Application of CCL2 / CCR2 signaling pathway blocker in relieving chemotherapeutic drug-induced cardiac injury

Abstract

The invention discloses an application of a CCL2 / CCR2 signal channel blocker in relieving chemotherapeutic drug-induced heart injury, and belongs to the technical field of biological medicines. The invention finds that a CCL2 / CCR2 signal channel blocker, particularly a CCL2 neutralizing antibody, has an obvious effect of improving the chronic cardiotoxicity induced by the chemotherapeutic drug adriamycin, and also can cooperate with the therapeutic effect of the chemotherapeutic drug at the same time. On the basis, the invention provides a combined treatment strategy based on a CCL2 neutralizing antibody, and the cardiotoxicity problem and tumor drug resistance challenge in the clinical application of the adriamycin are solved at the same time through targeted regulation and control of a CCL2 signal channel.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the application of a CCL2 / CCR2 signaling pathway blocker in alleviating chemotherapy drug-induced cardiac injury. Background Art

[0002] Cardiotoxicity is a serious complication during antitumor treatment. The cardiotoxic side effects caused by antitumor drugs seriously affect the quality of life of cancer patients. More importantly, the development of toxicity may lead to the adjustment or even termination of the antitumor treatment regimen, affecting the survival of patients. Therefore, effectively solving or preventing the cardiotoxic side effects of antitumor drugs can improve the quality of life of patients and improve the prognosis.

[0003] How to effectively solve chemotherapy-related cardiotoxicity is a practical problem faced by those skilled in the art. Studies have shown that, on the premise of ensuring the antitumor treatment effect, restricting the cumulative dose of chemotherapy drugs is a relatively effective method to reduce cardiotoxic reactions. Secondly, changing the administration route, such as continuously infusing the drug for 48 - 96 hours to reduce the peak drug concentration, is considered to reduce anthracycline toxicity without affecting the efficacy. Or, changing the dosing regimen, such as changing the 3-week dosing dose to weekly dosing, can reduce drug toxicity while ensuring the drug efficacy. However, the above-mentioned schemes are all for avoiding the occurrence of cardiotoxicity and do not solve the essence of the cardiotoxicity problem. Developing a drug that can improve the cardiac injury caused by drugs and has a cardioprotective effect is the key to solving the above problems.

[0004] The cardiotoxicity caused by antitumor drugs is complex and diverse. According to the different effects on cardiomyocytes, it is divided into type 1 and type 2: type 1 (chemotherapy drug-induced) cardiotoxicity refers to the induction of necrosis or apoptosis of cardiomyocytes, and this type is irreversible; type 2 (targeted drug-induced) cardiotoxicity refers to the induction of cardiomyocyte dysfunction rather than cell death, and this type is reversible. The present invention aims to solve type 1 cardiotoxicity caused by chemotherapy drugs.

[0005] Doxorubicin, as a representative drug of anthracycline antibiotics, has been a cornerstone drug in clinical oncology chemotherapy since its introduction in the 1960s. It exerts a broad-spectrum antitumor effect through multiple mechanisms such as intercalating into DNA double strands, inhibiting the activity of topoisomerase II, and generating reactive oxygen species (ROS), and occupies an important position in the treatment of various malignant tumors such as breast cancer, lymphoma, and soft tissue sarcoma. However, its clinical application is severely restricted by dose-limiting cardiotoxicity, and this side effect shows an obvious cumulative dose-dependent characteristic. Clinical data show that when the cumulative dose of doxorubicin exceeds 450 - 550 mg / m 2When this occurs, the risk of the patient developing congestive heart failure increases significantly, and this cardiac damage is often irreversible, seriously affecting the long-term quality of life of the patient.

[0006] In view of this, the present invention provides the following technical solutions. Summary of the Invention

[0007] The CCL2 / CCR2 signaling pathway is a key pathway mediating the recruitment of myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs). These immunosuppressive cells construct an immunosuppressive microenvironment by secreting immunosuppressive factors such as IL-10, TGF-β, etc., promoting tumor immune escape. Research shows that blocking the CCL2 / CCR2 signaling pathway can inhibit the growth and metastasis of malignant tumors, reduce postoperative recurrence, and improve survival rate. However, previous clinical trials of monotherapy targeting the CCL2 / CCR2 axis (such as carlumab) have shown poor efficacy, which may be related to compensatory mechanisms and complex tumor microenvironment regulatory networks. The present invention unexpectedly discovers that blocking the CCL2 / CCR2 signaling pathway can effectively improve the problem of cardiotoxicity induced by tumor chemotherapy drugs, and at the same time enhance the anti-tumor efficacy of chemotherapy drugs by regulating the immune response.

[0008] Based on the above discovery, the present invention provides the following specific technical solutions:

[0009] In the first aspect of the present invention, the present invention provides the use of a CCL2 / CCR2 signaling pathway blocker in at least one of the following:

[0010] a1) Use in the preparation of a drug for preventing cardiotoxicity induced by chemotherapy drugs;

[0011] a2) Use in the preparation of a drug for alleviating, treating or assisting in the treatment of cardiotoxicity induced by chemotherapy drugs.

[0012] In the present invention, prevention means administering a drug to a subject at risk of developing a disease to obtain a beneficial or desired result. The terms "treatment", "alleviation" or "improvement" as used in the present invention can be used interchangeably herein, and refer to the treatment process of administering a drug to a subject to achieve the eradication or improvement of the disease state.

[0013] The CCL2 / CCR2 signaling pathway blocker according to the present invention is selected from CCL2 inhibitors and / or CCR2 antagonists known to those skilled in the art.

[0014] In the present invention, the CCL2 inhibitor is selected from at least one of the following:

[0015] 1) The CCL2 inhibitor is selected from antibodies, polypeptides or small molecules that can bind to CCL2 protein, wherein the antibodies include but are not limited to humanized monoclonal antibodies, CCL2 neutralizing antibodies;

[0016] 2) The CCL2 inhibitor is selected from receptor antagonists with CCL2 as a ligand, and the receptor antagonist interferes with the binding of CCL2 to its receptor;

[0017] 3) The CCL2 inhibitor is selected from reagents that reduce or knock out the gene encoding the CCL2 protein, such as short hairpin RNA (shRNA) or small interfering RNA (siRNA).

[0018] In the present invention, the CCR2 antagonist is selected from at least one of the following:

[0019] 1) Chemical antagonists, including γ-aminobutyramide, glycinamide, thiazole, indole, disubstituted dipiperidinol, quaternary ammonium salts, or unsaturated heterocycles;

[0020] 2) Short hairpin RNA (shRNA), small interfering RNA (siRNA), or reagents related to CRISPER gene editing technology that can reduce or knock out the gene encoding CCR2.

[0021] In a specific embodiment of the present invention, the CCL2 / CCR2 signaling pathway blocker is selected from CCL2 neutralizing antibodies.

[0022] CCL2 (C-C motif chemokine ligand 2, also known as monocyte chemoattractant protein-1, MCP-1) belongs to the CC chemokine family and mainly exerts its function by binding to its receptor CCR2. The role of CCL2 in the body is very important and is mainly involved in immune responses and inflammatory processes. CCL2 can attract and recruit monocytes, memory T cells, and dendritic cells to the inflammatory site, helping the body resist infection and repair damage. In the immune system, when an infection or injury occurs in a certain part of the body, CCL2 will be released, guiding these immune cells to the places where they are needed to help clear pathogens or promote tissue repair. In the inflammatory response, CCL2 also plays a key role. It not only helps recruit immune cells but also regulates the intensity and duration of inflammation.

[0023] The chemotherapy drugs described in the present invention include anthracyclines, alkylating agents, anti-cytoskeletal agents, and antimetabolites.

[0024] In some embodiments of the present invention, the anthracyclines include, but are not limited to, doxorubicin (trade name adriamycin), epirubicin, idarubicin, or daunorubicin.

[0025] In some embodiments of the present invention, the alkylating agents include, but are not limited to, cyclophosphamide or ifosfamide.

[0026] In some embodiments of the present invention, the anti-cytoskeletal agent includes, but is not limited to, paclitaxel and docetaxel.

[0027] In some embodiments of the present invention, the antimetabolite includes, but is not limited to, fluorouracil.

[0028] In the present invention, the chemotherapy-induced myocardial injury includes the cardiac side effects caused by chemotherapy drugs during and after treatment known to those skilled in the art, including but not limited to myocardial injury, reduced left ventricular ejection fraction and fractional shortening, and cardiac atrophy.

[0029] The drug having a preventive, therapeutic or adjuvant therapeutic effect in the present invention has at least one of the following functions:

[0030] 1) Improve chemotherapy-induced cardiac injury;

[0031] 2) Alleviate chemotherapy-induced cardiac atrophy;

[0032] 3) Regulate the tumor immune microenvironment and enhance the efficacy of chemotherapy.

[0033] In a specific embodiment of the present invention, the improvement of chemotherapy-induced cardiac injury includes: 1) increasing the left ventricular ejection fraction (LVEF) and fractional shortening (FS); 2) reducing the levels of cardiac troponin T (cTnT) and creatine kinase isoenzyme (CK-MB).

[0034] In a specific embodiment of the present invention, the alleviation of chemotherapy-induced cardiac atrophy includes: 1) increasing the heart HW / TL ratio; 2) increasing the cross-sectional area of cardiomyocytes.

[0035] In a specific embodiment of the present invention, the regulation of the tumor immune microenvironment includes: 1) reducing granulocytic myeloid-derived suppressor cells (G-MDSC); 2) increasing the proportion of CD8+ T lymphocyte subsets; 3) increasing M1-type tumor-associated macrophages; 4) reducing M2-type tumor-associated macrophages.

[0036] In the present invention, the drug having a preventive, therapeutic or adjuvant therapeutic effect further includes an active substance having an effect of improving cardiac injury, including but not limited to dexrazoxane.

[0037] The drug further contains pharmaceutically acceptable excipients, and the pharmaceutically acceptable excipients are excipients and / or adjuvants conventionally used in the art for preparing pharmaceutical preparations.

[0038] In a specific embodiment of the present invention, the pharmaceutically acceptable excipients include one or a combination of two or more of carriers, excipients, fillers, disintegrants, emulsifiers, solubilizers, solubilizing agents, osmotic pressure regulators, surfactants, coating materials, binders, colorants, wetting agents, pH regulators, antioxidants, and bacteriostatic agents.

[0039] The dosage form of the drug is any pharmaceutically acceptable dosage form, including tablets, powders, suspensions, granules, capsules, and injections.

[0040] In a second aspect of the present invention, the present invention provides an application of a CCL2 / CCR2 signaling pathway blocker in the preparation of a tumor chemotherapy synergist.

[0041] The CCL2 / CCR2 signaling pathway blocker is as described in the first aspect of the present invention.

[0042] The tumor chemotherapy refers to the process of treating tumors with chemotherapy drugs, the chemotherapy drugs are as described in the first aspect of the present invention, and the tumors are malignant tumors, including but not limited to liver cancer, non-small cell lung cancer, breast cancer, lymphoma, and soft tissue sarcoma.

[0043] In a specific embodiment of the present invention, the application is selected from at least one of the following:

[0044] 1) Administering a therapeutically effective amount of the CCL2 / CCR2 signaling pathway blocker simultaneously with chemotherapy;

[0045] 2) Administering a therapeutically effective amount of the CCL2 / CCR2 signaling pathway blocker 12 - 36 hours before chemotherapy.

[0046] Preferably, the application is selected from administering a therapeutic dose of the CCL2 / CCR2 signaling pathway blocker 24 hours before chemotherapy.

[0047] In a third aspect of the present invention, the present invention provides a pharmaceutical composition, which includes a therapeutically effective amount of the CCL2 / CCR2 signaling pathway blocker and a chemotherapy drug.

[0048] Among them, the CCL2 / CCR2 signaling pathway blocker and the chemotherapy drug are as described in the first aspect of the present invention.

[0049] The drug further contains pharmaceutically acceptable excipients, and the pharmaceutically acceptable excipients are the excipients and / or auxiliaries conventionally used in the field for preparing pharmaceutical preparations.

[0050] In a specific embodiment of the present invention, the pharmaceutically acceptable excipients include one or a combination of two or more of a carrier, an excipient, a filler, a disintegrant, an emulsifier, a solubilizer, a solubilizing agent, an osmotic pressure regulator, a surfactant, a coating material, a binder, a colorant, a wetting agent, a pH regulator, an antioxidant, and an antibacterial agent.

[0051] Preferably, the pharmaceutical composition is a compound preparation or a medicine box with non-compound independent preparations.

[0052] In some embodiments of the present invention, the pharmaceutical composition is a compound preparation, and the compound preparation contains a therapeutically effective amount of a CCL2 / CCR2 signaling pathway blocker and a chemotherapeutic drug; wherein, the CCL2 / CCR2 signaling pathway blocker and the chemotherapeutic drug form a compound preparation through compounding.

[0053] In some embodiments of the present invention, the pharmaceutical composition is a medicine box, and the medicine box includes one or more doses of a pharmaceutical preparation of a CCL2 / CCR2 signaling pathway blocker and one or more doses of a pharmaceutical preparation of a chemotherapeutic drug; wherein, the pharmaceutical preparations of the CCL2 / CCR2 signaling pathway blocker and the chemotherapeutic drug are independently packaged.

[0054] The "one dose" as used in the present invention means that the content of the active ingredient in the pharmaceutical preparation is the single effective daily dose or the half-day dose.

[0055] The compound preparation or the independently packaged pharmaceutical preparation of the present invention can be a solid preparation, such as tablets, lozenges, and capsules, as well as solution preparations suitable for injection or oral administration.

[0056] The compound preparation or the independently packaged pharmaceutical preparation of the present invention can be administered by any suitable route to achieve the purpose of treating diseases. Such as by parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, oral, intrathecal, intracranial, nasal, or topical routes. As an alternative or in parallel, it can be administered orally. The dosage is determined by those skilled in the art according to the patient's age, health and weight, the type of concurrent treatment, the frequency of treatment, and the desired therapeutic benefit.

[0057] The compound preparation or the independently packaged pharmaceutical preparation of the present invention can be manufactured by methods known in the art, such as mixing, granulation, lozenge making, dissolution, freeze drying, and the like.

[0058] "Pharmaceutically acceptable" as used in the present invention means that a substance or composition must be chemically and / or toxicologically compatible with other components of the formulation and / or the mammal being treated therewith. Preferably, "pharmaceutically acceptable" means that it is approved by a federal regulatory agency or a national government or listed in the United States Pharmacopeia or other generally recognized pharmacopeias for use in animals, particularly in humans. The pharmaceutically acceptable excipients described in the present invention may include any solvent, solid excipient, diluent or other liquid excipient, etc., suitable for a particular target dosage form.

[0059] The pharmaceutical composition containing a CCL2 / CCR2 signaling pathway blocker and a chemotherapeutic drug according to the present invention can be used for the treatment of diseases and also for in vitro cell culture experiments. When used for the treatment of diseases, the pharmaceutical composition is usually administered in unit dosage form.

[0060] The disease treatment described in the present invention refers to administering a therapeutically effective amount of the pharmaceutical composition described in the third aspect of the present invention to a subject in need thereof. Further, the disease treatment also includes co-administering the pharmaceutical composition described in the third aspect of the present invention with other medicaments or conventional treatment methods commonly used in the art.

[0061] The inventors of the present invention unexpectedly found that a CCL2 / CCR2 pathway blocker, specifically a CCL2 neutralizing antibody, has a significant improvement in doxorubicin-induced chronic cardiotoxicity. On this basis, the present invention provides an application of a CCL2 / CCR2 pathway blocker including a CCL2 neutralizing antibody in improving cardiotoxicity of chemotherapeutic drugs. In future clinical practice, its specific application methods include the following key steps: First, on the basis of a standardized doxorubicin chemotherapy regimen, a therapeutic dose of the CCL2 neutralizing antibody is administered by intravenous infusion. It is recommended to administer the CCL2 neutralizing antibody 24 hours before doxorubicin administration to fully exert its cardioprotective effect. During the treatment, the following indicators need to be regularly monitored: ① Cardiac function parameters (left ventricular ejection fraction detected by echocardiogram); ② Serum myocardial injury markers (troponin I and CK-MB levels); ③ Tumor response (evaluated using the RECIST 1.1 standard). The combination treatment strategy provided by the present invention based on the CCL2 neutralizing antibody aims to simultaneously solve the problems of cardiotoxicity and tumor drug resistance challenges in the clinical application of doxorubicin by targeting and regulating the CCL2 signaling pathway.

[0062] Preclinical research data show that the combined application of the CCL2 neutralizing antibody provided by the present invention and doxorubicin can increase the maximum tolerated dose of doxorubicin while enhancing the tumor suppression effect. This combined treatment regimen is particularly suitable for patients with metastatic breast cancer, soft tissue sarcoma and other malignant tumors who need to receive high-dose doxorubicin treatment, significantly improving the clinical efficacy while ensuring treatment safety. All treatment procedures should be carried out in a medical center with the qualification of oncology cardiology diagnosis and treatment, and evaluated and monitored by a multidisciplinary team (MDT).

[0063] The treatment strategy provided by the present invention has the following two theoretical advantages: on the one hand, by blocking the CCL2 signaling pathway, it can reduce doxorubicin-induced myocardial inflammation and fibrosis and protect cardiac function; on the other hand, CCL2 inhibition may reshape the tumor immune microenvironment and enhance the anti-tumor effect of doxorubicin. In addition, doxorubicin can induce immunogenic cell death (ICD), release tumor-associated antigens and damage-associated molecular patterns (DAMPs), thereby activating anti-tumor immune responses, while the CCL2 neutralizing antibody may further enhance this process by regulating immune cell infiltration and function, achieving the synergistic effect of chemotherapy and immunomodulation.

[0064] The technical solution provided by the present invention has the following technical advantages:

[0065] 1) It realizes dual regulation in terms of action mechanism, not only significantly reducing the cardiotoxicity of doxorubicin, but also enhancing the anti-tumor efficacy of doxorubicin by targeting and regulating the immunosuppressive state in the tumor microenvironment.

[0066] 2) It proposes an innovative solution in terms of treatment strategy. By the sequential combined administration of the CCL2 neutralizing antibody and doxorubicin, a new "cardioprotection-tumor killing" synergistic treatment mode is established, providing an important theoretical basis for clinical translational research.

[0067] 3) It has significant expandability in terms of application scope. Given the core regulatory role of CCL2 in various pathological processes such as tumors, cardiovascular diseases, and fibrotic diseases, after appropriate optimization, this treatment regimen is expected to be extended to the treatment of various solid tumors such as breast cancer and non-small cell lung cancer, and provide new ideas for the toxicity management of other anthracycline chemotherapy drugs. These features make the present invention have important scientific value and clinical translational prospects in the field of comprehensive tumor treatment. Description of the Drawings

[0068] Figure 1A-C, CCL2 neutralizing antibody improved the left ventricular ejection fraction (LVEF) and fractional shortening (FS) in mice with doxorubicin-induced chronic cardiotoxicity, *P<0.05 vs. Dox+Vehicle; D-E, CCL2 neutralizing antibody reduced the levels of serum myocardial injury markers cTnT and CK-MB induced by doxorubicin, *P<0.05 vs. Dox+Vehicle.

[0069] Figure 2 A, H&E staining of mouse heart cross-sections; B, WGA staining; C, HW / TL and D, quantitative analysis of cardiomyocyte cross-sectional area, confirming that CCL2 neutralizing antibody improved the phenotypes related to chronic doxorubicin-induced cardiac atrophy in mice, *P<0.05 vs. Dox+Vehicle.

[0070] Figure 3 A, Establishment and experimental design of a subcutaneous tumor model of hepatocellular carcinoma in Hepa1-6 mice; B, Tumor growth curves of tumor-bearing mice treated with doxorubicin alone or in combination with CCL2 neutralizing antibody; C, Tumor weights of tumor-bearing mice treated with doxorubicin alone or in combination with CCL2 neutralizing antibody, *P<0.05 vs. Dox+Vehicle.

[0071] Figure 4 A, Flow cytometry gating strategy for G-MDSCs and M-MDSCs in mouse tumor tissues; B-C, Quantitative analysis of G-MDSCs and CD8+ T cells in tumor tissues based on flow cytometry (*P<0.05 vs. Dox+Vehicle); D, Representative immunohistochemical staining images of Ly6G, CD8, iNOS, and Arg-1 in mouse tumor tissue sections. Detailed implementation manners

[0072] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of them. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0073] In this embodiment, doxorubicin was purchased from Sigma-Aldrich, catalog number D1515. The CCL2 neutralizing antibody was purchased from antibody supplier BioXCell, catalog number BE0185.

[0074] I. Research methods

[0075] 1. Experimental animals

[0076] The male C57BL / 6 WT mice used in this experiment were purchased from Shanghai SLAC Laboratory Animal Center, Chinese Academy of Sciences, and Hepa1-6 cells were purchased from the American Type Culture Collection (ATCC). All animal experimental protocols were reviewed and approved by the Animal Experiment Ethics Committee of Zhongshan Hospital, Fudan University.

[0077] 2. Animal grouping

[0078] For the doxorubicin-induced chronic cardiac toxicity mouse model, the experimental mice were randomly divided into 3 groups, including: Control, Dox+Vehicle, Dox+anti-CCL2, with 10 mice in each group. Doxorubicin at 5 mg / kg was injected via the tail vein on the first day of each week for 4 consecutive weeks, and the control group was given an equal volume of normal saline. The CCL2 neutralizing antibody was intraperitoneally injected once every 3 days at a dose of 200 μg, and the control group was given an equal volume of normal saline. For the tumor-bearing model, the experimental mice were randomly divided into 3 groups, including: Control, Dox+Vehicle, Dox+anti-CCL2, with 10 mice in each group. Subsequently, a subcutaneous transplanted tumor liver cancer mouse model was established by subcutaneously inoculating the Hepa1-6 liver cancer cell line to simulate the growth and treatment effect of liver cancer, and the drug administration method was the same as above.

[0079] 3. Model preparation

[0080] In this study, male C57BL / 6 mice were used to establish a doxorubicin-induced chronic cardiac toxicity model and a Hepa1-6 cell liver cancer xenograft model. For the cardiac toxicity model, 30 mice were randomly divided into a Control group (injected with normal saline via the tail vein), a Dox+Vehicle group (5 mg / kg doxorubicin was injected via the tail vein once a week for 4 consecutive weeks and normal saline was intraperitoneally injected), and a Dox+anti-CCL2 group (doxorubicin as above + 200 μg CCL2 neutralizing antibody was intraperitoneally injected once every 3 days), with 10 mice in each group. To evaluate the anti-tumor effect, another liver cancer model was established. Mice were subcutaneously inoculated with 5×10 - 1×10 Hepa1-6 cells. On the 4th day after inoculation, the mice were grouped and given drugs according to the same protocol, and the tumor growth and cardiac function indexes were monitored simultaneously. All experimental operations complied with animal ethics norms.

[0081] 4. Main observation indexes

[0082] 4.1 Detection of mouse cardiac function and myocardial injury markers

[0083] Transthoracic echocardiography was performed using a Vevo 2100 system to evaluate cardiac function in mice. The left ventricular end-diastolic volume (LVEDV) and left ventricular end-systolic volume (LVESV) were calculated using the biplane area-length method. The left ventricular ejection fraction (LVEF) was calculated according to the formula: LVEF = [(LVEDV - LVESV) / LVEDV] × 100%. The two-dimensional guided M-mode curve of the left ventricle at the papillary muscle level was recorded in the short-axis and long-axis sections, and the left ventricular end-systolic internal diameter (LVID;s) and end-diastolic internal diameter (LVID;d) were measured. The left ventricular fractional shortening (LVFS) was calculated according to the formula LVFS = [(LVID;d - LVID;s) / LVID;d] × 100%. The contents of cardiac injury markers cTnT and CK-MB in mouse serum were detected by ELISA: after orbital blood collection, the blood was left to stand at 4°C for 30 minutes, and then centrifuged at 3000×g for 15 minutes to separate the serum, which was stored at -80°C for later use; mouse-specific cTnT and CK-MB ELISA kits were used in the experiment, and the operation was carried out strictly according to the instructions. The samples were diluted 50-fold before detection. The absorbance values were measured by an enzyme-linked immunosorbent assay reader at 450 nm (detection wavelength) and 630 nm (reference wavelength), and the concentrations of cTnT and CK-MB in the serum were calculated through the standard curve.

[0084] 4.2 Histological staining of the heart

[0085] The mice were sacrificed by cervical dislocation. After removing the heart, it was blotted dry on filter paper and weighed, and the heart weight / body weight ratio (HW / BW) was calculated; the tibia was removed, the tibia length was measured, and the heart weight / tibia length ratio (HW / TL) was calculated; the heart was fixed with 4% paraformaldehyde and then embedded in paraffin, and then sectioned into 4-μm-thick slices along the papillary muscle level of the heart. The sections were stained with hematoxylin-eosin (H&E) to observe the gross morphological structure of the left heart; wheat germ agglutinin (WGA) staining was used to observe the cross-sectional area of cardiomyocytes.

[0086] 4.3 Observation of the growth curve of subcutaneous transplanted tumors

[0087] In this study, a transplantable subcutaneous liver cancer animal model was established using a standardized experimental procedure. First, the Hepa1-6 mouse liver cancer cell line in the logarithmic growth phase was selected. After trypsin digestion and washing with serum-free medium, the cell viability was ensured to be >95% (detected by trypan blue staining). 5×10 - 1×10 cells were resuspended in PBS solution containing 20% Matrigel matrix gel and inoculated subcutaneously into the right inguinal region of C57BL / 6 mice (6 - 8 weeks old) using an insulin syringe in a laminar flow hood. The inoculation volume was strictly controlled at 100 μL per mouse. Starting from the 4th day after inoculation, daily observations were made. Two experimenters independently measured the longest diameter (a) and the perpendicular short diameter (b) of the tumor using a digital caliper (accuracy 0.01 mm). The tumor volume was calculated according to the formula V = 1 / 2×a×b 2 The final mass of the tumor was weighed and recorded during the final sample collection.

[0088] 4.4 Analysis of immune inflammatory cell infiltration in tumor tissues

[0089] For flow cytometry analysis, mouse tumor tissues were obtained and processed into single-cell suspensions using a mouse tumor cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's instructions. After excluding dead cells with BV510 viability dye, CD16 / 32 antibody was incubated to prevent non-specific binding. Subsequently, immune cells (CD45, CD11b, Ly6C, Ly6G) were stained darkly with relevant antibodies for 1 hour, washed, resuspended, and analyzed by machine. In immunohistochemical studies, mouse tumor tissues were fixed with 4% paraformaldehyde and embedded in paraffin. 4-μm thick sections were prepared and dewaxed and oven-dried. Microwave antigen retrieval was performed using sodium citrate antigen retrieval solution. After the sections were cooled to room temperature, they were rinsed with PBS and then blocked with 5% bovine serum albumin for 30 minutes to reduce background staining. Subsequently, the sections were incubated with anti-CD68, anti-Ly6G, anti-iNOS, and anti-Arg-1 antibodies at 37°C for 1 hour. Each section was then treated with 25 μL of horseradish peroxidase (HRP)-labeled secondary antibody at room temperature for 30 minutes, and a DAB HRP color development kit was used for color development. After the stained sections were counterstained with hematoxylin, observation and analysis were performed using a Leica DM LB2 microscope.

[0090] II. Research Results

[0091] 1. CCL2 neutralizing antibody improves cardiac function in mice with doxorubicin-induced chronic cardiotoxicity

[0092] To evaluate whether CCL2 neutralizing antibody can improve doxorubicin-induced chronic cardiotoxicity, mice were intravenously injected with doxorubicin (5 mg / kg) on days 0, 7, 14, and 21, with a cumulative dose of 20 mg / kg, which is equivalent to the standardized chemotherapy dose for clinical cancer patients.

[0093] Echocardiographic results showed that under chronic doxorubicin administration, the cardiac function parameters of mice in the CCL2 neutralizing antibody treatment group were significantly improved, as manifested by a significant increase in left ventricular ejection fraction (LVEF) and fractional shortening (FS) compared with the control group ( Figure 1 A-C). Further ELISA detection revealed that the levels of myocardial injury markers - cardiac troponin T (cTnT) and creatine kinase-MB (CK-MB) in the plasma of mice in the CCL2 neutralizing antibody group were significantly lower than those in the control group ( Figure 1 D-E). The above results indicate that blockade of the CCL2 signaling pathway can effectively reduce doxorubicin-induced chronic cardiotoxicity.

[0094] 2. CCL2 neutralizing antibody alleviates doxorubicin-induced cardiac atrophy phenotype in mice

[0095] To further explore the structural protective effect of CCL2 neutralizing antibody on chronic cardiac injury caused by doxorubicin, we further carried out cardiac tissue morphological analysis. Mice were randomly divided into two groups: Vehicle control group and CCL2 neutralizing antibody treatment group, and all mice were established with a chronic doxorubicin cardiotoxicity model according to the aforementioned protocol. Through H&E staining of cardiac tissue and quantitative analysis of heart weight / tibia length ratio (HW / TL), it was shown that chronic doxorubicin treatment led to significant pathological changes of cardiac atrophy in mice in the Vehicle group; while the cardiac atrophy in mice in the CCL2 neutralizing antibody intervention group was significantly alleviated, and the HW / TL ratio was significantly increased compared with the Vehicle group ( Figure 2 A and C), suggesting that CCL2 blockade can effectively relieve doxorubicin-induced cardiac atrophy. To further clarify the protective effect of CCL2 neutralizing antibody at the cardiomyocyte level, we used wheat germ agglutinin (WGA) staining to analyze the cross-sectional area of cardiomyocytes. The results showed that the cross-sectional area of cardiomyocytes in mice in the CCL2 neutralizing antibody group was significantly larger than that in the Vehicle group ( Figure 2 B and D). These histological evidences jointly confirmed at the organ level and cell level that blockade of the CCL2 signaling pathway can effectively improve doxorubicin-induced cardiac atrophy.

[0096] 3. CCL2 neutralizing antibody enhances the antitumor effect of doxorubicin

[0097] To evaluate the synergistic effect of CCL2 neutralizing antibody on the antitumor efficacy of doxorubicin, we established a Hepa1-6 hepatocarcinoma cell xenograft model. Specifically, a syngeneic xenograft model was constructed by subcutaneous inoculation of Hepa1-6 cells into C57BL / 6 mice. When the tumors grew to day 4, the tumor-bearing mice were randomly grouped and treatment was initiated. The treatment regimens included: (1) Vehicle control group; (2) Doxorubicin monotherapy group (5 mg / kg, intravenous injection once a week); (3) Doxorubicin combined with CCL2 neutralizing antibody treatment group (200 μg / time, intraperitoneal injection every 3 days). By dynamically monitoring the tumor growth curve, it was found that ( Figure 3 A), starting from day 4 after transplantation, the tumor volume was measured every 4 days. The results showed that compared with the Vehicle control group, doxorubicin monotherapy significantly inhibited tumor growth; while when combined with CCL2 neutralizing antibody treatment, tumor growth was more significantly inhibited. The results of sacrificing the mice at the experimental endpoint (day 21 after transplantation) and measuring the tumor weight further confirmed this finding: the average tumor weight in the doxorubicin monotherapy group was lower than that in the control group, and the tumor weight in the combination treatment group was further lower than that in the monotherapy group ( Figure 3 B and C). These data fully demonstrate that blocking the CCL2 signaling pathway can significantly enhance the antitumor efficacy of doxorubicin, suggesting the potential clinical value of combining CCL2 neutralizing antibody with chemotherapeutic drugs.

[0098] 4. CCL2 neutralizing antibody sensitizes the antitumor effect of doxorubicin by improving the tumor immune microenvironment

[0099] To further clarify the immune regulatory mechanism by which CCL2 neutralizing antibody enhances the antitumor efficacy of doxorubicin, we studied the changes in the tumor microenvironment through a multi-dimensional immune cell analysis system. First, flow cytometry was used to analyze the immune cell subsets in tumor tissues ( Figure 4 A). The quantitative results showed that after treatment with CCL2 neutralizing antibody, the infiltration ratio of granulocytic myeloid-derived suppressor cells (G-MDSCs) with immunosuppressive function was significantly reduced ( Figure 4 B), while the proportion of CD8+ T lymphocyte subsets with tumor-killing function was significantly increased ( Figure 4 C). Further, we performed immunohistochemical staining to analyze the immune cells in tumor tissues. The results showed that compared with the control group, after treatment with doxorubicin combined with CCL2 neutralizing antibody, the infiltration of tumor-suppressive G-MDSCs (labeled with Ly6G+) was significantly inhibited ( Figure 4(B), while the CD8+ T lymphocyte subset increased significantly. In addition, by analyzing the infiltration of tumor-associated macrophages, we found that the infiltration density of pro-inflammatory M1-type tumor-associated macrophages (TAMs, labeled with iNOS+) in the tumor tissue of the combination treatment group was significantly higher than that in the single-drug group, while the infiltration density of M2-type TAMs with immunosuppressive function (labeled with Arg-1+) decreased ( Figure 4 D). This significant reversal of the M1 / M2 macrophage ratio suggests that the CCL2 neutralizing antibody may enhance the efficacy of doxorubicin by remodeling the tumor immune microenvironment.

[0100] As a classic chemotherapy drug widely used clinically, doxorubicin has shown significant anti-tumor effects in the treatment of various malignancies. It has important therapeutic value especially for hematological malignancies and solid tumors. However, its clinical application has long been restricted by dose-limiting cardiotoxicity, and this serious side effect limits the full play of the drug's efficacy. Based on this key scientific problem, the present invention aims to systematically solve the inevitable cardiotoxicity problem during the treatment with doxorubicin, and at the same time deeply explore new strategies to enhance its anti-tumor efficacy. By targeting and intervening in the key regulatory molecule CCL2, the inventors of the present invention unexpectedly found that the CCL2 neutralizing antibody can not only significantly reduce the cardiotoxicity of doxorubicin, but also enhance the anti-tumor efficacy of doxorubicin by targeting and regulating the immunosuppressive state in the tumor microenvironment. The technical solution provided by the present invention has the important value of breaking through the treatment bottleneck of traditional chemotherapy drugs and providing a safer and more effective treatment plan for cancer patients.

[0101] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of a CCL2 / CCR2 signaling pathway blocker in at least one of the following: 1) Use in the preparation of a drug for preventing chemotherapy drug-induced myocardial injury; 2) Use in the preparation of a drug for alleviating, treating or assisting in the treatment of chemotherapy drug-induced myocardial injury.

2. The application according to claim 1, characterized in that, The CCL2 / CCR2 signaling pathway blocker is selected from CCL2 inhibitors and / or CCR2 antagonists; the chemotherapy drugs include anthracyclines, alkylating agents, anti-cytomicrotubule agents, and antimetabolic drugs.

3. The application according to claim 2, characterized in that, The CCL2 inhibitor is selected from at least one of the following: 1) The CCL2 inhibitor is selected from antibodies, polypeptides or small molecules that can bind to CCL2 protein, wherein the antibodies include but are not limited to humanized monoclonal antibodies and CCL2 neutralizing antibodies; 2) The CCL2 inhibitor is selected from receptor antagonists with CCL2 as a ligand, and the receptor antagonists interfere with the binding of CCL2 to its receptor; 3) The CCL2 inhibitor is selected from reagents that reduce or knock out the gene encoding CCL2 protein, such as short hairpin RNA or small interfering RNA; The CCR2 antagonist is selected from at least one of the following: 1) Chemical antagonists, including γ-aminobutyramide, glycinamide, thiazole, indole, disubstituted dipiperidinol, quaternary ammonium salts or unsaturated heterocycles; 2) Short hairpin RNA, small interfering RNA or reagents related to CRISPER gene editing technology that can reduce or knock out the CCR2 gene.

4. The application according to claim 3, wherein The CCL2 / CCR2 signaling pathway blocker is selected from CCL2 neutralizing antibodies.

5. The application according to claim 1, wherein The drug with preventive, therapeutic or adjuvant therapeutic effects has at least one of the following functions: 1) Improve chemotherapy-induced heart damage; 2) Alleviate chemotherapy-induced heart atrophy; 3) Regulate the tumor immune microenvironment and enhance the efficacy of chemotherapy.

6. The application according to claim 5, characterized in that, The improvement of chemotherapy-induced heart damage includes: 1) Increase the left ventricular ejection fraction (LVEF) and fractional shortening (FS); 2) Reduce the levels of cardiac troponin T (cTnT) and creatine kinase isoenzyme (CK-MB); The alleviation of chemotherapy-induced heart atrophy includes: 1) Increase the heart HW / TL ratio; 2) Increase the cross-sectional area of cardiomyocytes. The regulation of the tumor immune microenvironment includes: 1) Reduce granulocyte-like myeloid-derived suppressor cells (G-MDSC); 2) Increase the proportion of CD8+ T lymphocyte subsets; 3) Increase M1-type tumor-associated macrophages; 4) Reduce M2-type tumor-associated macrophages.

7. Use of the CCL2 / CCR2 signaling pathway blocker described in claim 1 in the preparation of a tumor chemotherapy synergist.

8. The application according to claim 7, characterized in that, The use is selected from at least one of the following: 1) Administer a therapeutically effective amount of the CCL2 / CCR2 signaling pathway blocker simultaneously with chemotherapy; 2) Administer a therapeutically effective amount of the CCL2 / CCR2 signaling pathway blocker 12 - 36 hours before chemotherapy.

9. The application according to claim 8, wherein, The use is selected from administering a therapeutic dose of the CCL2 / CCR2 signaling pathway blocker 24 hours before chemotherapy.

10. A pharmaceutical composition, characterized in that, The pharmaceutical composition includes a therapeutically effective amount of the CCL2 / CCR2 signaling pathway blocker described in claim 1 and a chemotherapy drug.

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