Application of biomarker combination in preparing drugs for fulminant myocarditis

Abstract

The application of the biomarker combination in the preparation of a drug for fulminant myocarditis belongs to the field of drugs. The application is characterized in that the biomarker combination comprises Siglec-5; the biomarker combination is further selected from the group consisting of sST2, PAI-1, Siglec-5, CD163, CD40, P-Cadherin, CD14, CTLA4. The application finds that the expression of the biomarker in the plasma of a patient with fulminant myocarditis is increased, and confirms the role of the biomarker as a drug target in relieving and / or improving fulminant myocarditis. The level of the biomarker can be controlled by a drug, the diagnosis and subsequent treatment of the disease can be more intensified, and the biomarker has great application value in clinical practice.

Description

[0001] This application is a divisional application of Chinese Patent Application CN 114414812 A, the application information of the parent application is as follows:

[0002] Application No.: 202111559565X

[0003] Filing Date: December 20, 2021

[0004] Priority No.: 202011518438.0

[0005] Priority Date: December 21, 2020

[0006] Invention Name: Application of Biomarker Combination in Preparation of Diagnosis Reagent and Drug for Fulminant Myocarditis TECHNICAL FIELD

[0007] The present application belongs to the field of medicine, and specifically relates to the application of a biomarker combination in the preparation of a drug for fulminant myocarditis. BACKGROUND

[0008] Fulminant myocarditis is the most severe and special type of myocarditis, and its main features are sudden onset and extremely rapid progression. Patients quickly develop hemodynamic abnormalities, and can also have respiratory failure and liver and kidney failure, with an early mortality rate of more than 50%.

[0009] The clinical features and treatment status of fulminant myocarditis have the following characteristics: 1. The clinical understanding and diagnostic ability of fulminant myocarditis are severely insufficient, resulting in a high rate of missed diagnosis and misdiagnosis of this type of patient, and treatment is often delayed. 2. There is no effective treatment program, consensus or guideline in the world. Fulminant myocarditis has been treated with empirical treatment programs in the past, including anti-infection, blood pressure elevation, and cardiac artificial assist devices, but the survival rate of patients has not been substantially improved. 3. The medical and social value of effective treatment of fulminant myocarditis is extremely great. Fulminant myocarditis often occurs in young and healthy people without underlying diseases, and due to the high early mortality rate, it will cause great harm to the patient's family and society. However, it is worth noting that once a patient with fulminant myocarditis survives the critical period, their long-term survival rate and quality of life are almost indistinguishable from the general population. A 11-year follow-up study showed that the survival rate of fulminant myocarditis was significantly higher than that of ordinary acute myocarditis.

[0010] The clinical onset of fulminant myocarditis is extremely dangerous, and its characteristics are fast disease progression and high mortality. The core of the past treatment is to enhance myocardial contraction and increase blood pressure, but it has not effectively reduced the mortality. The fundamental reason is that people lack correct understanding or even completely wrong understanding of the pathophysiology and pathogenesis of fulminant myocarditis, so they cannot propose effective treatment methods, which leads to the serious consequences of continuous progression of the disease and a large number of deaths. Therefore, it is urgent to fully study and understand the pathogenesis of fulminant myocarditis, find new treatment targets, and adopt more effective measures to improve or delay the progression of fulminant myocarditis, reduce the mortality and hospitalization rate of fulminant myocarditis; on the other hand, the diagnosis of fulminant myocarditis still lacks specific indicators, so it is necessary to find new biomarkers. SUMMARY

[0011] The present application aims at the problems of difficulty in diagnosis and treatment of fulminant myocarditis and insufficient risk assessment in the prior art, discovers a series of new biomarkers, and confirms their correlation with the onset of fulminant myocarditis, and the role of two of them (sST2 and CD163) as drug targets in relieving and / or improving fulminant myocarditis; therefore, by detecting the levels of inflammatory factors through a kit, fulminant myocarditis can be diagnosed or the prognosis of fulminant myocarditis can be evaluated, and by controlling the levels of sST2 and CD163 through drugs, the disease can be diagnosed and treated more intensively, which has great application value in clinic.

[0012] The application of the biomarker combination in the preparation of a fulminant myocarditis diagnostic reagent is characterized in that the biomarker combination comprises Siglec-5.

[0013] The biomarker combination is selected from the group consisting of sST2, PAI-1, Siglec-5, CD163, CD40, P-Cadherin, CD14, and CTLA4.

[0014] Specifically, the biomarker combination is selected from one or more than one of sST2, PAI-1, Siglec-5, CD163, CD40, P-Cadherin, CD14, and CTLA4.

[0015] More specifically, the biomarker combination is selected from one, or two, or three, or four, or five, or six, or seven, or eight of sST2, PAI-1, Siglec-5, CD163, CD40, P-Cadherin, CD14, and CTLA4.

[0016] The fulminant myocarditis diagnostic reagent comprises the biomarker combination and other diagnostically acceptable auxiliary reagents.

[0017] The said diagnostically acceptable auxiliary reagent is selected from the group consisting of: ELISA detection routine reagents, or, flow detection routine reagents.

[0018] The said diagnosis comprises judging whether suffering from fulminant myocarditis, or, evaluating the prognosis of fulminant myocarditis;

[0019] Preferably, the said diagnosis indicates judging whether suffering from fulminant myocarditis, or, evaluating the prognosis of fulminant myocarditis according to the expression level of the biomarker in the biomarker combination in the body of the subject;

[0020] Preferably, the expression level of Siglec-5 is ≥ 89.56 pg / mL, and / or,

[0021] The expression level of sST2 is ≥ 21.39 ng / mL, and / or,

[0022] The expression level of PAI-1 is ≥ 41.29 ng / mL, and / or,

[0023] The expression level of CD163 is ≥ 133.8 ng / mL, and / or,

[0024] The expression level of CD40 is ≥ 67.43 pg / mL, and / or,

[0025] The expression level of P-Cadherin is ≥ 2634 pg / mL, and / or,

[0026] The expression level of CD14 is ≥ 262.4 ng / mL, and / or,

[0027] The expression level of CTLA4 is ≥ 11.19 pg / mL, then the diagnosis of fulminant myocarditis is confirmed; any one of the above 8 markers can be used for the diagnosis of fulminant myocarditis, and if the expression level conditions of multiple markers are met at the same time, the accuracy of the confirmed diagnosis is higher.

[0028] Preferably, the expression level of Siglec-5 is < 89.56 pg / mL, and / or,

[0029] The expression level of sST2 is < 21.39 ng / mL, and / or,

[0030] The expression level of CD163 is < 133.8 ng / mL, the prognosis of fulminant myocarditis is good.

[0031] The said fulminant myocarditis is caused or triggered by virus, bacteria, allergy, drug, upper respiratory tract infection, and / or, intestinal infection.

[0032] Use of the biomarker combination in the preparation of a drug for preventing and treating fulminant myocarditis, characterized in that the biomarker combination comprises: Siglec-5.

[0033] The biomarker combination is selected from the group consisting of sST2, PAI-1, Siglec-5, CD163, CD40, P-Cadherin, CD14, CTLA4.

[0034] Specifically, the biomarker combination is selected from one or more of sST2, PAI-1, Siglec-5, CD163, CD40, P-Cadherin, CD14, CTLA4.

[0035] More specifically, the biomarker combination is selected from one, or two, or three, or four, or five, or six, or seven, or eight of sST2, PAI-1, Siglec-5, CD163, CD40, P-Cadherin, CD14, CTLA4.

[0036] The drug target for preventing and treating fulminant myocarditis is a biomarker in the biomarker combination;

[0037] Preferably, the drug for preventing and treating fulminant myocarditis comprises an active ingredient that can reduce, down-regulate, or inhibit the biomarker;

[0038] Preferably, the active ingredient that can reduce, down-regulate, or inhibit the biomarker is selected from an inhibitor, a neutralizing antibody, or a polypeptide of the biomarker.

[0039] The drug for preventing and treating fulminant myocarditis targets a biomarker in the biomarker combination, and achieves the effect of preventing and treating fulminant myocarditis by inhibiting the expression level of the biomarker;

[0040] Preferably, the drug for preventing and treating fulminant myocarditis reduces the expression level of the biomarker in the plasma of a patient with fulminant myocarditis by an inhibitor, a neutralizing antibody, or a polypeptide against the biomarker, thereby achieving the effect of preventing and treating fulminant myocarditis.

[0041] Preferably, the fulminant myocarditis is caused or triggered by a virus, bacteria, allergy, drug, upper respiratory tract infection, and / or intestinal infection.

[0042] The drug for preventing and treating fulminant myocarditis also comprises a pharmaceutically acceptable excipient.

[0043] The present application aims to provide an inflammatory factor biomarker for diagnosing and / or evaluating the prognosis of fulminant myocarditis.

[0044] The second object of the present application is to provide the use of the biomarker in diagnosing and / or evaluating the prognosis of fulminant myocarditis.

[0045] The third object of the present application is to provide a new therapeutic target of the above-mentioned biomarker, and to achieve the effect of relieving and / or improving fulminant myocarditis by inhibiting the level of sST2 in the plasma of a patient with fulminant myocarditis.

[0046] The above technical objects of the present application are achieved by the following technical solutions:

[0047] The first object of the present application is to provide a biomarker for diagnosing and / or evaluating the prognosis of fulminant myocarditis, which comprises: sST2, PAI-1, Siglec-5, CD163, CD40, P-Cadherin, CD14 and CTLA4.

[0048] Further, the fulminant myocarditis includes fulminant myocarditis caused by viruses, bacteria, allergies, drugs and various causes.

[0049] The second object of the present application is to provide the use of the biomarker in diagnosing and / or evaluating the prognosis of fulminant myocarditis.

[0050] Further, by detecting the level of the biomarker in the plasma of the human body, if the concentration of the biomarker in the plasma of the human body is significantly increased, it is considered that the human body has fulminant myocarditis and / or poor prognosis.

[0051] The biomarker is selected from: sST2, PAI-1, Siglec-5, CD163, CD40, P-Cadherin, CD14, CTLA4.

[0052] The present application finds that the levels of the above-mentioned inflammatory factors in the plasma of patients with fulminant myocarditis are significantly increased through large-sample clinical trials, which confirms that these inflammatory factors can be used as biomarkers for diagnosing fulminant myocarditis. Therefore, by detecting the level of the inflammatory factor in the plasma, fulminant myocarditis can be diagnosed or the prognosis of fulminant myocarditis can be evaluated.

[0053] The third object of the present application is to provide the use of the biomarker as a therapeutic target in the preparation of a drug for relieving and / or improving fulminant myocarditis, and to achieve the effect of relieving and / or improving fulminant myocarditis by inhibiting the increased level of the biomarker in the plasma of a patient with fulminant myocarditis.

[0054] Further, the biomarker neutralizing antibody reduces the level of the biomarker in the plasma of the fulminant myocarditis model mouse, thereby reducing the level of the biomarker in the plasma of the fulminant myocarditis model mouse, and then achieving the effect of alleviating and / or improving fulminant myocarditis.

[0055] The present application discloses the expression profile of a plurality of inflammatory factors in the plasma of fulminant myocarditis patients, especially the application of a biomarker (Suppression of Tumorigenicity 2) and a biomarker neutralizing antibody in fulminant myocarditis, and specifically relates to the application of a plurality of inflammatory factors in the diagnosis and prognosis evaluation of fulminant myocarditis, and the application of a biomarker neutralizing antibody in the treatment of fulminant myocarditis, belonging to the field of diagnosis, prevention and treatment of cardiovascular diseases.

[0056] The present application detects the expression profile of 122 inflammatory factors in a large sample of clinical fulminant myocarditis patients, screens out 8 inflammatory factors, and tests the efficiency of the 8 inflammatory factors as biomarkers of fulminant myocarditis. Further, the application of a biomarker neutralizing antibody in fulminant myocarditis model animals is detected, the application of the biomarker in fulminant myocarditis is determined, and a theoretical basis and evidence-based basis for alleviating and / or treating fulminant myocarditis are provided.

[0057] The present application has the following beneficial effects:

[0058] 1. The present application proves the correlation between specific inflammatory factors and fulminant myocarditis through a large number of experiments, and the level of a plurality of inflammatory factors is significantly increased in fulminant myocarditis patients, which indicates that the plurality of inflammatory factors can be used as biomarkers. Therefore, a reagent or kit for detecting biomarkers is prepared, and the level of specific inflammatory factors in the plasma outside the body can be used to assist in the diagnosis of fulminant myocarditis or the evaluation of the prognosis of fulminant myocarditis. The detection method is rapid and convenient, greatly improves the efficiency and accuracy of the diagnosis and prognosis evaluation of fulminant myocarditis, and is of great application value in clinical diagnosis and follow-up treatment of the disease.

[0059] 2. The present application not only provides a new diagnosis method for fulminant myocarditis, but also provides a new treatment target for fulminant myocarditis. The biomarker neutralizing antibody or inhibitor reduces the level of the biomarker in the plasma of the fulminant myocarditis patient by inhibiting the biomarker, improves the myocardial damage of fulminant myocarditis, helps to alleviate and improve fulminant myocarditis, and greatly reduces the hospital mortality rate of fulminant myocarditis patients. BRIEF DESCRIPTION OF DRAWINGS

[0060] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed in the embodiment description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other related drawings can be obtained by those skilled in the art without any creative effort.

[0061] Figure 1 Figure 4 is a graph showing the expression profile of inflammatory factors in peripheral blood of control population and fulminant myocarditis patients at admission; in the figure, control represents the control population, FM represents fulminant myocarditis; True represents greater than the threshold value, obvious difference; False represents less than the threshold value, insignificant difference.

[0062] Figure 2 Figure 5 is a graph showing the change profile of inflammatory factor levels in peripheral blood of fulminant myocarditis patients before and after treatment; in the figure, FM-admission represents the detection result of blood sample of fulminant myocarditis patients before discharge after recovery; FM-discharge represents the detection result of blood sample of fulminant myocarditis patients at admission without treatment; True represents greater than the threshold value, obvious difference; False represents less than the threshold value, insignificant difference.

[0063] Figure 3 Figure 6 is a graph showing the expression level values of specific inflammatory factors in fulminant myocarditis patients; in the figure, FM-admission represents the expression level values of inflammatory factors in blood sample of fulminant myocarditis patients before discharge after recovery; FM-discharge represents the expression level values of inflammatory factors in blood sample of fulminant myocarditis patients at admission without treatment; control represents the expression level values of inflammatory factors in blood sample of control population; folds refers to the fold of expression level values; Cytokines refers to inflammatory factors.

[0064] Figure 4 Figure 7 is a graph showing the content analysis of 8 inflammatory factors in blood samples of control population and fulminant myocarditis patients, which shows that the 8 inflammatory factors can be used for diagnosis and evaluation of fulminant myocarditis; in the figure, con represents the expression level values of inflammatory factors in blood sample of control population; FM represents the expression level values of inflammatory factors in blood sample of fulminant myocarditis patients.

[0065] Figure 5 Figure 8 is a graph showing the correlation analysis of 9 inflammatory factors and cardiac ejection fraction, in which 3 inflammatory factors can be used for prognosis evaluation of fulminant myocarditis; in the figure, Ejection Fraction represents ejection fraction, FM represents the expression level values of inflammatory factors in blood sample of fulminant myocarditis patients; hs-CRP represents positive control.

[0066] Figure 6The ROC curve of the diagnostic efficiency of peripheral blood specific inflammatory factor levels for fulminant myocarditis, wherein hs-CRP represents a positive control.

[0067] Figure 7 The column chart of the levels of each inflammatory factor of the four groups of mice in Experimental Example 4, indicating that the levels of various inflammatory factors in the peripheral blood of A / J mice with fulminant myocarditis are increased; wherein C57 represents C57 mice, A / J represents A / J mice, Balb / c represents Balb / c mice, C3H represents C3H mice, control represents a blank control group, and CVB3 represents a CVB3 myocarditis model group.

[0068] Figure 8 The numerical column chart of the maximum left ventricular pressure drop rate (-dp / dt min ) and the maximum left ventricular pressure rise rate (+dP / dt max ) of the three groups of mice in Experimental Example 5, indicating that sST2 can induce heart function damage in A / J mice.

[0069] Figure 9 The myocardial cell TUNEL staining photographs and the column chart of the TUNEL positive cell rate (TUNEL positive rate) per hundred myocardial cells of the three groups of mice in Experimental Example 5, indicating that sST2 can induce myocardial cell apoptosis in A / J mice; Figure 7 and Figure 8 , wherein control represents a blank control group, PBS represents a negative control group, and sST2 represents an experimental group.

[0070] Figure 10 The content analysis chart of sST2 in the blood samples of the four groups of mice in Experimental Example 6, indicating that sST2 neutralizing antibodies can inhibit the increased sST2 in the peripheral blood of A / J mice with fulminant myocarditis induced by CVB3.

[0071] Figure 11 The content analysis chart of sST2 in the myocardial tissue of the four groups of mice in Experimental Example 6, indicating that sST2 neutralizing antibodies can inhibit the increased sST2 in the heart of A / J mice with fulminant myocarditis induced by CVB3.

[0072] Figure 12 The survival rate analysis chart of the four groups of mice in Experimental Example 6, indicating that sST2 neutralizing antibodies can improve the survival rate of A / J mice with fulminant myocarditis induced by CVB3.

[0073] Figure 13 The myocardial tissue section staining chart of the four groups of mice in Experimental Example 6, indicating that sST2 neutralizing antibodies can improve the impaired heart function of A / J mice with fulminant myocarditis induced by CVB3.

[0074] Figure 14The myocardial tissue verification area proportion column chart of the 4 groups of mice in Experimental Example 6 shows that the sST2 neutralizing antibody can reduce the inflammatory cell infiltration in the CVB3-induced A / J fulminant myocarditis mouse heart.

[0075] Figure 15 The myocardial cell TUNEL staining photos and the column chart of the TUNEL positive cell rate per 100 myocardial cells (TUNEL positive rate) of the 4 groups of mice in Experimental Example 6 show that the sST2 neutralizing antibody can reduce the myocardial cell apoptosis in the CVB3-induced A / J fulminant myocarditis mouse heart.

[0076] Figures 10-15 In the above table, control represents the blank control group; PBS+goat IgG represents the negative control group; CVB3+PBS represents the CVB3 fulminant myocarditis model group; and CVB3+anti-sST2 or anti-sST2 represents the treatment group.

[0077] Figure 16 The expression level of the inflammatory factor in the peripheral blood of the fulminant myocarditis mouse in Experimental Example 7 shows that the anti-CD163 neutralizing antibody can inhibit the increase of CD163. In the figure, the vertical coordinate is the expression level value of the CD163 inflammatory factor, and the horizontal coordinate control represents the blank control group, PBS+IgG represents the negative control group, CVB3 represents the CVB3 fulminant myocarditis model group, CVB3+anti-CD163 represents the treatment group.

[0078]

[0079] Figure 17 The heart function of the mouse in Experimental Example 7 shows that the anti-CD163 neutralizing antibody can improve the CVB3-induced A / J fulminant myocarditis mouse heart dysfunction; in the figure, the vertical coordinate of the left figure cardiac output represents the heart output determination value, and the vertical coordinate of the right figure stroke volume represents the stroke output; control represents the blank control group, PBS+IgG represents the negative control group, CVB3 represents the CVB3 fulminant myocarditis model group, and CVB3+anti-CD163 represents the treatment group. DETAILED DESCRIPTION

[0080] The technical solutions of the present application will be described below in conjunction with specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments of the present application, all other embodiments obtained by a person of ordinary skill in the art without creative labor fall within the protection scope of the present application.

[0081] Source of the biological material ​

[0082] The mice used in the experimental examples of the present application are commercially available.

[0083] Application of the biomarker combination of the present application in the first group of examples

[0084] Application of the biomarker combination in the preparation of a diagnostic reagent for fulminant myocarditis, characterized in that the biomarker combination comprises: Siglec-5.

[0085] The biomarker combination is selected from the group consisting of sST2, PAI-1, Siglec-5, CD163, CD40, P-Cadherin, CD14, CTLA4.

[0086] Specifically, the biomarker combination is selected from one or more of sST2, PAI-1, Siglec-5, CD163, CD40, P-Cadherin, CD14, CTLA4;

[0087] More specifically, the biomarker combination is selected from one, or two, or three, or four, or five, or six, or seven, or eight of sST2, PAI-1, Siglec-5, CD163, CD40, P-Cadherin, CD14, CTLA4.

[0088] The diagnostic reagent for fulminant myocarditis comprises the biomarker combination and other diagnostically acceptable auxiliary reagents;

[0089] The diagnostically acceptable auxiliary reagents are selected from: ELISA detection routine reagents, or, flow detection routine reagents.

[0090] The diagnosis includes determining whether to suffer from fulminant myocarditis, or, evaluating the prognosis of fulminant myocarditis;

[0091] Preferably, the diagnosis indicates that whether to suffer from fulminant myocarditis, or, evaluate the prognosis of fulminant myocarditis is determined according to the expression level of the biomarker in the biomarker combination in the body of the subject;

[0092] Preferably, the expression level of Siglec-5 is ≥ 89.56 pg / mL, and / or,

[0093] The expression level of sST2 is ≥ 21.39 ng / mL, and / or,

[0094] The expression level of PAI-1 is ≥ 41.29 ng / mL, and / or,

[0095] The expression level of CD163 is ≥ 133.8 ng / mL, and / or,

[0096] an expression level of CD40 ≥ 67.43 pg / mL, and / or,

[0097] an expression level of P-Cadherin ≥ 2634 pg / mL, and / or,

[0098] an expression level of CD14 ≥ 262.4 ng / mL, and / or,

[0099] an expression level of CTLA4 ≥ 11.19 pg / mL, then the fulminant myocarditis is definitely diagnosed;

[0100] preferably, an expression level of Siglec-5 < 89.56 pg / mL, and / or,

[0101] an expression level of sST2 < 21.39 ng / mL, and / or,

[0102] an expression level of CD163 < 133.8 ng / mL, then the fulminant myocarditis has a good prognosis.

[0103] The fulminant myocarditis is caused or triggered by a virus, a bacterium, an allergy, a drug, an upper respiratory tract infection, and / or, an intestinal infection.

[0104] The above markers herein all have the conventional technical meanings commonly understood by those skilled in the art, for example:

[0105] Siglec-5 can be Siglec-5 described in patent application 201980062880.6;

[0106] sST2 is sST2 described in the “Guidelines for the Diagnosis and Treatment of Heart Failure in China”;

[0107] PAI-1 can be PAI-1 described in the “Application Research of PAI-1 Gene Detection in Prevention of Ischemic Stroke”;

[0108] CD163 can be CD163 described in the “Research Progress of M2 Macrophage Marker CD163 and Tumor”;

[0109] CD40 can be CD40 described in the “Correlation Study of Serum Soluble CD40 Ligand Level and Disease Activity of Crohn's Disease”;

[0110] P-Cadherin can be P-Cadherin described in the “Ultra-sensitive Detection of Fn14, P-Cadherin and N-Cadherin Based on Single Molecule Array Technology”;

[0111] The CD14 can be the CD14 described in the article "Correlation between CT signs of AIDS complicated with pulmonary tuberculosis and plasma soluble CD14".

[0112] The CTLA4 can be the CTLA4 described in the article "Correlation between serum GP73 and CTLA4 levels and liver fibrosis degree in patients with chronic hepatitis B".

[0113] The hs-CRP can be the hs-CRP described in the article "Relationship between serum PTX3, Cav-1, hs-CRP and disease condition and prognosis of patients with acute cerebral infarction".

[0114] The pharmaceutical use of the biomarker combination of the second embodiment of the application

[0115] The present embodiment provides the application of the biomarker combination in the preparation of a drug for preventing and treating fulminant myocarditis, characterized in that the biomarker combination comprises: Siglec-5.

[0116] The biomarker combination is selected from the group consisting of sST2, PAI-1, Siglec-5, CD163, CD40, P-Cadherin, CD14, and CTLA4.

[0117] Specifically, the biomarker combination is selected from one or more of sST2, PAI-1, Siglec-5, CD163, CD40, P-Cadherin, CD14, and CTLA4.

[0118] More specifically, the biomarker combination is selected from one, or two, or three, or four, or five, or six, or seven, or eight of sST2, PAI-1, Siglec-5, CD163, CD40, P-Cadherin, CD14, and CTLA4.

[0119] The drug target for preventing and treating fulminant myocarditis is the biomarker in the biomarker combination.

[0120] Preferably, the drug for preventing and treating fulminant myocarditis comprises an active ingredient that can reduce, down-regulate, or inhibit the biomarker.

[0121] Preferably, the active ingredient that can reduce, down-regulate, or inhibit the biomarker is selected from an inhibitor, a neutralizing antibody, or a polypeptide of the biomarker.

[0122] The drug for preventing and treating fulminant myocarditis takes the biomarker in the biomarker combination as a drug target, and achieves the effect of preventing and treating fulminant myocarditis by inhibiting the expression level of the biomarker.

[0123] Preferably, the drug for preventing and treating fulminant myocarditis reduces the expression level of the biomarker in the plasma of the patient with fulminant myocarditis by an inhibitor, a neutralizing antibody or a polypeptide against the biomarker, thereby achieving the effect of preventing and treating fulminant myocarditis.

[0124] Preferably, the fulminant myocarditis is caused or triggered by viruses, bacteria, allergies, drugs, upper respiratory tract infections, and / or intestinal infections.

[0125] The drug for preventing and treating fulminant myocarditis also includes a pharmaceutically acceptable excipient.

[0126] Preferably, the pharmaceutically acceptable excipient includes solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavorings, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, integration agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, antifoaming agents, thickening agents, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, release retardants.

[0127] Experimental Example 1

[0128] The expression level of a large number of inflammatory factors in the peripheral blood of patients with fulminant myocarditis is increased

[0129] Study population and study design: From April 2017 to March 2019, in Wuhan, Hubei Province, China, hospitalized patients with fulminant myocarditis were continuously enrolled in Tongji Hospital of Huazhong University of Science and Technology. The inclusion criteria include: ① age greater than 18 years old; ② diagnosed as fulminant myocarditis: myocardial biopsy suggests myocarditis or meets the clinical diagnosis (rapid onset of severe hemodynamic dysfunction symptoms, cTnI increase, echocardiography suggests rapid decrease of left ventricular EF value or lower than 40%). The exclusion criteria include: ① acute myocardial infarction; ② ordinary acute myocarditis; ③ severe nervous system diseases (Alzheimer's disease, Parkinson's syndrome in advanced stage), lower extremity amputation or deaf-mute patients.

[0130] Inflammatory factor expression profile detection: fasting peripheral blood was drawn into EDTA anticoagulant tube, immediately centrifuged at 3000 rpm for 8 minutes to take serum and frozen in-80℃ refrigerator for analysis. Plasma inflammatory factor expression profile was detected by RayBiotech Humancytokine arrays.

[0131] Experimental results: 4 cases of fulminant myocarditis patients were enrolled, and 4 cases of control group population samples were matched with no difference in baseline data such as gender and age. The test results show that, compared with the control group, the content of 58 kinds of inflammatory factors in the peripheral blood of patients with fulminant myocarditis is significantly increased (P<0.05). Figure 1). These results suggest that the increase of the levels of a large number of inflammatory factors in the plasma is associated with fulminant myocarditis. In the legend, control represents the control population, FM represents fulminant myocarditis, and the red dot indicates the inflammatory factor with a significant difference.

[0132] Experimental Example 2

[0133] The expression levels of specific inflammatory factors in the peripheral blood of patients with fulminant myocarditis decrease after treatment

[0134] Study population and study design: From April 2017 to March 2019, inpatients with fulminant myocarditis were continuously included in the Tongji Hospital of Huazhong University of Science and Technology in Wuhan, Hubei Province. The inclusion criteria include: ① Age greater than 18 years old; ② Diagnosed as fulminant myocarditis: myocardial biopsy suggests myocarditis or meets the clinical diagnosis (rapid onset of severe hemodynamic dysfunction symptoms, cTnI increase, echocardiography suggests rapid decrease of left ventricular EF value or lower than 40%). The exclusion criteria include: ① Acute myocardial infarction; ② Ordinary acute myocarditis; ③ Severe nervous system diseases (Alzheimer's disease, Parkinson's syndrome in advanced stage), lower limb amputation or deaf-mute patients.

[0135] Inflammatory factor expression profile detection: Fasting peripheral blood was drawn into an EDTA anticoagulant tube, and serum was immediately separated at 3000 rpm for 8 minutes and frozen in a -80°C refrigerator for analysis. Plasma inflammatory factor expression profile was detected by RayBiotech Humancytokine arrays.

[0136] Experimental results: 4 cases of fulminant myocarditis patients were included, and blood samples were stored at the time of admission without treatment and after treatment and before discharge. The test results show that after effective treatment, the content of some elevated inflammatory factors decreases significantly Figure 2 ). These results suggest that the increase of the levels of specific inflammatory factors in the peripheral blood is associated with fulminant myocarditis, namely sST2, PAI-1, Siglec-5, CD163, IL-17B, IL-4, VEGF-C, CD40, P-Cadherin, CD14, Angiostatin and CTLA4 Figure 3 ). * represents p<0.05.

[0137] Experimental Example 3

[0138] The level of specific inflammatory factors in peripheral blood for the diagnosis and prognosis evaluation of fulminant myocarditis

[0139] Study population and study design: From April 2017 to March 2019, consecutive inpatients with fulminant myocarditis were enrolled in Tongji Hospital, Huazhong University of Science and Technology, Wuhan, Hubei Province. The inclusion criteria included: ① age greater than 18 years old; ② diagnosis of fulminant myocarditis: myocardial biopsy prompted myocarditis or met the clinical diagnosis (rapid onset of severe hemodynamic dysfunction symptoms, cTnl increased, echocardiography prompted left ventricular EF value rapidly decreased or less than 40%). The exclusion criteria included: ① acute myocardial infarction; ② ordinary acute myocarditis; ③ severe nervous system diseases (Alzheimer's disease, Parkinson's syndrome in advanced stage), lower limb amputation or deaf-mute patients.

[0140] Inflammatory factor level detection: Peripheral blood was drawn into an EDTA anticoagulation tube on an empty stomach, and serum was immediately separated at 3000 rpm for 8 minutes and frozen at -80°C for analysis. Plasma inflammatory factor levels were detected using the corresponding ELISA kit from R&D Company.

[0141] Experimental results: 32 cases of fulminant myocarditis patients were enrolled, and 16 cases of control group population samples were matched with no difference in baseline data such as gender and age. The test results showed that for fulminant myocarditis, the contents of 8 inflammatory factors (sST2, PAI-1, Siglec-5, CD163, CD40, P-Cadherin, CD14 and CTLA4) in the peripheral blood of patients changed significantly, which had diagnostic value Figure 4 ). Among them, the contents of 3 inflammatory factors (sST2, Siglec-5 and CD163) had correlation with cardiac function EF value, which had prognostic evaluation value Figure 5 ). And compared with CRP, the content of specific inflammatory factors (sST2, Siglec-5 and CD163) in the peripheral blood of patients had better diagnostic efficiency Figure 6 ). * represents p<0.05.

[0142] Experimental Example 4

[0143] Increased levels of multiple inflammatory factors in the peripheral blood of fulminant myocarditis mice

[0144] Mouse model preparation: 8-week-old male C57, A / J, Balb / c and C3H mice were purchased from Nanjing University Model Animal Center. They were raised in the SPF level animal room of Huazhong University of Science and Technology Tongji Medical College. Different background mice were adapted to the animal room for one week, and then intraperitoneally injected with CVB3 virus (10 4 dissolved in 0.1 ml PBS) as the CVB3 myocarditis model group.

[0145] Inflammatory factor level detection: The corresponding ELISA Kit from R&D Company was used for detection.

[0146] Experimental results: CVB3 virus was injected intraperitoneally for 8 days, then heart catheter detection was performed, and then the animals were sacrificed, and tissue samples were collected for the above detection. The results showed that compared with the control group, the sST2, C163, P-Cadherin, CD14 and CTLA4 in the peripheral blood of A / J mice with fulminant myocarditis after CVB3 infection increased most significantly Figure 7 ) represents p<0.05.

[0147] Experimental Example 5

[0148] Taking the most significantly increased sST2 as an example, the effect of specific inflammatory factors on the heart function of A / J mice was observed

[0149] Preparation of mouse model: 8-week-old male A / J mice were purchased from the Model Animal Center of Nanjing University. They were raised in the SPF animal room of Tongji Medical College, Huazhong University of Science and Technology. After one week of adaptation in the animal room, the mice were intraperitoneally injected with recombinant sST2 protein (5ug dissolved in 0.1ml PBS) as the intervention group (experimental group). Another group of mice was treated with PBS as the negative control group, and the third group was the blank control group (control) without any treatment.

[0150] Detection of mouse cardiac hemodynamics: Millar pressure-volume system of Millar Instrument PowerLab Company was used. After the animals were anesthetized to an appropriate depth, a midline incision was made in the neck, the right common carotid artery was isolated, the distal end was ligated, the proximal end was clamped, a V-shaped incision was made on the artery with microscissors, a microcatheter was inserted into the left ventricle, and signals were recorded through the conduction system to obtain hemodynamic data such as heart rate (HR), left ventricular end-diastolic pressure (PED), left ventricular end-systolic pressure (PES), left ventricular pressure maximum descent rate (-dp / dt min ) and left ventricular pressure maximum rise rate (+dP / dt max ).

[0151] Histological detection: The myocardial tissue was placed in a embedding frame, soaked and fixed in neutral formaldehyde solution, dehydrated, and paraffin-embedded. The wax block was cut into 4pm-thick sections on a microtome. HE staining was used to observe the general appearance of myocardium, and picrosirius red staining was used to observe myocardial fibrosis. TUNEL staining was performed using a Roche kit.

[0152] Experimental results: After intraperitoneal injection of recombinant sST2 protein for 10 days, heart catheter detection was performed, and then the animals were sacrificed, and tissue samples were collected for the above detection. The results showed that compared with the control group, intraperitoneal injection of recombinant sST2 protein significantly decreased the heart function of mice Figure 8 ). And intraperitoneal injection of recombinant sST2 protein increased the TUNEL positive rate of myocardial cells in mice, suggesting that myocardial cell apoptosis increased Figure 9) p<0.05.

[0153] Experimental Example 6

[0154] Anti-sST2 neutralizing antibody can inhibit CVB3-induced myocardial injury in A / J mice with fulminant myocarditis

[0155] Preparation of mouse model: 8-week-old male A / J mice were purchased from the Model Animal Center of Nanjing University. They were raised in the SPF animal room of Tongji Medical College, Huazhong University of Science and Technology. After one week of adaptation in the animal room, the mice were intraperitoneally injected with CVB3 virus (10 4 dissolved in 0.1 ml PBS) as the CVB3 fulminant myocarditis model group. Another group of mice with fulminant myocarditis was treated with anti-sST2 neutralizing antibody (10 μg dissolved in 0.1 ml PBS) by intraperitoneal injection, as the treatment group; the third group was the blank control group without any treatment; the fourth group was the negative control group injected with sheep IgG antibody (10 4 dissolved in 0.1 ml PBS) intraperitoneally.

[0156] Detection of sST2 levels: R&D Mouse ST2 / IL-33R Quantikine ELISA Kit was used for detection.

[0157] Detection of hemodynamics in mouse heart: Millar pressure-volume system of Millar Instrument PowerLab was used. After the animals were anesthetized to an appropriate depth, a midline incision was made in the neck, the right common carotid artery was isolated, the distal end was ligated, the proximal end was clamped, a V-shaped incision was made on the artery with microscissors, a microcatheter was inserted into the left ventricle, and signals were recorded through the conduction system to obtain hemodynamic data such as heart rate (HR), left ventricular end-diastolic pressure (PED), left ventricular end-systolic pressure (PES), left ventricular pressure maximum descent rate (-dp / dt min ) and left ventricular pressure maximum rise rate (+dP / dt max ) and so on.

[0158] Histological examination: The myocardial tissue was placed in an embedding frame and soaked in neutral formaldehyde solution for fixation, dehydration, paraffin embedding, and the wax block was cut into a section of 4 μm thickness on a microtome. HE staining was used to observe the general appearance of myocardium, and picrosirius red staining was used to observe myocardial fibrosis. TUNEL staining was detected using a Roche kit.

[0159] Experimental results: After intraperitoneal injection of anti-sST2 neutralizing antibody for 8 days, cardiac catheterization was performed, and then the animals were sacrificed, and tissue specimens were collected for the above detection. The results showed that compared with the control group, sST2 in the peripheral blood of mice with fulminant myocarditis after CVB3 infection was increased, while anti-sST2 neutralizing antibody could reduce itFigure 10 ) sST2 level in heart of CVB3 infected mice with fulminant myocarditis increased, while anti-sST2 neutralizing antibody can reduce it ( Figure 11 ) Survival rate of CVB3 infected mice with fulminant myocarditis decreased significantly, while anti-sST2 neutralizing antibody can increase it ( Figure 12 ) Heart function of CVB3 infected mice with fulminant myocarditis decreased significantly, while anti-sST2 neutralizing antibody can enhance it ( Figure 13 ) Inflammatory cell infiltration in heart of CVB3 infected mice with fulminant myocarditis increased significantly, while anti-sST2 neutralizing antibody can reduce it ( Figure 14 ) Myocardial cell TUNEL positive rate of CVB3 infected mice with fulminant myocarditis increased significantly, while anti-sST2 neutralizing antibody can reduce it ( Figure 15 ) * represents p<0.05.

[0160] Experimental Example 7

[0161] Anti-CD163 neutralizing antibody can improve CVB3 induced heart dysfunction of A / J mice with fulminant myocarditis

[0162] Preparation of mouse model: 8-week-old male A / J mice were purchased from Nanjing University Model Animal Center. They were raised in the SPF animal room of Tongji Medical College, Huazhong University of Science and Technology. After one week of adaptation in the animal room, the mice were intraperitoneally injected with CVB3 virus (10 4 dissolved in 0.1 ml PBS) as the CVB3 fulminant myocarditis model group. Mice without any treatment were used as the blank control group, and mice intraperitoneally injected with sheep IgG antibody (10 4 dissolved in 0.1 ml PBS) were used as the negative control group. Another group of mice with fulminant myocarditis was intraperitoneally injected with anti-CD163 neutralizing antibody (10 μg dissolved in 0.1 ml PBS) as the treatment group.

[0163] Detection of inflammatory factor levels: R&D ELISA kit was used for detection.

[0164] Mouse heart ultrasound detection: Vevo 2100 small animal ultrasound of Visual Sonics Inc. was used for heart ultrasound detection. The main measurement indexes included heart rate (HR), left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), left ventricular diastolic or systolic interventricular septal thickness (LVIDd and LVIDs), and left ventricular posterior wall thickness (LVPWd and LVPWs).

[0165] The experimental results: after intraperitoneal injection of anti-CD163 neutralizing antibody for 8 days, the heart catheter detection is performed, then the animals are sacrificed, the tissue samples are collected, and the above detection is performed, and the results show that compared with the control group, the CD163 level in the peripheral blood of the mice with fulminant myocarditis after CVB3 infection is increased, and the anti-CD163 neutralizing antibody treatment can reduce the CD163 level( Figure 16 ). The heart function of the mice with fulminant myocarditis after CVB3 infection is significantly decreased, and the anti-CD163 neutralizing antibody treatment can enhance the function( Figure 17 ). * represents p<0.05.

Claims

1. Use of an anti-CD163 neutralizing antibody for the manufacture of a medicament for the prevention and treatment of fulminant myocarditis.

2. Use of an anti-CD163 neutralizing antibody according to claim 1 for the manufacture of a medicament for the prophylaxis and treatment of fulminant myocarditis, characterized in that, The medicament for the prevention and treatment of fulminant myocarditis can further comprise a pharmaceutically acceptable adjuvant.

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