Application of L-kynurenine in treatment of immune checkpoint inhibitor related myocarditis

Abstract

The invention discloses application of L-kynurenine in treatment of immune checkpoint inhibitor related myocarditis, and belongs to the field of biological medicine. It is found that by improving the level of L-kynurenine, phosphorylation of a JAK1 / STAT3 pathway of macrophages can be inhibited, proliferation of Tregs cells is promoted, and the progress of immune checkpoint inhibitor-related myocarditis is relieved; when the L-kynurenine is used for treatment, the immune checkpoint inhibitor related myocarditis caused by the PD-1 inhibitor can be obviously improved. Therefore, the L-kynurenine plays an important role in improving the immune checkpoint inhibitor related myocarditis and is expected to be developed into a medicine for treating the immune checkpoint inhibitor related myocarditis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to an application of L-kynurenine in treating immune checkpoint inhibitor-related myocarditis. Background Art

[0002] Immune checkpoint inhibitors (ICIs) have become an indispensable part of the comprehensive treatment strategy for cancer. ICIs enhance the immune killing effect of T lymphocytes on tumors by blocking the binding of T lymphocytes to tumor cell immunosuppressive signals (e.g., PD-L1). The targets of ICIs include anti-cytotoxic T lymphocyte-associated antigen (CTLA-4), anti-programmed death receptor 1 (PD-1), and anti-programmed death ligand 1 (PD-L1). With the development and use of ICIs, their unique immune-related adverse events (irAEs) have become a major problem currently faced in clinical practice. Big data shows that up to 60% of patients treated with ICIs will experience immune-related adverse events. Among them, the mortality rate of ICIs-related myocarditis ranks first, as high as 39.7%-66.0%.

[0003] As a powerful immunosuppressive organ, the heart expresses a large number of PD-L1 receptors on the surface of its vascular endothelial cells. By binding to PD1 on the surface of immune cells, T lymphocytes can be blocked on the vascular side. However, the use of ICIs blocks the binding of PD1 / PD-L1, which upregulates the expression of adhesion molecules on the surface of vascular endothelial cells and increases the endothelial gap, further promoting the infiltration of immune cells such as T lymphocytes into the myocardium, and promoting the progression of ICIs-related myocarditis by secreting cytokines and chemokines. Infiltration of a large number of CD8+T lymphocytes and CD68+ macrophages was found in myocardial biopsy tissues of patients with ICIs-related myocarditis. T lymphocytes infiltrating into myocardial tissue will be activated by antigen-presenting cells (including cardiomyocytes, fibroblasts, resident macrophages, etc.) to become cytotoxic T lymphocytes, which then secrete proinflammatory cytokines to directly damage the myocardium, or further recruit proinflammatory cells such as macrophages, resulting in an irreversible immune cascade reaction.

[0004] Therefore, providing a drug that can effectively prevent, control or improve ICIs-related myocarditis is a technical problem that urgently needs to be solved in the field of tumor immunotherapy. Summary of the invention

[0005] Based on the above background, the technicians of the present invention found that L-kynurenine can effectively alleviate the progression of immune checkpoint inhibitor-associated myocarditis (ICIs-associated myocarditis) and play a role in protecting the heart. It was verified by STAT agonists that L-kynurenine alleviates ICIs-associated myocarditis by inhibiting the phosphorylation of the macrophage JAK1 / STAT3 pathway. The present invention provides a solid research foundation for the development of a drug that can prevent and / or treat ICIs-associated myocarditis.

[0006] The present invention includes the following technical solutions:

[0007] In a first aspect of the present invention, the present invention provides a use of L-kynurenine in at least one of the following:

[0008] a1) Use in the preparation of drugs for preventing diseases related to phosphorylation of the macrophage JAK1 / STAT3 pathway;

[0009] a2) Use in the preparation of drugs for treating or assisting in treating diseases related to the phosphorylation of the macrophage JAK1 / STAT3 pathway.

[0010] In a second aspect of the present invention, the present invention provides a use of L-kynurenine in at least one of the following:

[0011] b1) Use in the preparation of a medicament for preventing immune checkpoint inhibitor-associated myocarditis;

[0012] b2) Use in the preparation of drugs for the treatment and / or adjuvant treatment of immune checkpoint inhibitor-associated myocarditis.

[0013] Furthermore, the drug has at least one of the following functions:

[0014] c1) Improve heart function;

[0015] c2) Reduce myocardial damage;

[0016] c3) Improve the degree of myocardial fibrosis;

[0017] c4) inhibiting CD8+T lymphocyte infiltration in cardiac tissue;

[0018] c5) Promote the transformation of CD3+CD4+T lymphocytes in cardiac tissue into anti-inflammatory CD3+CD4+Foxp3+Tregs cells;

[0019] c6) Reduce the infiltration of pro-inflammatory CD80+CD163-M1 macrophages in cardiac tissue;

[0020] c7) Reduce the infiltration of pro-inflammatory CD86+M1 macrophages in cardiac tissue and promote the transformation of macrophages into anti-inflammatory CD206+M2 macrophages.

[0021] Specifically, the improvement of cardiac function refers to increasing the left ventricular ejection fraction (LVEF) and the left ventricular short-axis contraction fraction (LVFS); the reduction of myocardial damage refers to reducing the levels of myocardial markers troponin I (cTnI) and troponin T (cTnT).

[0022] In the third aspect of the present invention, the present invention provides a use of L-kynurenine combined with an immune checkpoint inhibitor in at least one of the following:

[0023] d1) Use in the preparation of drugs for preventing tumor combined with immune checkpoint inhibitor-related myocarditis;

[0024] d2) Use in the preparation of drugs for the treatment or adjuvant treatment of tumors combined with immune checkpoint inhibitor-related myocarditis.

[0025] The drug is an anti-tumor drug.

[0026] Specifically, the drug is used for tumor immunotherapy, and at the same time prevents and improves immune-related adverse events occurring during the treatment process, wherein the immune-related adverse event is immune checkpoint inhibitor-related myocarditis.

[0027] In an embodiment of the present invention, the immune checkpoint inhibitor refers to a drug that enhances the attack power of immune cells on tumors by inhibiting inhibitory signaling pathways in the immune system, and the drug includes anti-PD-1 antibodies, anti-PD-L1 antibodies or anti-CTLA-4 antibodies.

[0028] Preferably, the immune checkpoint inhibitor is selected from anti-PD-1 antibodies.

[0029] In a specific embodiment of the present invention, the anti-PD-1 antibodies include but are not limited to pembrolizumab, sintilimab, nivolumab, carrelizumab and toripalimab.

[0030] In a possible embodiment of the present invention, the tumor includes any tumor known in the art that can be immunotherapeutic by immune checkpoint inhibitors.

[0031] Exemplarily, the tumor includes melanoma, lung cancer, lymphoma, hemangioma, lymphangioma, kidney cancer, gastric cancer, liver cancer, biliary tract cancer, pancreatic cancer, ovarian cancer, cervical cancer, breast cancer, colorectal cancer, bladder cancer, cervical squamous cell carcinoma, esophageal squamous cell carcinoma, Hodgkin's lymphoma, mesothelioma, prostate cancer or urothelial carcinoma.

[0032] In a fourth aspect of the present invention, the present invention provides a pharmaceutical composition comprising an effective amount of an immune checkpoint inhibitor and L-kynurenine.

[0033] The immune checkpoint inhibitor refers to a drug that enhances the attack power of immune cells on tumors by inhibiting inhibitory signaling pathways in the immune system. The drug includes anti-PD-1 antibodies, anti-PD-L1 antibodies or anti-CTLA-4 antibodies.

[0034] Preferably, the immune checkpoint inhibitor is selected from anti-PD-1 antibodies.

[0035] In a specific embodiment of the present invention, the anti-PD-1 antibodies include but are not limited to pembrolizumab, sintilimab, nivolumab, carrelizumab and toripalimab.

[0036] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0037] Exemplarily, the pharmaceutically acceptable carrier is an adjuvant and / or auxiliary agent conventionally used in the art for preparing pharmaceutical preparations, including but not limited to a binder, a filler, a disintegrant, a lubricant, an isotonicity regulator, a solubilizer, a solubilizing agent, a preservative, a colorant, a suspending agent, a wetting agent, an emulsifier or a surfactant.

[0038] Preferably, the pharmaceutical composition is a compound preparation or a medicine box packaged as a non-compound independent preparation.

[0039] In some embodiments of the present invention, the pharmaceutical composition is a compound preparation, which contains a therapeutically effective amount of an immune checkpoint inhibitor and L-kynurenine; wherein the pharmaceutical preparation of the immune checkpoint inhibitor and L-kynurenine are compounded to form a compound preparation.

[0040] In some embodiments of the present invention, the pharmaceutical composition is a medicine kit, which includes one or more doses of a pharmaceutical preparation of an immune checkpoint inhibitor and one or more doses of a pharmaceutical preparation of L-kynurenine; wherein the pharmaceutical preparation of the immune checkpoint inhibitor and the pharmaceutical preparation of L-kynurenine are packaged separately.

[0041] The "one dose" mentioned in the present invention refers to the content of the active ingredient contained in the pharmaceutical preparation as a single effective daily dose or half a daily dose.

[0042] The compound preparation or independently packaged pharmaceutical preparation of the present invention can be a solid preparation, such as tablets, lozenges and capsules, and a solution preparation suitable for injection or oral administration. The preparation contains an active ingredient with a mass fraction of 0.25-95% and a pharmaceutically acceptable carrier.

[0043] The compound preparation of the present invention or the independently packaged pharmaceutical preparation 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 external route administration. As an alternative or in parallel, oral administration can be performed. The dosage is determined by those skilled in the art according to the age, health and weight of the patient, the type of concurrent treatment, the frequency of treatment, and the required therapeutic benefit.

[0044] The compound preparation or independently packaged pharmaceutical preparation of the present invention can be manufactured by methods known in the art, such as mixing, granulating, tableting, dissolving, freeze-drying and the like.

[0045] The term "pharmaceutically acceptable" as used herein means that the substance or composition must be chemically and / or toxicologically compatible with the other ingredients of the formulation and / or the mammal to be treated with it. Preferably, the term "pharmaceutically acceptable" means approved by a federal regulatory agency or a national government or listed in the United States Pharmacopoeia or other generally recognized pharmacopoeia for use in animals, especially humans. The pharmaceutically acceptable excipients described herein may include any solvent, solid excipient, diluent or other liquid excipient, etc., suitable for a specific target dosage form.

[0046] The L-kynurenine or the pharmaceutical composition composed of the immune checkpoint inhibitor and L-kynurenine of the present invention can be used for the treatment of diseases and can also be used for in vitro cell culture experiments. When used for the treatment of diseases, L-kynurenine or the pharmaceutical composition is usually administered in the form of a unit dose.

[0047] The disease treatment described in the present invention refers to administering a therapeutically effective amount of L-kynurenine or the pharmaceutical composition described in the present invention to a subject in need thereof. Further, the disease treatment also includes administering the L-kynurenine or the pharmaceutical composition described in the present invention in combination with other medicaments or conventional treatment methods conventionally used in the art.

[0048] The technical solution provided by the present invention has the following beneficial technical effects:

[0049] (1) The present invention has experimentally verified that increasing the level of L-kynurenine inhibits the phosphorylation of the macrophage JAK1 / STAT3 pathway, promotes the proliferation of Tregs cells, and alleviates the progression of immune checkpoint inhibitor-related myocarditis.

[0050] (2) The present invention confirms through in vivo and in vitro experiments that L-kynurenine treatment is expected to significantly improve ICIs-related myocarditis caused by PD-1 inhibitors and play a role in protecting the heart.

[0051] (3) In the present invention, L-kynurenine can be used as a cardioprotectant in conjunction with first-line clinical immune checkpoint inhibitors for tumor immunotherapy, which can significantly reduce the symptoms and symptoms of immune checkpoint inhibitor-related myocarditis that occur during treatment. The combination of L-kynurenine and immune checkpoint inhibitors provided by the present invention can be used as a new medication regimen for tumor immunotherapy, providing a solution for the management of adverse reactions that occur in tumor immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 A is the test result of L-Kynurenine level in plasma of patients with ICIs-related myocarditis and non-ICIs-related myocarditis; Figure 1 B is the expression level of IDO1 in mouse heart tissue; *: P<0.05; **: P<0.01; ***: P<0.001; ****: P<0.0001.

[0053] Figure 2 These are the echocardiograms, left ventricular ejection fraction (LVEF), left ventricular short-axis contraction fraction (LVFS) and mouse serum myocardial markers (cTNI, cTNT) test results of mice in the Control group, L-Kynurenine group, TNI+PD-1inhibitor group and TNI+PD-1inhibitor+L-Kynurenine group on the 21st day of modeling.

[0054] Figure 3 These are photos of HE staining and Masson staining of mouse heart sections.

[0055] Figure 4 The results of flow cytometry detection of T lymphocytes in mouse heart tissue.

[0056] Figure 5 These are the results of flow cytometry and immunofluorescence detection of macrophages in mouse heart tissue.

[0057] Figure 6 Bulk-RNA sequencing results of mouse heart tissue.

[0058] Figure 7 These are the results of flow cytometry and Western blot detection of polarization of the macrophage cell line RAW264.7.

[0059] Figure 8 The results of Western blot detection of macrophage cell line RAW264.7. DETAILED DESCRIPTION

[0060] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only some embodiments of the present invention, not all. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0061] It should be understood that these examples are merely illustrative and are not intended to limit the scope of the present invention. Unless otherwise specified, the materials and experimental methods used in the examples are understood to be those conventionally used in the art.

[0062] Example 1 Increased L-Kynurenine levels in plasma of patients with ICIs-related myocarditis

[0063] Peripheral venous blood was collected from tumor patients who received ICIs treatment and developed ICIs-related myocarditis (n=18) and patients who did not develop ICIs-related myocarditis (n=15), and the level of L-Kynurenine in the plasma of the two groups of patients was detected.

[0064] See also Figure 1 A, The plasma L-Kynurenine level in patients with ICIs-related myocarditis was significantly higher than that in patients with non-ICIs-related myocarditis (P<0.05).

[0065] Example 2 Study on the Mechanism of L-Kynurenine's Effect on ICIs-Related Myocarditis

[0066] SPF male Balb / c mice 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 5 mice in each group: Control group (control group, marked as PBS in the figure); L-Kynurenine group (Kyn group, marked as L-Kynurenine in the figure); TNI+PD-1inhibitor group (modeling group, marked as ICIs in the figure); TNI+PD-1inhibitor+L-Kynurenine group (treatment group, marked as ICIs+L-Kynurenine in the figure).

[0067] The TNI+PD-1inhibitor group and the TNI+PD-1inhibitor+L-Kynurenine group were subcutaneously injected with TNI+complete fluorogenic reagent (TNI 250 μg / mouse, Sangon Biotech (Shanghai) Co., Ltd., designed TnI sequence: HARVDKVDEERYDVEAKVTKNITEIADLTQKIYDLRGKFKRPTLRRVRIS (troponin I); complete fluorogenic reagent 100 μL / mouse, Sigma, catalog number: F5881) on the 8th day of modeling. PD-1 inhibitor (5 mg / kg, InVivoMab anti-mouse PD-1, CD279) was injected intraperitoneally every two days for a total of 5 times to establish a PD-1 inhibitor-induced myocarditis mouse model. In addition, the TNI+PD-1inhibitor+L-Kynurenine group and the L-Kynurenine group were intraperitoneally injected with L-Kynurenine (300 mg / kg, medchemexpress, catalog number: HY-104026) every two days starting from the 8th day of modeling; the Control group was injected with an equal amount of PBS starting from the 8th day of modeling.

[0068] On the 21st day of modeling, the mouse hearts were detected by color Doppler ultrasound, serum was collected to detect myocardial markers, heart specimens were collected for HE and Masson staining, Western Blot was used to detect the level of IDO1 in mouse heart tissue, immunofluorescence was used to detect the infiltration of CD86 and CD206 positive macrophages in myocardial tissue, flow cytometry was used to analyze the proportion of CD4, CD8, and Foxp3 positive T lymphocytes in myocardial tissue, as well as the proportion of CD80 and CD163 positive macrophages, and bulk-RNA sequencing of myocardial tissue was used to explore the mechanism by which L-Kynurenine protects cardiac function.

[0069] 2.1) Detection of rate-limiting enzyme IDO1 in mouse heart tissue

[0070] The myocardial tissue blocks were added to the protein lysis buffer prepared in proportion, the total protein of the myocardial tissue was extracted, and the expression of the rate-limiting enzyme IDO1 in the heart tissue of the control group and the model group was detected. Figure 1 B, The expression level of IDO1 in the hearts of mice in the model group was significantly higher than that in the control group.

[0071] 2.2) Echocardiographic testing of mice

[0072] On the 21st day of modeling, the echocardiograms of mice in each group were detected, and the Vevo2100 ultrasound system (Visual Sonics) and 30MHz high-frequency scanning probe were used to collect images. The mice with skin preparation in the chest area were anesthetized by inhalation of isoflurane, and then they were placed in a supine position and fixed on the echocardiogram detector platform. When the heart rate of the mice was maintained at 450-500 times / 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 include: left ventricular ejection fraction (EF) and left ventricular short-axis contraction rate (FS). The changes in cardiac function of mice in each group were compared, and all measured values ​​were the average of 5 consecutive cardiac cycles.

[0073] See also Figure 2 The experimental results showed that the cardiac function indicators of mice in the TNI+PD-1inhibitor group were significantly lower than those in the Control group (LVEF: 49.30±9.85% vs 82.55±9.14%, P<0.0001; LVFS: 24.92±6.20% vs 51.02±9.30%, P<0.001); after treatment with L-Kynurenine, the cardiac function of mice was significantly improved. The LVEF of mice in the TNI+PD-1inhibitor+L-Kynurenine group was 71.69±11.48% (P<0.05), and the LVFS was 40.86±9.33% (P<0.05).

[0074] 2.3) Detection of mouse myocardial markers

[0075] After isoflurane anesthesia of mice, blood was collected from the inner canthus of mice. About 600 μl of blood was collected from the inner canthus of mice and centrifuged. After the supernatant was aspirated, 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.

[0076] See also Figure 2The results showed that the myocardial markers of mice in the TNI+PD-1inhibitor group were significantly higher than those in the Control group (cTnI: 181.80±9.55pg / mL vs 123.80±15.43pg / mL, P<0.0001; cTnT: 163.90±6.21pg / mLvs 138.10±6.98pg / mL, P<0.001); while the myocardial markers of mice in the TNI+PD-1inhibitor+L-Kynurenine group were improved compared with those in the TNI+PD-1inhibitor group (cTnI: 167.5±6.23pg / mL vs 181.80±9.55pg / mL, P<0.05; cTnT: 148.2±5.66pg / mL vs 163.90±6.21pg / mL, P<0.01).

[0077] 2.4) HE and Masson staining of mouse myocardial tissue

[0078] After euthanizing the mice, they were fixed to the operating table. Following animal ethics, the mouse hearts were removed and the tissues were fixed in tissue fixative, embedded in paraffin, and then cut into 5 μm thick sections for HE and Masson staining. Representative images under a light microscope (magnification 10 times) were selected to observe the infiltration of inflammatory cells and the degree of fibrosis.

[0079] See also Figure 3 The results showed that the cardiac inflammatory infiltration in the TNI+PD-1inhibitor group was significantly higher than that in the Control group, while the cardiac inflammatory infiltration in the TNI+PD-1inhibitor+L-Kynurenine group was improved. The myocardial fibrosis in the TNI+PD-1inhibitor group was significantly higher than that in the Control group, while the myocardial fibrosis in the TNI+PD-1inhibitor+L-Kynurenine group was improved.

[0080] 2.5) Flow cytometry detection of immune cell infiltration in myocardial tissue

[0081] 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 3Place the fragments of different sizes into an EP tube containing 1.5 mL of digestion solution (0.15 mg / ml LiberaseTM, 20 μg / ml DNA enzyme 1, prepared in serum-free DMEM medium) 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 4°C and 350 g for 5 minutes, discard the supernatant and resuspend with 100 μL PBS to obtain a single cell suspension. Tubes: blank tube, single staining tube, sample tube. Dead and live cell staining: BD Horizon TM Fixable Viability Stain 700 (BD Pharmingen, Catalog No.: 564997, 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), PE Rat Anti-MouseCD11b (BD Pharmingen, Catalog No.: 557397), BV605 Rat Anti-Mouse F4 / 80 (BD Pharmingen, Catalog No.: 743281), BV421 Hamster Anti-Mouse CD80 (BD Pharmingen, Catalog No.: 562611), ANTI-MOCD163 TNKUPJ APC (Thermo, Catalog No.: 17-1631-82), Ms CD3e BV510 (BD Pharmingen, Catalog No.: 740113), BB700 Rat Anti-Mouse CD4 (BD Pharmingen, Catalog No.: 566407), Rat Anti-MouseCD8a-FITC (Miltenyi, catalog number: 130-118-468). After washing with PBS, centrifuge at 4℃350g for 5min, discard the supernatant and fix and permeabilize the membrane with Fixation / Permeablization Kit (BD Pharmingen, catalog number: 554714), wash and stain the nucleus with PERat anti-Mouse Foxp3 (BD ​​Pharmingen, catalog number: 563101) for 30min. After washing with PBS, centrifuge at 4℃350g for 5min, discard the supernatant and resuspend with 300uL PBS, sieve and load on the machine. Flow data were analyzed by Flowjo.

[0082] See also Figure 4 A. The results showed that compared with the Control group, the number of CD8+T lymphocytes in the heart tissue of mice in the TNI+PD-1inhibitor group was significantly increased (P<0.01), while the number of CD8+T lymphocytes in the heart tissue of mice in the TNI+PD-1inhibitor+L-Kynurenine group was decreased compared with that in the TNI+PD-1inhibitor group.

[0083] See also Figure 4 B. The results showed that compared with the Control group, the CD3+CD4+Foxp3+Tregs cells in the TNI+PD-1inhibitor group and the TNI+PD-1inhibitor+L-Kynurenine group were significantly increased (P<0.0001); among them, the CD3+CD4+Foxp3+Tregs cells in the TNI+PD-1inhibitor+L-Kynurenine group were increased compared with those in the TNI+PD-1inhibitor group (P<0.05).

[0084] See also Figure 5 A. The results showed that compared with the Control group, the infiltration of CD80+CD163-M1 macrophages in the heart tissue of mice in the TNI+PD-1inhibitor group was significantly increased (P<0.01). After the protection of L-Kynurenine, the infiltration of CD80+CD163-M1 macrophages in the heart tissue of the TNI+PD-1inhibitor+L-Kynurenine group was reduced (P<0.05).

[0085] 2.6) Immunofluorescence staining of mouse myocardial tissue

[0086] After dewaxing and hydrating the heart slides, place them in a citric acid (PH6.0) antigen retrieval solution in a water bath at 92°C for 20 minutes for antigen retrieval. After cooling naturally, wash the slides in PBS for 3 times, 5 minutes each time. After the slides are dried, add buffer reagent to the tissue surface, incubate at room temperature for 10 minutes, and wash again in PBS for 3 times, 5 minutes each time. Incubate the primary antibody at 4°C overnight: anti-CD86 (servicebio, catalog number: GB115630-50, 1:200), anti-CD206 (cell signallingtechnology, catalog number: 24595, 1:400). After washing with PBS, add fluorescent secondary antibodies and incubate at room temperature in the dark for 50 minutes: CY3-goat anti-rabbit IgG (servicebio, catalog number: GB21303, 1:300), Alexa Fluor 488-labeled goat anti-rabbit IgG (servicebio, catalog number: GB25303, 1:300). After washing with PBS, add DAPI staining solution, incubate at room temperature in the dark for 10 minutes, wash again with PBS, and seal with anti-fluorescence quenching sealing agent. Observe and collect images under a fluorescence microscope (magnification 5 times).

[0087] See also Figure 5 B. The results showed that: in the heart tissue of mice in the TNI+PD-1inhibitor group, there were a large number of CD86+M1 macrophages infiltrating, and relatively few CD206+M2 macrophages; after L-Kynurenine protection, the infiltration of CD86+M1 macrophages in the heart tissue of the TNI+PD-1inhibitor+L-Kynurenine group was reduced.

[0088] 2.7) Mouse heart bulk RNA sequencing

[0089] Total RNA from the heart tissues of mice in the TNI+PD-1 inhibitor group and the control group was extracted for sequencing. The integrity of the extracted total RNA was tested using an Agilent Bioanalyzer 2100 (Agilent technologies, Santa Clara, CA, US) and the The concentration and purity of total RNA were tested by 3.0 Fluorometer (Life Technologies, CA, USA) and Nanodrop Onespectrophotometer (Thermo Fisher Scientific Inc, USA). After the quality inspection was qualified, the next step of RNA library construction was carried out, and the specific steps included: ployA enrichment → mRNA fragmentation → first-strand cDNA synthesis → magnetic bead purification of the first strand → second-strand cDNA synthesis → strand end repair → 3' end addition of A → addition of connector → purification and library amplification → sequencing sample library construction was completed. RNA sequencing was performed according to the sequencing process and its results were analyzed.

[0090] See also Figure 6 Compared with the Control group, there were 2751 differentially expressed genes in the TNI+PD-1inhibitor group, of which 1666 differentially expressed genes were significantly up-regulated and 1085 differentially expressed genes were significantly down-regulated. GO enrichment analysis of differentially expressed genes showed that differentially expressed genes were mainly involved in regulating initial and adaptive immune responses, cytokine pathways, cell-cell adhesion, leukocyte migration and other pathways. KEGG pathway enrichment analysis of differentially expressed genes showed that differentially expressed genes were mainly enriched in JAK-STAT pathway, NF-kappa B pathway, TNF and other signaling pathways.

[0091] 2.8) In vitro experimental cell grouping and model preparation

[0092] Mouse macrophage cell line RAW264.7 was purchased from the Cell Bank of Type Culture Collection Committee of Chinese Academy of Sciences and divided into 4 groups: Control group, L-Kynurenine group, LPS group, and LPS+L-Kynurenine group. The LPS group and LPS+L-Kynurenine group were treated with 100 ng / mL LPS for 24 hours, the L-Kynurenine group and LPS+L-Kynurenine group were treated with 10 nmol L-Kynurenine for 24 hours, and the Control group was treated with an equal amount of PBS for 24 hours. The cells were collected, and the polarization of macrophages among the groups was analyzed by flow cytometry, and the expression of related proteins was detected by Western blot.

[0093] See also Figure 7The results showed that compared with the Control group, the CD80+CD206-M1 macrophages in the LPS group increased significantly (30.27±2.14% vs 1.90±0.63%, P<0.0001); and after L-Kynurenine treatment, the proportion of M1 macrophages decreased significantly (21.03±2.73% vs 30.27±2.14%, P<0.01). In addition, the expression level of iNOS, a marker of M1 macrophages, was significantly upregulated in the LPS group, while after L-Kynurenine treatment, its expression level decreased.

[0094] See also Figure 8 A, The results showed that compared with the Control group, the phosphorylation level of JAK1 / STAT3 protein in the LPS group was upregulated; after treatment with L-Kynurenine, the phosphorylation level of JAK1 / STAT3 protein in the LPS+L-Kynurenine group was significantly downregulated compared with the LPS group.

[0095] 2.9) In vitro experiments verify the mechanism of L-Kynurenine in protecting ICIs-related myocarditis

[0096] Cell grouping and model preparation: divided into 5 groups: Control group, LPS group, LPS+L-Kynurenine group, LPS+RO8191 group, LPS+L-Kynurenine+RO8191 group. Except for the Control group, the other 4 groups were treated with 100ng / mL LPS for 24 hours, the LPS+L-Kynurenine group and the LPS+L-Kynurenine+RO8191 group were treated with 10nmoL L-Kynurenine for 24 hours, the LPS+RO8191 group and the LPS+L-Kynurenine+RO8191 group were treated with 10uM RO8191 for 24 hours, and the Control group was treated with an equal amount of PBS for 24 hours. Total cell protein was collected, and the expression of related proteins was detected by Western blot.

[0097] See also Figure 8 B. The results showed that compared with the Control group, the JAK1 / STAT3 protein phosphorylation level in the LPS group was upregulated, and after treatment with the L-Kynurenine group, the JAK1 / STAT3 protein phosphorylation expression was downregulated; after administration of the STAT agonist RO8191, the JAK1 / STAT3 protein phosphorylation level in the LPS+RO8191 group was further upregulated compared with the LPS group, while the protein phosphorylation expression in the LPS+L-Kynurenine+RO8191 group was downregulated.

[0098] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of L-kynurenine in at least one of the following: a1) Use in the preparation of drugs for preventing diseases related to phosphorylation of the macrophage JAK1 / STAT3 pathway; a2) Use in the preparation of drugs for treating or assisting in treating diseases related to the phosphorylation of the macrophage JAK1 / STAT3 pathway.

2. Use of L-kynurenine in at least one of the following: b1) Use in the preparation of a medicament for preventing immune checkpoint inhibitor-associated myocarditis; b2) Use in the preparation of drugs for the treatment and / or adjuvant treatment of immune checkpoint inhibitor-associated myocarditis.

3. The use according to claim 2, characterized in that: The drug has at least one of the following functions: c1) Improve heart function; c2) Reduce myocardial damage; c3) Improve the degree of myocardial fibrosis; c4) inhibiting CD8+T lymphocyte infiltration in cardiac tissue; c5) Promote the transformation of CD3+CD4+T lymphocytes in cardiac tissue into anti-inflammatory CD3+CD4+Foxp3+Tregs cells; c6) Reduce the infiltration of pro-inflammatory CD80+CD163-M1 macrophages in cardiac tissue; c7) Reduce the infiltration of pro-inflammatory CD86+M1 macrophages in cardiac tissue and promote the transformation of macrophages into anti-inflammatory CD206+M2 macrophages.

4. Use of L-kynurenine in combination with an immune checkpoint inhibitor in at least one of the following: d1) Use in the preparation of drugs for preventing tumor combined with immune checkpoint inhibitor-related myocarditis; d2) Use in the preparation of drugs for the treatment or adjuvant treatment of tumors combined with immune checkpoint inhibitor-related myocarditis.

5. The use according to claim 4, wherein the drug is an anti-tumor drug, which is used for tumor immunotherapy and prevents and improves immune-related adverse events occurring during the treatment process, wherein the immune-related adverse event is immune checkpoint inhibitor-related myocarditis.

6. A pharmaceutical composition comprising an effective amount of an immune checkpoint inhibitor and L-kynurenine.

7. The pharmaceutical composition according to claim 6, characterized in that The immune checkpoint inhibitor refers to a drug that enhances the attack power of immune cells on tumors by inhibiting inhibitory signaling pathways in the immune system. The drug includes anti-PD-1 antibodies, anti-PD-L1 antibodies or anti-CTLA-4 antibodies.

8. The pharmaceutical composition according to claim 6, characterized in that The pharmaceutical composition also includes a pharmaceutically acceptable carrier.

9. The pharmaceutical composition according to claim 6, characterized in that The pharmaceutical composition is a medicine box packaged as a compound preparation or a non-compound independent preparation.

10. The pharmaceutical composition according to claim 9, characterized in that The pharmaceutical composition is a compound preparation, which contains a therapeutically effective amount of an immune checkpoint inhibitor and L-kynurenine; wherein the pharmaceutical preparation of the immune checkpoint inhibitor and L-kynurenine are compounded to form the compound preparation.

11. The pharmaceutical composition according to claim 9, characterized in that The pharmaceutical composition is a medicine kit, which includes one or more doses of a pharmaceutical preparation of an immune checkpoint inhibitor and one or more doses of a pharmaceutical preparation of L-kynurenine; wherein the pharmaceutical preparation of the immune checkpoint inhibitor and the pharmaceutical preparation of L-kynurenine are packaged independently.

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