Pharmaceutical composition for preventing and / or treating tumors

Abstract

A pharmaceutical composition for preventing and / or treating tumors, which pharmaceutical composition contains the following topoisomerase II inhibitors as active ingredients: doxorubicin, etoposide, idarubicin, amsacrine, aclarubicin, teniposide, epirubicin, and pirarubicin. The pharmaceutical composition, compared with any one of the components therein, has significantly enhanced tumor inhibitory effects and significantly reduced toxic side effects, wherein a synergistic effect is present among the components.

Description

A pharmaceutical composition for preventing and / or treating tumors TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and particularly relates to a pharmaceutical composition for preventing and / or treating tumors. BACKGROUND

[0002] DNA topoisomerases

[0003] DNA topoisomerases are a class of enzymes existing in the cell nucleus, which can catalyze the breakage and reunion of DNA strands, thereby controlling the topological state of DNA and participating in the regulation of supercoiled structure, and are of great significance for the survival of organisms. The existence of topoisomerases is to solve the problem of entanglement in the process of DNA replication, which is carried out through a series of safe and effective mechanisms, and the whole process is a simple topological change. Its biological action mainly embodies in two ways: one is to regulate and control the supercoiled state of DNA and the catenation state of knotted or unknotted DNA, thereby indirectly affecting the nucleic acid metabolic process in cells; the other is to directly participate in the processes of DNA recombination, repair, transcription and replication.

[0004] For mammals, there are two types of topoisomerase I (TOPO I) and topoisomerase II (TOPO II), which respectively catalyze the generation of single-strand and double-strand gaps on DNA molecules. They mediate the transient breakage and reconnection of single-strand or double-strand DNA, so as to achieve the purpose of changing the topological structure of DNA, and the breakage of single-strand or double-strand is the standard for distinguishing TOPO I and TOPO II. First, the single strand is broken, and then the ends of the bond are rotated along the helical axis in the direction of untwisting supercoiling, and then the broken single strand is connected, so that TOPO I changes the topological structure. While TOPO II breaks double-strand at the same time, and then connects the broken ends after passing through the cut, so as to change the topological structure.

[0005] Topoisomerase I can be divided into type IA and type IB, which are similar in function but not related, and different in action mode. From the sequence or structure, the two are not similar, and there are obvious differences in function. TOPO I binds tightly with DNA substrate, which can wrap DNA. The hydrolysis reaction catalyzed by TOPO I leads to the breakage of DNA, and the active site of TOPO I is covalently connected with the broken DNA to form a transient covalent complex, and then the reconnection reaction of DNA is completed.

[0006] Topoisomerase II also exists in two types, i.e. type IIA and type II B; in mammals, TOPO II is also divided into two subtypes, TOPO II alpha and TOPO II beta; the two subtypes are different in structure, distribution and catalytic activity.

[0007] Topoisomerase is an important target of antitumor drugs, because it presents high level expression in tumor cells, and is not affected by other factors, so inhibiting the topoisomerase activity of tumor cells can curb the rapid proliferation of tumor cells, and thus play a role in killing tumor cells.

[0008] DNA topoisomerase inhibitors

[0009] Topoisomerase inhibitors destroy the activity of the enzyme through each stage of the enzyme action, rather than a certain stage. At the same time, its mode of action is flexible, which can directly act on DNA or topoisomerase, or act on topoisomerase-DNA break complex. The most common is to form a DNA-enzyme-drug ternary complex, which blocks the last step of enzyme and DNA reaction (i.e. the rejoining of single or double stranded DNA at the nick site) to achieve. The inhibitor actually makes the topoisomerase become a substance that breaks the DNA, and eventually the cell dies due to the error repair of DNA strand breakage or the formation of a breakable complex. The mechanism of action of topoisomerase inhibitors is divided into two categories: toxic mechanism and catalytic inhibition mechanism. Toxic mechanism refers to the formation of a larger ternary complex with TOPO-DNA complex by the inhibitor. By increasing the steady-state concentration of TOPO-DNA covalent complex, TOPO enzyme achieves "poisoning" effect. Unlike the toxic mechanism, the catalytic inhibition mechanism refers to the inhibitor by blocking a certain function of TOPO enzyme or a step in the catalytic reaction, thereby inhibiting the total catalytic activity of TOPO enzyme. Topoisomerase inhibitors mainly include anthracycline drugs, camptothecin drugs, and podophyllotoxin drugs, etc. Currently, TOPO inhibitors are the main chemotherapy means for treating cancer in clinic, and they have good clinical effects in leukemia, breast cancer, and lung cancer.

[0010] Topoisomerase I inhibitors are broadly classified into two categories: TOPO I poisons and TOPO I catalytic inhibitors. TOPO I poisons can be further classified into DNA intercalators, groove binders, and DNA binding and TOPO I inhibitors. DNA intercalators include: (1) benzoanthracenequinone compounds such as saintopin and its analog UCE26, which have a common structural feature of a flat naphtacendione structure that allows the drug to intercalate between base pairs; (2) benzophenanthridine compounds such as narwedine and fagaronine, which can trap TOPO I-DNA cleavage complexes and are effective DNA intercalators; and (3) other compounds such as intoplicine, which inhibits both TOPO I and TOPO II and has good antitumor activity. Aclacinomycin A, a trisaccharide derivative of an anthracycline compound, effectively stabilizes TOPO I-DNA covalent complexes and is a typical intercalator that inhibits both TOPO I and TOPO II. Groove binders are further classified into minor groove binders and major groove binders. The former are mainly benzimidazole compounds, and the latter are acridine compounds. Inhibitors that bind DNA and TOPO I include camptothecin and its analogs. Catalytic inhibitors of TOPO I are relatively rare and include aclacinomycin A and shikonin acyl analogs, but require high concentrations for efficacy.

[0011] Topoisomerase II inhibitors are classified into TOPO II catalytic inhibitors and TOPO II poisons based on whether the drug directly causes DNA breaks. TOPO II catalytic inhibitors block a step in the catalytic reaction and inhibit the overall catalytic activity of TOPO II by acting on different stages of the TOPO II catalytic cycle. Such drugs include aclarubicin, novobiocin, fostriecin, and ICRF-159. TOPO II poisons trap TOPO II-mediated DNA break complexes, thereby increasing the number of DNA double-strand breaks and ultimately causing cell death. Such drugs include etoposide, doxorubicin, mitoxantrone, and idarubicin.

[0012] TOPO II inhibitors can be divided into DNA intercalators and DNA non-intercalators according to the binding mode of the drug to DNA. Intercalation refers to the planar part of the molecular structure (similar to the polycyclic structure of purine or pyrimidine base) intercalating between the double strands of the binding site of topoisomerase II and DNA, thereby interfering with the enzyme's reaction of rejoining the DNA break and causing DNA damage, leading to cell death. Such drugs include actinomycin class (actinomycin D), anthracycline class (doxorubicin), anthracenedione class (mitoxantrone), acridine class (aclarubicin). The mode of action of non-intercalation inhibitors is not very clear, and it may be directly acting on TOPO II or only acting on one strand of DNA to affect the function of the enzyme. The main drugs are isoflavones and podophyllotoxin (etoposide, teniposide) and the like. As a large class of compounds with antitumor activity, TOPO II inhibitors are limited by toxic side effects while exerting antitumor effects. For example, doxorubicin (adriamycin), etoposide, idarubicin, aclarubicin, arabinosyl cytosine, teniposide, epirubicin, pirarubicin are eight common antitumor drugs, and their common feature is that the adverse reactions are serious in clinical practice. For example, doxorubicin has main toxic side effects of myelosuppression, cardiotoxicity and gastrointestinal reactions. Etoposide mainly has myelosuppression and gastrointestinal reactions. Idarubicin mainly has severe myelosuppression and cardiotoxicity. Aclarubicin mainly has adverse reactions of myelosuppression and gastrointestinal reactions. Arabinosyl cytosine mainly has adverse reactions of gastrointestinal reactions and myelosuppression, and occasionally has alopecia. Teniposide mainly has adverse reactions of myelosuppression and gastrointestinal reactions. Epirubicin commonly has alopecia (80% of patients), myelosuppression (60%, white blood cells can be reduced to the lowest point 10-14 days after administration, and gradually recover in about 3 weeks, anemia and obvious thrombocytopenia are rare), anorexia, nausea, vomiting, but the degree is lighter than that of doxorubicin in comparison with the same dose of doxorubicin; the cardiotoxicity is also lighter than that of doxorubicin, and the incidence and severity are proportional to the cumulative amount of itself. Pirarubicin mainly has adverse reactions of myelosuppression and is dose-limiting toxicity, mainly granulocytopenia, and the average minimum value appears in 14 days; the cardiotoxicity is lower than that of doxorubicin, and the acute cardiotoxicity mainly includes reversible electrocardiogram changes such as arrhythmia or non-specific ST-T abnormalities, and the chronic cardiotoxicity is dose-cumulative.

[0013] In addition to topoisomerase I inhibitors and topoisomerase II inhibitors, there are also topoisomerase I and II dual inhibitors. Such TOPO I / TOPO II dual inhibitors can simultaneously act on two key enzymes in the cell cycle, further improving drug antitumor activity and reducing drug resistance. The main compounds are as follows: irinotecan, SN-38 (irinotecan metabolite; irinotecan itself is a prodrug, which is converted into active metabolite SN-38 in vivo under the action of carboxylase), pixantrone, mitoxantrone, daunorubicin, doxorubicin, idarubicin, aclarubicin, epirubicin, pirarubicin, intoplicine, aclacinomycin A, tafluposide (derivative of etoposide), salvicine, batracylin (analogue of ellipticine), trabectedin, lurbinectedin (analogue of trabectedin), phenazine derivatives XR11576 and XR5944 (MLN944), and prodigiosin, curcumin, etc. They all have their own characteristics and advantages. In general, dual inhibitors are the focus of research on TOPO enzyme-related antitumor drugs.

[0014] So far, there is still no antitumor chemotherapy drug with good efficacy and low toxicity. SUMMARY

[0015] In order to overcome the defects of the prior art, the applicant has made an in-depth study and unexpectedly found a pharmaceutical composition for preventing and / or treating tumors, which comprises a plurality of topoisomerase II inhibitors. The antitumor effect of the pharmaceutical composition is excellent, especially better than that of a single compound (i.e. a single topoisomerase II inhibitor), and the toxicity and side effects of the pharmaceutical composition are greatly reduced. And the applicant found that the excellent effect and greatly reduced toxicity were produced when each single compound (i.e. a single topoisomerase II inhibitor) was combined at a non-effective dose, which was a combination effect of ultra-low concentration (dose), and the toxicity of the pharmaceutical composition of the present application was lower than that of other similar drug combinations, which was completely unexpected.

[0016] The above-mentioned object of the present application is achieved by adopting the following technical solutions.

[0017] In one aspect, the present application provides a pharmaceutical composition for preventing and / or treating tumors, which comprises the following topoisomerase II inhibitors as active ingredients:

[0018] doxorubicin, etoposide, idarubicin, amsacrine, aclarubicin, teniposide, epirubicin, pirarubicin. Most of these active ingredients are also dual inhibitors of topoisomerase I and II, and among them also TOPO enzyme catalytic inhibitors and TOPO enzyme poisons, whose targets are complex.

[0019] Preferably, the pharmaceutical composition comprises as active ingredient, in parts by mass:

[0020] 0.38 to 0.70 parts of doxorubicin, 1.37 to 3.84 parts of etoposide, 0.12 to 0.18 parts of idarubicin, 0.08 to 0.91 parts of amsacrine, 0.30 to 1.02 parts of aclarubicin, 0.95 to 1.78 parts of teniposide, 0.41 to 0.68 parts of epirubicin, 0.59 to 2.16 parts of pirarubicin.

[0021] Preferably, the pharmaceutical composition consists of as active ingredient, in parts by mass:

[0022] 0.38 to 0.70 parts of doxorubicin, 1.37 to 3.84 parts of etoposide, 0.12 to 0.18 parts of idarubicin, 0.08 to 0.91 parts of amsacrine, 0.30 to 1.02 parts of aclarubicin, 0.95 to 1.78 parts of teniposide, 0.41 to 0.68 parts of epirubicin, 0.59 to 2.16 parts of pirarubicin.

[0023] Preferably, the pharmaceutical composition comprises as active ingredient, in parts by mass:

[0024] 0.4 to 0.63 parts of doxorubicin, 2.41 to 3.84 parts of etoposide, 0.12 to 0.18 parts of idarubicin, 0.63 to 0.91 parts of amsacrine, 0.64 to 1.02 parts of aclarubicin, 1.18 to 1.78 parts of teniposide, 0.41 to 0.67 parts of epirubicin, 0.59 to 0.88 parts of pirarubicin.

[0025] Preferably, the pharmaceutical composition consists of as active ingredient, in parts by mass:

[0026] 0.4 to 0.63 parts of doxorubicin, 2.41 to 3.84 parts of etoposide, 0.12 to 0.18 parts of idarubicin, 0.63 to 0.91 parts of amsacrine, 0.64 to 1.02 parts of aclarubicin, 1.18 to 1.78 parts of teniposide, 0.41 to 0.67 parts of epirubicin, 0.59 to 0.88 parts of pirarubicin.

[0027] Preferably, the pharmaceutical composition comprises as active ingredient, by mass fraction, a topoisomerase II inhibitor:

[0028] 0.5 parts of doxorubicin, 3.2 parts of etoposide, 0.15 parts of idarubicin, 0.75 parts of amsacrine, 0.81 parts of aclarubicin, 1.48 parts of teniposide, 0.54 parts of epirubicin, 0.70 parts of pirarubicin.

[0029] Preferably, the pharmaceutical composition consists of as active ingredient, by mass fraction, a topoisomerase II inhibitor:

[0030] 0.5 parts of doxorubicin, 3.2 parts of etoposide, 0.15 parts of idarubicin, 0.75 parts of amsacrine, 0.81 parts of aclarubicin, 1.48 parts of teniposide, 0.54 parts of epirubicin, 0.70 parts of pirarubicin.

[0031] Preferably, the pharmaceutical composition comprises as active ingredient, by mass fraction, a topoisomerase II inhibitor:

[0032] 0.38 to 0.70 mg of doxorubicin, 1.37 to 3.84 mg of etoposide, 0.12 to 0.18 mg of idarubicin, 0.08 to 0.91 mg of amsacrine, 0.30 to 1.02 mg of aclarubicin, 0.95 to 1.78 mg of teniposide, 0.41 to 0.68 mg of epirubicin, 0.59 to 2.16 mg of pirarubicin.

[0033] Preferably, the pharmaceutical composition consists of as active ingredient, by mass fraction, a topoisomerase II inhibitor:

[0034] 0.38 to 0.70 mg of doxorubicin, 1.37 to 3.84 mg of etoposide, 0.12 to 0.18 mg of idarubicin, 0.08 to 0.91 mg of amsacrine, 0.30 to 1.02 mg of aclarubicin, 0.95 to 1.78 mg of teniposide, 0.41 to 0.68 mg of epirubicin, 0.59 to 2.16 mg of pirarubicin.

[0035] Preferably, the pharmaceutical composition comprises as active ingredient, by mass fraction, a topoisomerase II inhibitor:

[0036] 0.4 to 0.63 mg of doxorubicin, 2.41 to 3.84 mg of etoposide, 0.12 to 0.18 mg of idarubicin, 0.63 to 0.91 mg of amsacrine, 0.64 to 1.02 mg of aclarubicin, 1.18 to 1.78 mg of teniposide, 0.41 to 0.67 mg of epirubicin, 0.59 to 0.88 mg of pirarubicin.

[0037] Preferably, the pharmaceutical composition consists of the following topoisomerase II inhibitors as active ingredients:

[0038] 0.4-0.63 mg of doxorubicin, 2.41-3.84 mg of etoposide, 0.12-0.18 mg of idarubicin, 0.63-0.91 mg of amsacrine, 0.64-1.02 mg of aclarubicin, 1.18-1.78 mg of teniposide, 0.41-0.67 mg of epirubicin, 0.59-0.88 mg of pirarubicin.

[0039] Preferably, the pharmaceutical composition comprises the following topoisomerase II inhibitors as active ingredients:

[0040] 0.5 mg of doxorubicin, 3.2 mg of etoposide, 0.15 mg of idarubicin, 0.75 mg of amsacrine, 0.81 mg of aclarubicin, 1.48 mg of teniposide, 0.54 mg of epirubicin, 0.70 mg of pirarubicin.

[0041] In one specific embodiment, the active ingredients of the pharmaceutical composition of the present application are as follows:

[0042] Preferably, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.

[0043] Preferably, the pharmaceutical composition can be prepared into a suitable dosage form, for example, into an injection, a tablet, etc.

[0044] In another aspect, the present application provides use of the above pharmaceutical composition in the preparation of a medicament for preventing and / or treating a tumor.

[0045] Preferably, the tumor is selected from the group consisting of liver cancer, leukemia, lymphoma, breast cancer, gastric cancer, lung cancer, ovarian cancer, bladder cancer, etc., preferably liver cancer, leukemia or lymphoma.

[0046] In still another aspect, the present application also relates to a method for preventing and / or treating a tumor, which comprises administering to a patient in need thereof a therapeutically effective amount of the above pharmaceutical composition.

[0047] Preferably, the patient is a mammal, which can be a rodent (e.g., mouse, rat, etc.), a primate (e.g., rhesus monkey, human, etc.), etc.

[0048] Preferably, the tumor is selected from the group consisting of liver cancer, leukemia, lymphoma, breast cancer, gastric cancer, lung cancer, ovarian cancer, bladder cancer, etc., preferably liver cancer, leukemia or lymphoma.

[0049] Compared with the prior art, the application has at least the following beneficial technical effects:

[0050] The pharmaceutical composition of the application has a significant anti-tumor effect and a significantly reduced toxicity compared with any of the components, and the components have a synergistic effect (combination effect). Moreover, the dosage of each single compound in the pharmaceutical composition is the non-effective dosage when the compound is administered alone.

[0051] The eight drugs described above have a good tumor inhibition effect (tumor inhibition rate 54.3%) and no serious toxic reactions (no death, no liver, heart or kidney damage) when combined at an unexpectedly ineffective dosage, which has never been seen in chemotherapy drug treatment. Correspondingly, the eight drugs described above in the parallel experiment (the dosage is 5 times the dosage of each drug in the combined drugs) do not show an effective anti-tumor effect (tumor inhibition rate only 24.6%-38.1%; it is generally considered that a tumor inhibition rate of 50% is effective) but show serious toxicity, with 10%-50% of the mice in each group dying and causing liver, heart or kidney damage in the surviving mice.

[0052] The eight drugs in the pharmaceutical composition of the application are toxic when administered alone at the effective dosage, but the application greatly reduces the toxicity by combining these specific drugs and controlling the content (dosage) within a specific range, and the toxicity of the pharmaceutical composition of the application is lower than that of other similar drug combinations.

[0053] The eight drugs are used alone in the prior art, and there is no report on the combination of the eight drugs. In addition, it is known that the eight drugs have toxicity when used alone. The strategy for reducing toxicity in the prior art mainly focuses on the control of toxicity when the drug takes effect, and there is no consideration of the toxicity problem when the treatment is ineffective. In addition, the existing researches mainly focus on the mechanism of action and the target of the compounds, and less on the relationship between the toxicity target or the chemical structure and the toxicity. Therefore, although there is a theory that the combination of multiple drugs can produce additive efficacy, due to the lack of clear understanding of the toxicity mechanism (such as the toxicity target or the structure-activity relationship), it is difficult to predict the toxicity after combination, and even the same drug combination, but the toxicity may be significantly different under the control of different proportions. As shown in Example 5 of the present application, even the same combination of the eight drugs, and the same low-dose combination, if the proportion of each drug in the combination exceeds a certain range, the combination of these drugs will also show great toxicity; for example, in Example 5, compared with Prescription 1, Prescriptions 2-5 all show great toxicity, resulting in 10%-50% of mice dying, and the surviving mice showing significant liver, heart and / or kidney toxicity, and also showing serious damage to the immune system. These results show that even the same combination of the eight drugs also needs appropriate proportioning of each component to achieve good antitumor efficacy and low toxicity.

[0054] The present application researches find that the combination of the eight drugs in a specific proportion not only can significantly improve the efficacy, but also can effectively reduce the overall toxicity. BRIEF DESCRIPTION OF DRAWINGS

[0055] Hereinafter, the embodiments of the present application will be described in detail with reference to the accompanying drawings, in which:

[0056] Figure 1: Effect of eight TOPO II inhibitors and their combinations on the volume of H22 solid tumor in mice;

[0057] Figure 2: Effect of eight TOPO II inhibitors and their combinations on the tumor index of H22 solid tumor in mice, wherein the difference has statistical significance (p<0.01) compared with the model group ## The difference has statistical significance (p<0.05) compared with the model group # The difference has statistical significance (p<0.01) compared with the combination drug group △△ The difference has statistical significance (p<0.05) compared with the combination drug group △ ;

[0058] Figure 3: Effect of eight TOPO II inhibitors and their combinations on the inhibition rate of H22 solid tumor in mice, wherein the difference has statistical significance (p<0.01) compared with the combination drug group △△The difference has statistical significance (p<0.05) compared with the combination drug group △ ;

[0059] Figure 4: Effect of eight TOPO II inhibitors and their combinations on the survival days of H22 ascitic tumor mice, wherein the difference has statistical significance (p<0.01) compared with the model group ## ;

[0060] Figure 5: Effect of eight TOPO II inhibitors and their combinations on the life extension rate of H22 ascitic tumor mice, wherein the difference has statistical significance (p<0.01) compared with the model group △△ ;

[0061] Figure 6: Survival curve of the effect of eight TOPO II inhibitors and their combinations on H22 solid tumor mice;

[0062] Figure 7: Effect of eight TOPO II inhibitors and their combinations on the body weight change trend of H22 solid tumor mice;

[0063] Figure 8: Effect of eight TOPO II inhibitors and their combinations on the single-day food intake of H22 solid tumor mice;

[0064] Figure 9: Effect of eight TOPO II inhibitors and their combinations on the single-day water intake of H22 solid tumor mice;

[0065] Figure 10: Effect of eight TOPO II inhibitors and their combinations on the white blood cell count of H22 solid tumor mice, wherein the difference has statistical significance (p<0.01) compared with the model group ## The difference has statistical significance (p<0.05) compared with the model group # ;

[0066] Figure 11: Effect of eight TOPO II inhibitors and their combinations on the platelet count of H22 solid tumor mice, wherein the difference has statistical significance (p<0.01) compared with the model group ## The difference has statistical significance (p<0.05) compared with the model group # ;

[0067] Figure 12: Effect of eight TOPO II inhibitors and their combinations on the thymus index of H22 solid tumor mice, wherein the difference has statistical significance (p<0.01) compared with the model group ## The difference has statistical significance (p<0.05) compared with the model group # The difference has statistical significance (p<0.05) compared with the combination drug group △ ;

[0068] Figure 13: Effects of eight TOPO II inhibitors and their combinations on the spleen index of H22 solid tumor mice, wherein the difference has statistical significance (p<0.01) compared with the blank group; the difference has statistical significance (p<0.01) compared with the model group ## The difference has statistical significance (p<0.05) compared with the model group # ;

[0069] Figure 14: Effects of eight TOPO II inhibitors and their combinations on the heart index of H22 solid tumor mice

[0070] Figure 15: Effects of eight TOPO II inhibitors and their combinations on the LDH of H22 solid tumor mice; the difference has statistical significance (p<0.01) compared with the blank group; the difference has statistical significance (p<0.01) compared with the model group ## The difference has statistical significance (p<0.01) compared with the combination drug group △△ ;

[0071] Figure 16: Effects of eight TOPO II inhibitors and their combinations on the CK of H22 solid tumor mice; wherein the difference has statistical significance (p<0.01) compared with the model group ## The difference has statistical significance (p<0.05) compared with the model group # The difference has statistical significance (p<0.01) compared with the combination drug group △△ The difference has statistical significance (p<0.05) compared with the combination drug group △ ;

[0072] Figure 17: Effects of eight TOPO II inhibitors and their combinations on the liver index of H22 solid tumor mice; the difference has statistical significance (p<0.01) compared with the blank group; the difference has statistical significance (p<0.05) compared with the model group # ;

[0073] Figure 18: Effects of eight TOPO II inhibitors and their combinations on the ALT of H22 solid tumor mice; the difference has statistical significance (p<0.01) compared with the blank group; the difference has statistical significance (p<0.01) compared with the model group ## The difference has statistical significance (p<0.01) compared with the combination drug group △△ ;

[0074] Figure 19: Effects of eight TOPO II inhibitors and their combinations on the AST of H22 solid tumor mice; the difference has statistical significance (p<0.01) compared with the blank group; the difference has statistical significance (p<0.01) compared with the model group## The difference has statistical significance (p<0.05) compared with the model group # The difference has statistical significance (p<0.01) compared with the combination drug group △△ ;

[0075] Figure 20: Effect of eight TOPO II inhibitors and their combinations on ALP of H22 solid tumor mice; the difference has statistical significance (p<0.01) compared with the model group ## The difference has statistical significance (p<0.05) compared with the combination drug group △ ;

[0076] Figure 21: Effect of eight TOPO II inhibitors and their combinations on TP of H22 solid tumor mice; the difference has statistical significance (p<0.05) compared with the model group # ;

[0077] Figure 22: Effect of eight TOPO II inhibitors and their combinations on ALB of H22 solid tumor mice; the difference has statistical significance (p<0.01) compared with the combination drug group △△ The difference has statistical significance (p<0.05) compared with the combination drug group △ ;

[0078] Figure 23: Effect of eight TOPO II inhibitors and their combinations on kidney index of H22 solid tumor mice;

[0079] Figure 24: Effect of eight TOPO II inhibitors and their combinations on UREA of H22 solid tumor mice; the difference has statistical significance (p<0.01) compared with the model group ## ;

[0080] Figure 25: Effect of eight TOPO II inhibitors and their combinations on SCr of H22 solid tumor mice; the difference has statistical significance (p<0.01) compared with the model group ## The difference has statistical significance (p<0.05) compared with the model group # The difference has statistical significance (p<0.01) compared with the combination drug group △△ The difference has statistical significance (p<0.05) compared with the combination drug group △ ;

[0081] Figure 26: Time schedule of inoculation, administration and measurement in mouse experiment. (A) solid tumor study of combination drug; (B) ascites tumor study of combination drug;

[0082] Figure 27: Effect of different combinations of A and B drugs on cell viability of HL-60 tumor cells; ** means P<0.01, **** means P<0.0001 compared with control, n=3, Mean±SD; I, cell viability of HL-60 cells under the effect of each combination of drugs in group A; II, cell viability of HL-60 cells under the effect of each combination of drugs in group B;

[0083] Figure 28: Inhibition of HL-60 tumor cells and toxicity on H9C2 normal cells by A and B groups; **** means P<0.0001 compared with control, n=3, Mean±SD; I, cell viability of HL-60 tumor cells under the effect of each combination of drugs in groups A and B (this is the information cited from Figure 27); II, toxicity (cell viability of normal cells) of the corresponding combination of drugs on H9C2 normal cells;

[0084] Figure 29: Anti-tumor proliferation effect and toxicity of the combination of drugs; * means P<0.05, **** means P<0.0001 compared with control, n=3, Mean±SD; ns means no statistical difference. DETAILED DESCRIPTION

[0085] The present application will be described in detail below with reference to specific examples. Those skilled in the art will understand that these examples are only used to illustrate the present application, and do not limit the scope of the present application in any way.

[0086] Example 1: Pharmaceutical composition of the present application

[0087] The formula of the pharmaceutical composition is as follows:

[0088] Example 2: Inhibition experiment of the pharmaceutical composition of the present application in H22 (liver cancer) solid tumor mice

[0089] 1.1. Experimental materials, reagents and instruments

[0090] 1.1.1 Experimental animals

[0091] Healthy ICR male mice (body weight 19-20 g) were provided by the Experimental Animal Science Department of the Medical Department of Peking University. They were bred in an environment with constant temperature of 23±2℃, humidity of 0.50-0.60, and standard feed and tap water available at will.

[0092] H22 tumor-bearing mice were provided by the Experimental Animal Science Department of the Medical Department of Peking University.

[0093] Animal experiments were approved by the Beijing University Biomedical Ethics Committee (Approval No: SYXK2011-0039), and the experiment animal practitioner's certificate number was 2014062000055.

[0094] 1.1.2 Main reagents and materials

[0095] Doxorubicin (5927S, CST) was purchased from Beijing Zhongke Keao Biological Technology Co., Ltd;

[0096] Etoposide (E1383-100MG, Sigma) was purchased from San Chemical Technology (Shanghai) Co., Ltd;

[0097] Idarubicin (CC1384-10mg, ChemCatch) was purchased from Beijing Lebo Biological Technology Co., Ltd;

[0098] Amsacrine (HY-13551-10mg, MCE) was purchased from Beijing Lebo Biological Technology Co., Ltd;

[0099] Aclarubicin (TRC, 5mg, A190160) was purchased from Aimer;

[0100] Teniposide (T3109, 20mg, TCI) was purchased from Inokai;

[0101] Epirubicin (Santa Cruz, 25mg, SC-279016) was purchased from Huazhong Haiwei;

[0102] Pirarubicin (Selleck, 10mg, S1393) was purchased from Beijing Xinseng Technology Co., Ltd;

[0103] Dimethyl Sulfoxide (DMSO, DH105-9, 100mL, Sigma) was purchased from Beijing Dingguo Biological Technology Co., Ltd;

[0104] Physiological saline (7647-14-5, 500g, National Pharmaceutical Reagent) was purchased from Beijing Dingguo Biological Technology Co., Ltd;

[0105] Picric acid (74069, 25g, Sigma) was purchased from Beijing Wanlan Shitousu Biological Technology Co., Ltd;

[0106] Medical alcohol (HQ001249, 500mL) was purchased from Beijing Jinghe Gas Technology Co., Ltd;

[0107] Blood cell analyzer diluent, purchased from Shanghai Optoelectronic Medical Electronic Instrument Co., Ltd.

[0108] The selected drugs (eight topoisomerase II inhibitors) in this study are all raw materials of the above drugs, in the form of solid powder.

[0109] 1.1.3 Main instruments

[0110] Inverted microscope (OLYMPUS, CKX41);

[0111] Centrifuge (LD5-2A, Jingli);

[0112] Autoclave (G154DWS, Qimeixia Instruments Co., Ltd.);

[0113] MILLI-Q ultrapure water system (Millipore, USA);

[0114] Electronic analytical balance (Sartorius, Germany);

[0115] SHA-C water bath constant temperature oscillator (Changzhou Guohua Electrical Appliance Co., Ltd.);

[0116] Thermostatic water tank (SHHW21-420, Tianjin Test Instrument Co., Ltd.);

[0117] Ultrasonic cleaner (Ningbo Xinzhi Biological Technology Co., Ltd.);

[0118] Vortex mixer (Its Linbeier);

[0119] Full-automatic biochemical detector (BS-350, Shenzhen Mindray Biomedical Electronics Co., Ltd.).

[0120] 1.2. Experimental method

[0121] 1.2.1 Preparation of main reagents

[0122] Drug solution: first use a pipette to add 50 μL DMSO to the ep tube containing the drug, dissolve the drug, then add physiological saline, prepare the solution into 4 mg / mL stock solution (clear solution), and freeze to-20℃ refrigerator. Before use, take out to melt, then dilute the stock solution to the required concentration with physiological saline. After the drug solution is prepared, ultrasonic oscillation for 10 min to ensure thorough mixing. (Note: the intraperitoneal injection concentration of DMSO is less than 10%).

[0123] The drug concentration of each administration group is set according to its LD 50 . That is, the drug concentration of each drug in the single administration group in the solid tumor (eight drugs) experiment is its LD 50of the LD50. The concentration of each drug in the combination group is one-twentieth of the LD50. 50 The preparation method of the combination group is as follows:

[0124] For example, the total injection volume of the combination group is 4 mL, and the doxorubicin needs 0.5 mg / kg. Then the doxorubicin concentration is expanded by eight times to be 4 mg / kg, 0.5 mL. Eight drugs are mixed in the same volume according to this method, i.e. diluted by eight times again, and the concentration of each drug in the final mixed solution is the required concentration for the experiment.

[0125] 1.2.2 Preparation of animal models and grouping, and administration

[0126] 1.2.2.1 Drug selection

[0127] The drugs selected for this study are all anti-tumor drugs that have been used in clinical practice. The selected drugs are: doxorubicin, etoposide, idarubicin, amsacrine, aclarubicin, teniposide, epirubicin, and pirarubicin.

[0128] 1.2.2.2 Tumor inoculation

[0129] Inoculation of solid tumors: Under sterile conditions, ascites from tumor-bearing mice was extracted, and tumor cells were counted under a microscope. The tumor cells were adjusted to 3 x 10 6 cells / mL with normal saline, and then mixed. Each mouse was subcutaneously injected with 0.2 mL / 20 g of the mixture in the right armpit.

[0130] 1.2.2.3 Animal grouping

[0131] In the solid tumor experiment, ICR mice were inoculated with tumor cells, and 48 h after inoculation, the animals were randomly divided into groups after weighing, with 10 mice in each group, and a total of 11 groups. The groups were: blank control group, model group, doxorubicin group, etoposide group, idarubicin group, amsacrine group, aclarubicin group, teniposide group, epirubicin group, pirarubicin group, and combination drug group (prescription 1, including doxorubicin group, etoposide group, idarubicin group, amsacrine group, teniposide group, aclarubicin group, pirarubicin group, and epirubicin group, a total of eight low-dose mixtures).

[0132] 1.2.2.4 Administration method, route, and concentration

[0133] The concentration of each drug in each administration group is set according to the LD50. In the solid tumor experiment, the concentration of each drug in the single administration group is one-fourth of the LD50. The concentration of each drug in the combination drug group is one-twentieth of the LD50. 50 50 The concentration of each drug in the combination drug group is one-twentieth of the LD50. 50 The specific values are as follows:

[0134] ​Solid tumor experiment: blank control group, no tumor cells, from the third day, every other day intraperitoneal injection of normal saline 0.2 mL / 20 g; model group, after tumor cells from the third day, every other day intraperitoneal injection of normal saline 0.2 mL / 20 g; each drug group, after tumor cells from the third day, every other day intraperitoneal injection of corresponding drugs, 0.2 mL / 20 g; combination drug group, after tumor cells from the third day, every other day intraperitoneal injection of prescription 1 drugs, 0.2 mL / 20 g; a total of 5 times, the second day after the last administration, all animals were sacrificed, a total of 12 days (see Figure 26A for administration process). The specific administration concentration is as follows Table 1:

[0135] Table 1: Eight TOPO II inhibitors and combination (prescription 1) for H22 solid tumor experiment administration concentration (dose)

[0136] 1.2.3 Index detection

[0137] 1.2.3.1 Observation of animal growth state

[0138] At 8:30 every morning, observe the mental state, appetite, water intake, fur, activity and excretion growth state, and make records. The solid tumor experiment animal growth state observation is 12 days. The measurement and observation period of all the indicators is consistent with the animal growth state observation period.

[0139] 1.2.3.2 Body weight measurement:

[0140] All groups of mice were weighed from 9:30 to 10:30 every morning, and the changes in body weight were observed.

[0141] 1.2.3.3 Daily food and water intake determination

[0142] Each group of mice was given 150 g of food per day, and the remaining food was weighed at 9 o'clock the next day. 200 mL of drinking water was given daily, and the remaining drinking water was measured at 9 o'clock the next day.

[0143] 1.2.3.4 In vitro measurement of tumor volume:

[0144] All tumor cell inoculated mice were measured once every two days (measurement frequency: 1 time / 2 days, measurement started from the third day; see Figure 26A), the change of tumor volume was observed, and the tumor growth curve was drawn. The tumor volume formula:

[0145] V = L (mm) x I 2 (mm) / 2, calculated, wherein L is the long diameter of the tumor, and I is the short diameter of the tumor.

[0146] 1.2.3.5 Blood test:

[0147] All animals of solid tumor experiment were killed. Before the animals were killed, the eyeball was taken out to get blood, 20 μL / mouse, and added to diluent (pure water, anhydrous sodium sulfate, Tris), and sent to the animal science department to detect blood items. It was used to reflect bone marrow suppression. Detection indexes: white blood cells, platelets.

[0148] 1.2.3.6 Serum enzyme indexes:

[0149] Before the animals were killed, the eyeball was taken out to get blood, centrifuged to get supernatant, and the serum was reserved to detect lactate dehydrogenase (LDH), creatine kinase (CK), and to reflect myocardial injury. At the same time, glutamic-pyruvic transaminase (ALT), glutamic-oxalacetic transaminase (AST), alkaline phosphatase (ALP), total protein (TP), albumin (ALB), and other indexes reflecting liver function, and blood urea (UREA), serum creatinine (SCr), and other indexes reflecting kidney function were detected.

[0150] 1.2.3.7 Tumor mass weighing:

[0151] After the animals were killed, the tumor part was separated, washed with normal saline, dried with filter paper, weighed, the tumor volume was measured, and a photo was taken. It was used to reflect tumor suppression.

[0152] 1.2.3.8 Photographing:

[0153] After the animals were killed, they were dissected and photographed, and whether the organs were congested was observed to reflect the toxic side effects.

[0154] 1.2.3.9 Weighing of each organ:

[0155] After the animals were killed, they were dissected, and the heart, liver, thymus, spleen, and kidney were taken out and weighed. It was used to reflect the damage of each organ.

[0156] 1.2.3.10 Experimental data processing and analysis

[0157] All data were expressed as mean ± standard deviation (x ± s), and SPSS 17.0 software was used for difference analysis of experimental results (p value detection), and p<0.05 was statistically significant. Mortality was compared by chi-square tests. Other parameters were compared by one-way ANOVA.

[0158] The initial number of animals in each group of solid tumor (eight) experiment was 10. After the experiment, statistical analysis was performed. The number of animals in the blank group, model group, and combination drug group was 10 (n=10); the number of animals in the pirarubicin group was 9 (n=9); the number of animals in the doxorubicin group, amsacrine group, and ararubicin group was 8 (n=8); the number of animals in the idarubicin group and teniposide group was 7 (n=7); the number of animals in the epirubicin group was 6 (n=6); and the number of animals in the idarubicin group was 5 (n=5).

[0159] Calculation of each change rate:

[0160] 1.3. Experimental results

[0161] 1.3.1 Anti-tumor efficacy

[0162] 1.3.1.1 Tumor inhibition effect in solid tumor (eight drugs) experiment

[0163] (1) Tumor volume and tumor weight

[0164] The results of tumor volume and tumor weight are shown in Figure 1 and Table 2.

[0165] Table 2: Effect of eight TOPO II inhibitors and their combinations on H22 solid tumor Note: The dosages are shown in Table 1. The dosages in the following tables of this example are the same. The difference compared with the model group has statistical significance (p<0.01) ## The difference compared with the model group has statistical significance (p<0.05) # The difference compared with the combination drug group has statistical significance (p<0.01) △△ The difference compared with the combination drug group has statistical significance (p<0.05) △ .

[0166] The tumor volume of each drug administration group compared with the model group had statistical significance (p<0.01, and p<0.05 for the ararubicin group and the pirarubicin group). This indicates that after the intervention of each drug administration group, the tumor volume significantly decreased.

[0167] Among them, in terms of the decrease in tumor volume, the combination drug group (Prescription 1) was 69.6%, 26.2%, 42.5%, 47.3%, 163.1%, 136.5%, 85.2%, and 168.3% greater than the doxorubicin group, topoisomerase group, idarubicin group, amsacrine group, ararubicin group, teniposide group, epirubicin group, and pirarubicin group, respectively, indicating that the combination drug group had the best effect on inhibiting tumor volume growth.

[0168] As shown in Figure 1, the tumor volume of each group increased over time, and the combination drug group had the slowest growth rate.

[0169] As shown in Table 2, in terms of tumor weight, each administration group had a statistically significant difference (p<0.01) compared with the model group. This indicates that after the intervention of each administration group, the tumor weight was significantly reduced. In terms of the reduction amplitude, the combined drug group was increased by 95.2%, 41.4%, 57.7%, 70.8%, 115.8%, 95.2%, 95.2%, and 86.4% compared with the doxorubicin group, etoposide group, idarubicin group, amsacrine group, aclarubicin group, teniposide group, epirubicin group, and pirarubicin group, respectively. This indicates that the combined drug group has the best effect on inhibiting tumor weight growth.

[0170] (2) Tumor index and tumor inhibition rate

[0171] The results of the tumor index and tumor inhibition rate are shown in FIG. 2, FIG. 3, and Table 3.

[0172] Table 3: Effects of eight TOPO II inhibitors and their combinations on H22 solid tumors Note: The difference has statistical significance (p<0.01) compared with the model group ## The difference has statistical significance (p<0.05) compared with the model group # The difference has statistical significance (p<0.01) compared with the combined drug group △△ The difference has statistical significance (p<0.05) compared with the combined drug group △ .

[0173] As shown in FIG. 2 and Table 3, the tumor index of the combined drug group (p<0.01), amsacrine group (p<0.01), and pirarubicin group (p<0.05) had a statistically significant difference compared with the model group. Among them, the tumor index of the combined drug group was the smallest, indicating that the combined drug group had the best tumor inhibition effect.

[0174] As shown in FIG. 3 and Table 3, the tumor inhibition rate of the combined drug group had a statistically significant difference (p<0.01, corresponding p<0.05 for the idarubicin group) compared with other administration groups (except the etoposide group). The tumor inhibition rate of the combined drug group was 54.3%, which was much higher than that of other single administration groups, specifically: 101.1% higher than the doxorubicin group, 57.4% higher than the idarubicin group, 74.6% higher than the amsacrine group, 120.7% higher than the aclarubicin group, 87.2% higher than the teniposide group, 93.9% higher than the epirubicin group, and 91.2% higher than the pirarubicin group. This indicates that the anti-tumor effect of the combined drug group is significantly better than that of each single administration group.

[0175] The difference of the tumor inhibition rate between the combination drug group and the etoposide group was not statistically significant (P>0.05), but compared with the specific value, the tumor inhibition rate of the combination drug group was 42.5% higher than that of the etoposide group, indicating that the anti-tumor activity of the combination drug group was also superior to that of the etoposide group.

[0176] In summary, the tumor volume, tumor weight, tumor index and tumor inhibition rate showed that the anti-tumor effect of the combination drug group was significantly better than that of each single drug group.

[0177] In summary, combined with the four indicators of tumor volume, tumor weight, tumor index and tumor inhibition rate, it is suggested that the drug concentration of each single drug group is only one fourth of its LD 50 , but its anti-tumor effect is still significantly lower than that of the combination drug group (the dose of each drug is only one twentieth of its LD 50 ).

[0178] 1.3.2 Study of toxic side effects

[0179] 1.3.2.1 Toxic side effects of eight drug solid tumor experiments

[0180] (1) Survival rate

[0181] Among all the indicators of reaction toxic side effects, the survival rate is the most important one, and the survival of each group is shown in Table 4 and Figure 6, and the specific time of death of each single drug group is shown in Table 5.

[0182] Table 4: Effect of eight TOPO II inhibitors and their combinations on the survival of H22 solid tumor mice Note: The difference is statistically significant (p<0.01) compared with the blank group ** , the difference is statistically significant (p<0.05) compared with the blank group * ; the difference is statistically significant (p<0.01) compared with the model group ## , the difference is statistically significant (p<0.05) compared with the model group # , the difference is statistically significant (p<0.01) compared with the combination drug group △△ , the difference is statistically significant (p<0.05) compared with the combination drug group △ .

[0183] Table 5: Effect of eight TOPO II inhibitors and their combinations on the number of deaths of H22 solid tumor mice

[0184] From the survival rate of each group in Table 4 and Figure 6, it can be seen that no animal died in the combination drug group, the blank group and the model group during the 10 days of administration (12 days of observation from the day of tumor inoculation), and there was no difference in survival rate. However, 1-5 mice died in each of the other administration groups from the 10th day after tumor inoculation. Compared with the doxorubicin group, the etoposide group, the idarubicin group, the amsacrine group, the aclarubicin group, the teniposide group, the epirubicin group and the pirarubicin group, the survival rate of the combination drug group (Prescription 1) was increased by 25.0%, 42.9%, 100.0%, 25.0%, 42.9%, 25.0%, 66.7% and 11.1%, respectively. This suggests that the administration method of the combination drug group can greatly reduce the toxic side effects of each drug and improve the survival rate.

[0185] (2) Growth state

[0186] The right limb axillary tumor of the mice after successful inoculation of tumor cells was obvious, and the mental state of the mice in the blank group and the model group was good. Compared with the model group and the blank group, the activity of the mice in the combination drug group was more flexible, the fur was bright and beautiful, the body state was good, and there was no obvious difference in the state of the mice.

[0187] However, the other administration groups showed a sharp contrast, i.e. the fur was not smooth, the mental state gradually deteriorated during the administration period, the resistance gradually weakened when being grabbed, and phenomena such as less movement, piloerection and reduced food intake were common, and the number of deaths gradually increased, suggesting that the toxic side effects of each individual administration group were obvious.

[0188] (3) Gastrointestinal reaction

[0189] Gastrointestinal reaction is a common toxic side effect of antitumor drugs. In order to evaluate the gastrointestinal reaction of each administration group, three indicators closely related to the gastrointestinal tract, i.e. body weight, daily food intake and daily water intake, were selected for evaluation, and the specific results are shown in Table 6 and Figures 7, 8 and 9.

[0190] Table 6: Effect of eight TOPO II inhibitors and their combinations on the gastrointestinal tract of H22 solid tumor mice Note: The difference was statistically significant (p<0.01) compared with the blank group ** The difference was statistically significant (p<0.05) compared with the blank group * The difference was statistically significant (p<0.01) compared with the model group ## The difference was statistically significant (p<0.05) compared with the model group # The difference was statistically significant (p<0.01) compared with the combination drug group △△ The difference was statistically significant (p<0.05) compared with the combination drug group △ .

[0191] 1) As can be seen from Table 6, the weight loss of the combination drug group is obvious compared with the blank group and the model group, and the difference is statistically significant (p<0.01). It is suggested that the mice in the combination drug group are indeed troubled by gastrointestinal side effects to a certain extent, and the weight loss is 28.5% compared with the blank group and 22.2% compared with the model group.

[0192] 2) As can be seen from Figure 7, the weight loss of the idarubicin group is the most in the single drug administration group, and the weight loss is 26.7% compared with the blank group and 20.3% compared with the model group, and the difference is statistically significant (p<0.01), which suggests that idarubicin is also troubled by gastrointestinal reactions to a certain extent.

[0193] The weight loss of the other administration groups (except the amsacrine group) is certain compared with the model group, that is, the weight loss of the doxorubicin group is 10.5%, the etoposide group is 17.3%, the idarubicin group is 20.3%, the aclarubicin group is 17.0%, the teniposide group is 11.4%, the epirubicin group is 17.0%, and the pirarubicin group is 7.8%, and the difference is statistically significant (p<0.01). At the same time, combined with the growth state of the mice in these groups, it is judged that the mice in these groups are also affected by side effects.

[0194] 3) As can be seen from Table 6, the daily food intake of a single mouse in the combination drug group is statistically different compared with the blank group and the model group (p<0.05). It is suggested that the weight loss of the mice in the combination drug group is obvious, which may be related to the decrease of food intake.

[0195] 4) In the single drug administration group, the daily food intake of a single mouse in the epirubicin group is the smallest, and the decrease is 35.0% compared with the model group, and the difference is statistically significant (p<0.01). Combined with the weight of this group, it is suggested that the epirubicin group is indeed troubled by gastrointestinal reactions to a certain extent.

[0196] As can be seen from Figure 8, from the seventh day, the food intake of the combination drug group shows an obvious upward trend and is better than that of the idarubicin group and the epirubicin group, which shows that the gastrointestinal reactions of the mice in the combination drug group are gradually alleviated.

[0197] 5) As can be seen from Table 6, the water intake of each group is not statistically different (p>0.05), which suggests that the gastrointestinal reactions of each group are different, but have not caused serious impact. The water intake of epirubicin is the least, and the combination drug group is the second.

[0198] As can be seen from Figure 9, the water intake of the combination drug group shows an obvious upward trend from the seventh day, and is better than that of the idarubicin group and the epirubicin group, which shows that the gastrointestinal reactions of the mice in the combination drug group are gradually alleviated.

[0199] Combining weight, daily food intake and daily water intake, it is shown that idarubicin and epirubicin can cause certain gastrointestinal reactions, and the combination drug group also has certain gastrointestinal reactions, but the degree is not serious. The cause of this side effect may be from idarubicin and epirubicin in the combination drug group.

[0200] Compared with idarubicin and epirubicin, the gastrointestinal reaction of the combination drug group is better than the two groups.

[0201] (4) Myelosuppression

[0202] Myelosuppression is the most common side effect of antitumor drugs. Previous literature research shows that the drugs selected in this study will cause different degrees of myelosuppression. In this experiment, blood routine test was used to evaluate whether myelosuppression occurred in each drug group. The decrease of white blood cell count and platelet count indicates the occurrence of myelosuppression. The specific results are shown in Table 7 and Figures 10-11.

[0203] Table 7: Effect of eight TOPO II inhibitors and their combinations on myelosuppression of H22 solid tumor mice Note: The difference is statistically significant (p<0.01) compared with the blank group ** The difference is statistically significant (p<0.05) compared with the blank group * The difference is statistically significant (p<0.01) compared with the model group ## The difference is statistically significant (p<0.05) compared with the model group # The difference is statistically significant (p<0.01) compared with the combination drug group △△ The difference is statistically significant (p<0.05) compared with the combination drug group △ .

[0204] 1) As shown in Table 7, compared with the model group, the white blood cell (Figure 10) and platelet (Figure 11) of the blank group had no statistically significant difference (p>0.05), indicating that after tumor inoculation, no myelosuppression occurred;

[0205] 2) Compared with the model group, the white blood cell of the combination drug group decreased by 25.5% and the platelet decreased by 18.5%, which had statistical significance (p<0.01) and no statistical significance (p>0.05), respectively, indicating that the combination drug group had a slight trend of myelosuppression;

[0206] The other groups were as follows: the idarubicin group had statistically significant differences in white blood cell count and platelet count compared with the model group (p<0.01), which decreased by 40.4% and 35.4%, respectively; the amsacrine group also had statistically significant differences in white blood cell count and platelet count compared with the model group (p<0.05), which decreased by 26.6% and 21.3%, respectively; the doxorubicin group (p<0.05) and the pirarubicin group (p<0.01) had statistically significant differences in white blood cell count compared with the model group, which decreased by 21.3% and 24.5%, respectively; the etoposide group and the epirubicin group had statistically significant differences in platelet count compared with the model group (p<0.05), which decreased by 13.9% and 22.2%, respectively; the aclarubicin group and the teniposide group had no statistically significant differences in white blood cell count and platelet count compared with the model group (p>0.05). This suggests that the idarubicin and amsacrine groups have severe bone marrow suppression, and the toxicities are greater than those of the combined drug group; the doxorubicin group, the pirarubicin group, the etoposide group, and the epirubicin group have a tendency of bone marrow suppression, and the toxicities are comparable to those of the combined drug group. The aclarubicin group and the teniposide group have a slight tendency of bone marrow suppression, and the toxicities are weaker than those of the combined drug group.

[0207] The degree of bone marrow suppression of each group above is higher or lower than that of the combined drug group, and the difference is not statistically significant (p>0.05). Although the difference between the idarubicin group and the combined drug group is not statistically significant (p>0.05), the white blood cell count of the idarubicin group is 20.0% less than that of the combined drug group, and the platelet count is 20.7% less than that of the combined drug group, which indicates that the combined drug group has a great relief in bone marrow suppression compared with the idarubicin group, which has the most severe bone marrow suppression.

[0208] In summary, the combined drug group has a certain tendency of bone marrow suppression, and each drug group also has a certain degree of bone marrow suppression. In terms of severity, the idarubicin group is the most severe, and the combined drug group has a certain relief compared with the idarubicin group.

[0209] (5) Immune system damage

[0210] After the mice are inoculated with tumors, the immune system will respond to the intervention process and may also be damaged by the drugs. The immune system involves the following indicators: thymus index (Figure 12) and spleen index (Figure 13). This study will evaluate these two indicators, and a decrease in the two indicators indicates that the immune system is damaged. The specific results are as follows in Table 8:

[0211] Table 8: Effects of eight TOPO II inhibitors and their combinations on the immune system of H22 solid tumor mice Note: The difference compared with the blank group is statistically significant (p<0.01) **The difference has statistical significance (p<0.05) compared with the blank group * The difference has statistical significance (p<0.01) compared with the model group ## The difference has statistical significance (p<0.05) compared with the model group # The difference has statistical significance (p<0.01) compared with the combination drug group △△ The difference has statistical significance (p<0.05) compared with the combination drug group △ .

[0212] From Table 8, it can be seen that,

[0213] 1) Compared with the blank group, the thymus index of the model group increased by 17.6%, and the spleen index increased by 93.6%, indicating that after inoculation of tumor, the immune system was stimulated by foreign substances, and the immunity was improved to cope with the invasion of foreign substances;

[0214] 2) Compared with the model group, the thymus index of each administration group decreased, and the numerical value had statistical significance (p<0.01, p<0.05 for the pirarubicin group). It can be seen that after administration, the immune system was damaged, and the damage degree of each administration group was different. In order, the combination drug group decreased by 35.0%, the doxorubicin group decreased by 55.0%, the etoposide group decreased by 65.0%, the idarubicin group decreased by 45.0%, the amsacrine group decreased by 50.0%, the aclarubicin group decreased by 60.0%, the teniposide group decreased by 60.0%, the epirubicin group decreased by 55.0%, and the pirarubicin group decreased by 25.0%. It is indicated that the toxicity of the etoposide group and the aclarubicin group is greater than that of the combination drug group; the toxicity of the remaining administration groups is greater than that of the combination drug group except for the pirarubicin group.

[0215] 3) In the spleen index, the combination drug group decreased by 23.1% (but the difference with the model group had no statistical significance), the doxorubicin group decreased by 38.5%, the etoposide group decreased by 40.7%, the idarubicin group decreased by 40.7%, the amsacrine group decreased by 29.7%, the aclarubicin group decreased by 28.6%, the teniposide group increased by 1.1%, the epirubicin group decreased by 16.5%, and the pirarubicin group decreased by 26.4%. It can be seen that the toxicity of the remaining administration groups is greater than that of the combination drug group except for the teniposide group and the epirubicin group. In terms of the thymus index and the spleen index, the damage of the combination drug group is the lightest.

[0216] 4) Compared with the combination drug group, the thymus index of each administration group showed that the difference between the etoposide group, the aclarubicin group and the combination drug group had statistical significance (p<0.05), which was 46.2% and 38.5% lower than the combination drug group, respectively. The spleen index showed that the difference between each administration group and the combination drug group had no statistical significance (p>0.05). It is indicated that the toxicity of the combination drug group is smaller in terms of immune damage.

[0217] In summary, the immune system of each administration group was damaged to some extent, and the combined drug group was the least damaged.

[0218] (6) Cardiotoxicity

[0219] Previous literature research showed that doxorubicin, idarubicin, amsacrine, arabinosyl aclarubicin, epirubicin, pirarubicin and other drugs have cardiotoxicity (no cardiotoxicity was reported for etoposide and teniposide). In the experiment, the heart index (Figure 14) and LDH (Figure 15) and CK (Figure 16) were used to evaluate the cardiotoxicity of each group of mice, and the results were as follows:

[0220] 1) Heart index

[0221] As shown in Table 9, the heart index of the combined drug group had no statistically significant difference (p>0.05) compared with the blank group, the model group and the other individual administration groups, indicating that the combined drug group did not show myocardial damage, and the other administration groups also did not show significant myocardial damage.

[0222] Table 9: Effect of eight TOPO II inhibitors and their combinations on the heart index of H22 solid tumor mice Note: The difference was statistically significant (p<0.01) compared with the blank group ** The difference was statistically significant (p<0.05) compared with the blank group * The difference was statistically significant (p<0.01) compared with the model group ## The difference was statistically significant (p<0.05) compared with the model group # The difference was statistically significant (p<0.01) compared with the combined drug group △△ The difference was statistically significant (p<0.05) compared with the combined drug group △ .

[0223] 2) Serum enzymatic indicators

[0224] The increase of LDH and CK indicates heart damage. As shown in Table 10:

[0225] (i) The difference in LDH between the model group and the blank group was statistically significant (p<0.01), and LDH, as a common tumor marker, increased in value, indicating successful inoculation. The difference in CK between the model group and the blank group was not statistically significant (p>0.05). The two indicators of LDH and CK (Figures 15-16) together indicated that the model group did not have myocardial damage.

[0226] (ii) Compared with the model group, only the doxorubicin group and the idarubicin group had statistically significant differences in LDH and CK, suggesting that the two groups would cause cardiac injury at the corresponding concentration. Specifically, compared with the model group, the LDH of the doxorubicin group increased by 48.5%, and the CK increased by 25.5%. Compared with the model group, the LDH of the idarubicin group increased by 115.5%, and the CK increased by 29.7%, suggesting that the two groups had severe myocardial injury. The combined drug group had no statistically significant difference in LDH and CK compared with the model group (p>0.05), suggesting that the combined drug group did not have myocardial injury.

[0227] (iii) Compared with the combined drug group, the LDH of the doxorubicin group and the idarubicin group had statistically significant differences (p<0.01). The CK of the doxorubicin group and the idarubicin group had statistically significant differences (p<0.05). Specifically, the doxorubicin group and the combined drug group had a large difference in the amplitude of increase compared with the model group. The combined drug group had a lower increase in LDH and CK than the doxorubicin group by 89.1% and 92.6%, respectively. The idarubicin group and the combined drug group also had a large difference in the amplitude of increase compared with the model group. The combined drug group had a lower increase in LDH and CK than the idarubicin group by 95.4% and 93.6%, respectively. This suggests that the combined drug group reduces the degree of myocardial injury by about 90% compared with the doxorubicin group and the idarubicin group, which have severe myocardial injury, and does not show myocardial injury.

[0228] In summary, the doxorubicin group and the idarubicin group have the most severe damage, the combined drug group does not have myocardial injury, and the remaining drug groups also do not show myocardial injury.

[0229] Table 10 Effects of eight TOPO II inhibitors and their combinations on myocardial injury-related serum enzymes in H22 solid tumor mice Note: The difference was statistically significant compared with the blank group (p<0.01) ** The difference was statistically significant compared with the blank group (p<0.05) * The difference was statistically significant compared with the model group (p<0.01) ## The difference was statistically significant compared with the model group (p<0.05) # The difference was statistically significant compared with the combined drug group (p<0.01) △△ The difference was statistically significant compared with the combined drug group (p<0.05) △ .

[0230] (7) Hepatotoxicity

[0231] Liver is the site of drug metabolism, relatively easy to be injured by drugs. In this experiment, liver index, serum enzyme indicators AST, ALT, ALP, TP, ALB were used to evaluate whether the liver was damaged by each drug group.

[0232] 1) Liver index

[0233] The liver index (Figure 17) can be used to compare and intuitively judge whether the liver is damaged. From Table 11, we can know that:

[0234] (i) Compared with the blank group, the difference in liver index was statistically significant (p<0.01), and the liver index increased by 28.6%, indicating that after inoculation of tumor, the liver was slightly swollen;

[0235] (ii) Compared with the model group, the liver index of the aclarubicin group and the teniposide group had statistically significant difference (p<0.05) compared with the model group. Among them, the liver index of the aclarubicin group decreased by 12.3%, and the teniposide group decreased by 13.2%, indicating that the liver function of the aclarubicin group and the teniposide group had an abnormal trend, and the liver index of the rest of the groups compared with the model group did not show abnormality;

[0236] (iii) The difference in liver index between each single drug group and the combination drug group was not statistically significant, indicating that compared with the single drug group, the liver of the combination drug group was not abnormal.

[0237] Table 11 Effect of eight TOPO II inhibitors and their combinations on liver index of H22 solid tumor mice Note: The difference was statistically significant compared with the blank group (p<0.01) * The difference was statistically significant compared with the blank group (p<0.05) * The difference was statistically significant compared with the model group (p<0.01) ## The difference was statistically significant compared with the model group (p<0.05) # The difference was statistically significant compared with the combination drug group (p<0.01) △△ The difference was statistically significant compared with the combination drug group (p<0.05) △ .

[0238] 2) Serum enzyme indicators

[0239] Alanine aminotransferase (ALT) (Figure 18) and aspartate aminotransferase (AST) (Figure 19), distributed within glutamate cells, increase when hepatocellular cells are damaged, and the degree of increase corresponds to the severity of hepatocellular damage. Significantly elevated alkaline phosphatase (ALP) (Figure 20) often indicates biliary obstruction, suggesting possible cholestatic hepatitis or extrahepatic biliary obstruction. Decreased total protein (TP) (Figure 21) and albumin (ALB) (Figure 22) values ​​often indicate weakened hepatic protein synthesis capacity, and the degree of reduction is directly proportional to the severity of liver disease.

[0240] As shown in Table 12:

[0241] (i) Compared with the control group, the differences in ALP, TP, and ALB (p>0.05) were not statistically significant, while the differences in ALT and AST were statistically significant (p<0.01). This suggests that tumor inoculation can cause liver inflammation and lead to liver swelling.

[0242] (ii) Differences were observed in various indicators between the doxorubicin and model groups. Specifically, compared with the model group, the doxorubicin group showed statistically significant differences in ALT (p < 0.01) and AST (p < 0.05); the idarubicin group showed statistically significant differences in AST (p < 0.05) and ALP.

[0243] The differences in the two indicators (p < 0.01) were statistically significant; the differences in AST (p < 0.01) in the arubicin group and in AST (p < 0.05) in the pirarubicin group were statistically significant; the difference in TP (p < 0.05) in the epirubicin group was statistically significant. This indicates that all groups showed varying degrees of liver damage, with the most severe cases in the doxorubicin and idarubicin groups. Among the five indicators reflecting liver function, there were no statistically significant differences between the combination drug group and the model group, suggesting that the combination drug group did not cause liver damage.

[0244] (iii) Compared with the combination drug group, the changes in the five liver function indicators differed among the single-drug groups, as follows: For ALT, the difference between the combination drug group and the model group was 82.9% smaller than that between the doxorubicin group and the model group, meaning that the liver damage caused by the combination drug group was reduced by 82.9% compared to the doxorubicin group (the same applies below); for ALB, the difference between the combination drug group and the model group was 32.8% smaller than that between the doxorubicin group and the model group. For AST, the change in the combination drug group was 78.3% smaller than that in the alarubicin group, and for ALP, the change in the combination drug group was 95.2% smaller than that in the idarubicin group. These results suggest that the combination drug group significantly reduced liver damage compared to the single-drug groups with severe liver injury.

[0245] In summary, each individual administration group showed a certain degree of liver injury, among which the doxorubicin group and the idarubicin group were the most serious, while the combined drug group did not show obvious liver injury.

[0246] Table 12 Effects of eight TOPO II inhibitors and their combinations on serum enzymatic indexes related to liver injury of H22 solid tumor mice Note: The difference has statistical significance (p<0.01) compared with the blank group ** The difference has statistical significance (p<0.05) compared with the blank group * The difference has statistical significance (p<0.01) compared with the model group ## The difference has statistical significance (p<0.05) compared with the model group # The difference has statistical significance (p<0.01) compared with the combined drug group △△ The difference has statistical significance (p<0.05) compared with the combined drug group △ .

[0247] (8) Nephrotoxicity

[0248] The kidney is relatively easy to be damaged by drugs, just like the liver. In this experiment, the kidney index (Figure 23), serum urea (UREA) (Figure 24), and serum creatinine (SCr) (Figure 25) were used to evaluate whether each administration group caused damage to the kidney.

[0249] 1) Kidney index

[0250] From Table 13, it can also be seen that:

[0251] (i) The difference in kidney index between each administration group and the model group was not statistically significant (p>0.05), indicating that each administration group did not show abnormalities in kidney index;

[0252] (ii) The difference in kidney index between each individual administration group and the combined drug group was not statistically significant (p>0.05), indicating that each administration group did not show abnormalities in kidney index.

[0253] Table 13 Effects of eight TOPO II inhibitors and their combinations on kidney index of H22 solid tumor mice Note: The difference has statistical significance (p<0.01) compared with the blank group ** The difference has statistical significance (p<0.05) compared with the blank group * The difference has statistical significance (p<0.01) compared with the model group ## The difference has statistical significance (p<0.05) compared with the model group# The difference has statistical significance (p<0.01) compared with the combination drug group △△ The difference has statistical significance (p<0.05) compared with the combination drug group △ .

[0254] 2) Serum enzyme indicators

[0255] The increase of UREA and SCr indicates abnormal kidney. As shown in Table 14,

[0256] (i) Among the two indicators of UREA and SCr, the difference between the model group and the blank group has no statistical significance (P>0.05), indicating that inoculation of tumor does not cause abnormal kidney.

[0257] (ii) Compared with the combination drug group, different drugs show differences in different indicators in each single drug group. Specifically, compared with the model group, the doxorubicin group has statistical significance in UREA (p<0.01) and SCr (p<0.05); the aclarubicin group has statistical significance in UREA (p<0.01); the etoposide group has statistical significance in SCr (p<0.01); the idarubicin group has statistical significance in SCr (p<0.05). It indicates that each group has different degrees of kidney dysfunction, and the most serious one is the doxorubicin group. In the two indicators reflecting kidney function, the difference between the combination drug group and the model group has no statistical significance, indicating that the combination drug group does not cause abnormal kidney function.

[0258] (iii) Compared with the combination drug group, the data of each single drug group and the difference from the model group fluctuate. Among them, the statistically significant fluctuations are: in the SCr indicator, the fluctuation of the combination drug group is 80.4% lower than that of the etoposide group and 85.0% lower than that of the idarubicin group. It indicates that compared with each drug with serious kidney damage, the combination drug group has a significant decrease in kidney damage.

[0259] In summary, each drug group shows a certain degree of kidney damage, among which the doxorubicin group is the most serious, while the combination drug group does not show obvious kidney damage.

[0260] Table 14 Effect of eight TOPO II inhibitors and their combinations on serum indicators related to kidney damage in H22 solid tumor mice Note: The difference has statistical significance (p<0.01) compared with the blank group ** The difference has statistical significance (p<0.05) compared with the blank group * The difference has statistical significance (p<0.01) compared with the model group ##The difference has statistical significance (p<0.05) compared with the model group # The difference has statistical significance (p<0.01) compared with the combination drug group △△ The difference has statistical significance (p<0.05) compared with the combination drug group △ .

[0261] In conclusion, in the selected eight types of toxic side effects, the growth state, cardiac toxicity, liver toxicity, kidney toxicity, mortality and the like, the combination drug group and each single administration group do not show toxic side effects in most indexes, or the toxic side effects are not obviously enhanced compared with the single administration group, and the overall trend is that the toxicity is reduced. It is proved that the drug composition of the application has low toxic side effects.

[0262] Example 3: Anti-tumor effect of the drug composition of the application in mouse ascitic tumor experiment

[0263] 1.1 Experimental materials, reagents and instruments

[0264] Referring to Example 2, if not specifically stated, it is basically the same as Example 2.

[0265] 1.2. Experimental method

[0266] Referring to Example 2, if not specifically stated, it is basically the same as Example 2.

[0267] 1.2.1 Preparation of main reagents

[0268] The administration concentration of each drug in the single administration group of the ascitic tumor (eight drugs) experiment is one-twentieth of its LD 50 The administration concentration of each drug in the single administration group of the ascitic tumor (eight drugs) experiment is one-twentieth of its LD 50 .

[0269] 1.2.2 Tumor inoculation

[0270] Ascitic tumor inoculation: under sterile conditions, the ascitic fluid of tumor-bearing mice was extracted, tumor cells were counted under a microscope, and the tumor cells were adjusted to 3×10 6 cells / mL with normal saline, and then 0.2 mL / 20 g was injected into the abdominal cavity of each mouse.

[0271] 1.2.3 Animal grouping

[0272] Ascitic tumor experiment: ICR mice were inoculated with tumor cells, 48 h after inoculation, the animals were weighed and randomly divided into groups, 10 animals in each group, a total of 10 groups. Model group, doxorubicin group, etoposide group, idarubicin group, amsacrine group, arabinosyl cytosine group, teniposide group, epirubicin group, pirarubicin group, combined drug group (doxorubicin group, etoposide group, idarubicin group, amsacrine group, teniposide group, arabinosyl cytosine group, pirarubicin group, epirubicin group, a total of eight groups).

[0273] 1.2.4 Administration method, route and concentration

[0274] The concentration of each drug in the ascitic tumor experiment alone administration group was 1 / 20 of its LD 50 The concentration of each drug in the ascitic tumor experiment alone administration group was 1 / 20 of its LD 50 The specific values are as follows:

[0275] Ascitic tumor experiment: model group, after inoculation with tumor cells, from the third day, intraperitoneal injection of normal saline 0.2 mL / 20g every other day; each administration group, after inoculation with tumor cells, from the third day, intraperitoneal injection of the corresponding drug, 0.2 mL / 20g every other day; combined drug group, after inoculation with tumor cells, from the third day, intraperitoneal injection of the corresponding drug, 0.2 mL / 20g every other day; a total of 5 times of administration, a total of 10 days (see Figure 26B for administration procedure). The administration concentration is as follows:

[0276] Table 15 Eight TOPO Ⅱ inhibitors and their combinations for ascitic tumor experiment

[0277] 1.2.5 Index detection

[0278] The growth state of ascitic tumor (eight drugs) experiment animals was observed for 20 days.

[0279] 1.2.5.1 Survival days of ascitic tumor model mice, life extension rate

[0280] The death time of animals in each group was counted to determine the survival days, and the average life extension rate of each group was calculated.

[0281] 1.3 Experimental results

[0282] (1) Death record of mice in each group

[0283] The death of each group of mice was recorded, and is shown in Table 16. From the 6th day of the experiment, mice began to die gradually, and by the 10th day, 10 animals in three groups (including the model group) had all died. The combination drug group began to die on the 13th day, while the rest of the groups began to die on the 6th to 8th day. On the last day that the combination drug group did not have any deaths (the 12th day), only one animal in total was still alive in the other groups.

[0284] Table 16: Summary of the death of ascitic tumor mice

[0285] (2) Survival time of each group of mice

[0286] As shown in Figure 4, the average survival time of the combination drug group was 16 days, while the average survival time of the rest of the groups was no more than 10 days. The difference in survival time between the combination drug group and the model group was statistically significant (p<0.01). The differences in survival time between the rest of the groups and the model group were not statistically significant (p>0.05). This indicates that the survival time of mice in the combination drug group was significantly increased after drug intervention. The rest of the groups did not have this effect, indicating that the drugs in each of the individual drug groups did not have an anti-tumor effect at this dosage.

[0287] (3) Survival time extension rate of each group of mice

[0288] As shown in Figure 5, the life extension rate of the combination drug group was 92.8%, and the differences between the combination drug group and the other groups were statistically significant (p<0.01).

[0289] This indicates that the life extension rate of mice in the combination drug group was significantly improved after drug intervention. The rest of the individual drug groups did not have this effect.

[0290] Combining the average survival time and the life extension rate, it is indicated that the combination drug group can significantly extend the life cycle of ascitic tumor mice, and the anti-tumor effect is significantly better than that of each of the individual drug groups (in fact, each of the individual drug groups did not have an anti-tumor effect).

[0291] Example 4: Anti-tumor effect of different prescription drug combinations in mouse solid tumor experiments

[0292] In addition to Prescription 1 described in Examples 2-3, the applicant also studied the anti-tumor effect and toxicity of each of the combinations of the same eight drugs in different proportions (Prescriptions 2-5) in mouse solid tumor experiments. The specific prescriptions are shown in Table 17, and the experimental scheme was the same as in Example 2. The results are shown in Table 18.

[0293] Table 17. Comparison of the prescription doses (μmol / kg) of different prescription combinations

[0294] Table 18. Comparison of anti-tumor effect and toxicity between different formulations of the composition * Values are the percentage change relative to the model group (except for mortality). Percentage = (model group data - each drug group data) / model group data, indicating toxicity when the index of the drug group is reduced, or percentage = (each drug group data - model group data) / model group data, indicating toxicity when the index of the drug group is increased. Bold numbers indicate toxicity. Negative numbers indicate better measurement than the model group. # P < 0.05 and ## P < 0.01. ALB, albumin; ALP, alkaline phosphatase; ALT, glutamic-pyruvic transaminase; AST, aspartate aminotransferase; CK, creatine kinase; LDH, lactate dehydrogenase; SCr, creatinine; TP, total protein.

[0295] As can be seen from the above Table 18, Formulation 2 resulted in 20% of the mice dying while causing severe damage to the liver, heart, kidney, and immune system of the surviving mice, compared to Formulation 1, while the tumor inhibition rate was substantially the same. In summary, the toxicity of Formulation 2 was significantly greater than that of Formulation 1.

[0296] Formulation 3 resulted in 30% of the mice dying while causing severe damage to the liver, heart, kidney, and immune system of the surviving mice, compared to Formulation 1, while the tumor inhibition rate was substantially the same. In summary, the toxicity of Formulation 3 was significantly greater than that of Formulation 1.

[0297] Formulation 4 resulted in 50% of the mice dying while causing the most severe damage to the liver, heart, kidney, and immune system of the surviving mice, compared to Formulation 1, while the tumor inhibition rate was substantially the same or even less. In summary, the toxicity of Formulation 4 was much greater than that of Formulation 1.

[0298] Formulation 5 resulted in 10% of the mice dying while causing severe damage to the immune system of the surviving mice and some damage to the liver and heart, compared to Formulation 1, while the tumor inhibition rate was substantially the same. In summary, the toxicity of Formulation 5 was significantly greater than that of Formulation 1.

[0299] In summary, even if the same eight drugs are combined, and even if the same low doses are used, the combination will exhibit greater toxicity if the ratio of the individual drugs in the combination is outside a certain range. For example, Formulation 2 through Formulation 5 all exhibit significant toxicity, resulting in 10% to 50% mortality in mice, and the surviving mice exhibit significant liver, heart and / or kidney toxicity, and significant immune system damage. These results show that even if the same eight drugs are combined, the individual components need to be properly proportioned to achieve good anti-tumor efficacy and low toxicity.

[0300] Example 5: Comparison of Toxicity of Different Drug Combinations

[0301] 1.1 Purpose of Experiment

[0302] This example compares the pharmacodynamics and toxicology of the A group combination drugs (topotecan, 10-hydroxy camptothecin, 9-amino camptothecin, irinotecan, idarubicin, daunorubicin, doxorubicin, etoposide, teniposide, epirubicin) and the B group combination drugs (doxorubicin, etoposide, idarubicin, amsacrine, aclarubicin, teniposide, epirubicin, pirarubicin) at the cellular level. The focus is on the inhibition of the proliferation of tumor cells (human acute myeloid leukemia cells, HL-60 cells) by the two groups of combination drugs (i.e., anti-tumor efficacy), and the damage to normal cells (rat myocardial cell line, H9C2 cells) (i.e., cardiotoxicity). By comparing the differences in efficacy (against HL-60 cells) and toxicity (against H9C2 cells) between the A group and the B group, the goal is to provide experimental evidence for determining which group of combination drugs is superior in terms of the balance between anti-tumor efficacy and safety.

[0303] The technical ideas and technical routes of this experiment are as follows:

[0304] ① Chemotherapeutic drugs all have significant toxicity, and both groups of combination drugs have some toxicity. To compare which group of drugs has more application prospects, the toxicity of the two groups of drugs can be compared.

[0305] ②Because the number of drug types and the composition of drugs in the two groups of combined drugs are not the same, the total amount of drugs in the two groups of combined drugs is also different. Therefore, it is not easy to obtain an objective and reasonable conclusion by directly comparing the size of the tumor inhibition effect of the two groups of combined drugs. Therefore, we designed a technical solution for comparison experiments, that is, first, we screened the respective drug concentrations of the two groups of drugs that have the same inhibition rate on tumor cells. Then, under the respective concentrations of the two groups of drugs, we compared the toxicity of the two groups of combined drugs to normal cells (expressed by the value of cell survival rate, the larger the value, the smaller the toxicity). The size of the toxicity is used to determine which group of combined drugs has higher application value: that is, in the case of the same anti-tumor effect, the combined drug with smaller toxicity has higher application value.

[0306] 1.2 Experimental materials

[0307] 1.2.1 Experimental consumables

[0308] Human promyelocytic leukemia cells (HL-60 cells; CC-Y1236; ATCC; Shanghai Zephyn Biotech Co., Ltd.); Rat myocardial cells (H9C2 cells; YCL-0349; ATCC; Shanghai Yizhefeng Biotech Co., Ltd.); Streptomycin-penicillin mixture (100x) (P1400, Solabio); Dimethyl sulfoxide (DMSO; D8371, Solabio); Cell Counting Kit-8 kit (Lianji Biological); 1x0.01M phosphate buffer (PBS, pH 7.2-7.4) (P1200, Solabio); 0.25% trypsin (containing EDTA) digestion solution; DMEM complete medium (Shanghai Yizhefeng Biotech Co.); Culture dishes (430167, Corning), 96-well plates (3599, Corning) were purchased from Beijing Shizhi Technology Co., Ltd.; Fetal Bovine Serum (Gibco, A5669701, Thermo Fisher Scientific Co., Ltd.); DMEM basal medium (Gibco, C11960500BT, Thermo Fisher Scientific Co., Ltd.); IMDM basal medium (Gibco, 12440053, Thermo Fisher Scientific Co., Ltd.); 10-Hydroxycamptothecin (HY-N0095), 9-Aminocamptothecin (HY-100309), Topotecan hydrochloride (HY-13768A), Irinotecan (HY-16562); Idarubicin hydrochloride (HY-17381), Mitoxantrone dihydrochloride (HY-13502A), Aclacinomycin A hydrochloride (HY-N2306A), Epirubicin hydrochloride (HY-13624A), Doxorubicin hydrochloride (HY-15142), Pirarubicin Hydrochloride (HY-13725A), Daunorubicin hydrochloride (HY-13062); Teniposide (HY-13761), Etoposide (HY-13629); Amsacrine (HY-13551), all the above drugs were purchased from Shanghai Haoyuan Biological Medicine Technology Co., Ltd., and the purity of each drug was >99% (company provided test report).

[0309] 1.2.2 Experimental instruments

[0310] Multiskan FC (Thermo Scientific); inverted microscope (OLYMPUS, CKX41); centrifuge (LD5-2A, Jingli); autoclave (G154DWS, Qimeixia) high pressure; CO2 incubator (CCL-170B-8, ESCO); MILLI-Q ultrapure water system (ZRXQ010T0, MilliPore); electronic analytical balance (MSA125P-1CE-DU, Sartorius); water bath constant temperature oscillator (SHA, Changzhou Guohua); constant temperature water tank (SHHW21-420, Tianjin Test); cell incubator: ESEO (cell culture; CO2; incubator); Multiskan FC enzyme-labeled instrument (Thermo Fisher Scientific; 51119000).

[0311] 1.3 Experimental steps and content

[0312] 1.3.1 Investigation of tumor cell inhibition rate and normal cell combined drug toxicity using CCK-8 method

[0313] 1 mL of 0.25% trypsin digestion solution containing EDTA was used to aspirate the logarithmic growth period of H9C2 cells in a T25 cell culture bottle for 1 minute and 30 seconds, and then 4 mL of fresh culture medium was added to terminate the digestion and transferred to a 15 mL centrifuge for centrifugation (1000 rpm, 5 min). HL-60 cells as suspended cells do not need to be digested, so the centrifugation operation is directly performed. After centrifugation, fresh culture medium was added for resuspension, and the concentration of HL-60 cell suspension was adjusted to 4x10 5 / mL, and the concentration of H9C2 cell suspension was adjusted to 2x10 4 / mL; 100 μL of each was inoculated into a cell 96-well plate using a pipette. To prevent evaporation of liquid in the edge wells, 100 μL of (PBS) was added to the outer circle of the 96-well plate, and then incubated in the cell incubator for 24 H before drug treatment.

[0314] Take the 96-well plate after 24 h of stable cell growth, set up a blank well without cells, a control group containing cells without drug treatment, and each drug group, with 6 parallel holes in each group. The stock solution of the aforementioned drug was diluted with the base culture medium according to the designed concentration, and was prepared immediately before use. Each well was given 100 μL of drug treatment.

[0315] After drug administration, the 96-well plate was placed in the cell incubator for 24 h, and the next day the color development treatment was performed.

[0316] After 24h of drug reaction, the cell well plate was taken out, 10% CCK-8 solution reagent was added to each well with a total volume of 10%, and it was placed in an incubator for 1.5h, and the color development degree was checked constantly to prevent over incubation. After the incubation was completed, the absorbance OD value was measured at 450nm using a microplate reader.

[0317] The inhibition rate of cells was calculated using the following formula, and the corresponding dose-effect curve of each drug was calculated and plotted using GraPhPad Prism 10.1.2 data processing software.

[0318] 1.3.2 Specific experimental content and steps

[0319] 1.3.2.1 Drug information A group: topotecan, 10-hydroxy camptothecin, 9-amino camptothecin, irinotecan, idarubicin, doxorubicin, daunorubicin, etoposide, teniposide, epirubicin (10 kinds).

[0320] B group: doxorubicin, etoposide, idarubicin, amsacrine, aclarubicin, teniposide, epirubicin, pirarubicin (8 kinds).

[0321] The concentration ratio of the prescription drugs in group A is shown in Tables 19 and 20; the concentration ratio of the prescription drugs in group B is shown in Tables 19 and 21.

[0322] 1.3.2.2 Experimental content

[0323] (1) First, according to the dose-effect curves of the 13 drugs in groups A and B measured in HL-60 tumor cells, different concentrations of each combination drug of groups A and B were combined, and the concentration was adjusted by constant attempts to make the inhibition of the two groups of combination drugs on HL-60 cells reach the same inhibition rate, so as to find out the combination drug groups of groups A and B with the same inhibition rate.

[0324] (2) According to the drug concentration that can exert the same inhibitory effect on tumor cell proliferation, the effects of the combination drugs of groups A and B on H9C2 normal cells were observed using the corresponding concentration, and the toxicity and safety differences of the two combination drugs under the same tumor inhibition rate were observed.

[0325] 1.4 Experimental results

[0326] 1.4.1 Concentration exploration of groups A and B for exerting the same inhibition rate on HL-60 tumor cells

[0327] The study aimed to investigate the interaction between different numbers and drugs of DNA TOPO inhibitors in two groups (A and B) after combination therapy, using the inhibition rate of HL-60 tumor cells by combined drugs as the target observation. First, dose-response curves of 13 drugs in groups A and B on HL-60 cells were used to obtain inhibition rates of 5%, 10%, 20%, and 30% (IC50, IC50, IC50, IC50). 10 IC 20 IC 30 The corresponding concentration values ​​are shown in Table 19, and the corresponding concentrations at each inhibition rate are diluted at different ratios. That is, the combination concentrations of each drug in group A are as follows: IC50 values ​​for each drug. 05 The corresponding concentrations were diluted 0.25 times, 0.5 times, and 0.75 times respectively, and the IC50 values ​​of each drug were analyzed. 20 The corresponding concentrations were diluted 0.25, 0.5, and 0.75 times and combined accordingly (Table 20); the concentrations of each combination in group B were as follows: IC50 of each drug. 10 The corresponding concentrations were determined by combining 0.25-fold, 0.5-fold, and original concentrations, and the IC50 values ​​of each drug were analyzed. 20 The corresponding concentrations were diluted 0.25 times and 0.5 times respectively for combination, and the IC50 of each drug was evaluated. 30 The drugs were combined by diluting them 0.25 times at the corresponding concentrations (Table 21). Next, the drugs were combined according to different dilution ratios and administered to HL-60 cells to observe cell viability (Figures 27-28).

[0328] Table 19. Concentrations (μM) corresponding to the respective inhibition rates of the two drug groups.

[0329] Table 20. Dosage concentration (μM) for Group A

[0330] Table 21. Dosage concentration (μM) in Group B

[0331] Figure 27 shows the survival rate of HL-60 tumor cells in groups A and B under different combinations. Figure 27-I shows the inhibition of HL-60 tumor cells by combination drug A at different concentrations, and Figure 27-II shows the inhibition of HL-60 tumor cells by different combinations of combination drug B. As can be seen from Figure 27, combination A, with an IC50 concentration of [missing information], [missing information]. 20 The inhibitory effect of combination administration after 0.5-fold dilution at the corresponding concentration on HL-60 tumor cells and the effect of combination B on IC50 tumor cells. 30 The combination of 0.25-fold dilutions at the corresponding concentrations showed comparable inhibitory effects on HL-60 tumor cells, i.e., [A(IC 20survival rate of HL-60 tumor cells treated with [A(IC 30 )×0.75] was 44.05%, and the survival rates of the two combinations had no statistical difference (P>0.05). The other two combinations: [A(IC 20 )×0.75] and [B(IC 10 )], also inhibited the production of HL-60 tumor cells with similar survival rates (P>0.05), i.e. the survival rate of HL-60 tumor cells treated with [A(IC 20 )×0.75] was 22.77%, and the survival rate of HL-60 tumor cells treated with [B(IC 10 )×0.75] was 22.5% (Fig. 28-I).

[0332] 1.4.2 Effects of A group and B group on H9C2 normal cells under the same inhibitory efficacy of tumor cells

[0333] Fig. 28-I is the inhibitory effect of combinations [A(IC 20 )×0.5] and [B(IC 30 )×0.75], [A(IC 20 )×0.75] and [B(IC 10 )] on HL-60 tumor cells, and Fig. 28-II shows the toxic effect of [A(IC 20 )×0.5] and [B(IC 30 )×0.75], [A(IC 20 )×0.75] and [B(IC 10 )] on H9C2 normal cells, wherein the survival rates of H9C2 normal cells treated with [A(IC 20 )×0.5] and [B(IC 30 )×0.75] were 48.93% and 78.93%, respectively, and the survival rates of H9C2 normal cells treated with [A(IC 20 )×0.75] and [B(IC 10 )] were 35.34% and 67.86%, respectively.

[0334] Figure 29 brings together the survival rates of HL-60 tumor cells (anti-tumor efficacy) and H9C2 normal cells (toxicity) under the action of each combination drug for easy comparison with each other. It can be seen that the two groups A and B, in the case of exerting the same inhibitory effect on HL-60 tumor cells (anti-tumor efficacy), the cardiotoxicity of each combination drug of group B is far less than that of group A (the higher the survival rate of H9C2 myocardial cells, the smaller the toxicity). Secondly, the inhibitory rate of each combination drug on HL-60 tumor cells is significantly higher than that on H9C2 normal cells, and the difference of the two combination drugs of group B is far greater than that of the combination drugs of group A under the same tumor inhibition rate; therefore, from the difference in the survival rates of HL-60 and H9C2 cells of the same combination drug and the difference in the inhibition of H9C2 myocardial cells of groups A and B in the case of exerting the same anti-tumor efficacy, the combination drugs of group B have less cardiotoxicity (H9C2 cells) than the combination drugs of group A.

[0335] 1.5 Summary

[0336] Under the premise that the combination drugs of groups A and B exert the same inhibitory effect on tumor cells (HL-60 leukemia cells), the toxicity of the combination drugs of groups A and B on normal cells (H9C2 myocardial cells) was observed, and it was found that the toxicity of the combination drugs of group B on H9C2 myocardial cells was far less than that of the combination drugs of group A.

[0337] The specific case is as follows: first, two types of combination drugs of groups A and B that can exert the same inhibitory rate on HL-60 tumor cells were determined: [A(IC 20 ) x 0.5] treated HL-60 cells, the survival rate was 45.36%, [B(IC 30 ) x 0.75] treated HL-60 cells, the survival rate was 44.05%; [A(IC 20 ) x 0.75] treated HL-60, the survival rate was 22.77%, [B(IC 10 ) x 0.75] treated HL-60, the survival rate was 22.5%. Then, the two types of combination drugs were treated on H9C2 cells, and the inhibition was as follows: [A(IC 20 ) x 0.5] and [B(IC 30 ) x 0.75] treated H9C2 cells, the survival rates were 48.93% and 78.93% respectively (inhibition rates were 51.1% and 21.1% respectively), that is, the toxicity of B was reduced by 58.7% compared with A; [A(IC 20 ) x 0.75] and [B(IC 10The survival rates of H9C2 cells treated with B are 35.34% and 67.86% (inhibition rates are 64.66% and 32.14%, respectively), i.e., the toxicity of B is reduced by 50.3% as compared with A. In addition, B (IC 10 ) is compared with A (IC 20 ) x 0.5, the toxicity of B (IC 10 ) (H9C2 survival rate is 67%) is obviously lower than that of A (IC 20 ) x 0.5 (survival rate is 49%), but the tumor inhibition rate of B (IC 10 ) (78%) is much higher than that of A (IC 20 ) x 0.5 (55%). These results suggest that the therapeutic window of the combination drugs of B group is much wider than that of A group.

[0338] Therefore, from the difference in the inhibition of HL-60 tumor cells and H9C2 normal cells between A and B groups, it can be seen that the combination drugs of B group have less toxicity and better safety than those of A group.

[0339] Table 22 shows the administration concentration and concentration range of B group which has anti-tumor efficacy and low toxicity. According to Table 22, we obtain the proportion of each drug included in the eight combinations of topoisomerase II inhibitors which have anti-tumor efficacy and low toxicity, and the proportion is in the range of 0.38-0.70 parts of doxorubicin, 1.37-2.0 parts of etoposide, 0.15-0.16 parts of idarubicin, 0.076-0.28 parts of amsacrine, 0.30-0.43 parts of aclarubicin, 0.95-1.60 parts of teniposide, 0.67-0.68 parts of epirubicin, and 1.21-2.16 parts of pirarubicin, in terms of mass fraction.

[0340] Table 22 shows the administration concentration and concentration range of B group which has anti-tumor efficacy and low toxicity a, mass concentration (μg) = molar concentration (μM) x molecular weight; b, mass concentration ÷ 300; c, mass concentration ÷ 100

Claims

1. A pharmaceutical composition of a topoisomerase II inhibitor for preventing and / or treating a tumor, the pharmaceutical composition comprising, as an active ingredient, a topoisomerase II inhibitor selected from the group consisting of doxorubicin, etoposide, idarubicin, amsacrine, aclarubicin, teniposide, epirubicin, pirarubicin. The pharmaceutical composition comprises, as an active ingredient, 0.38 to 0.70 parts by mass of doxorubicin, 1.37 to 3.84 parts by mass of etoposide, 0.12 to 0.18 parts by mass of idarubicin, 0.08 to 0.91 parts by mass of amsacrine, 0.30 to 1.02 parts by mass of aclarubicin, 0.95 to 1.78 parts by mass of teniposide, 0.41 to 0.68 parts by mass of epirubicin, and 0.59 to 2.16 parts by mass of pirarubicin.

2. The pharmaceutical composition according to claim 1, wherein, Preferably, the pharmaceutical composition consists of, as an active ingredient, 0.38 to 0.70 parts by mass of doxorubicin, 1.37 to 3.84 parts by mass of etoposide, 0.12 to 0.18 parts by mass of idarubicin, 0.08 to 0.91 parts by mass of amsacrine, 0.30 to 1.02 parts by mass of aclarubicin, 0.95 to 1.78 parts by mass of teniposide, 0.41 to 0.68 parts by mass of epirubicin, and 0.59 to 2.16 parts by mass of pirarubicin. The pharmaceutical composition comprises, as an active ingredient, 0.4 to 0.63 parts by mass of doxorubicin, 2.41 to 3.84 parts by mass of etoposide, 0.12 to 0.18 parts by mass of idarubicin, 0.63 to 0.91 parts by mass of amsacrine, 0.64 to 1.02 parts by mass of aclarubicin, 1.18 to 1.78 parts by mass of teniposide, 0.41 to 0.67 parts by mass of epirubicin, and 0.59 to 0.88 parts by mass of pirarubicin. Preferably, the pharmaceutical composition consists of, as an active ingredient, 0.4 to 0.63 parts by mass of doxorubicin, 2.41 to 3.84 parts by mass of etoposide, 0.12 to 0.18 parts by mass of idarubicin, 0.63 to 0.91 parts by mass of amsacrine, 0.64 to 1.02 parts by mass of aclarubicin, 1.18 to 1.78 parts by mass of teniposide, 0.41 to 0.67 parts by mass of epirubicin, and 0.59 to 0.88 parts by mass of pirarubicin. The pharmaceutical composition comprises, as an active ingredient, 0.50 parts by mass of doxorubicin, 3.20 parts by mass of etoposide, 0.15 parts by mass of idarubicin, 0.75 parts by mass of amsacrine, 0.81 parts by mass of aclarubicin, 1.48 parts by mass of teniposide, 0.54 parts by mass of epirubicin, and 0.70 parts by mass of pirarubicin.

3. The pharmaceutical composition according to claim 1 or 2, wherein, Preferably, the pharmaceutical composition consists of, as an active ingredient, 0.50 parts by mass of doxorubicin, 3.20 parts by mass of etoposide, 0.15 parts by mass of idarubicin, 0.75 parts by mass of amsacrine, 0.81 parts by mass of aclarubicin, 1.48 parts by mass of teniposide, 0.54 parts by mass of epirubicin, and 0.70 parts by mass of pirarubicin. ​ ​ ​ 4. The pharmaceutical composition according to any one of claims 1 to 3, wherein, ​ ​ ​ 0.5 parts of doxorubicin, 3.2 parts of etoposide, 0.15 parts of idarubicin, 0.75 parts of amsacrine, 0.81 parts of aclarubicin, 1.48 parts of teniposide, 0.54 parts of epirubicin, 0.70 parts of pirarubicin.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein, The pharmaceutical composition comprises as an active ingredient a topoisomerase II inhibitor: 0.38 to 0.70 mg of doxorubicin, 1.37 to 3.84 mg of etoposide, 0.12 to 0.18 mg of idarubicin, 0.08 to 0.91 mg of amsacrine, 0.30 to 1.02 mg of aclarubicin, 0.95 to 1.78 mg of teniposide, 0.41 to 0.68 mg of epirubicin, 0.59 to 2.16 mg of pirarubicin; Preferably, the pharmaceutical composition consists of as an active ingredient a topoisomerase II inhibitor: 0.38 to 0.70 mg of doxorubicin, 1.37 to 3.84 mg of etoposide, 0.12 to 0.18 mg of idarubicin, 0.08 to 0.91 mg of amsacrine, 0.30 to 1.02 mg of aclarubicin, 0.95 to 1.78 mg of teniposide, 0.41 to 0.68 mg of epirubicin, 0.59 to 2.16 mg of pirarubicin; Preferably, the pharmaceutical composition comprises as an active ingredient a topoisomerase II inhibitor: 0.4 to 0.63 mg of doxorubicin, 2.41 to 3.84 mg of etoposide, 0.12 to 0.18 mg of idarubicin, 0.63 to 0.91 mg of amsacrine, 0.64 to 1.02 mg of aclarubicin, 1.18 to 1.78 mg of teniposide, 0.41 to 0.67 mg of epirubicin, 0.59 to 0.88 mg of pirarubicin; Preferably, the pharmaceutical composition consists of as an active ingredient a topoisomerase II inhibitor: 0.4 to 0.63 mg of doxorubicin, 2.41 to 3.84 mg of etoposide, 0.12 to 0.18 mg of idarubicin, 0.63 to 0.91 mg of amsacrine, 0.64 to 1.02 mg of aclarubicin, 1.18 to 1.78 mg of teniposide, 0.41 to 0.67 mg of epirubicin, 0.59 to 0.88 mg of pirarubicin; Preferably, the pharmaceutical composition comprises as an active ingredient a topoisomerase II inhibitor: 0.5 mg of doxorubicin, 3.2 mg of etoposide, 0.15 mg of idarubicin, 0.75 mg of amsacrine, 0.81 mg of aclarubicin, 1.48 mg of teniposide, 0.54 mg of epirubicin, 0.70 mg of pirarubicin; Preferably, the pharmaceutical composition consists of as an active ingredient a topoisomerase II inhibitor: 0.5 mg of doxorubicin, 3.2 mg of etoposide, 0.15 mg of idarubicin, 0.75 mg of amsacrine, 0.81 mg of aclarubicin, 1.48 mg of teniposide, 0.54 mg of epirubicin, 0.70 mg of pirarubicin.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein, The pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.

7. Use of the pharmaceutical composition of any one of claims 1 to 6 in the manufacture of a medicament for preventing and / or treating a tumor.

8. Use according to claim 7, wherein, The tumor is selected from the group consisting of liver cancer, leukemia, lymphoma, breast cancer, gastric cancer, lung cancer, ovarian cancer, bladder cancer, etc., preferably liver cancer, leukemia or lymphoma.

9. A method for preventing and / or treating a tumor, the method comprising administering to a patient in need thereof a therapeutically effective amount of the pharmaceutical composition of any one of claims 1 to 6; Preferably, the patient is a mammal, which can be a rodent (e.g., mouse, rat, etc.), a primate (e.g., macaque, human, etc.), etc. Preferably, the tumor is selected from the group consisting of liver cancer, leukemia, lymphoma, breast cancer, gastric cancer, lung cancer, ovarian cancer, bladder cancer, etc., preferably liver cancer, leukemia or lymphoma.

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