Pharmaceutical composition for preventing and / or treating tumors

Abstract

The invention relates to a pharmaceutical composition for preventing and / or treating tumors. The pharmaceutical composition comprises the following topoisomerase II inhibitors as active ingredients: doxorubicin, etoposide, idarubicin, anacridine, acarubicin, teniposide, epirubicin and pirarubicin. Compared with any one of the components, the pharmaceutical composition disclosed by the invention has a remarkable tumor inhibition effect, the toxic and side effects are obviously reduced, and all the components have a synergistic effect.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a pharmaceutical composition for the prevention and / or treatment of tumors. Background Technology

[0002] Regarding DNA topoisomerase

[0003] DNA topoisomerases are a class of enzymes found in the cell nucleus. They catalyze the breaking and binding of DNA strands, thereby controlling the topological state of DNA and participating in the regulation of supercoiled structures, which is of great significance for the survival of organisms. The existence of topoisomerases is to solve the problem of DNA strand entanglement during replication. They do this through a series of safe and efficient mechanisms; the entire process is a simple topological change. Their biological functions are mainly manifested in two ways: first, regulating and controlling the supercoiled state of DNA and the knotting or untying of DNA loops, thereby indirectly affecting intracellular nucleic acid metabolism; second, directly participating in DNA recombination, repair, transcription, and replication processes.

[0004] In mammals, there are two types of topoisomerases: TOPOI and TOPOII. They catalyze the creation of single-strand and double-strand breaks in DNA molecules, respectively. TOPOI mediates the transient breakage and rejoining of single-stranded or double-stranded DNA, thereby altering the DNA's topological structure. This breaking of the single or double strand is the distinguishing feature between TOPOI and TOPOII. TOPOI involves breaking the single strand first, causing the bond ends to rotate along the helical axis in the direction of loosening the supercoil, and then rejoining the broken single strands, thus altering the topological structure. TOPOII, on the other hand, involves simultaneously breaking the double strand, allowing it to pass through the cut site, and then rejoining the severed ends, thus altering the topological structure.

[0005] Topoisomerase I can be divided into type IA and type IB. While their functions are similar, they are unrelated and their mechanisms of action differ. Sequencely and structurally, they are not similar, and their functions are significantly different. TOPOI binds tightly to DNA substrates, encapsulating them completely. The hydrolysis catalyzed by TOPOI leads to DNA breakage. The active site of TOPOI covalently links to the broken DNA, forming a transient covalent complex, which then completes the DNA rejoining reaction.

[0006] Topoisomerase II also exists in two types, namely type IIA and type IIB; in mammals, TOPOII is further divided into two subtypes, TOPOIIα and TOPOIIβ; the two subtypes differ in structure, distribution, catalytic activity and other aspects.

[0007] Topoisomerases are an important target for anti-tumor drugs because they are highly expressed in tumor cells and are not affected by other factors. Therefore, inhibiting the activity of topoisomerases in tumor cells can curb the rapid proliferation of tumor cells and thus kill them.

[0008] Regarding DNA topoisomerase inhibitors

[0009] Topoisomerase inhibitors disrupt enzyme activity through various stages of enzyme action, rather than a single stage. Their mechanisms of action are flexible; they can act directly on DNA or the topoisomerase itself, or on the topoisomerase-DNA breakage complex. The most common mechanism is through the formation of a DNA-enzyme-drug ternary complex, blocking the final step of the enzyme-DNA reaction (i.e., the rejoining of single-stranded or double-stranded DNA at the nick site). In essence, the inhibitor transforms the topoisomerase into a substance that breaks DNA, ultimately leading to programmed cell death due to incorrect repair of DNA strand breaks or the formation of breakable complexes. The mechanisms of action of topoisomerase inhibitors are divided into two categories: toxic mechanisms and catalytic inhibition mechanisms. Toxicity mechanisms involve the inhibitor forming a larger ternary complex with the TOPO-DNA complex. This increases the steady-state concentration of the TOPO-DNA covalent complex, achieving a "poisoning" effect on the TOPO enzyme. In contrast, catalytic inhibition mechanisms involve the inhibitor inhibiting a specific function or step in the catalytic reaction of the TOPO enzyme, thereby inhibiting its overall catalytic activity. Topoisomerase inhibitors mainly include anthracyclines, camptothecins, and podophyllotoxins. Currently, TOPO inhibitors are the main chemotherapy treatment for cancer, and they have shown good clinical efficacy in leukemia, breast cancer, and lung cancer.

[0010] Topoisomerase I inhibitors broadly conform to the previously mentioned "toxicity mechanism and catalytic inhibition mechanism" and are divided into two main categories: TOPOI poisons and TOPOI catalytic inhibitors. For TOPOI poisons, they can be further subdivided into DNA inserters, groove binding agents, and those with both DNA binding and TOPO I inhibitory effects. DNA inserters include the following types: (1) Benzoanthraquinone compounds, such as saintopin and its analogue UCE26, share a common structural feature of having a flat naphtacendione structure, which allows saintopin drugs to insert into the middle of the base pair to exert their effect; (2) Benzophenanthridine compounds, such as zanthoxylin and zanthoxylin, can capture TOPOI-DNA cleavage complexes and are effective DNA inserters; (3) Others, such as intoplicine, can simultaneously inhibit TOPO I and TOPO II and have good anti-tumor effects. Aclacinomycin A is a trisaccharide derivative of anthracycline compounds that effectively stabilizes the TOPOI-DNA covalent complex, acting as a typical intercalation agent for both TOPOI and TOPOII. Groove binders are further divided into microgroove binders and macrogroove binders. Microgroove binders are mainly benzimidazole compounds, while macrogroove binders are acridine compounds. Inhibitors that bind both DNA and TOPOI are primarily camptothecin and its analogues. For TOPOI catalytic inhibitors, these drugs are relatively few, mainly aclacinomycin A and shikonin acyl analogues, but they require relatively high effective concentrations.

[0011] Topoisomerase II inhibitors are classified into TOPOII catalytic inhibitors and TOPOII poisons based on whether they directly cause DNA breaks. Topoisomerase II catalytic inhibitors work by blocking a specific step in the catalytic reaction, acting at different stages of the TOPOII catalytic cycle, thereby inhibiting the overall catalytic activity of TOPOII. Examples of these drugs include ararubicin, neomycin, fostracin, and ICRF-159. TOPOII poisons, on the other hand, increase the number of DNA double-strand breaks by capturing TOPOII-mediated DNA breakage complexes, ultimately leading to cell death. Examples of these drugs include etoposide, doxorubicin, mitoxantrone, and idarubicin.

[0012] TOPOII inhibitors can be further classified into DNA intercalators and DNA non-intercalators based on their binding mechanism with DNA. Intercalating inhibitors insert their planar portions (similar to polycyclic structures of purine or pyrimidine bases) into the double strands of DNA at the binding site of topoisomerase II, interfering with the enzyme's reconnection of DNA ends and causing DNA damage, ultimately leading to cell death. Examples of these drugs include actinomycins (actinomycin D), anthracyclines (doxorubicin), anthraquinones (mitoxantrone), and acridines (acrimidine). The mechanism of action of non-intercalating inhibitors is not fully understood; they may act directly on TOPOII or only on one strand of DNA to affect enzyme function. Major drugs in this category include isoflavones and podophyllotoxins (etoposide, teniposide), etc. As a large class of compounds with antitumor activity, TOPOII inhibitors are limited by their toxic side effects while exerting their antitumor effects. For example, doxorubicin (Axorubicin), etoposide, idarubicin, acridine, arubicin, teniposide, epirubicin, and pirarubicin are eight common anti-tumor drugs, all characterized by severe adverse reactions in clinical practice. For instance, doxorubicin primarily causes bone marrow suppression, cardiotoxicity, and gastrointestinal reactions. Etoposide mainly causes bone marrow suppression and gastrointestinal reactions. Idarubicin primarily causes severe bone marrow suppression and cardiotoxicity. Acridine's main adverse reactions are bone marrow suppression and gastrointestinal reactions. Arubicin's main adverse reactions are gastrointestinal reactions and bone marrow suppression, with occasional hair loss. Teniposide's main adverse reactions are bone marrow suppression and gastrointestinal reactions. Epirubicin commonly causes hair loss (80% of patients), bone marrow suppression (60%, white blood cell count reaches its lowest point 10-14 days after administration and usually recovers gradually within about 3 weeks; anemia and significant thrombocytopenia are rare), loss of appetite, nausea, and vomiting, but these are less severe than those of doxorubicin at equivalent doses. Cardiotoxicity is also less severe than with doxorubicin, and its incidence and severity are proportional to its cumulative dose. Pirarubicin's main adverse reaction is bone marrow suppression, which is a dose-limiting toxicity, primarily granulocytopenia, with the average lowest value appearing at 14 days. Cardiotoxicity is lower than with doxorubicin; acute cardiotoxicity mainly manifests as reversible ECG changes, such as arrhythmias or nonspecific ST-T abnormalities, while chronic cardiotoxicity is dose-cumulative.

[0013] In addition to topoisomerase I and topoisomerase II inhibitors, there are also dual inhibitors of topoisomerase I and II. These TOPOI / TOPOII dual inhibitors can act on two key enzymes in the cell cycle simultaneously, further enhancing the antitumor activity of the drug and reducing drug resistance. The main compounds include: irinotecan, SN-38 (irinotecan metabolite; irinotecan itself is its prodrug, converted to the active metabolite SN-38 in vivo by carboxylesterase), pixantrone, mitoxantrone, daunorubicin, doxorubicin, idarubicin, arubicin, epirubicin, pirarubicin, intoplicine, and aclacinomycin A. A) Tafluposide (a derivative of etoposide), salvicine, batracylin (an analog of rosehip alkaloid), trabectedin, lurbinectedin (an analog of trabectedin), phenazine derivatives XR11576 and XR5944 (MLN944), as well as strychnine, curcumin, etc., each have their own characteristics and advantages. In general, dual inhibitors are a hot topic in TOPO enzyme-related antitumor drug research.

[0014] To date, there is still no anti-tumor chemotherapy drug that is both effective and has very low toxicity. Summary of the Invention

[0015] To overcome the shortcomings of existing technologies, the applicant, through in-depth research, unexpectedly discovered a pharmaceutical composition for the prevention and / or treatment of tumors, comprising multiple topoisomerase II inhibitors. This pharmaceutical composition exhibits superior antitumor efficacy, particularly compared to the efficacy of a single compound (i.e., a single topoisomerase II inhibitor), and its toxic side effects are significantly reduced. Furthermore, the applicant discovered that this superior efficacy and significantly reduced toxicity occur when each individual compound (i.e., a single topoisomerase II inhibitor) is combined at ineffective doses, representing an effect of ultra-low concentration (dose) combination. Moreover, the toxicity of the pharmaceutical composition in this application is lower than that of other similar drug combinations, which was entirely unexpected.

[0016] The above-mentioned objective of the present invention is achieved by the following technical solution.

[0017] On one hand, the present invention provides a pharmaceutical composition for the prevention and / or treatment of tumors, the pharmaceutical composition comprising the following topoisomerase II inhibitor as an active ingredient:

[0018] Doxorubicin, etoposide, idarubicin, acridine, arubicin, teniposide, epirubicin, and pirarubicin. Most of these active ingredients are dual inhibitors of topoisomerase I and II, and some also include TOPO enzyme catalytic inhibitors and TOPO enzyme poisons, making their targets quite complex.

[0019] Preferably, the pharmaceutical composition comprises, by weight parts, the following topoisomerase II inhibitors as active ingredients:

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

[0021] Preferably, the pharmaceutical composition comprises, by weight parts, the following topoisomerase II inhibitor as the active ingredient:

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

[0023] Preferably, the pharmaceutical composition comprises, by weight parts, the following topoisomerase II inhibitors as active ingredients:

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

[0025] Preferably, the pharmaceutical composition comprises, by weight parts, the following topoisomerase II inhibitor as the active ingredient:

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

[0027] Preferably, the pharmaceutical composition comprises, by weight parts, the following topoisomerase II inhibitors as active ingredients:

[0028] 0.5 parts doxorubicin, 3.2 parts etoposide, 0.15 parts idarubicin, 0.75 parts acridine, 0.81 parts arubicin, 1.48 parts teniposide, 0.54 parts epirubicin, and 0.70 parts pirarubicin.

[0029] Preferably, the pharmaceutical composition comprises, by weight parts, the following topoisomerase II inhibitor as the active ingredient:

[0030] 0.5 parts doxorubicin, 3.2 parts etoposide, 0.15 parts idarubicin, 0.75 parts acridine, 0.81 parts arubicin, 1.48 parts teniposide, 0.54 parts epirubicin, and 0.70 parts pirarubicin.

[0031] Preferably, the pharmaceutical composition comprises the following topoisomerase II inhibitor as an active ingredient:

[0032] Doxorubicin 0.38–0.70 mg, etoposide 1.37–3.84 mg, idarubicin 0.12–0.18 mg, acridine 0.08–0.91 mg, arubicin 0.30–1.02 mg, teniposide 0.95–1.78 mg, epirubicin 0.41–0.68 mg, pirarubicin 0.59–2.16 mg.

[0033] Preferably, the pharmaceutical composition comprises the following topoisomerase II inhibitor as the active ingredient:

[0034] Doxorubicin 0.38–0.70 mg, etoposide 1.37–3.84 mg, idarubicin 0.12–0.18 mg, acridine 0.08–0.91 mg, arubicin 0.30–1.02 mg, teniposide 0.95–1.78 mg, epirubicin 0.41–0.68 mg, pirarubicin 0.59–2.16 mg.

[0035] Preferably, the pharmaceutical composition comprises the following topoisomerase II inhibitor as an active ingredient:

[0036] Doxorubicin 0.4–0.63 mg, etoposide 2.41–3.84 mg, idarubicin 0.12–0.18 mg, acridine 0.63–0.91 mg, arubicin 0.64–1.02 mg, teniposide 1.18–1.78 mg, epirubicin 0.41–0.67 mg, and pirarubicin 0.59–0.88 mg.

[0037] Preferably, the pharmaceutical composition comprises the following topoisomerase II inhibitor as the active ingredient:

[0038] Doxorubicin 0.4–0.63 mg, etoposide 2.41–3.84 mg, idarubicin 0.12–0.18 mg, acridine 0.63–0.91 mg, arubicin 0.64–1.02 mg, teniposide 1.18–1.78 mg, epirubicin 0.41–0.67 mg, and pirarubicin 0.59–0.88 mg.

[0039] Preferably, the pharmaceutical composition comprises the following topoisomerase II inhibitor as an active ingredient:

[0040] 0.5 mg doxorubicin, 3.2 mg etoposide, 0.15 mg idarubicin, 0.75 mg acridine, 0.81 mg arubicin, 1.48 mg teniposide, 0.54 mg epirubicin, and 0.70 mg pirarubicin.

[0041] In one specific embodiment, the active ingredient of the pharmaceutical composition of the present invention is as follows:

[0042]

[0043] Preferably, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.

[0044] Preferably, the pharmaceutical composition can be prepared into a suitable dosage form, such as an injection, tablet, or other dosage form.

[0045] On the other hand, the present invention provides the use of the above-described pharmaceutical composition in the preparation of a medicament for the prevention and / or treatment of tumors.

[0046] Preferably, the tumor is selected from liver cancer, leukemia, lymphoma, breast cancer, stomach cancer, lung cancer, ovarian cancer, bladder cancer, etc., with liver cancer, leukemia or lymphoma being the most preferred.

[0047] Compared with the prior art, this application has at least the following beneficial technical effects:

[0048] The pharmaceutical composition of the present invention exhibits significantly reduced antitumor efficacy and toxicity compared to any one of its components, and the components demonstrate synergistic effects (combination effect). Furthermore, the dosage of each individual compound in the pharmaceutical composition is the dose at which it would not be effective against tumors when administered alone.

[0049] Even at unexpectedly ineffective doses, the combination of these eight drugs produced a significant tumor-suppressing effect (tumor inhibition rate of 54.3%) without serious toxic reactions (no deaths, and no liver, heart, or kidney damage). Such an effect is unprecedented in chemotherapy. In contrast, the eight individual drug groups in parallel experiments (at doses five times that of the individual drugs in the combination) showed no effective anti-tumor effect (tumor inhibition rates of only 24.6%–38.1%; while a tumor inhibition rate of 50% is generally considered effective for anti-tumor treatment), but instead exhibited severe toxicity, with 10%–50% of mice dying in each group, and liver, heart, or kidney damage in the surviving mice.

[0050] The eight drugs in the pharmaceutical composition of this application are toxic individually at their effective dose. However, by combining these specific drugs and controlling the content (dose) within a specific range, this application greatly reduces the toxicity. Furthermore, the toxicity of the pharmaceutical composition of this application is lower than that of other combinations of similar drugs.

[0051] All eight drugs mentioned above are currently used individually; there are no reports of combinations of all eight. Furthermore, each of these eight drugs is known to be toxic when used alone. Existing strategies for reducing toxicity primarily focus on controlling toxicity during drug onset, without considering toxicity issues when treatment is ineffective. In addition, current research largely focuses on the mechanisms of action and targets of the compounds, with less exploration of the relationship between toxic targets or chemical structures and toxicity. Therefore, although there is a theoretical possibility that multi-drug combinations may produce synergistic effects, the lack of a clear understanding of toxicity mechanisms (such as toxic targets or structure-activity relationships) makes it difficult to predict the level of toxicity after combination. Even with the same drug combination, the level of toxicity can vary significantly depending on the controlled ratios. As shown in Example 5 of this application, even with the same combination of eight drugs, using the same low dosage, if the proportions of the individual drugs in the composition exceed a certain range, these drug compositions will still exhibit significant toxicity. For example, in Example 5, compared to Formula 1, Formulas 2 through 5 all exhibited significant toxicity, leading to the death of 10% to 50% of mice. The surviving mice all showed significant liver, heart, and / or kidney toxicity, and all showed severe immune system damage. These results demonstrate that even with the same combination of eight drugs, appropriate proportions of the components are necessary to achieve good antitumor efficacy and low toxicity.

[0052] This study found that using these eight drugs in a specific combination not only significantly improves efficacy but also effectively reduces overall toxicity. Attached Figure Description

[0053] Figure 1Effects of eight TOPOII inhibitors and their combinations on the volume of H22 solid tumors in mice;

[0054] Figure 2 The effects of eight TOPOII inhibitors and their combinations on the tumor index of H22 solid tumors in mice were investigated, with statistically significant differences compared to the model group (p < 0.01). ## The difference compared to the model group was statistically significant (p < 0.05). # The difference compared to the combination drug group was statistically significant (p < 0.01). △△ The difference compared to the combination drug group was statistically significant (p < 0.05). △ ;

[0055] Figure 3 The effects of eight TOPOII inhibitors and their combinations on the inhibition rate of H22 solid tumors in mice were investigated. Among them, the differences compared with the combination drug group were statistically significant (p < 0.01). △△ The difference compared to the combination drug group was statistically significant (p < 0.05). △ ;

[0056] Figure 4 The effects of eight TOPOII inhibitors and their combinations on the survival days of H22 ascites tumor mice were investigated, with statistically significant differences compared to the model group (p < 0.01). ## ;

[0057] Figure 5 The effects of eight TOPOII inhibitors and their combinations on the life extension rate of H22 ascites tumor mice were investigated, with statistically significant differences compared to the model group (p < 0.01). △△ ;

[0058] Figure 6 Survival curves of eight TOPOII inhibitors and their combinations on H22 solid tumor mice;

[0059] Figure 7 Effects of eight TOPOII inhibitors and their combinations on the body weight change trend in H22 solid tumor mice;

[0060] Figure 8 Effects of eight TOPOII inhibitors and their combinations on the daily food intake of a single H22 solid tumor mouse;

[0061] Figure 9 Effects of eight TOPOII inhibitors and their combinations on daily water intake in a single H22 solid tumor mouse;

[0062] Figure 10The effects of eight TOPOII inhibitors and their combinations on white blood cell count in H22 solid tumor mice were investigated, with statistically significant differences compared to the model group (p < 0.01). ## The difference compared to the model group was statistically significant (p < 0.05). # ;

[0063] Figure 11 The effects of eight TOPOII inhibitors and their combinations on platelet count in H22 solid tumor mice were investigated, with statistically significant differences compared to the model group (p < 0.01). ## The difference compared to the model group was statistically significant (p < 0.05). # ;

[0064] Figure 12 The effects of eight TOPOII inhibitors and their combinations on the thymus index in H22 solid tumor mice were investigated, with statistically significant differences compared to the model group (p < 0.01). ## The difference compared to the model group was statistically significant (p < 0.05). # The difference compared to the combination drug group was statistically significant (p < 0.05). △ ;

[0065] Figure 13 The effects of eight TOPOII inhibitors and their combinations on the spleen index of H22 solid tumor mice were investigated. The differences between the control group and the model group were statistically significant (p < 0.01)**. ## The difference compared to the model group was statistically significant (p < 0.05). # ;

[0066] Figure 14 Effects of eight TOPOII inhibitors and their combinations on cardiac index in H22 solid tumor mice;

[0067] Figure 15 Effects of eight TOPOII inhibitors and their combinations on LDH in H22 solid tumor mice; the differences compared with the control group were statistically significant (p < 0.01)**; the differences compared with the model group were statistically significant (p < 0.01). ## The difference compared to the combination drug group was statistically significant (p < 0.01). △△ ;

[0068] Figure 16 Effects of eight TOPOII inhibitors and their combinations on the control group (CK) of H22 solid tumor mice; among them, the differences compared with the model group were statistically significant (p < 0.01). ##The difference compared to the model group was statistically significant (p < 0.05). # The difference compared to the combination drug group was statistically significant (p < 0.01). △△ The difference compared to the combination drug group was statistically significant (p < 0.05). △ ;

[0069] Figure 17 Effects of eight TOPOII inhibitors and their combinations on liver index in H22 solid tumor mice; the difference was statistically significant compared with the blank group (p < 0.01)**; the difference was statistically significant compared with the model group (p < 0.05). # ;

[0070] Figure 18 Effects of eight TOPOII inhibitors and their combinations on ALT levels in H22 solid tumor mice; statistically significant differences were observed compared to the control group (p < 0.01)**; statistically significant differences were observed compared to the model group (p < 0.01). ## The difference compared to the combination drug group was statistically significant (p < 0.01). △△ ;

[0071] Figure 19 Effects of eight TOPOII inhibitors and their combinations on AST in H22 solid tumor mice; the differences compared with the control group were statistically significant (p < 0.01)**; the differences compared with the model group were statistically significant (p < 0.01). ## The difference compared to the model group was statistically significant (p < 0.05). # The difference compared to the combination drug group was statistically significant (p < 0.01). △△ ;

[0072] Figure 20 Effects of eight TOPOII inhibitors and their combinations on ALP in H22 solid tumor mice; the differences compared with the model group were statistically significant (p < 0.01). ## The difference compared to the combination drug group was statistically significant (p < 0.05). △ ;

[0073] Figure 21 Effects of eight TOPOII inhibitors and their combinations on total thrombosis (TP) in H22 solid tumor mice; the differences compared with the model group were statistically significant (p < 0.05). # ;

[0074] Figure 22 Effects of eight TOPOII inhibitors and their combinations on ALB in H22 solid tumor mice; the differences compared with the combination drug group were statistically significant (p < 0.01). △△The difference compared to the combination drug group was statistically significant (p < 0.05). △ ;

[0075] Figure 23 Effects of eight TOPOII inhibitors and their combinations on the kidney index in H22 solid tumor mice;

[0076] Figure 24 Effects of eight TOPOII inhibitors and their combinations on UREA in H22 solid tumor mice; the differences compared with the model group were statistically significant (p < 0.01). ## ;

[0077] Figure 25 Effects of eight TOPOII inhibitors and their combinations on SCr in H22 solid tumor mice; the differences compared with the model group were statistically significant (p < 0.01). ## The difference compared to the model group was statistically significant (p < 0.05). # The difference compared to the combination drug group was statistically significant (p < 0.01). △△ The difference compared to the combination drug group was statistically significant (p < 0.05). △ ;

[0078] Figure 26 Timeline of inoculation, administration, and measurement in mouse experiments. (A) Solid tumor study of combination drugs; (B) Ascites tumor study of combination drugs;

[0079] Figure 27 Effects of different combinations of drugs A and B on HL-60 tumor cell viability; compared with the control group, ** indicates P < 0.01, **** indicates P < 0.0001, n = 3, Mean ± SD; I, HL-60 cell viability under the action of each combination of drugs in group A; II, HL-60 cell viability under the action of each combination of drugs in group B;

[0080] Figure 28 The inhibitory effects of groups A and B on HL-60 tumor cells and the cytotoxic effects on H9C2 normal cells. Compared with the control group, **** indicates P < 0.0001, n = 3, Mean ± SD; I. Survival rate of HL-60 tumor cells under the action of each combination of drugs in groups A and B (this is a reference). Figure 27 Information); II, the toxic effects of the corresponding combination drugs on normal H9C2 cells (survival rate of normal cells);

[0081] Figure 29The antitumor proliferation and toxicity of the combination drugs were compared with those of the control group. * indicates P < 0.05, **** indicates P < 0.0001, n = 3, Mean ± SD; ns indicates no statistical difference. Detailed Implementation

[0082] The technical solution of the present invention will be further described below in conjunction with specific implementation methods.

[0083] Example 1: The pharmaceutical composition of the present invention

[0084] The formulation of this pharmaceutical composition is as follows:

[0085]

[0086] Example 2: Inhibition experiment of the pharmaceutical composition of the present invention in H22 (hepatocellular carcinoma) solid tumor mice.

[0087] 1.1. Experimental materials, reagents and instruments

[0088] 1.1.1 Laboratory Animals

[0089] Healthy male ICR mice (weighing 19-20g) were provided by the Department of Laboratory Animal Science, Peking University School of Medicine. They were housed in an environment with a constant temperature of 23±2℃ and humidity of 0.50-0.60%, with standard feed and tap water available for their own use.

[0090] H22 tumor-bearing mice were provided by the Department of Laboratory Animal Science, Peking University School of Medicine.

[0091] The animal experiments were approved by the Biomedical Ethics Committee of Peking University (Approval No.: SYXK2011-0039), and the certificate number for laboratory animal practitioners is: 2014062000055.

[0092] 1.1.2 Main Reagents and Materials

[0093] Doxorubicin (5927S, CST) was purchased from Beijing Zhongke Keao Biotechnology Co., Ltd.

[0094] Etoposide (E1383-100MG, Sigma) was purchased from Saen Chemical Technology (Shanghai) Co., Ltd.

[0095] Idarubicin (CC1384-10mg, ChemCatch) was purchased from Beijing Lebo Biotechnology Co., Ltd.

[0096] Amsacrine (HY-13551-10mg, MCE) was purchased from Beijing Lebo Biotechnology Co., Ltd.

[0097] Aclarubicin (TRC, 5mg, A190160), purchased from Amelin;

[0098] Teniposide (T3109, 20 mg, TCI), purchased from Inokai;

[0099] Epirubicin (Santa Cruz, 25mg, SC-279016), purchased from Huazhong Haiwei;

[0100] Pirarubicin (Selleck, 10mg, S1393) was purchased from Beijing Xinshengke Technology Co., Ltd.

[0101] Dimethyl sulfoxide (DMSO, DH105-9, 100 mL, Sigma) was purchased from Beijing Dingguo Biotechnology Co., Ltd.

[0102] Physiological saline (7647-14-5, 500g, Sinopharm Reagent) was purchased from Beijing Dingguo Biotechnology Co., Ltd.

[0103] Picric acid (74069, 25g, Sigma), purchased from Beijing Wanlan Shitu Biotechnology Co., Ltd.;

[0104] Medical alcohol (HQ001249, 500mL) was purchased from Beijing Jinghe Auto Technology Co., Ltd.

[0105] The blood cell analyzer diluent was purchased from Shanghai Optoelectronic Medical Electronic Instruments Co., Ltd.

[0106] The drugs selected in this study (eight topoisomerase II inhibitors) were all raw materials of the aforementioned drugs, in the form of solid powder.

[0107] 1.1.3 Main Instruments

[0108] Inverted microscope (OLYMPUS, CKX41);

[0109] Centrifuge (LD5-2A, Jingli);

[0110] Autoclave (G154DWS, Zhiwei Xiamen Instrument Co., Ltd.);

[0111] MILLI-Q ultrapure water system (Millipore, USA);

[0112] Electronic analytical balance (Sartorius GmbH, Germany);

[0113] SHA-C water bath constant temperature oscillator (Changzhou Guohua Electric Appliance Co., Ltd.);

[0114] Thermostatic water tank (SHHW21-420, Tianjin Tester);

[0115] Ultrasonic cleaning machine (Ningbo Xinzhi Biotechnology Co., Ltd.);

[0116] Vortex mixer (Qilinbell);

[0117] Fully automated biochemical analyzer (BS-350, Shenzhen Mindray Bio-Medical Electronics Co., Ltd.)

[0118] 1.2. Experimental Methods

[0119] 1.2.1 Preparation of main reagents

[0120] Drug solution: First, use a pipette to add 50 μL of DMSO to the Eppendorf tube containing the drug to dissolve it. Then, add physiological saline to prepare a 4 mg / mL stock solution (a clear solution), and freeze it at -20°C. Before use, remove it and allow it to thaw, then dilute the stock solution with physiological saline to the required concentration. After the drug solution is prepared, sonicate for 10 minutes to ensure thorough mixing. (Note: The intraperitoneal injection concentration of DMSO is less than 10%).

[0121] The drug concentrations in each administration group are based on their LD50. 50 This is a pre-defined concentration. Specifically, it refers to the LD50 of each drug in the single-drug group during the solid tumor (eight drugs) trial. 50 One-quarter of that, while the administered concentration of each drug in the combination drug group is its LD50. 50 One-twentieth. The preparation method of the combination drug group is as follows:

[0122] For example: If the total injection volume of the combination drug group is 4 mL, and doxorubicin requires 0.5 mg / kg, then first increase the doxorubicin concentration eightfold to prepare 4 mg / kg, 0.5 mL. Mix the eight drugs in equal volumes using this method, i.e., dilute them eight times again. The final concentration of each drug in the mixed solution is the concentration required for the experiment.

[0123] 1.2.2 Animal model preparation and grouping, and drug administration

[0124] 1.2.2.1 Drug Selection

[0125] The drugs selected in this study are all anti-tumor drugs that are already in clinical use. The selected drugs are: doxorubicin, etoposide, idarubicin, acridine, arubicin, teniposide, epirubicin, and pirarubicin.

[0126] 1.2.2.2 Tumor inoculation:

[0127] Solid tumor inoculation: Under aseptic conditions, ascites fluid was extracted from tumor-bearing mice, and tumor cells were counted under a microscope. The solution was then adjusted to 3×10⁻⁶ saline. 6 One tumor cell / mL, after mixing, 0.2mL / 20g was subcutaneously injected into the right axilla of each mouse.

[0128] 1.2.2.3 Animal Grouping

[0129] Solid tumor experiment: ICR mice were inoculated with tumor cells. 48 hours after inoculation, the animals were weighed and randomly divided into 11 groups of 10 mice each. The groups were: blank control group, model group, doxorubicin group, etoposide group, idarubicin group, acridine group, arubicin group, teniposide group, epirubicin group, pirarubicin group, and combination drug group (Formula 1, including eight low-dose mixtures of doxorubicin group, etoposide group, idarubicin group, acridine group, teniposide group, arubicin group, pirarubicin group, and epirubicin group).

[0130] 1.2.2.4 Administration method, route, and concentration

[0131] The concentrations of each administration group are based on their LD50. 50 This is a pre-defined concentration. Specifically, it refers to the LD50 of each drug in the monotherapy group during solid tumor trials. 50 One-quarter of that, while the administered concentration of each drug in the combination drug group is its LD50. 50 One-twentieth of that. The specific value is as follows:

[0132] Solid tumor experiment: In the blank control group, no tumor cells were introduced; starting from day 3, 0.2 mL / 20 g of physiological saline was injected intraperitoneally every other day. In the model group, starting from day 3 after tumor cell introduction, 0.2 mL / 20 g of physiological saline was injected intraperitoneally every other day. In each drug-treated group, starting from day 3 after tumor cell introduction, the corresponding drug was injected intraperitoneally every other day, at 0.2 mL / 20 g. In the combination drug group, starting from day 3 after tumor cell introduction, the drug of prescription 1 was injected intraperitoneally every other day, at 0.2 mL / 20 g. A total of 5 administrations were administered. All animals were sacrificed the day after the last administration, for a total of 12 days (see administration procedure below). Figure 26 A). Specific dosage concentrations are shown in Table 1 below:

[0133] Table 1: Dosage (concentration) of eight TOPO II inhibitors and their combinations (Formula 1) in H22 solid tumor experiments

[0134]

[0135] 1.2.3 Indicator Testing

[0136] 1.2.3.1 Observation of animal growth status

[0137] Every morning at 8:30, observe and record the animals' mental state, appetite, water intake, coat, activity, excretion, and growth status. The growth status of animals with solid tumors is observed for 12 days. All the following measurements and observation periods are consistent with the animal growth status observation period.

[0138] 1.2.3.2 Weight Measurement:

[0139] Mice in all groups were weighed every morning from 9:30 to 10:30 to observe changes in body weight.

[0140] 1.2.3.3 Measurement of daily food and water intake

[0141] Each group of mice was given 150g of food daily, and the remaining food was collected and weighed at 9:00 AM on the second day. Each group of mice was given 200mL of drinking water daily, and the remaining drinking water was collected and measured at 9:00 AM on the second day.

[0142] 1.2.3.4 In vitro measurement of tumor volume:

[0143] All mice inoculated with tumor cells were measured every two days using vernier calipers (measurement frequency: once every 2 days, starting from the third day; see [link]). Figure 26 A) Observe the changes in tumor volume and plot the tumor growth curve. Tumor volume formula:

[0144] V = L(mm) x I 2 (mm) / 2, which is calculated as follows, where L is the long diameter of the tumor and I is the short diameter of the tumor.

[0145] 1.2.3.5 Blood tests:

[0146] All animals used in the solid tumor experiment were euthanized. Before euthanasia, blood was collected by gouging out the eyeballs, 20 μL / mouse, and diluted with a diluent (pure water, anhydrous sodium sulfate, Tris). The blood samples were sent to the Animal Science Department for analysis to reflect bone marrow suppression. The measured parameters were: white blood cells and platelets.

[0147] 1.2.3.6 Serum enzyme indicators:

[0148] Before the animals were euthanized, blood was collected by enucleation of the eyeballs, centrifuged, and the supernatant was collected. The serum was retained for the detection of lactate dehydrogenase (LDH) and creatine kinase (CK), reflecting the degree of myocardial damage. Simultaneously, indicators reflecting liver function, such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total protein (TP), and albumin (ALB), as well as indicators reflecting kidney function, such as blood urea nitrogen (UREA) and serum creatinine (SCr), were measured.

[0149] 1.2.3.7 Weighing the tumor mass:

[0150] After the animals were euthanized, the tumor was separated, washed with saline solution, blotted dry with filter paper, weighed, and its volume was measured and photographed. This reflects the tumor suppression effect.

[0151]

[0152] 1.2.3.8 Taking photos:

[0153] After the animal is killed, it is dissected, photographed, and its organs are observed for congestion, reflecting the extent of toxic side effects.

[0154] 1.2.3.9 Weighing of each organ:

[0155] After the animal is killed, it is dissected, and the heart, liver, thymus, spleen, and kidneys are removed and weighed. This reflects the extent of damage to each organ.

[0156]

[0157] 1.2.3.10 Experimental Data Processing and Analysis

[0158] All data are expressed as mean ± standard deviation (x ± s). SPSS 17.0 software was used for analysis of differences in experimental results (p-value test), and a p-value < 0.05 was considered statistically significant. Mortality rates were compared using the chi-square test. Other parameters were compared using one-way ANOVA.

[0159] In the solid tumor (eight types) experiment, the initial number of animals in each group 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, acridine group, and arubicin 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).

[0160] Calculation of each rate of change:

[0161]

[0162] 1.3. Experimental Results

[0163] 1.3.1 Antitumor efficacy

[0164] 1.3.1.1 Experimental tumor-suppressing effects of eight drugs on solid tumors

[0165] (1) Tumor volume and tumor weight

[0166] The results of tumor volume and weight are shown in Figure 1 And Table 2.

[0167] Table 2: Effects of eight TOPO II inhibitors and their combinations on H22 solid tumors

[0168]

[0169]

[0170] Note: Dosage is shown in Table 1; the dosages in the following tables of this embodiment are the same. The differences compared to the model group were statistically significant (p < 0.01). ## The difference compared to the model group was statistically significant (p < 0.05). # The difference compared to the combination drug group was statistically significant (p < 0.01). △△ The difference compared to the combination drug group was statistically significant (p < 0.05). △ .

[0171] The tumor volume in each treatment group was significantly different from that in the model group (p < 0.01, with p < 0.05 in the ararubicin and pirarubicin groups). This suggests that the tumor volume was significantly reduced after drug intervention in each treatment group.

[0172] In terms of the reduction in tumor volume, the combination drug group (Prescription 1) showed a reduction of 69.6%, 26.2%, 42.5%, 47.3%, 163.1%, 136.5%, 85.2%, and 168.3% greater than the eight single-drug groups, namely doxorubicin, topposide, idarubicin, acridine, arubicin, teniposide, epirubicin, and pirarubicin, respectively, indicating that the combination drug group had the best effect in inhibiting tumor growth.

[0173] Depend on Figure 1 The tumor volume in each group increased over time, with the combination drug group showing the slowest growth rate.

[0174] Table 2 shows that, in terms of tumor weight, the differences between each treatment group and the model group were statistically significant (p < 0.01). This indicates that the tumor weight was significantly reduced in all treatment groups after intervention. Regarding the magnitude of reduction, the combination drug group showed increases of 95.2%, 41.4%, 57.7%, 70.8%, 115.8%, 95.2%, 95.2%, and 86.4% compared to the doxorubicin, etoposide, idarubicin, acridine, arubicin, teniposide, epirubicin, and pirarubicin groups, respectively. This suggests that the combination drug group had the best effect in inhibiting tumor weight growth.

[0175] (2) Tumor index and tumor inhibition rate

[0176] The results of tumor index and tumor inhibition rate are shown in Figure 2 , Figure 3And Table 3.

[0177] Table 3: Effects of eight TOPO II inhibitors and their combinations on H22 solid tumors

[0178]

[0179] Note: The difference compared with the model group was statistically significant (p < 0.01). ## The difference compared to the model group was statistically significant (p < 0.05). # The difference compared to the combination drug group was statistically significant (p < 0.01). △△ The difference compared to the combination drug group was statistically significant (p < 0.05). △ .

[0180] Depend on Figure 2 As can be clearly seen from Table 3, the differences in tumor index between the combination drug group (p < 0.01), the acridine group (p < 0.01), and the pirarubicin group (p < 0.05) and the model group were statistically significant. Among them, the combination drug group had the lowest tumor index, indicating that the combination drug group had the best tumor-suppressing effect.

[0181] Depend on Figure 3 Comparison with Table 3 shows that the tumor inhibition rate of the combination drug group was significantly different from that of each other drug group (except for the etoposide group) (p < 0.01, p < 0.05 for the idarubicin group). The tumor inhibition rate of the combination drug group was 54.3%, which was much higher than that of the other single drug groups. Specifically, it was higher than the doxorubicin group by 101.1%, higher than the idarubicin group by 57.4%, higher than the acridine group by 74.6%, higher than the arubicin group by 120.7%, higher than the teniposide group by 87.2%, higher than the epirubicin group by 93.9%, and higher than the pirarubicin group by 91.2%, indicating that the antitumor effect of the combination drug group was significantly better than that of each single drug group.

[0182] The difference in tumor inhibition rate between the combination drug group and the etoposide group was not statistically significant (P>0.05). However, comparing specific values, the tumor inhibition rate of the combination drug group was 42.5% higher than that of the etoposide group, suggesting that the antitumor activity of the combination drug group also has a significant advantage over that of the etoposide group.

[0183] In summary, considering tumor volume, tumor weight, tumor index, and tumor inhibition rate, it is evident that the antitumor effect of the combination drug group is significantly better than that of each individual drug group.

[0184] In summary, considering the four indicators of tumor volume, tumor weight, tumor index, and tumor inhibition rate, it is suggested that although the drug concentration in each individual treatment group was its LD50, the overall drug concentration was still within the range of 100%. 50 It was only one-quarter of the effective dose, but its anti-tumor effect was still significantly lower than that of the combination drug group (where the dose of each drug was only one-quarter of its LD50).50 One-twentieth of it.

[0185] 1.3.2 Study on toxic side effects

[0186] 1.3.2.1 Toxic side effects of eight drugs in solid tumor experiments

[0187] (1) Survival rate

[0188] Of all the indicators of adverse reactions, survival rate is the most important. The survival status of each group is shown in Table 4. Figure 6 The specific time of death for each individual drug administration group is shown in Table 5.

[0189] Table 4: Effects of eight TOPO II inhibitors and their combinations on survival in mice with H22 solid tumors

[0190]

[0191]

[0192] Note: The difference compared with the control group was statistically significant (p < 0.01). ** The difference compared with the control group was statistically significant (p < 0.05). * The difference compared to the model group was statistically significant (p < 0.01). ## The difference compared to the model group was statistically significant (p < 0.05). # The difference compared to the combination drug group was statistically significant (p < 0.01). △△ The difference compared to the combination drug group was statistically significant (p < 0.05). △ .

[0193] Table 5: Effects of eight TOPOII inhibitors and their combinations on the number of deaths in H22 solid tumor mice

[0194]

[0195] From Table 4 and Figure 6Survival rates were assessed across groups. During the 10-day observation period (12 days from tumor inoculation), no animals died in the combination drug group, the control group, or the model group, showing no difference in survival rates. However, in all other drug groups, 1–5 mice died starting from day 10 after tumor inoculation. Compared to the doxorubicin, etoposide, idarubicin, acridine, arubicin, teniposide, epirubicin, and pirarubicin groups, the survival rates of the combination drug group (Formula 1) were increased by 25.0%, 42.9%, 100.0%, 25.0%, 42.9%, 25.0%, 66.7%, and 11.1%, respectively. This suggests that the combination drug administration method significantly reduces the toxic side effects of individual drugs and improves survival rates.

[0196] (2) Growth status

[0197] Mice that successfully received tumor cell inoculation showed obvious tumor growths in the right axilla of their right limbs. Mice in the control group and model group were in good spirits. Compared with the model group and control group, mice in the combination drug group were more agile, had glossy fur, and were in good physical condition; there were no significant differences in the overall condition of the mice.

[0198] In stark contrast, the other treatment groups showed signs of rough fur, a gradual decline in mental state during the treatment period, a weakening of resistance when grabbed, and generally exhibited reduced movement, bristling hair, and decreased food intake. Furthermore, the number of deaths gradually increased, indicating that each individual treatment group had significant toxic side effects.

[0199] (3) Gastrointestinal reactions

[0200] Gastrointestinal reactions are a common toxic side effect of antitumor drugs. To evaluate the gastrointestinal reactions in each treatment group, three indicators closely related to the gastrointestinal tract—body weight, daily food intake, and daily water intake—were selected for evaluation. Specific results are shown in Table 6 and... Figure 7 , Figure 8 , Figure 9 .

[0201] Table 6: Effects of eight TOPO II inhibitors and their combinations on the gastrointestinal tract of H22 solid tumor mice

[0202]

[0203] Note: The difference compared with the control group was statistically significant (p < 0.01). ** The difference compared with the control group was statistically significant (p < 0.05). * The difference compared to the model group was statistically significant (p < 0.01). ## The difference compared to the model group was statistically significant (p < 0.05). # The difference compared to the combination drug group was statistically significant (p < 0.01).△△ The difference compared to the combination drug group was statistically significant (p < 0.05). △ .

[0204] 1) As shown in Table 6, the combined drug group showed a significant decrease in body weight compared to the blank group and the model group, with statistically significant differences (p < 0.01). This suggests that although the mice in the combined drug group were in good growth condition, they were indeed suffering from some degree of gastrointestinal toxicity, resulting in a decrease in body weight, which was 28.5% lower than that of the blank group and 22.2% lower than that of the model group.

[0205] 2) From Figure 7 It can be seen that among the single-drug groups, the idarubicin group had the largest weight loss, with a weight loss of 26.7% compared to the control group and a weight loss of 20.3% compared to the model group. The differences were statistically significant (p < 0.01), suggesting that idarubicin was also affected by some gastrointestinal reactions.

[0206] In the other treatment groups (excluding the acridine group), compared with the model group, the body weight decreased by 10.5% in the doxorubicin group, 17.3% in the etoposide group, 20.3% in the idarubicin group, 17.0% in the arubicin group, 11.4% in the teniposide group, 17.0% in the epirubicin group, and 7.8% in the pirarubicin group. All differences were statistically significant (p < 0.01). Combined with the growth status of these groups of mice, it was determined that these groups of mice were also affected by the side effects.

[0207] 3) Table 6 also shows that the daily food intake per mouse in the combination drug group was significantly different from that in the blank group and the model group (p < 0.05). This suggests that the significant decrease in body weight in the combination drug group may be related to the reduced food intake.

[0208] 4) Among the monotherapy groups, the epirubicin group had the lowest food intake, with a 35.0% decrease in daily food intake per mouse compared to the model group, a statistically significant difference (p < 0.01). Combined with the body weight of this group, this suggests that the epirubicin group experienced gastrointestinal discomfort.

[0209] Depend on Figure 8 It can be seen that from the seventh day onwards, the food intake of the combination drug group showed a significant upward trend, which was better than that of the idarubicin group and the epirubicin group, indicating that the gastrointestinal reaction of the mice in the combination drug group was gradually reduced.

[0210] 5) As can be seen from Table 6, there was no statistically significant difference in water intake among the groups (p > 0.05), indicating that although there were differences in gastrointestinal reactions among the groups, they did not cause serious effects. The group with epirubicin had the lowest water intake, followed by the combination drug group.

[0211] from Figure 9 As can be seen, similar to the trend in food intake, the water intake in the combination drug group showed a significant upward trend starting from the seventh day, and was better than that in the idarubicin group and the epirubicin group, indicating that the gastrointestinal reactions of the mice in the combination drug group were gradually reduced.

[0212] Based on data on body weight, daily food intake, and daily water intake, it is indicated that idarubicin and epirubicin can cause certain gastrointestinal reactions. Similarly, the combination drug group also experienced some gastrointestinal reactions, but they were not severe. The cause of these side effects may be idarubicin and epirubicin in the combination drug group.

[0213] Compared with epirubicin, the combination drug group had a better severity of gastrointestinal reactions than either of the two groups.

[0214] (4) Bone marrow suppression

[0215] Myelosuppression is the most common toxic side effect of antitumor drugs. Previous literature review showed that all the drugs selected in this study caused varying degrees of myelosuppression. This experiment evaluated the occurrence of myelosuppression in each treatment group using complete blood count (CBC) tests. Decreased white blood cell count and platelet count both indicated the occurrence of myelosuppression. Specific results are shown in Table 7 and... Figure 10-11 .

[0216] Table 7: Effects of eight TOPOII inhibitors and their combinations on myelosuppression in H22 solid tumor mice

[0217]

[0218] Note: The difference compared with the control group was statistically significant (p < 0.01). ** The difference compared with the control group was statistically significant (p < 0.05). * The difference compared to the model group was statistically significant (p < 0.01). ## The difference compared to the model group was statistically significant (p < 0.05). # The difference compared to the combination drug group was statistically significant (p < 0.01). △△ The difference compared to the combination drug group was statistically significant (p < 0.05). △ .

[0219] 1) As shown in Table 7, compared with the control group, the white blood cell count ( Figure 10 ) and platelets ( Figure 11 The difference was not statistically significant (p>0.05), suggesting that no bone marrow suppression occurred after tumor inoculation.

[0220] 2) Compared with the model group, the combination drug group showed a statistically significant decrease in white blood cell count (p < 0.01) but not a statistically significant decrease in platelet count (p > 0.05). Specifically, the white blood cell count decreased by 25.5% and the platelet count decreased by 18.5%, suggesting a slight tendency for myelosuppression in the combination drug group.

[0221] The results for the other treatment groups are as follows: The idarubicin group showed statistically significant differences in white blood cell and platelet counts compared to the model group (p < 0.01), with decreases of 40.4% and 35.4%, respectively. The acridine group also showed statistically significant differences in white blood cell and platelet counts compared to the model group (p < 0.05), with decreases of 26.6% and 21.3%, respectively. The doxorubicin group (p < 0.05) and pirarubicin group (p < 0.01) showed statistically significant differences in white blood cell counts compared to the model group, with decreases of 21.3% and 24.5%, respectively. The etoposide group and epirubicin group showed statistically significant differences in platelet counts compared to the model group (p < 0.05), with decreases of 13.9% and 22.2%, respectively. The arubicin group and teniposide group showed no statistically significant differences in white blood cell and platelet counts compared to the model group (p > 0.05). The results indicated that the idarubicin and acridine groups caused severe bone marrow suppression, with greater toxicity than the combination drug group; while the doxorubicin, pirarubicin, etoposide, and epirubicin groups showed a tendency towards bone marrow suppression, with toxicity comparable to the combination drug group. The arubicin and teniposide groups showed a mild tendency towards bone marrow suppression, with less toxicity than the combination drug group.

[0222] The degree of bone marrow suppression in each group varied compared to the combination drug group, with no statistically significant difference (p > 0.05). While the difference between the idarubicin group and the combination drug group was not statistically significant (p > 0.05), the idarubicin group had a 20.0% lower white blood cell count and a 20.7% lower platelet count, indicating that the combination drug group showed significant relief from bone marrow suppression compared to the idarubicin group, which had the most severe bone marrow suppression.

[0223] In summary, the combination drug group showed a certain tendency towards myelosuppression, and all drug administration groups also exhibited some degree of myelosuppression. In terms of severity, the idarubicin group was the most severe, while the combination drug group provided some relief compared to idarubicin.

[0224] (5) Damage to the immune system

[0225] When mice are inoculated with tumors and simultaneously treated with drugs, their immune system responds to this intervention, but may also be damaged by the drugs. Indicators related to the immune system include: thymus index (…). Figure 12 ), spleen index ( Figure 13This study will use these two indicators for evaluation. A decrease in either indicator suggests damage to the immune system. The specific results are shown in Table 8 below:

[0226] Table 8: Effects of eight TOPO II inhibitors and their combinations on the immune system of H22 solid tumor mice

[0227]

[0228] Note: The difference compared with the control group was statistically significant (p < 0.01). ** The difference compared with the control group was statistically significant (p < 0.05). * The difference compared to the model group was statistically significant (p < 0.01). ## The difference compared to the model group was statistically significant (p < 0.05). # The difference compared to the combination drug group was statistically significant (p < 0.01). △△ The difference compared to the combination drug group was statistically significant (p < 0.05). △ .

[0229] As shown in Table 8,

[0230] 1) Compared with the control group, the thymus index of the model group increased by 17.6% and the spleen index increased by 93.6%, suggesting that after tumor inoculation, the immune system is stimulated by external factors and the immunity is enhanced to cope with the invasion of foreign substances.

[0231] 2) Compared with the model group, the thymus index decreased in all treatment groups, and the values ​​were statistically significant (p < 0.01, p < 0.05 for the pirarubicin group). This indicates that the immune system was damaged after administration, with varying degrees of damage across the treatment groups. The decreases were as follows: combination drug group (35.0%), doxorubicin group (55.0%), etoposide group (65.0%), idarubicin group (45.0%), acridine group (50.0%), arubicin group (60.0%), teniposide group (60.0%), epirubicin group (55.0%), and pirarubicin group (25.0%). This suggests that the etoposide and arubicin groups have greater toxicity than the combination drug group; all other treatment groups, except the pirarubicin group, tended to have greater toxicity than the combination drug group.

[0232] 3) Among the spleen indices, the combination drug group decreased by 23.1% (but the difference from the model group was not statistically significant), the doxorubicin group decreased by 38.5%, the etoposide group decreased by 40.7%, the idarubicin group decreased by 40.7%, the acridine group decreased by 29.7%, the arubicin 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 is evident that, except for the teniposide and epirubicin groups, the toxicity of the other treatment groups tended to be greater than that of the combination drug group. Considering both the thymus index and spleen index, the combination drug group experienced the least damage.

[0233] 4) Comparison of the single-drug groups and the combination drug groups: Thymus index showed statistically significant differences between the etoposide group and the arubicin group and the combination drug group (p < 0.05), with the former being 46.2% and 38.5% lower than the latter, respectively. Spleen index showed no statistically significant differences between any of the single-drug groups and the combination drug group (p > 0.05). This suggests that the combination drug group has lower toxicity in terms of immunosuppression.

[0234] In summary, all treatment groups suffered some degree of damage to the immune system, with the combination drug group experiencing the least damage.

[0235] (6) Cardiotoxicity

[0236] Preliminary literature review indicates that drugs such as doxorubicin, idarubicin, acridine, arubicin, epirubicin, and pirarubicin have cardiotoxicity (etoposide and teniposide have not been reported to have cardiotoxicity). Cardiac indexes were used in experiments (…). Figure 14 ) and LDH Figure 15 ), CK Figure 16 The cardiotoxicity of each group of mice was evaluated, and the results are as follows:

[0237] 1) Cardiac index

[0238] As shown in Table 9, the cardiac index of the combination drug group was not significantly different from that of the blank group, the model group, and other single-drug groups (p>0.05), indicating that the combination drug group did not show myocardial damage, and the other drug groups also did not show significant myocardial damage.

[0239] Table 9: Effects of eight TOPOII inhibitors and their combinations on cardiac index in H22 solid tumor mice

[0240]

[0241] Note: The difference compared with the control group was statistically significant (p < 0.01). ** The difference compared with the control group was statistically significant (p < 0.05). *The difference compared to the model group was statistically significant (p < 0.01). ## The difference compared to the model group was statistically significant (p < 0.05). # The difference compared to the combination drug group was statistically significant (p < 0.01). △△ The difference compared to the combination drug group was statistically significant (p < 0.05). △ .

[0242] 2) Serum enzyme indicators

[0243] Elevated levels of LDH and CK indicate heart damage. Table 10 shows that:

[0244] (i) Compared with the control group, the difference in LDH levels was statistically significant (p < 0.01). As a common tumor marker, elevated LDH levels indicate successful inoculation. Compared with the control group, the difference in CK levels was not statistically significant (p > 0.05). LDH and CK ( Figure 15-16 Both indicators suggest that no myocardial damage occurred in the model group.

[0245] (ii) Compared with the model group, only the doxorubicin and idarubicin groups showed statistically significant differences in LDH and CK levels, suggesting that these two groups would cause cardiac injury at the corresponding concentrations. Specifically, compared with the model group, the doxorubicin group showed a 48.5% increase in LDH and a 25.5% increase in CK, while the idarubicin group showed a 115.5% increase in LDH and a 29.7% increase in CK, indicating severe myocardial injury in these two groups. The combination drug groups showed no statistically significant differences in LDH and CK levels compared with the model group (p > 0.05), suggesting that no myocardial injury occurred in the combination drug groups.

[0246] (iii) Comparison of each single-drug group and the combination drug group: LDH showed statistically significant differences between the doxorubicin group, idarubicin group, and the combination drug group (p < 0.01). CK showed statistically significant differences between the doxorubicin group, idarubicin group, and the combination drug group (p < 0.05). Specifically, the increases in LDH and CK levels in the doxorubicin group and the combination drug group compared to the model group were significantly different. The increases in LDH and CK levels in the combination drug group were 89.1% and 92.6% lower than those in the doxorubicin group, respectively. Similarly, the increases in LDH and CK levels in the idarubicin group and the combination drug group compared to the model group were also significantly different. The increases in LDH and CK levels in the combination drug group were 95.4% and 93.6% lower than those in the idarubicin group, respectively. This suggests that the combination drug group reduced the degree of myocardial injury by approximately 90% compared to the doxorubicin and idarubicin groups, which experienced severe myocardial injury, and did not exhibit myocardial injury.

[0247] In summary, the doxorubicin and idarubicin groups showed the most severe myocardial damage, the combination drug group did not experience myocardial damage, and none of the other drug groups showed myocardial damage.

[0248] Table 10 Effects of eight TOPO II inhibitors and their combinations on serum enzymes associated with myocardial injury in H22 solid tumor mice.

[0249]

[0250]

[0251] Note: The difference compared with the control group was statistically significant (p < 0.01). ** The difference compared with the control group was statistically significant (p < 0.05). * The difference compared to the model group was statistically significant (p < 0.01). ## The difference compared to the model group was statistically significant (p < 0.05). # The difference compared to the combination drug group was statistically significant (p < 0.01). △△ The difference compared to the combination drug group was statistically significant (p < 0.05). △ .

[0252] (7) Hepatotoxicity

[0253] The liver is the site of drug metabolism and is relatively susceptible to drug damage. This experiment used liver indexes and serum enzyme indicators such as AST, ALT, ALP, TP, and ALB to evaluate whether each drug administration group caused liver damage.

[0254] 1) Liver index

[0255] Using liver index ( Figure 17 This allows for a relatively intuitive assessment of whether the liver is damaged. As shown in Table 11:

[0256] (i) Compared with the control group, the liver index of the model group was significantly different (p<0.01), specifically, the liver index increased by 28.6%, indicating that the liver was slightly swollen after tumor inoculation;

[0257] (ii) Compared with the model group, the liver index differences in the arubicin group and the teniposide group were statistically significant (p < 0.05). Specifically, the liver index decreased by 12.3% in the arubicin group and by 13.2% in the teniposide group, suggesting a trend of abnormal liver function in these two groups. The liver indexes in the other groups did not show any abnormalities compared with the model group.

[0258] (iii) There were no statistically significant differences in liver index between the single-drug groups and the combination drug group, suggesting that no abnormalities were found in the liver of the combination drug group compared with the single-drug groups.

[0259] Table 11 Effects of eight TOPO II inhibitors and their combinations on liver index in H22 solid tumor mice

[0260]

[0261] Note: The difference compared with the control group was statistically significant (p < 0.01). * The difference compared with the control group was statistically significant (p < 0.05). * The difference compared to the model group was statistically significant (p < 0.01). ## The difference compared to the model group was statistically significant (p < 0.05). # The difference compared to the combination drug group was statistically significant (p < 0.01). △△ The difference compared to the combination drug group was statistically significant (p < 0.05). △ .

[0262] 2) Serum enzyme indicators

[0263] Alanine aminotransferase (ALT) distributed within glutamate cells Figure 18 ) and aspartate aminotransferase (AST) Figure 19 Alkaline phosphatase (ALP) levels rise when liver cells are damaged, and the degree of increase corresponds to the extent of liver cell damage. Figure 20 A significantly elevated level of total protein (TP) often indicates obstructed biliary drainage, suggesting possible cholestatic hepatitis or extrahepatic biliary obstruction. Figure 21 ) and albumin (ALB) Figure 22 A decrease in the value often indicates a weakened ability of the liver to synthesize proteins, and the degree of reduction is directly proportional to the severity of liver disease.

[0264] As shown in Table 12:

[0265] (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.

[0266] (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.

[0267] 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.

[0268] (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.

[0269] In summary, each of the single-drug groups showed some degree of liver damage, with the doxorubicin and idarubicin groups showing the most severe damage, while the combination drug groups did not show significant liver damage.

[0270] Table 12 Effects of eight TOPO II inhibitors and their combinations on serum enzyme indicators related to liver injury in H22 solid tumor mice

[0271]

[0272]

[0273] Note: The difference compared with the control group was statistically significant (p < 0.01). ** The difference compared with the control group was statistically significant (p < 0.05). * The difference compared to the model group was statistically significant (p < 0.01). ## The difference compared to the model group was statistically significant (p < 0.05). # The difference compared to the combination drug group was statistically significant (p < 0.01). △△ The difference compared to the combination drug group was statistically significant (p < 0.05). △ .

[0274] (8) Nephrotoxicity

[0275] Like the liver, the kidneys are relatively susceptible to drug damage. This experiment used the kidney index (KPI) to investigate this. Figure 23 ), serum enzyme marker Urea (UREA) Figure 24 ), serum creatinine (SCr) ( Figure 25 To evaluate whether each treatment group caused kidney damage.

[0276] 1) Kidney index

[0277] As can also be seen from Table 13:

[0278] (i) There were no statistically significant differences in renal index between the drug administration groups and the model group (p>0.05), indicating that no abnormalities were observed in renal index in any of the drug administration groups;

[0279] (ii) There were no statistically significant differences in renal index between the single-drug groups and the combination drug groups (p>0.05), indicating that no abnormalities were observed in renal index in any of the drug groups.

[0280] Table 13 Effects of eight TOPO II inhibitors and their combinations on the kidney index in H22 solid tumor mice

[0281]

[0282] Note: The difference compared with the control group was statistically significant (p < 0.01). ** The difference compared with the control group was statistically significant (p < 0.05). * The difference compared to the model group was statistically significant (p < 0.01). ## The difference compared to the model group was statistically significant (p < 0.05). # The difference compared to the combination drug group was statistically significant (p < 0.01). △△ The difference compared to the combination drug group was statistically significant (p < 0.05). △ .

[0283] 2) Serum enzyme indicators

[0284] Elevated levels of UREA and SCr indicate kidney abnormalities. As shown in Table 14,

[0285] (i) Among the two indicators UREA and SCr, there was no statistically significant difference between the model group and the blank group (P>0.05), suggesting that tumor inoculation does not cause kidney abnormalities.

[0286] (ii) Comparison of the single-drug groups and the combination drug groups revealed differences in different indicators among the various drugs. Specifically, compared with the model group, the doxorubicin group showed statistically significant differences in UREA (p < 0.01) and SCr (p < 0.05); the arubicin group showed a statistically significant difference in UREA (p < 0.01); the etoposide group showed a statistically significant difference in SCr (p < 0.01); and the idarubicin group showed a statistically significant difference in SCr (p < 0.05). This indicates that all groups exhibited varying degrees of renal dysfunction, with the doxorubicin group showing the most severe abnormalities. In these two indicators reflecting renal function, there were no statistically significant differences between the combination drug group and the model group, suggesting that the combination drug did not induce renal dysfunction.

[0287] (iii) The differences between the data in each single-drug group and the combination drug group fluctuated. Among them, the statistically significant fluctuations were as follows: In the SCr index, the fluctuation in the combination drug group was 80.4% lower than that in the etoposide group and 85.0% lower than that in the idarubicin group. This suggests that, compared with the drugs that caused severe renal damage, the combination drug group showed a significant reduction in renal function indicators.

[0288] In summary, all treatment groups showed some degree of kidney damage, with the doxorubicin group showing the most severe damage, while the combination drug group did not show significant kidney damage.

[0289] Table 14 Effects of eight TOPO II inhibitors and their combinations on serum markers related to kidney injury in H22 solid tumor mice

[0290]

[0291] Note: The difference compared with the control group was statistically significant (p < 0.01). ** The difference compared with the control group was statistically significant (p < 0.05). * The difference compared to the model group was statistically significant (p < 0.01). ## The difference compared to the model group was statistically significant (p < 0.05). # The difference was statistically significant compared with the combination drug group (p < 0.01) and statistically significant compared with the combination drug group (p < 0.05). △ .

[0292] In summary, among the eight selected categories of toxic side effects, including growth status, cardiotoxicity, hepatotoxicity, nephrotoxicity, and mortality, the combination drug group showed no toxic side effects in most indicators compared to the individual drug groups, or the toxic side effects were not significantly enhanced compared to the individual drug groups, and the overall trend showed a decrease in toxicity. This demonstrates that the pharmaceutical composition of the present invention has low toxic side effects.

[0293] Example 3: Antitumor effect of the pharmaceutical composition of the present invention in mouse ascites tumor experiment

[0294] 1.1 Experimental Materials, Reagents and Instruments

[0295] Unless otherwise specified, the same principle applies to Example 2.

[0296] 1.2. Experimental Methods

[0297] Unless otherwise specified, the same principle applies to Example 2.

[0298] 1.2.1 Preparation of main reagents

[0299] Ascites tumor (eight drugs) experiment: the LD50 of each drug in the single-drug group was as follows 50 One-twentieth of the LD50, the dosage concentration of each drug in the combination drug group is also its LD50. 50 One-twentieth of it.

[0300] 1.2.2 Tumor inoculation:

[0301] Ascites tumor inoculation: Under aseptic conditions, ascites fluid was extracted from tumor-bearing mice, and tumor cells were counted under a microscope. The solution was then adjusted to 3 × 10⁻⁶ saline. 6 One tumor cell / mL, mixed well, and injected intraperitoneally into each mouse with 0.2mL / 20g.

[0302] 1.2.3 Animal Grouping

[0303] Ascites tumor experiment: ICR mice were inoculated with tumor cells. 48 hours after inoculation, the animals were weighed and randomly divided into 10 groups of 10 mice each. The groups were: model group, doxorubicin group, etoposide group, idarubicin group, acridine group, arubicin group, teniposide group, epirubicin group, pirarubicin group, and combination drug group (a mixture of eight groups: doxorubicin group, etoposide group, idarubicin group, acridine group, teniposide group, arubicin group, pirarubicin group, and epirubicin group).

[0304] 1.2.4 Administration method, route, and concentration

[0305] The drug concentrations in the ascites tumor experimental group administered alone were their LD50 concentrations. 50 One-twentieth of the LD50, the dosage concentration of each drug in the combination drug group is also its LD50. 50 One-twentieth of that. The specific value is as follows:

[0306] Ascites tumor experiment: In the model group, starting from the third day after tumor cell inoculation, 0.2 mL / 20 g of normal saline was injected intraperitoneally every other day; in each drug treatment group, starting from the third day after tumor cell inoculation, 0.2 mL / 20 g of the corresponding drug was injected intraperitoneally every other day; in the combination drug group, starting from the third day after tumor cell inoculation, 0.2 mL / 20 g of the corresponding drug was injected intraperitoneally every other day; a total of 5 administrations were given, for a total of 10 days (see the administration procedure). Figure 26 B). The dosage concentrations are shown in Table 15 below:

[0307] Table 15 Experimental drug concentrations of eight TOPO II inhibitors and their combinations against H22 ascites tumors.

[0308]

[0309] 1.2.5 Indicator Testing

[0310] The growth status of experimental animals with ascites tumors (eight drugs) was observed for 20 days.

[0311] 1.2.5.1 Survival days and life extension rate of ascites tumor model mice

[0312] The time of death for each group of animals was recorded to determine the number of days of survival, and the average life extension rate for each group was calculated.

[0313]

[0314] 1.3 Experimental Results

[0315] (1) Records of mouse mortality in each group

[0316] The mortality records of mice in each group are shown in Table 16. Starting from day 6 of the experiment, mice gradually began to die, and by day 10, all 10 mice in three groups (including the model group) had died. The combination drug group began dying on day 13, while deaths in the other groups began between days 6 and 8. On day 12, the last day without any deaths in the combination drug group, only one animal survived in all other groups.

[0317] Table 16: Summary of Mortality in Mice with Ascites Tumors

[0318]

[0319]

[0320] (2) Survival time of mice in each group

[0321] Depend on Figure 4The results showed that the average survival time in the combination drug group was 16 days, while the average survival time in the other groups was less than 10 days. The survival time in the combination drug group was significantly different from that in the model group (p < 0.01). The survival time in the other drug groups was not significantly different from that in the model group (p > 0.05). This indicates that the combination drug intervention significantly increased the survival time of mice. The other drug groups did not show this effect, suggesting that the individual drugs at this dosage had no anti-tumor efficacy.

[0322] (3) Survival time extension rate of mice in each group

[0323] Depend on Figure 5 It can be seen that the life extension rate of the combination drug group was 92.8%, which was statistically significant compared with other drug groups (p<0.01).

[0324] The results showed that the combined drug group significantly increased the life expectancy of mice after intervention, while none of the other individual drug groups exhibited this effect.

[0325] Based on the combined indicators of average survival time and life extension rate of mice, it is suggested that the combination drug group can significantly prolong the life cycle of mice with ascites tumors, and its anti-tumor effect is significantly better than that of each individual drug group (in fact, none of the individual drug groups had anti-tumor effects).

[0326] Example 4: Antitumor effects of drug compositions with different formulations in mouse solid tumor experiments

[0327] In addition to Formula 1 mentioned in Examples 2-3, the applicant also studied the antitumor effects and toxic side effects of various formulas (Formulas 2 to 5) in mouse solid tumor experiments after the combination of each drug in different proportions of the same eight drugs. The specific formulas are shown in Table 17, the experimental scheme is the same as in Example 2, and the results are shown in Table 18.

[0328] Table 17. Comparison of prescription dosages (μmol / kg) of different formulations of the composition

[0329]

[0330] Table 18. Comparison of antitumor effects and adverse reactions among different formulations*

[0331]

[0332]

[0333] *Values ​​represent percentage changes relative to the model group (excluding mortality). Percentage = (Model group data - Data for each drug group) / Model group data. A decrease in the index of a drug group indicates toxicity. Alternatively, percentage = (Data for each drug group - Model group data) / Model group data. An increase in the index of a drug group indicates toxicity. Bold numbers indicate toxicity. Negative numbers indicate that the measured value is better than the model group. # P<0.05 and ## P<0.01. ALB, albumin; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; CK, creatine kinase; LDH, lactose dehydrogenase; SCr, creatinine; TP, total protein.

[0334] As shown in Table 18 above, while maintaining a similar tumor inhibition rate, prescription 2 resulted in 20% mortality in mice compared to prescription 1, and also caused severe damage to the liver, heart, kidneys, and immune system in surviving mice. In conclusion, prescription 2 is significantly more toxic than prescription 1.

[0335] While maintaining a similar tumor inhibition rate, Formula 3 resulted in 30% mortality in mice compared to Formula 1, and also caused severe damage to the liver, heart, kidneys, and immune system in surviving mice. In conclusion, Formula 3 was significantly more toxic than Formula 1.

[0336] While formula 4 showed a similar or even lower tumor inhibition rate, compared to formula 1, it resulted in 50% mortality in mice, and caused the most severe damage to the liver, heart, kidneys, and immune system in the surviving mice. In conclusion, the toxicity of formula 4 was far greater than that of formula 1.

[0337] While maintaining a similar tumor inhibition rate, prescription 5 resulted in 10% mortality in mice compared to prescription 1. It also caused severe damage to the immune system in surviving mice and some damage to the liver and heart. In conclusion, prescription 5 was significantly more toxic than prescription 1.

[0338] In summary, even with the same combination of eight drugs, using low doses, if the proportions of the individual drugs in the composition exceed a certain range, these drug combinations can still exhibit significant toxicity. For example, formulations 2 through 5 mentioned above all showed high toxicity, leading to the death of 10% to 50% of mice. The surviving mice all exhibited significant liver, heart, and / or kidney toxicity, and severe immune system damage. These results demonstrate that even with the same eight-drug combination, appropriate proportions of the components are necessary to achieve good antitumor efficacy and low toxicity.

[0339] Example 5: Comparison of toxicity of different drug compositions

[0340] 1.1 Experimental Objective

[0341] This embodiment conducts parallel pharmacodynamic and toxicological evaluations at the cellular level of the combination drugs in group A (topotecan, 10-hydroxycamptothecin, 9-aminocamptothecin, irinotecan, idarubicin, daunorubicin, doxorubicin, etoposide, teniposide, epirubicin) and group B (doxorubicin, etoposide, idarubicin, acridine, arubicin, teniposide, epirubicin, pirarubicin). The focus is on investigating the inhibitory effects of these two combination drugs on the proliferation of tumor cells—human acute myeloid leukemia cells (HL-60 cells) (i.e., antitumor efficacy) and their damaging effects on normal cells—rat cardiomyocytes (H9C2 cells) (i.e., cardiotoxicity). By comparing the differences in efficacy (targeting HL-60 cells) and toxicity (targeting H9C2 cells) between group A and group B, the aim is to provide experimental evidence to determine which combination drug offers a better balance between antitumor efficacy and safety.

[0342] The technical approach and methodology of this experiment are as follows:

[0343] ① All chemotherapy drugs have significant toxicity, and both combinations of drugs have some degree of toxicity. To compare which combination of drugs has greater application potential, the relative toxicity of the two combinations can be compared.

[0344] ② Because the number of drug types and the composition of the drugs in groups A and B are not entirely the same, the total amount of drugs in the two groups is also different. Therefore, directly comparing the tumor-inhibiting effects of the two groups of drugs is not easy to obtain an objective and reasonable conclusion. Therefore, we designed a comparative experimental technique. First, we screened out the drug concentrations of each group that have the same inhibitory rate on tumor cells. Then, we compared the toxicity of the two groups of drugs to normal cells at their respective concentrations (expressed as cell survival rate values; the higher the value, the lower the toxicity). We used this toxicity to determine which group of drugs has higher application value: that is, under the condition of the same anti-tumor effect, the combination drug with less toxicity has higher application value.

[0345] 1.2 Experimental Materials

[0346] 1.2.1 Experimental Consumables

[0347] Human promyelocytic leukemia cells (HL-60 cells; CC-Y1236; ATCC; Shanghai Enzyme Research Biotechnology Co., Ltd.); rat cardiomyocytes (H9C2 cells; YCL-0349; ATCC; Shanghai Yizefeng Biotechnology Co., Ltd.); penicillin-streptomycin mixture (100×) (P1400, Solarbio); dimethyl sulfoxide (DMSO; D8371, Solarbio); Cell Counting Kit-8 kit (Liji Biotechnology); 1×0.01M phosphate-buffered saline (PBS, pH 7.2-7.4) (P1200, Solarbio); 0.25% trypsin (containing EDTA) digestion solution; DMEM complete medium (Shanghai Yizefeng Biotechnology Co., Ltd.); culture dishes (430167, Corning) and 96-well plates (3599, Corning) were purchased from Beijing Shuangzhi Technology Co., Ltd.; Fetal Bovine Serum (Gibco, A5669701, Thermo Fisher Scientific); DMEM basal medium (Gibco, C11960500BT, Thermo Fisher Scientific); IMDM basal medium (Gibco, 12440053, Thermo Fisher Scientific); 10-Hydroxycamptothecin (HY-N0095), 9-Aminocamptothecin (HY-100309), Topotecan hydrochloride (HY-13768A), Irinotecan (HY-16562); Idarubicin hydrochloride (HY-17381), Mitoxantrone hydrochloride The following drugs were purchased from Shanghai Haoyuan Biomedical Technology Co., Ltd.: aclacinomycin 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), and amsacrine (HY-13551). All drugs were purchased with a purity >99% (test reports provided by the company).

[0348] 1.2.2 Experimental Apparatus

[0349] Microplate reader (Multiskan FC, Thermo Scientific); Inverted microscope (OLYMPUS, CKX41); Centrifuge (LD5-2A, Jingli); Autoclave (G154DWS, Zhiwei Xiamen); Carbon dioxide incubator (CCL-170B-8, ESCO); Milli-Q ultrapure water system (ZRXQ010T0, MilliPore); Electronic analytical balance (MSA125P-1CE-DU, Sartorius); Water bath constant temperature shaker (SHA, Changzhou Guohua); Constant temperature water bath (SHHW21-420, Tianjin Taist); Cell culture incubator: ESEO (cell cuture; CO2; INCUBATOR); Multiskan FC microplate reader (Thermo Fisher Scientific; 51119000).

[0350] 1.3 Experimental Procedures and Content

[0351] 1.3.1 The CCK-8 assay was used to investigate the tumor cell inhibition rate and the toxicity of the combination drug to normal cells.

[0352] Using a pipette, 1 mL of 0.25% trypsin digestion solution containing EDTA was used to digest H9C2 cells in logarithmic growth phase in a T25 cell culture flask for 1 minute and 30 seconds. Then, 4 mL of fresh culture medium was added to stop the digestion, and the cells were transferred to a 15 mL flask for centrifugation (1000 rpm, 5 min). HL-60 cells, being suspension cells, did not require digestion and were directly centrifuged. After centrifugation, fresh culture medium was added to resuspend the cells, and the HL-60 cell suspension concentration was adjusted to 4 × 10⁻⁶ cells / mL. 5 / mL, H9C2 cell suspension concentration was 2×10 4 / mL; use a pipette to seed 100μL of each cell into a 96-well plate. To prevent evaporation of liquid from the edge wells, add 100μL of (PBS) around the outer edge of the 96-well plate, then incubate in a cell culture incubator for 24 hours before drug administration.

[0353] Cells that had reached stable growth status after 24 hours were collected from 96-well plates. Cell-free culture medium blank wells, a control group containing cells but not treated, and each treated group were set up, with 6 parallel wells in each group. The stock solution of the aforementioned drug was diluted with basal culture medium to the designed concentration and prepared fresh for each use. 100 μL was administered to each well.

[0354] After drug administration, the 96-well plate was placed in a cell culture incubator to allow the drug to act for 24 hours, and then the color development process was performed the next day.

[0355] After 24 hours of drug reaction, remove the cell plates and add 10% (total volume) of CCK-8 solution to each well. Incubate for 1.5 hours, continuously monitoring the color development to prevent over-incubation. After incubation, measure the absorbance (OD) at 450 nm using a microplate reader.

[0356] The cell inhibition rate was calculated using the following formula, and the dose-response curves for each drug were calculated and plotted using GraPhPad Prism 10.1.2 data processing software.

[0357]

[0358] 1.3.2 Specific experimental content and procedures

[0359] 1.3.2.1 Drug Information Group A: Topotecan, 10-hydroxycamptothecin, 9-aminocamptothecin, irinotecan, idarubicin, daunorubicin, doxorubicin, etoposide, teniposide, epirubicin (10 kinds).

[0360] Group B: Doxorubicin, Etoposide, Idarubicin, Acridine, Arubicin, Teniposide, Epirubicin, Pirarubicin (8 types).

[0361] The concentration ratios of prescription drugs in Group A are shown in Tables 19 and 20; the concentration ratios of prescription drugs in Group B are shown in Tables 19 and 21.

[0362] 1.3.2.2 Experiment Content

[0363] (1) First, based on the dose-response curves of 13 drugs in HL-60 tumor cells in groups A and B, different concentrations of combination drugs in groups A and B were formed. By continuously trying and adjusting the concentration, the two groups of combination drugs were made to achieve the same inhibition rate on HL-60 cells, so as to find the combination drug groups of groups A and B with the same inhibition rate.

[0364] (2) Based on the drug concentration that achieves the same inhibitory effect on tumor cell proliferation, the effects of the combination drugs of Group A and Group B on H9C2 normal cells were observed using the corresponding concentrations, and then the differences in toxicity and safety of the two combination drugs under the same tumor inhibition rate were observed.

[0365] 1.4 Experimental Results

[0366] 1.4.1 Exploration of concentrations in groups A and B that exhibit the same inhibitory rate against HL-60 tumor cells

[0367] 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 after being diluted 0.25 times at their corresponding concentrations (Table 21). Next, the various drugs were combined according to different dilution ratios and administered to HL-60 cells to observe cell viability. Figures 27-28 ).

[0368] Table 19. Concentrations (μM) corresponding to the respective inhibition rates of the two drug groups.

[0369]

[0370] Table 20A: Dosage Concentration (μM)

[0371]

[0372]

[0373] Table 21. Drug concentration (μM) in Group B

[0374]

[0375] Figure 27 This indicates the survival rate of HL-60 tumor cells in groups A and B under different combinations. Figure 27-I This indicates the inhibition of HL-60 tumor cells by combination drug A at different concentrations. Figure 27-II This indicates the results of different combinations of drug B inhibiting HL-60 tumor cells. From Figure 27 It can be seen from this that combination A uses IC 20The 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 20 The survival rate of HL-60 tumor cells after treatment with [B(IC)×0.5] was 45.36%. 30 The survival rate of HL-60 tumor cells after treatment with [A(IC)×0.75] was 44.05%, and there was no statistically significant difference in survival rate between the two combinations (P>0.05). The other two combinations: [A(IC)×0.75] 20 [B(IC)×0.75] and [B(IC)] 10 [A(IC)] can also inhibit HL-60 tumor cells from producing similar survival rates (P>0.05), i.e., [A(IC)] 20 The survival rate of HL-60 tumor cells after treatment with [B(IC)×0.75] was 22.77%. 10 The survival rate of HL-60 tumor cells after treatment was 22.5%. Figure 28 -I).

[0376] 1.4.2 Effects of Group A and Group B on H9C2 normal cells under the same tumor cell inhibitory efficacy

[0377] Figure 28-I For the combination [A(IC 20 [B(IC)×0.5] and [B(IC)] 30 [)×0.75]、[A(IC 20 [B(IC)×0.75] and [B(IC)] 10 [Regarding the inhibitory effect on HL-60 tumor cells, while] Figure 28-II Indicates [A(IC)] 20 [B(IC)×0.5] and [B(IC)] 30 [)×0.75]、[A(IC 20 [B(IC)×0.75] and [B(IC)] 10 The toxic effects exhibited by [A(IC)] on normal H9C2 cells, of which [A(IC)] 20 [B(IC)×0.5] and [B(IC)] 30 After treatment with [A(IC)×0.75], the survival rates of normal H9C2 cells were 48.93% and 78.93%, respectively, while [A(IC)×0.75]... 20 [B(IC)×0.75] and [B(IC)] 10 The survival rates of H9C2 normal cells after treatment were 35.34% and 67.86%, respectively.

[0378] Figure 29The survival rates of HL-60 tumor cells (anti-tumor efficacy) and H9C2 normal cells (toxicity) under the action of each combination drug were combined for comparison. It can be seen that, when both groups A and B exerted the same inhibitory effect on HL-60 tumor cells (anti-tumor efficacy), the cardiocytotoxicity of any combination drug in group B was significantly lower than that in group A (the higher the survival rate of H9C2 cardiomyocytes, the lower the toxicity). Secondly, the inhibition rate of each combination drug on HL-60 tumor cells was significantly higher than that on H9C2 normal cells, and this difference between the two combination drugs in group B was much greater than that in group A at the same tumor inhibition rate. Therefore, considering the difference in the survival rates of HL-60 and H9C2 cells under the same combination drug, and the difference in the inhibition of H9C2 cardiomyocytes between groups A and B when exerting the same anti-tumor efficacy, the combination drugs in group B have less cardiotoxicity (H9C2 cells) than those in group A.

[0379] 1.5 Summary

[0380] Under the premise that the two combination drugs A and B exert the same inhibitory effect on tumor cells (HL-60 leukemia cells), the toxicity of the two combination drugs on normal cells (H9C2 cardiomyocytes) was observed. The results showed that the toxicity of the combination drug B on H9C2 cardiomyocytes was much less than that of the combination drug A.

[0381] The specific details are as follows: First, two types of combination drugs in groups A and B, each capable of achieving the same HL-60 tumor cell inhibition rate, were identified: [A(IC 20 The survival rate of HL-60 cells after treatment with [B(IC)×0.5] was 45.36%. 30 The survival rate of HL-60 cells after treatment with [A(IC)×0.75] was 44.05%; 20 The survival rate after treatment with HL-60 was 22.77% (B(IC)×0.75). 10 The survival rate of HL-60 cells after treatment was 22.5%. Then, the combined drugs were used to treat H9C2 cells, and the inhibition results were as follows: [A(IC 20 [B(IC)×0.5] and [B(IC)] 30 After treatment with [A(IC)×0.75], the survival rates of H9C2 cells were 48.93% and 78.93% respectively (inhibition rates were 51.1% and 21.1% respectively), meaning that the toxicity of B was reduced by 58.7% compared to A; [A(IC)×0.75] 20 [B(IC)×0.75] and [B(IC)] 10After treatment with B, the survival rates of H9C2 cells were 35.34% and 67.86% respectively (inhibition rates were 64.66% and 32.14% respectively), meaning that B's toxicity was reduced by 50.3% compared to A. Furthermore, B (IC 10 ) and A(IC 20 Compare with B(IC) × 0.5, in B(IC) 10 The toxicity of H9C2 (survival rate 67%) was significantly lower than that of A (IC). 20 With a survival rate of 49% (%), B(IC) × 0.5 10 The tumor inhibition rate of HL-60 (78%) of A (IC) was much greater than that of HL-60. 20 The tumor inhibition rate was 55% (55%). These results suggest that the therapeutic window of the combination drugs in group B may be much wider than that in group A.

[0382] Therefore, based on the difference in inhibition between the two combinations A and B on HL-60 tumor cells and H9C2 normal cells, the combination drug in group B has less toxicity and better safety than the combination drug in group A.

[0383] Table 22 shows the dosage concentrations and concentration ranges of Group B, which exhibits antitumor efficacy and low toxicity. Based on Table 22, we obtained the following proportions of each drug in the compositions of eight topoisomerase II inhibitors exhibiting antitumor efficacy and low toxicity, by mass: 0.38–0.70 parts doxorubicin, 1.37–2.0 parts etoposide, 0.15–0.16 parts idarubicin, 0.076–0.28 parts acridine, 0.30–0.43 parts arubicin, 0.95–1.60 parts teniposide, 0.67–0.68 parts epirubicin, and 1.21–2.16 parts pirarubicin.

[0384] Table 22 shows the dosage concentrations and concentration ranges for Group B, which has antitumor efficacy and low toxicity.

[0385]

[0386]

[0387] a. Mass concentration (μg) = molar concentration (μM) × molecular weight; b. Mass concentration ÷ 300; c. Mass concentration ÷ 100.

Claims

1. A pharmaceutical composition for the prevention and / or treatment of tumors using a topoisomerase II inhibitor, said pharmaceutical composition comprising, as an active ingredient, a topoisomerase II inhibitor: Doxorubicin, etoposide, idarubicin, acridine, arubicin, teniposide, epirubicin, pirarubicin.

2. The pharmaceutical composition according to claim 1, wherein, The pharmaceutical composition comprises, by weight parts, the following topoisomerase II inhibitors as active ingredients: 0.38–0.70 parts of doxorubicin, 1.37–3.84 parts of etoposide, 0.12–0.18 parts of idarubicin, 0.08–0.91 parts of acridine, 0.30–1.02 parts of arubicin, 0.95–1.78 parts of teniposide, 0.41–0.68 parts of epirubicin, and 0.59–2.16 parts of pirarubicin; Preferably, the pharmaceutical composition comprises, by weight parts, the following topoisomerase II inhibitor as the active ingredient: 0.38–0.70 parts of doxorubicin, 1.37–3.84 parts of etoposide, 0.12–0.18 parts of idarubicin, 0.08–0.91 parts of acridine, 0.30–1.02 parts of arubicin, 0.95–1.78 parts of teniposide, 0.41–0.68 parts of epirubicin, and 0.59–2.16 parts of pirarubicin.

3. The pharmaceutical composition according to claim 1 or 2, wherein, The pharmaceutical composition comprises, by weight parts, the following topoisomerase II inhibitors as active ingredients: 0.4–0.63 parts of doxorubicin, 2.41–3.84 parts of etoposide, 0.12–0.18 parts of idarubicin, 0.63–0.91 parts of acridine, 0.64–1.02 parts of arubicin, 1.18–1.78 parts of teniposide, 0.41–0.67 parts of epirubicin, and 0.59–0.88 parts of pirarubicin; Preferably, the pharmaceutical composition comprises, by weight parts, the following topoisomerase II inhibitor as the active ingredient: 0.4–0.63 parts of doxorubicin, 2.41–3.84 parts of etoposide, 0.12–0.18 parts of idarubicin, 0.63–0.91 parts of acridine, 0.64–1.02 parts of arubicin, 1.18–1.78 parts of teniposide, 0.41–0.67 parts of epirubicin, and 0.59–0.88 parts of pirarubicin.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein, The pharmaceutical composition comprises, by weight parts, the following topoisomerase II inhibitors as active ingredients: 0.50 parts doxorubicin, 3.20 parts etoposide, 0.15 parts idarubicin, 0.75 parts acridine, 0.81 parts arubicin, 1.48 parts teniposide, 0.54 parts epirubicin, and 0.70 parts pirarubicin; Preferably, the pharmaceutical composition comprises, by weight parts, the following topoisomerase II inhibitor as the active ingredient: 0.5 parts doxorubicin, 3.2 parts etoposide, 0.15 parts idarubicin, 0.75 parts acridine, 0.81 parts arubicin, 1.48 parts teniposide, 0.54 parts epirubicin, and 0.70 parts pirarubicin.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein, The pharmaceutical composition comprises the following topoisomerase II inhibitor as an active ingredient: Doxorubicin 0.38–0.70 mg, etoposide 1.37–3.84 mg, idarubicin 0.12–0.18 mg, acridine 0.08–0.91 mg, arubicin 0.30–1.02 mg, teniposide 0.95–1.78 mg, epirubicin 0.41–0.68 mg, pirarubicin 0.59–2.16 mg; Preferably, the pharmaceutical composition comprises the following topoisomerase II inhibitor as the active ingredient: Doxorubicin 0.38–0.70 mg, etoposide 1.37–3.84 mg, idarubicin 0.12–0.18 mg, acridine 0.08–0.91 mg, arubicin 0.30–1.02 mg, teniposide 0.95–1.78 mg, epirubicin 0.41–0.68 mg, pirarubicin 0.59–2.16 mg; Preferably, the pharmaceutical composition comprises the following topoisomerase II inhibitor as an active ingredient: Doxorubicin 0.4–0.63 mg, etoposide 2.41–3.84 mg, idarubicin 0.12–0.18 mg, acridine 0.63–0.91 mg, arubicin 0.64–1.02 mg, teniposide 1.18–1.78 mg, epirubicin 0.41–0.67 mg, pirarubicin 0.59–0.88 mg; Preferably, the pharmaceutical composition comprises the following topoisomerase II inhibitor as the active ingredient: Doxorubicin 0.4–0.63 mg, etoposide 2.41–3.84 mg, idarubicin 0.12–0.18 mg, acridine 0.63–0.91 mg, arubicin 0.64–1.02 mg, teniposide 1.18–1.78 mg, epirubicin 0.41–0.67 mg, pirarubicin 0.59–0.88 mg; Preferably, the pharmaceutical composition comprises the following topoisomerase II inhibitor as an active ingredient: 0.5 mg doxorubicin, 3.2 mg etoposide, 0.15 mg idarubicin, 0.75 mg acridine, 0.81 mg arubicin, 1.48 mg teniposide, 0.54 mg epirubicin, and 0.70 mg pirarubicin; Preferably, the pharmaceutical composition comprises the following topoisomerase II inhibitor as the active ingredient: 0.5 mg doxorubicin, 3.2 mg etoposide, 0.15 mg idarubicin, 0.75 mg acridine, 0.81 mg arubicin, 1.48 mg teniposide, 0.54 mg epirubicin, and 0.70 mg 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 preparation of a medicament for the prevention and / or treatment of tumors.

8. The use according to claim 7, wherein, The tumor is selected from liver cancer, leukemia, lymphoma, breast cancer, stomach cancer, lung cancer, ovarian cancer, bladder cancer, etc., preferably liver cancer, leukemia or lymphoma.

Images