Application of CCR2 antagonist in preparation of heart protection medicine

Abstract

The invention provides an application of a CCR2 antagonist in myocarditis treatment, in particular to an application in immune checkpoint inhibitor related myocarditis treatment. The invention also provides a pharmaceutical composition containing the CCR2 antagonist and the 5-aminosalicylic acid and application of the pharmaceutical composition in immune checkpoint inhibitor related myocarditis, and when the CCR2 antagonist and the 5-aminosalicylic acid are combined for administration, the myocarditis treatment effect is enhanced.

Description

Technical Field

[0001] The present invention relates to the use of chemokine receptor 2 (CCR2) antagonists in the treatment of myocarditis, especially in the treatment of immune checkpoint inhibitor-related 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 immune checkpoint inhibitor-related myocarditis. Background Art

[0002] Immune checkpoint inhibitors (ICIs) are now used to treat a variety of malignant tumors. Compared with traditional chemotherapy drugs, ICIs have achieved amazing results in improving the long-term survival rate of some tumor patients. Therefore, the expansion of their indications is also under constant exploration. With the increase in the number of people using ICIs, the incidence of corresponding immune-related adverse events (irAEs) has also increased. The most common irAEs are fatigue, itching, diarrhea, and rash. Although most irAEs are mild or moderate, no special treatment is required, and they can be relieved after drug withdrawal, some serious irAEs, such as ICIs-related pneumonia, ICIs-related myocarditis, ICIs-related neurotoxicity, and fatal diarrhea, will not only hinder treatment and thus affect the treatment effect, but may also directly lead to patient death. Among them, although the incidence of ICIs-related myocarditis is low, it is the adverse event with the highest mortality rate among irAEs. Big data shows that the incidence of ICIs-related myocarditis is 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 occurrence of ICIs-related myocarditis is associated with the infiltration of a large number of immune cells, mainly CD68-positive macrophages, CD4 and CD8-positive T lymphocytes. However, the molecular mechanism of why related immune cells specifically attack the heart remains unclear. TCR sequencing results show that there are a large number of T cell clones in cardiac tissue and tumor tissue. It is speculated that myocardial tissue has the same antigenic epitopes as tumor tissue, which can be recognized by the same T cells. Some researchers also believe that the TCR clones of cardiac tissue overlap only a small part with tumor tissue, and most TCR clones are completely different from those in tumor tissue. At present, the preferred treatment for immune myocarditis is shock therapy with sufficient glucocorticoids, but some patients do not respond well to hormone therapy. The relevant guidelines define such patients as hormone-resistant myocarditis and recommend enhanced immunosuppressive therapy. However, while enhancing immunosuppressive therapy, it will not only promote the progression of tumors, but also weaken the body's resistance to the invasion of microorganisms such as bacteria and viruses, causing serious infections. Therefore, there is an urgent need to develop new treatments for ICIs-related myocarditis in clinical practice. 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 medicament 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 1The echocardiograms, left ventricular ejection fraction (LVEF), left ventricular short axis contraction rate (LVFS), left ventricular internal diameter (Diameter, s), left ventricular internal diameter (Diameter, d), left ventricular anterior wall thickness (LVAW; s), left ventricular anterior wall thickness (LVAW; d), left ventricular posterior wall thickness (LVPW; s), left ventricular posterior wall thickness (LVPW; d) of the mice in the Control group, RS504393 group, TNI+PD-1inhibitor group and TNI+PD-1inhibitor+RS504393 group on the 21st day of the experiment in Example 1 are shown. *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001, the same below. The significant difference results of the specific inter-group comparisons not shown by horizontal lines are the results relative to the Control group, the same below.

[0016] Figure 2 The results of the detection of mouse serum myocardial markers (cTNI, cTNI) 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 body weights 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 in Example 3 on the 21st day of the experiment are shown.

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

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

[0024] Fig.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 meanings 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 activities. CCR2 antagonists can prevent the binding of CCL2 to CCR2, thus showing the effect of CCR2 inhibitors.

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

[0028] "Pharmaceutically acceptable carrier" in this article refers to solid or liquid diluents, fillers, antioxidants, stabilizers and other substances that can be safely administered to animals or humans, which are suitable for administration to humans and / or animals without excessive adverse side effects, and are suitable for maintaining the vitality of the drugs or active agents located therein. Depending on the route of administration, various different 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, emulsifiers, isotonic saline, and / or pyrogen-free water, etc. Suitable routes of administration include, for example, oral, intravenous infusion, intramuscular injection, subcutaneous injection, subperitoneal, rectal, sublingual, or by inhalation, transdermal and other routes. The drug and a pharmaceutically acceptable carrier can be formulated into any clinically acceptable pharmaceutical composition or dosage form, such as tablets, granules, powders, capsules, injection preparations, 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] The present invention demonstrates the role of the CCL2-CCR2 axis in ICIs-related myocarditis through animal experiments, and verifies that inhibition of CCR2 can alleviate ICIs-related myocarditis through the CCR2 antagonist RS504393. This discovery can not only alleviate ICIs-related myocarditis, but also provide a new solution for the management of adverse reactions in tumor immunotherapy. In particular, when we conducted animal experiments, we unexpectedly found that the combined administration of 5-aminosalicylic acid and RS504393 can enhance the myocarditis treatment effect of RS504393.

[0034] The present invention is further illustrated by the following 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 body 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 (modeling group); TNI+PD-1inhibitor+RS504393 group (treatment group).

[0037] The mice in the TNI+PD-1inhibitor group and the TNI+PD-1inhibitor+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, catalog number: F5881) on the 1st day and the 7th day, respectively. From the 8th day on, the PD-1 inhibitor (5 mg / kg, InVivoMab anti-mouse PD-1, CD279) was intraperitoneally injected once every two days for a total of 5 times to construct a mouse model of myocarditis induced by PD-1 inhibitor (the modeling method was carried out with reference to Chinese patent publication CN113424800A). For mice in the TNI+PD-1inhibitor+RS504393 group, RS504393 (5 mg / kg, MedChemExpress, Cat. No.: HY-15418, injected together with the PD-1 inhibitor on the day of PD-1 inhibitor injection) was injected intraperitoneally once every two days starting from the 8th day. Mice in the RS504393 group were injected with an equal amount of RS504393 every two days starting from the 8th day; mice in the Control group were injected with an equal amount of saline every two days starting from the 8th day. On the 21st day, the mouse hearts were examined by color Doppler ultrasound, serum was collected to detect myocardial markers, heart specimens were collected for HE staining, immunofluorescence was used to detect the infiltration of CD68-positive macrophages in myocardial tissue, flow cytometry was used to analyze the proportion of CCR2-positive macrophages in myocardial tissue, and RT-PCR was used to detect the expression of chemokine genes such as CCL2 in myocardial tissue.

[0038] 1.1 Echocardiographic testing of mice

[0039] On the 21st day, the echocardiograms of mice in each group were detected, and the images were collected using the Vevo2100 ultrasound system (Visual Sonics) and a 30MHz high-frequency scanning probe. The mice with the skin prepared in the chest area were anesthetized by inhalation of isoflurane, and then they were placed in a supine position and fixed on the echocardiogram platform. When the heart rate of the mice was maintained at 450-500 beats / min, M-mode images were recorded at the maximum left ventricular internal diameter of the parasternal left ventricular long-axis outflow tract section, and the images were collected continuously for 15s. Functional indicators 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). The changes in cardiac function of mice in each group were compared, and all measured values ​​were the average values ​​of 5 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-1inhibitor 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 that in the Control group (systole: 2.58±0.29mm vs 1.50±0.25mm, p<0.0001; diastole: 3.71±0.38mm vs 1.50±0.25mm, p<0.0001). 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 the mice were anesthetized with isoflurane, blood was collected from the inner canthus of the mice. About 600 μL of blood was collected and centrifuged. The supernatant was aspirated and the serum cTnI (Mouse cTnI ELISA Kit, E-EL-M1203, Elabscience) and cTnT (Mouse cTnT ELISA Kit, E-EL-M1801, Elabscience) concentrations were determined using ELISA kits according to the method provided by the kit.

[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 / mLvs2.45±2.48pg / mL, p<0.01); and the myocardial markers of mice in the TNI+PD-1inhibitor+RS504393 group were improved compared with those in 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 to 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 light microscopy (magnification 10 times) were selected to observe the infiltration of inflammatory cells.

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

[0047] 1.4 Immunofluorescence staining of mouse myocardial tissue

[0048] After dewaxing and hydration, the heart slides were placed in a citric acid (pH 6.0) antigen retrieval solution in a water bath at 92°C for 20 minutes for antigen retrieval. After cooling naturally, the slides were washed in PBS for 3 times, 5 minutes each time. After the slides were dried, the buffer reagent was added to the tissue surface, incubated at room temperature for 10 minutes, and then washed again in PBS for 3 times, 5 minutes each time. TUNEL reagents 1 and 2 were added and incubated at 37°C for 1 hour. After washing with PBS, serum was added to block at room temperature for 30 minutes. The primary antibody was incubated at 4°C overnight: anti-CD68 (servicebio, item number: GB113109, 1:200). After washing with PBS, the fluorescent secondary antibody was added and incubated at room temperature in the dark for 50 minutes: CY3-goat anti-rabbit (servicebio, item number: GB21303, 1:300). After washing with PBS, DAPI staining solution was added, incubated at room temperature in the dark for 10 minutes, and then washed with PBS again and sealed with anti-fluorescence quenching sealing agent. Observe and collect images under a fluorescence microscope (magnification 10 times).

[0049] See also Figure 3 The results of the lower row of pictures show that there was a large number of macrophage infiltration in 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 of mouse myocardial tissue

[0051] Cut the heart tissue (about 5*5*5mm in size), use the cell / tissue total RNA extraction kit (YEASEN, catalog number: 19221ES50) to extract the total RNA of the heart tissue, use the reverse transcription kit (PrimeScript RT Reagent Kit WithgDNA Eraser) to reverse transcribe the RNA into cDNA, and use the real-time fluorescence quantitative PCR kit (TBGreenPremixExTaq) for the next PCR amplification. The PCR reaction conditions are: pre-denaturation at 95℃ for 30s, 95℃ for 5s, 60℃ for 30s, and a total of 40 cycles. Detect the expression of cytokine and chemokine related genes. -ΔΔCt The values ​​represent the relative expression level of each target gene.

[0052] Figure 4The representative test results of 2 mice in each of the Control group and TNI+PD-1inhibitor group are shown. The results showed that the genes of cytokines and chemokines in the heart tissue of mice in the TNI+PD-1inhibitor group were significantly upregulated compared with those in the Control group, among which the CCL2 chemokine gene was significantly upregulated compared with the Control group.

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

[0054] After the mice were euthanized, the hearts 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 The heart was digested by placing 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 medium) and shaking gently at 37°C for 15 minutes. After filtering the cells with a 70 μm filter, 2 volumes of staining buffer containing 2% FBS were added to terminate the digestion. The cells were centrifuged at 4°C and 350 g for 5 minutes. The supernatant was discarded and resuspended with 100 μL PBS to obtain a single cell suspension. The tubes are divided into: blank tube, single staining tube, and sample tube. Dead and live cell staining: BD Horizon TMFixable Viability Stain 510 (BD Pharmingen, Catalog No.: 564406, 1:1000) was incubated at room temperature in the dark for 15 min. After washing with staining buffer, the cells were blocked with mouse Fc receptor blocker (Catalog No.: abs9477-200T, 2 μL / tube) at 4°C for 5 min. 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 Fluor647 Rat Anti-Mouse IA / IE (MHC II) (BD Pharmingen, Catalog No.: 562367). After washing with PBS, centrifuge at 350 g for 5 min at 4°C, discard the supernatant, resuspend in 300 μL PBS, sieve and load onto the machine. 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 + The infiltration of macrophages was significantly increased (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 it was observed in Example 1 that the expression of CCL2 chemokine was upregulated in mice in the TNI+PD-1 inhibitor group, we performed cytological experiments to observe the effect of PD-1 inhibitor (InVivoMab anti-mouse PD-1) on the secretion of cellular CCL2 chemokine.

[0058] Cell grouping and model preparation: Human umbilical vein endothelial cells (HUVECs) were purchased from the cell bank of the Chinese Academy of Sciences Type Culture Collection Committee and divided into 2 groups: PBS group (control group) and PD-1 inhibitor group (treatment group). The PD-1 inhibitor group was treated with different concentrations of PD-1 inhibitor for 24 hours; the PBS group was treated with the same amount of PBS for 24 hours. The cell supernatant was collected and the secretion of CCL2 chemokine was detected 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 in Example 1. During one experiment, two mice in the TNI+PD-1inhibitor+RS504393 group showed mild colitis symptoms. We treated them by adding 5-aminosalicylic acid (5-ASA) to their feed and raised them separately. The results showed that the weight of these two mice at the end of the experiment was significantly higher than that of other mice in the same group, and the symptoms of myocarditis (such as erect hair, curling, reduced activity, etc.) were weakened. After excluding the influence of other factors, we realized that 5-aminosalicylic acid may also have the effect of treating mouse myocarditis, and conducted the following experiment.

[0062] The experimental process and the dosage of reagents were basically carried out as described in Example 1. Male Balb / c mice of about 6 weeks old and weighing 21-23g were divided into 5 groups, 8 in each group: Control group; TNI+PD-1inhibitor group (model group); TNI+PD-1inhibitor+5-ASA group (group A); TNI+PD-1inhibitor+RS504393 group (group B); TNI+PD-1inhibitor+RS504393+5-ASA group (group C). For mice in the model group, group A, group B, and group C, TNI+complete Freund's reagent was subcutaneously injected on the 1st and 7th days. From the 8th day, PD-1 inhibitor (5 mg / kg) was injected intraperitoneally every two days for a total of 5 times. For mice in group A, 5-ASA (2 mg / kg, Sigma, catalog number: A3537) was injected intraperitoneally every two days starting from day 8; for mice in group B, RS504393 (5 mg / kg) was injected intraperitoneally every two days starting from day 8; for mice in group C, RS504393 (5 mg / kg) and 5-ASA (2 mg / kg) were injected intraperitoneally every two days starting from day 8. Mice in the control group were injected with an equal amount of normal saline every two days starting from day 8.

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

[0064] Figure 7 The results of mouse 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 weight of mice in Group A treated with 5-ASA alone was not significantly different from that in the Model group (p>0.05); the weight of mice in Group C treated with 5-ASA and RS504393 increased significantly, which was significantly different from that in Group B treated with RS504393 alone (p<0.05). This result is consistent with the previous study that found that adding 5-aminosalicylic acid to the feed can promote weight gain in mice with myocarditis.

[0065] Figure 8The results of LVEF and LVFS of mice were 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%. The results showed that the use of 5-ASA made the results of mice in group C better than those in group B in terms of LVEF and LVFS, although the LVEF results did not show a significant difference (p>0.05).

[0066] Fig. 9 The results of mouse serum cTnI and cTnT detection are shown. cTnI: control group, 12.13±4.17pg / mL; model group, 97.51±14.19pg / mL; group A 102.12±15.33pg / mL; group B 42.43±6.17pg / mL; group C 22.38±3.84%. cTnT: control group, 4.66±2.11pg / mL; model group 38.46±10.36pg / mL; group A 42.02±9.95pg / mL; group B, 28.12±6.25pg / mL; group C, 7.81±4.62pg / mL. The results show that the use of 5-ASA significantly improved the symptoms of myocardial injury. It is worth noting that the cTnT test results were no significantly different from those of the control group (p>0.05).

[0067] Fig.10 CCR2 + MHC II + Flow cytometric results of macrophage infiltration (CD64 + The control group, 24.44±5.54%; the model group, 69.53±6.77%; group A, 63.71±8.03%; group B, 39.65±8.46%; group C, 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 for the treatment of colitis, it has little effect on the treatment of myocarditis in mice. When 5-ASA is used in combination with the CCR2 antagonist RS504393, the myocarditis treatment effect of RS504393 is improved.

Claims

1. Use of CCR2 antagonists in the preparation of drugs for cardioprotection.

2. The use according to claim 1, wherein the drug is a drug for myocarditis.

3. The use according to claim 1 or 2, wherein the myocarditis is immune checkpoint inhibitor-associated myocarditis.

4. The use according to any one of claims 1 to 3, wherein the CCR2 antagonist is RS504393.

5. The use according to any one of claims 1 to 4, wherein the drug further comprises 5-aminosalicylic acid.

6. The use according to any one of claims 1 to 5, wherein the medicine is an injection.

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

8. The pharmaceutical composition of claim 7, wherein the CCR2 antagonist is RS504393.

9. A drug kit comprising a CCR2 antagonist and 5-aminosalicylic acid.

10. The pharmaceutical composition of claim 9, wherein the CCR2 antagonist is RS504393.

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