the lung cancer cell line PC9, the use of cisplatin alone only slightly reduces the survival rate of PC9 cells, while the combination of cisplatin and trozastilboside greatly reduces the survival rate of PC9 cells. At the same time, the combined index CI obtained by the combined application of multiple concentrations of the two is lower than 1, indicating that trozastilboside can sensitize cisplatin and improve its anti-tumor efficacy. From the results of in vivo experiments, in the human lung cancer H1975 mouse transplant tumor model, when the two are used in combination, the tumor inhibition rate is greatly increased, the synergistic effect is significant, and there is a high degree of consistency with the in vitro experiment. After relatively detailed in vitro and in vivo activity tests, it is proved that the combination of trozastilboside and the first-line chemotherapy drug cisplatin can play a synergistic role and effectively improve the anti-tumor activity of cisplatin.

[0026] 2. The present invention has found through experiments that the combination of tadalafil and cisplatin can not only effectively improve the anti-tumor activity of cisplatin, but also significantly reduce the toxic and side effects of cisplatin, and can alleviate the side effects such as sudden weight loss, acute liver and kidney damage and decreased appetite caused by the chemotherapy drug cisplatin, and has a good protective effect on the toxic and side effects such as liver and kidney damage caused by cisplatin.

[0027] 3. Trizalin can be extracted from plants with high yield, can be used for industrial production, and the drugs used are easily available, with low cost and good market prospects. The present invention not only provides a new use of trizalin, but also provides a new scientific basis for the development of new anti-tumor auxiliary drugs, which is of great significance for the development and utilization of traditional Chinese medicine; it also provides a new therapeutic drug regimen for the treatment of lung cancer, and has good application prospects in the prevention and treatment of lung cancer.

[0028] The specific implementation modes of the present invention are further described in detail below in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The accompanying drawings are part of the present invention and are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention, but do not constitute an improper limitation of the present invention. Obviously, the drawings described below are only some embodiments. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work. In the accompanying drawings:

[0030] Figure 1 This is the inhibitory effect of cisplatin alone and in combination with cisplatin on PC9 cells in Experimental Example 1 of the present invention.

[0031] It should be noted that these drawings and textual descriptions are not intended to limit the conceptual scope of the present invention in any way, but are intended to illustrate the concept of the present invention for those skilled in the art by referring to specific embodiments. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. The following embodiments are used to illustrate the present invention but are not used to limit the scope of the present invention.

[0033] Unless otherwise specified, the reagents and materials used in the examples of the present invention are commercially available.

[0034] Pharmaceutical composition

[0035] Drug composition components: 100-300 mg of trazalin, 5-10 mg of cisplatin

[0036] Prescription of pharmaceutical composition 1: 100 mg of trozalin, 5 mg of cisplatin, excipients: 90 g of sodium chloride, 75 g of mannitol, and an appropriate amount of water for injection to make 1000 ml.

[0037] According to the above prescription ratio, sodium chloride, mannitol and an appropriate amount of water for injection are added to the drug composition, stirred evenly, 0.1% activated carbon for injection is added for adsorption, filtered for decarbonization, water for injection is added to the specified amount, filtered through a microporous filter membrane, packaged at 2 mL / vial, freeze-dried, packaged, and obtained after passing the inspection.

[0038] Prescription 2 of the pharmaceutical composition: 300 mg of trozastilboside, 10 mg of cisplatin, the composition accounts for 10%; auxiliary materials: 70% starch, 12% microcrystalline cellulose, 3% magnesium stearate, and 5% sodium carboxymethyl starch.

[0039] According to the above prescription ratio, the pharmaceutical composition is mixed with starch, microcrystalline cellulose, sodium carboxymethyl starch and magnesium stearate, and then filled into a hard gelatin capsule to obtain the product.

[0040] Test Example 1 Antitumor Activity Test Method

[0041] 1. Experimental Materials

[0042] 1.1 Test samples: trozastilboside (Kunming Pharmaceutical Group Co., Ltd. Research Institute), batch number: 020A21S04-F, content: 99.1%; cisplatin (Shanghai Yuanye), batch number: Y21F12T139647, purity 98%.

[0043] 1.2 Cell line: Human lung cancer cell PC9 cell line, Guangzhou Geneo Biotechnology Co., Ltd.

[0044] 1.3 Reagents and consumables

[0045] 1640 culture medium, PBS buffer, FBS, Trypsin-EDTA were all purchased from Gibco; MTS (Promega).

[0046] 1.4 Equipment

[0047] Biological safety cabinet (Thermo), carbon dioxide incubator (Thermo), electric constant temperature water tank (Shanghai Zuo Le Instrument Co., Ltd.), ultra-low temperature refrigerator (Thermo Fisher Scientific, USA), Haier refrigerator (Qingdao Haier Co., Ltd.), water purifier (Millipore), multi-function microplate reader (BioTek, USA), ultrasonic cleaner (Ningbo Xinzhi Biotechnology Co., Ltd.), small MiniSpin centrifuge (Eppendorf), inverted biological microscope (Nanjing Jiangnan Yongxin Optics Co., Ltd.).

[0048] 2. Experimental Methods

[0049] 2.1 Preparation of test products

[0050] The raw material of quzaqi glycoside was completely dissolved in DMSO to prepare a 50 mM stock solution;

[0051] Completely dissolve cisplatin in physiological saline to prepare a 1 mg / ml stock solution.

[0052] 2.2 Determination of the antitumor activity of compounds against PC9

[0053] PC9 in the logarithmic growth phase was plated to a 96-well plate at 6000 cells / well, and single-drug dosing wells, combined dosing wells, control wells, and blank wells were set up at the same time. Three replicate wells were set up for each group, and the cells were cultured in a 37°C, 5% CO2 incubator for 24 hours to adhere to the wall. Quzaqi glycoside and cisplatin were prepared in serum-free medium to different concentration gradients of 100, 33.33, 11.11, 3.70, 1.23 μM and 53, 17.67, 5.89, 1.96, and 0.65 μM, respectively. Three replicate wells were set up for each concentration. Different concentrations of Quzaqi glycoside or cisplatin were added to the single-drug group, and different concentrations of Quzaqi glycoside and cisplatin were added to the combined drug group at the same time. After drug addition, the cells continued to be cultured. After the cells were treated with drugs for 48 hours, the supernatant of the culture medium containing the drug solution was aspirated, and the culture medium containing MTS was added to make each well contain 100 μL of culture medium and 20 μL of MTS. After incubation in an incubator away from light for 1 hour, the absorbance value was measured at 490 nm using an enzyme marker, and the inhibition rate of the drug on the cells was calculated according to the following formula:

[0054] Inhibition rate = (OD value of control well - OD value of sample well) / OD value of control well

[0055] 3. Experimental results

[0056] The results are as follows Figure 1 As shown, where:

[0057] A represents 100 μM trozacitidine alone, 53 μM cisplatin alone, and the combination of the two;

[0058] B represents 33.33 μM trozacitin alone, 17.67 μM cisplatin alone, and the combination of the two;

[0059] C represents 11.11 μM trozacitidine alone, 5.89 μM cisplatin alone, and the combination of the two;

[0060] D represents 3.70 μM trozacitin alone, 1.96 μM cisplatin alone, and the combination of the two;

[0061] E represents 1.23 μM trozacitidine alone, 0.65 μM cisplatin alone, and the combination of the two;

[0062] Results: Cisplatin had a significant inhibitory effect on PC9 cells after 48 hours of treatment, but trozaqi glycoside had almost no inhibitory effect on PC9 cells; the combined effect of cisplatin and trozaqi glycoside for 48 hours could significantly enhance the inhibitory effect of cisplatin on PC9 cells.

[0063] CompuSyn software was used to calculate the synergistic index of the combined action of the two drugs, see Table 1; CompuSyn software was used to calculate the combination index (CI) of drug interactions. Evaluation criteria: CI = 1, additive interaction; CI> 1, antagonistic interaction; CI< 1, synergistic interaction.

[0064] The results showed that within the effective concentration, the synergistic index of the combination of cisplatin and trozastilbin was less than 1, indicating that the combination of cisplatin and trozastilbin had a certain synergistic effect. Trozastilbin could significantly improve the cisplatin chemotherapy efficacy of tumor cells and increase the sensitivity of cisplatin.

[0065] Table 1. Synergistic index of the combined effect of cisplatin and trozalin on PC9 cells

[0066]

[0067] Experimental Example 2: Efficacy on human lung cancer H1975 transplanted tumors in nude mice

[0068] 1. Experimental Materials

[0069] 1.1 Test samples: trozastilbin (Kunming Pharmaceutical Group Co., Ltd. Research Institute); cisplatin (Shanghai Yuanye);

[0070] 1.2 Experimental animals: BALB / C nude mice (SPF grade), male, 18-20 g, source: Shanghai Slake Laboratory Animal Co., Ltd.; production license number: SCXK (Shanghai) 2012-0002, use license number: SYXK (Shanghai) 2009-0068.

[0071] 1.3 Transplanted tumor: Human lung cancer H1975, preserved by Shanghai Institute of Medical Engineering.

[0072] 2. Test methods

[0073] H1975 cells in the logarithmic growth phase were digested with 0.25% trypsin and adjusted to a concentration of 1×10 7 The cell suspension of 100 cells / ml was inoculated subcutaneously in the left axilla of nude mice, 0.2 ml for each mouse. When the diameter of the tumor mass was about 3 mm, 40 nude mice with relatively uniform tumor volume were selected and randomly divided into 4 groups: model control group (NS), cisplatin group (2 mg / kg; administration on d1, 3, and 5), trozastilboside group (50 mg / kg, d1--14 days), trozastilboside + cisplatin group (cisplatin: 2 mg / kg, d1, 3, and 5 days; trozastilboside: 50 mg / kg, d1--14 days). The administration method was intraperitoneal injection. After d14 of administration in each group, 0.5 mL of peripheral blood was collected and centrifuged at 3000 r / min for 5 min. The supernatant was used for blood biochemical detection. The animals were killed and the tumor mass was dissected and weighed.

[0074] During the treatment, the mice's response to treatment, activity, mental state, and hair loss were observed daily, and the significance of the combined drug therapy was comprehensively evaluated based on the body weight change curve, tumor inhibition rate, and liver and kidney function indicators.

[0075] 3. Test results

[0076] 3.1 Treatment response and body weight changes of mice

[0077] During the administration period, the hair of mice in the trozastilbin group was smooth and shiny, and they were in good spirits; the cisplatin group had greater toxic and side effects, and the mice had reduced activity and were listless; the model control group had less shiny hair and less activity than the trozastilbin group; while the combined administration group had smoother hair, good spirits, and more activity than the cisplatin group alone; by the end of the experiment, one animal died in the cisplatin group, and no animal died in the other groups. Comparison of the weight change trend shows that the weight of mice in the model group and the trozastilbin group increased slowly, while the weight of the cisplatin group and the combined group increased slowly, which may be related to the accumulation of platinum in the later stage of administration, which affects the living conditions of mice, as shown in Table 2.

[0078] Table 2 Changes in mouse body weight ( g)

[0079]

[0080] 3.2 Inhibitory effect on H1975 lung cancer

[0081] After blood collection, the animals were killed by cervical dislocation, and the tumor tissue was quickly taken out and weighed. The tumor inhibition rate was calculated according to the following formula: tumor inhibition rate = [1-average tumor weight of treatment group / average tumor weight of NS] × 100%.

[0082] As shown in Table 3, the combination of trozastilboside and cisplatin showed a better anti-tumor effect, which was stronger than cisplatin or trozastilboside alone (P<0.05); the anti-tumor effect of the cisplatin group was stronger than that of the trozastilboside group (P<0.05).

[0083] Table 3 Antitumor effect on human lung cancer H1975 transplanted into nude mice ( )

[0084]

[0085] Compared with the model group: *P<0.05, **P<0.01.

[0086] 3.3 Evaluation of synergistic effects of combined medication

[0087] The effect of combined medication is evaluated according to King's formula: q = E a+b / (E a +E b 1E a ·E b ), where Ea+b is the inhibition rate of the two drugs combined, E a and E b is the inhibition rate of each drug when used alone. When q>1, it indicates that the two drugs have a synergistic effect. As shown in Table 3, 2 mg / kg of cisplatin was injected intraperitoneally for 3 times, and the relative tumor proliferation inhibition rate of human lung cancer H1975 was 49.71%; 50 mg / kg of trozastilbene was intraperitoneally administered for 14 consecutive days, and the relative tumor proliferation inhibition rate of human lung cancer H1975 was 4.83% at the end of the experiment; the relative tumor proliferation inhibition rate of trozastilbene combined with cisplatin on human lung cancer H1975 was 69.03%. According to King's formula, q>1 indicates that trozastilbene combined with cisplatin has a synergistic effect.

[0088] 3.4 Blood biochemical analysis results

[0089] As can be seen from Table 4, the serum ALT, AST, BUN, and Cre levels of mice in the cisplatin group increased significantly, indicating that cisplatin caused certain damage to the kidney and liver. In the combination group, the combination weakened the damage caused by cisplatin, significantly improved biochemical indicators, and reduced the hepatorenal toxicity caused by cisplatin, indicating that trozastilbin has a significant protective effect on the liver and kidney damage caused by cisplatin.

[0090] Table 4 Effects on serum biochemical indices ( )

[0091]

[0092]

[0093] Compared with the model group: * / **P<0.05 / 0.01; Compared with the cisplatin group: # / # P<0.05 / 0.01

[0094] The main toxic target organs of cisplatin are the liver, kidneys, and ears, which seriously affect the chemotherapy effect and the quality of life of patients. The current research hotspot is to reduce the toxicity of cisplatin and enhance the chemotherapy effect through synergistic sensitization. In this experimental case, we explored whether the combination of trozastilbin and cisplatin in the H1975 lung cancer mouse model has the advantage of reducing toxicity while synergizing.

[0095] in conclusion

[0096] It can be seen from Experimental Examples 1 and 2 that the combination of trozastilbin and cisplatin has a significant synergistic effect on inhibiting the proliferation of lung cancer cells PC9; in the human lung cancer H1975 mouse transplant tumor model, when the two are used in combination, the tumor inhibition rate is significantly increased, and the synergistic effect is significant, which is highly consistent with the in vitro experiment. At the same time, in the safety assessment, one animal died in the cisplatin monotherapy group, and no animal died in the combination group, and the animal status was better than that of the cisplatin monotherapy group; the weight data showed that after trozastilbin combined with cisplatin, there was no further weight loss, showing the advantage of reducing the toxicity of cisplatin; in the main accumulation target organs of cisplatin-liver and kidney function-related tests, the damage to the target tissues in the combination group was significantly lower than that in the cisplatin monotherapy group, showing a significant reduction in toxicity, allowing more patients who use cisplatin clinically to benefit, and having higher clinical value.

[0097] The above is only a preferred embodiment of the present invention, and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technician familiar with the present invention can make some changes or modify the technical contents suggested above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the solution of the present invention.

Claims

1. An anti-tumor pharmaceutical composition, characterized in that: The pharmaceutical composition comprises trozastilboside and cisplatin; the molar ratio of trozastilboside to cisplatin is 1 μM-100 μM: 0.5 μM-60 μM.

2. The pharmaceutical composition according to claim 1, characterized in that The molar ratio of trazastilboside to cisplatin is 10-100 μM:5-60 μM.

3. The pharmaceutical composition according to claim 1, characterized in that The pharmaceutical composition also includes pharmaceutically acceptable excipients.

4. The pharmaceutical composition according to claim 3, characterized in that The auxiliary material is selected from one or more of fillers, disintegrants, binders, lubricants, flavoring agents, preservatives, antioxidants, and colorants.

5. The pharmaceutical composition according to any one of claims 1 to 4, characterized in that The dosage form of the pharmaceutical composition is selected from injection, tablet, capsule, powder, pill, granule or oral solution.

6. The pharmaceutical composition according to any one of claims 1 to 4, characterized in that The pharmaceutical composition is a single compound preparation, which contains trazalin and cisplatin at the same time.

7. The pharmaceutical composition according to any one of claims 1 to 4, characterized in that The pharmaceutical composition is divided into two separate preparations of trazosin and cisplatin, and the two separate preparations are administered simultaneously, separately or sequentially.

8. The pharmaceutical composition according to any one of claims 1 to 4, characterized in that The tumor is lung cancer.

9. Use of a combination of trazastilboside and cisplatin in the preparation of a drug for treating tumors, characterized in that: The molar ratio of trazastilboside to cisplatin is 1 μM-100 μM: 0.5 μM-60 μM; and the tumor is lung cancer.

10. The use of trozastilbin in the preparation of an anti-tumor drug for enhancing the efficacy of cisplatin and reducing the toxicity of cisplatin, characterized in that: The molar ratio of trazastilboside to cisplatin is 1 μM-100 μM: 0.5 μM-60 μM; and the tumor is lung cancer.

Images