Application of CCR2 antagonists in the preparation of cardioprotective drugs

Abstract

The present invention provides the use of a CCR2 antagonist in the treatment of myocarditis, particularly in the treatment of immune checkpoint inhibitor-associated myocarditis. The present invention also provides a pharmaceutical composition comprising a CCR2 antagonist and 5-aminosalicylic acid, and its use in the treatment of immune checkpoint inhibitor-associated myocarditis. When the CCR2 antagonist and 5-aminosalicylic acid are administered in combination, the therapeutic effect of myocarditis is enhanced.

Description

Technical Field

[0001] The present invention relates to the use of chemokine receptor 2 (CCR2) antagonists in the treatment of myocarditis, particularly in the treatment of immune checkpoint inhibitor-associated myocarditis. The present invention also relates to a pharmaceutical composition comprising a chemokine receptor 2 (CCR2) antagonist and 5-aminosalicylic acid and its use in the treatment of immune checkpoint inhibitor-associated myocarditis. Background Art

[0002] Immune checkpoint inhibitors (ICIs) are now used to treat a variety of malignancies. Compared to traditional chemotherapy drugs, ICIs have achieved remarkable results in improving the long-term survival of some cancer patients. Therefore, the expansion of their indications is under constant exploration. As the number of people using ICIs increases, the incidence of immune-related adverse events (irAEs) has also increased. The most common irAEs are fatigue, pruritus, diarrhea, and rash. Although most irAEs are mild or moderate, do not require specific treatment, and resolve after drug discontinuation, some severe irAEs, such as ICI-related pneumonitis, ICI-related myocarditis, ICI-related neurotoxicity, and fatal diarrhea, not only hinder treatment and thus affect therapeutic efficacy, but may also directly lead to patient death. Among these, ICI-related myocarditis, despite its low incidence, is the adverse event with the highest mortality rate among irAEs. Data show that the incidence of ICI-related myocarditis ranges from 0.06% to 3.8%, while the mortality rate is as high as 39.7% to 66.0%.

[0003] Related studies have shown that the development of ICI-related myocarditis is associated with the infiltration of a large number of immune cells, primarily CD68-positive macrophages and CD4- and CD8-positive T lymphocytes. However, the molecular mechanism by which these immune cells specifically attack the heart remains unclear. TCR sequencing results have shown the presence of numerous T cell clones in cardiac and tumor tissues, suggesting that myocardial tissue harbors the same antigenic epitopes as tumor tissue, which can be recognized by the same T cells. Some researchers also believe that there is only a small overlap between the TCR clones in cardiac tissue and tumor tissue, with the majority of TCR clones being completely different from those in tumor tissue. Currently, the preferred treatment for immune myocarditis is pulse therapy with adequate glucocorticoids, but some patients do not respond well to steroid therapy. Guidelines define such patients as having steroid-resistant myocarditis and recommend intensive immunosuppressive therapy. However, intensive immunosuppressive therapy not only promotes tumor progression but also weakens the body's defenses against invasion by microorganisms such as bacteria and viruses, leading to serious infection. Therefore, there is an urgent need to develop new treatments for ICI-related myocarditis. Summary of the Invention

[0004] In one aspect, provided herein is the use of a CCR2 antagonist in the preparation of a medicament for cardioprotection.

[0005] In some embodiments, the drug is a myocarditis drug.

[0006] In some embodiments, the myocarditis is immune checkpoint inhibitor-associated myocarditis.

[0007] In some embodiments, the CCR2 antagonist is RS504393.

[0008] In some embodiments, the medicament further comprises 5-aminosalicylic acid.

[0009] In some embodiments, the drug is an injection.

[0010] In another aspect, provided herein is a pharmaceutical composition comprising a CCR2 antagonist, 5-aminosalicylic acid, and a pharmaceutically acceptable carrier.

[0011] In some embodiments, the CCR2 antagonist is RS504393.

[0012] In another aspect, provided herein is a pharmaceutical kit comprising a CCR2 antagonist and 5-aminosalicylic acid.

[0013] In some embodiments, the CCR2 antagonist is RS504393.

[0014] Thus, the present invention provides the use of CCR2 antagonists for cardioprotection in immune checkpoint inhibitor-associated myocarditis, and in particular, when administered in combination with 5-aminosalicylic acid, the therapeutic effect of myocarditis is enhanced. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1Shown are echocardiograms of mice in the control, RS504393, TNI+PD-1 inhibitor, and TNI+PD-1 inhibitor+RS504393 groups on day 21 of the experiment. Results are presented for left ventricular ejection fraction (LVEF), left ventricular short-axis contraction fraction (LVFS), systolic left ventricular diameter (Diameter, s), diastolic left ventricular diameter (Diameter, d), systolic left ventricular anterior wall thickness (LVAW; s), diastolic left ventricular anterior wall thickness (LVAW; d), systolic left ventricular posterior wall thickness (LVPW; s), and diastolic left ventricular posterior wall thickness (LVPW; d) from Example 1. *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001, same below. Results without a horizontal line indicating significant differences between groups are relative to the control group, same below.

[0016] Figure 2 The results of the detection of myocardial markers (cTNI, cTNI) in mouse serum in Example 1 are shown.

[0017] Figure 3 The photographs of HE staining and immunofluorescence staining of mouse heart sections in Example 1 are shown.

[0018] Figure 4 The RNA detection results of mouse heart tissue in Example 1 are shown.

[0019] Figure 5 The flow cytometry results of immune cells in mouse heart tissue in Example 1 are shown.

[0020] Figure 6 The results of CCL2 secretion by HUVECs human umbilical vein endothelial cells in Example 2 are shown.

[0021] Figure 7 The weight of mice in the Control group, TNI+PD-1inhibitor group, TNI+PD-1inhibitor+5-ASA group, TNI+PD-1inhibitor+RS504393 group, and TNI+PD-1inhibitor+RS504393+5-ASA group on the 21st day of the experiment in Example 3 are shown.

[0022] Figure 8 The results of left ventricular ejection fraction (LVEF) and left ventricular fractional contraction (LVFS) tests in mice in Example 3 are shown.

[0023] Figure 9 The results of the detection of mouse myocardial markers (cTNI, cTNI) in Example 3 are shown.

[0024] Figure 10 The results of flow cytometry detection of immune cells in mouse heart tissue in Example 3 (CCR2 + MHC II + macrophage percentage). DETAILED DESCRIPTION

[0025] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0026] "CCR2 antagonist" herein refers to an agent that can bind to the CCR2 receptor with high affinity but does not produce chemokine 2 (CCL2)-related activity. CCR2 antagonists can prevent the binding of CCL2 to CCR2, thereby exhibiting the effect of CCR2 inhibitors.

[0027] "Pharmaceutical kit" herein refers to packaging containing at least one drug, such as a paper box, glass container, etc. The different drugs in the pharmaceutical kit can be present separately and can be formulated into different pharmaceutical compositions or different dosage forms. The pharmaceutical kit typically also includes instructions for use.

[0028] "Pharmaceutically acceptable carrier" herein refers to a solid or liquid diluent, filler, antioxidant, stabilizer or other substance that can be safely administered to an animal or human body, and is suitable for administration to humans and / or animals without excessive adverse side effects, and is suitable for maintaining the activity of the drug or active agent located therein. Depending on the route of administration, various carriers well known in the art can be used, including, but not limited to, sugars, starch, cellulose and its derivatives, maltose, gelatin, talc, calcium sulfate, vegetable oils (such as castor oil), synthetic oils, polyols, alginic acid, phosphate buffer, emulsifier, isotonic saline, and / or pyrogen-free water. Suitable routes of administration include, for example, oral, intravenous infusion, intramuscular injection, subcutaneous injection, subperitoneal, rectal, sublingual, or by inhalation, transdermal or other routes. The drug and pharmaceutically acceptable carrier can be formulated into any clinically acceptable pharmaceutical composition or dosage form, such as tablets, granules, powders, capsules, injections, suppositories, drops, external ointments, ointments, medicated oils, or sprays, etc.

[0029] The CCR2 antagonist RS504393 has the following structure (cas number: 300816-15-3):

[0030]

[0031] 5-aminosalicylic acid has the following structure (CAS number: 89-57-6)

[0032]

[0033] This study demonstrates the role of the CCL2-CCR2 axis in ICI-associated myocarditis through animal studies. Using the CCR2 antagonist RS504393, we demonstrate that inhibiting CCR2 can alleviate ICI-associated myocarditis. This finding not only mitigates ICI-associated myocarditis but also provides a new approach for managing adverse reactions associated with tumor immunotherapy. In particular, during animal studies, we unexpectedly discovered that the combined administration of 5-aminosalicylic acid and RS504393 enhances the therapeutic effect of RS504393 in treating myocarditis.

[0034] The present invention is further illustrated below by means of specific examples.

[0035] Example 1

[0036] The SPF-grade male Balb / c mice used were purchased from Shanghai Jiesijie Experimental Animal Co., Ltd., with an age of 6-8 weeks and a weight of 20-25 g. They were divided into 4 groups, with 8 mice in each group: Control group; RS504393 group (CCR2 antagonist group); TNI+PD-1inhibitor group (model group); TNI+PD-1inhibitor+RS504393 group (treatment group).

[0037] Mice in the TNI+PD-1 inhibitor group and the TNI+PD-1 inhibitor+RS504393 group were subcutaneously injected with TNI+complete Freund's reagent (TNI 250 μg / mouse, Sangon Biotech (Shanghai) Co., Ltd., TnI sequence: HARVDKVDEERYDVEAKVTKNITEIADLTQKIYDLRGKFKRPTLRRVRIS (troponin I); complete Freund's reagent 100 μL / mouse, Sigma, cat. no. F5881) on days 1 and 7, respectively. Starting on day 8, mice were intraperitoneally injected with a PD-1 inhibitor (5 mg / kg, InVivoMab anti-mouse PD-1, CD279) every two days for a total of five times to establish a PD-1 inhibitor-induced myocarditis mouse model (this modeling method was based on Chinese Patent Publication CN113424800A). Mice in the TNI+PD-1 inhibitor+RS504393 group received intraperitoneal injections of RS504393 (5 mg / kg, MedChemExpress, Cat. No. HY-15418) every two days starting on day 8, co-injected with the PD-1 inhibitor on the day of PD-1 inhibitor injection. Mice in the RS504393 group received an equal dose of RS504393 every two days starting on day 8; mice in the control group received an equal dose of saline every two days starting on day 8. On day 21, cardiac ultrasound was performed on the mice, serum was collected for myocardial marker analysis, and heart specimens were collected for hematoxylin and eosin staining. Myocardial infiltration of CD68-positive macrophages was assessed by immunofluorescence, the proportion of CCR2-positive macrophages in myocardial tissue was analyzed by flow cytometry, and the expression of chemokine genes such as CCL2 in myocardial tissue was assessed by RT-PCR.

[0038] 1.1 Echocardiographic examination of mice

[0039] On day 21, echocardiography was performed on mice in each group using a Vevo 2100 ultrasound system (Visual Sonics) with a 30 MHz high-frequency scanning probe. Mice were anesthetized with isoflurane inhalation after pre-thoracic skin preparation, placed in a supine position, and secured to the echocardiography platform. When the heart rate was maintained at 450-500 beats / min, M-mode images were acquired continuously for 15 seconds from the parasternal left ventricular long-axis outflow tract view at the point of maximum left ventricular internal diameter. Functional indices included left ventricular ejection fraction (LVEF), left ventricular short-axis contraction fraction (LVFS), systolic left ventricular internal diameter (Diameter; s), diastolic left ventricular internal diameter (Diameter; d), systolic left ventricular anterior wall thickness (LVAW; s), diastolic left ventricular anterior wall thickness (LVAW; d), systolic left ventricular posterior wall thickness (LVPW; s), and diastolic left ventricular posterior wall thickness (LVPW; d). Cardiac function changes were compared among the mice in each group. All measurements were averaged over five consecutive cardiac cycles.

[0040] See also Figure 1 The experimental results showed that the cardiac function indicators (LVEF, LVFS) of mice in the TNI+PD-1 inhibitor group were significantly lower than those in the control group (LVEF: 58.51±4.04% vs 80.66±1.74%, p<0.0001; LVFS: 30.38±2.71% vs 47.89±1.79%, p<0.0001), and the left ventricular internal diameter was significantly higher in both systole and diastole than in the control group (systole: 2.58±0.29mm vs 1.50±0.25mm, p<0.0001; diastole: 3.71±0.38mm vs 2.88±0.46mm, p<0.01); after treatment with RS504393, the cardiac function of mice was significantly improved compared with that of mice in the TNI+PD-1inhibitor group. The LVEF of mice in the TNI+PD-1inhibitor+RS504393 group was 71.03±2.59% (p<0.001), and the LVFS was 39.33±2.15% (p<0.001). In addition, the left ventricular internal diameter, anterior and posterior wall thickness and other indicators were also significantly improved compared with those in the TNI+PD-1inhibitor group.

[0041] 1.2 Detection of mouse myocardial markers

[0042] After isoflurane anesthesia of mice, approximately 600 μL of blood was collected from the medial canthus of the mice and centrifuged. The supernatant was aspirated and serum cTnI (Mouse cTnI ELISA Kit, E-EL-M1203, Elabscience) and cTnT (Mouse cTnT ELISA Kit, E-EL-M1801, Elabscience) concentrations were measured using ELISA kits according to the methods provided in the kits.

[0043] See also Figure 2 The results showed that the myocardial markers of mice in the TNI+PD-1inhibitor group were significantly higher than those in the control group (cTnI: 83.86±24.39pg / mL vs 11.65±6.99pg / mL, p<0.0001; cTnT: 49.77±28.00pg / mL vs 2.45±2.48pg / mL, p<0.01); and the myocardial markers of mice in the TNI+PD-1inhibitor+RS504393 group were improved compared with the TNI+PD-1inhibitor group (cTnI: 30.43±3.34pg / mL vs 83.86±24.39pg / mL, p<0.01; cTnT: 20.12±10.25pg / mL vs 49.77±28.00pg / mL, p=0.057).

[0044] 1.3 HE staining of mouse myocardial tissue

[0045] After euthanizing the mice, they were fixed on the operating table in accordance with animal ethics. After removing the mouse heart, the tissue was fixed in tissue fixative, embedded in paraffin, and then cut into 5 μm thick sections for HE staining. Representative images under the light microscope (magnification 10 times) were selected to observe the infiltration of inflammatory cells.

[0046] See also Figure 3 The results in the upper row of pictures show that the cardiac inflammatory infiltration in mice in the TNI+PD-1inhibitor group was significantly more severe than that in the Control group; while the cardiac inflammatory infiltration in mice in the TNI+PD-1inhibitor+RS504393 group was improved.

[0047] 1.4 Immunofluorescence staining of mouse myocardial tissue

[0048] After dewaxing and hydrating, cardiac slides were placed in citric acid (pH 6.0) antigen retrieval solution in a water bath at 92°C for 20 minutes for antigen retrieval. After cooling, the slides were washed three times in PBS for 5 minutes each. After drying, the slides were dripped with buffer and incubated at room temperature for 10 minutes. The slides were then washed again in PBS for 5 minutes each. TUNEL reagents 1 and 2 were added and incubated at 37°C for 1 hour. After washing with PBS, the slides were blocked with serum for 30 minutes at room temperature. The primary antibody was incubated overnight at 4°C: anti-CD68 (servicebio, catalog number: GB113109, 1:200). After washing with PBS, the slides were incubated with a fluorescent secondary antibody: CY3-goat anti-rabbit (servicebio, catalog number: GB21303, 1:300) for 50 minutes at room temperature in the dark. After washing with PBS, the slides were stained with DAPI and incubated at room temperature for 10 minutes in the dark. After washing with PBS, the slides were mounted with anti-quenching mounting medium. Images were acquired under a fluorescence microscope (10x magnification).

[0049] See also Figure 3 The results in the lower row of pictures show that a large number of macrophages infiltrated the heart tissue of mice in the TNI+PD-1inhibitor group, accompanied by cardiomyocyte apoptosis; while the macrophage infiltration and cardiomyocyte apoptosis in the heart tissue of mice in the TNI+PD-1inhibitor+RS504393 group were improved.

[0050] 1.5 RNA extraction, reverse transcription, and RT-PCR detection from mouse myocardial tissue

[0051] Heart tissue (about 5*5*5mm in size) was cut and total RNA from heart tissue was extracted using a cell / tissue total RNA extraction kit (YEASEN, catalog number: 19221ES50). RNA was reverse transcribed into cDNA using a reverse transcription kit (PrimeScript RT Reagent Kit With gDNA Eraser) and then amplified using a real-time fluorescence quantitative PCR kit (TBGreen Premix ExTaq). PCR reaction conditions were as follows: pre-denaturation at 95°C for 30s, 95°C for 5s, and 60°C for 30s, for a total of 40 cycles. The expression of cytokine and chemokine-related genes was detected. -ΔΔCt The values ​​represent the relative expression levels of each target gene.

[0052] Figure 4Representative test results from two mice each in the control and TNI+PD-1 inhibitor groups are shown. The results showed that cytokine and chemokine genes were significantly upregulated in the heart tissues of mice in the TNI+PD-1 inhibitor group compared with those in the control group, with the CCL2 chemokine gene being significantly upregulated.

[0053] 1.6 Flow cytometry detection of CCR2 in myocardial tissue + MHC II + Macrophage infiltration

[0054] After euthanasia, the mice were exposed and perfused with pre-cooled saline until the liver turned white. The hearts were separated, the atria and valves were removed, and the ventricular tissue was cut into 1 mm 3 Place the fragments of different sizes into an EP tube containing 1.5 mL of digestion solution (0.15 mg / mL LiberaseTM, 20 μg / mL DNase I, prepared in serum-free DMEM) and gently shake at 37°C for 15 minutes to digest the heart. After filtering the cells with a 70 μm filter, add 2 volumes of staining buffer containing 2% FBS to terminate the digestion. Centrifuge at 350g for 5 minutes at 4°C, discard the supernatant, and resuspend in 100 μL PBS to obtain a single-cell suspension. Tubes: blank tube, single-staining tube, sample tube. Dead cell staining: BD Horizon TMIncubate with Fixable Viability Stain 510 (BD Pharmingen, Catalog No. 564406, 1:1000) at room temperature in the dark for 15 minutes. After washing with staining buffer, block with mouse Fc receptor blocker (Catalog No. abs9477-200T, 2 μL / tube) at 4°C for 5 minutes. The primary antibodies were incubated at room temperature in the dark for 15 min (according to the recommended dose in the instructions): APC-Cy7 Rat Anti-Mouse CD45 (BD Pharmingen, Catalog No.: 557659), FITC Rat Anti-Mouse Ly-6C (BD Pharmingen, Catalog No.: 553104), PE-Cyanine7 Rat Anti-Mouse Ly-6G (Thermo, Catalog No.: 25-9668-82), PE ANTI-MO CD64 X54-5 / 7.1 (Thermo, Catalog No.: 12-0641-82), BV421 Rat Anti-Mouse CD192 (CCR2) (BD Pharmingen, Catalog No.: 747963), Alexa Fluor 647 Rat Anti-Mouse IA / IE (MHC II) (BD Pharmingen, Catalog No.: 562367). After washing with PBS, the cells were centrifuged at 350 g for 5 min at 4°C. The supernatant was discarded and the cells were resuspended in 300 μL of PBS, sieved, and loaded onto the flow cytometer. Flow cytometry data were analyzed using FlowJo.

[0055] See also Figure 5 The results showed that compared with the Control group, the CCR2 + MHC II + Macrophage infiltration increased significantly (84.20±3.03% vs 28.87±6.50%, p<0.0001). + MHC II + Macrophage infiltration was significantly improved compared with the TNI+PD-1inhibitor group (44.90±11.14% vs 84.20±3.03%, p<0.01).

[0056] Example 2

[0057] Since upregulation of CCL2 chemokine expression was observed in mice in the TNI+PD-1 inhibitor group in Example 1, we performed cytological experiments to observe the effect of PD-1 inhibitor (InVivoMab anti-mouse PD-1) on cellular CCL2 chemokine secretion.

[0058] Cell Grouping and Model Preparation: Human umbilical vein endothelial cells (HUVECs) were purchased from the Cell Bank of the Chinese Academy of Sciences and divided into two groups: PBS (control group) and PD-1 inhibitor (treatment group). Cells in the PD-1 inhibitor group were treated with different concentrations of PD-1 inhibitor for 24 hours; cells in the PBS group were treated with the same amount of PBS for 24 hours. Cell supernatants were collected, and CCL2 chemokine secretion was assayed by ELISA.

[0059] The concentration of CCL2 in the supernatant was determined by enzyme-linked immunosorbent assay (ELISA) using a kit (proteintech, catalog number: KE00091). Figure 6 The results showed that after HUVECs were treated with different concentrations of PD-1 inhibitors, the secretion of CCL2 was significantly increased compared with the PBS group (p<0.0001).

[0060] Example 3

[0061] We conducted multiple animal experiments as described in Example 1. During one experiment, two mice in the TNI+PD-1 inhibitor+RS504393 group developed mild colitis. We treated them by adding 5-aminosalicylic acid (5-ASA) to their feed and housing them individually. The results showed that these two mice had significantly increased body weight at the end of the experiment compared to the other mice in the same group, and their myocarditis symptoms (such as piloerection, huddling, and decreased activity) were reduced. After eliminating other factors, we realized that 5-aminosalicylic acid might also be effective in treating myocarditis in mice and conducted the following experiments.

[0062] The experimental procedures and reagent dosages were essentially as described in Example 1. Male Balb / c mice, approximately 6 weeks old and weighing 21-23 g, were divided into five groups, each with eight mice: control group; TNI + PD-1 inhibitor group (model group); TNI + PD-1 inhibitor + 5-ASA group (Group A); TNI + PD-1 inhibitor + RS504393 group (Group B); and TNI + PD-1 inhibitor + RS504393 + 5-ASA group (Group C). Mice in the model group, Groups A, B, and C were subcutaneously injected with TNI + complete Freund's reagent on days 1 and 7. Starting on day 8, a PD-1 inhibitor (5 mg / kg) was intraperitoneally injected every two days for a total of five times. Mice in Group A were intraperitoneally injected with 5-ASA (2 mg / kg, Sigma, Catalog No. A3537) every two days starting on day 8. Mice in Group B were intraperitoneally injected with RS504393 (5 mg / kg) every two days starting on day 8. Mice in Group C were intraperitoneally injected with RS504393 (5 mg / kg) and 5-ASA (2 mg / kg) every two days starting on day 8. Mice in the Control group were injected with an equal volume of saline every two days starting on day 8.

[0063] On the 21st day, the mice were weighed and relevant tests were performed according to the method described in Example 1. The results are as follows.

[0064] Figure 7 The results of mouse body weight are shown. Control group, 26.8±1.33g; model group, 21.9±1.56g; Group A, 21.4±1.43g; Group B, 23.6±1.72g; Group C, 25.3±2.03g. The body weight of mice in Group A, treated only with 5-ASA, was not significantly different from that in the model group (p>0.05). Group C, treated with both 5-ASA and RS504393, showed a significant increase in body weight, which was significantly different from that in Group B, treated only with RS504393 (p<0.05). This result is consistent with previous studies that found that adding 5-aminosalicylic acid to the feed promoted weight gain in mice with myocarditis.

[0065] Figure 8The results of LVEF and LVFS in mice are shown. LVEF: control group, 78.32±3.14%; model group, 49.87±4.12%; group A, 55.42±3.54%; group B, 70.66±2.79%; group C, 74.34±3.12%. LVFS: control group, 49.19±2.21%; model group, 28.35±2.61%; group A, 26.38±1.60%; group B, 36.07±2.82%; group C, 42.07±3.11%. These results demonstrate that the use of 5-ASA resulted in better LVEF and LVFS in mice in group C than in group B, although the LVEF results did not show a significant difference (p>0.05).

[0066] Figure 9 The results of mouse serum cTnI and cTnT assays are shown. cTnI: Control group, 12.13±4.17 pg / mL; Model group, 97.51±14.19 pg / mL; Group A, 102.12±15.33 pg / mL; Group B, 42.43±6.17 pg / mL; Group C, 22.38±3.84%. cTnT: Control group, 4.66±2.11 pg / mL; Model group, 38.46±10.36 pg / mL; Group A, 42.02±9.95 pg / mL; Group B, 28.12±6.25 pg / mL; Group C, 7.81±4.62 pg / mL. These results demonstrate that 5-ASA administration significantly improved symptoms of myocardial injury. Notably, cTnT assay results were no significantly different from those in the control group (p>0.05).

[0067] Figure 10 CCR2 + MHC II + Flow cytometric results of macrophage infiltration (CD64 + The percentage of cells in the control group was 24.44±5.54%; the model group was 69.53±6.77%; the group A was 63.71±8.03%; the group B was 39.65±8.46%; and the group C was 35.78±6.11%. + MHC II + The macrophage infiltration was also better than that in group B, although there was no significant difference (p>0.05).

[0068] The above results show that although 5-ASA itself can be used as an anti-inflammatory agent to treat colitis, it has little therapeutic effect on myocarditis in mice. When 5-ASA is used in combination with the CCR2 antagonist RS504393, the myocarditis therapeutic effect of RS504393 is improved.

Claims

1. Use of a CCR2 antagonist in the preparation of a drug for cardioprotection, wherein the drug is an immune checkpoint inhibitor-associated myocarditis drug, the CCR2 antagonist is RS504393, and the drug also includes 5-aminosalicylic acid.

2. The use according to claim 1, wherein the medicine is an injection.

3. A pharmaceutical composition comprising the CCR2 antagonist RS504393, 5-aminosalicylic acid and a pharmaceutically acceptable carrier.

4. A drug kit comprising the CCR2 antagonist RS504393 and 5-aminosalicylic acid.

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