CN114984014A - Inhibitor for treating sepsis and application thereof - Google Patents

Abstract

The invention discloses an inhibitor for treating sepsis, which is CP-673451 and achieves the purpose of treating sepsis by reducing cytokine release of circulating immune cells. The invention discovers for the first time that CP-673451(PDGFR inhibitor) can effectively treat sepsis, especially early sepsis; the invention discovers the treatment effect and mechanism of CP-673451 for adjusting early-stage excessive immune inflammatory response in sepsis for the first time, and proves that CP-673451 is a medicine for effectively treating sepsis, can reduce the organ failure degree and death rate of sepsis, and can be used as a novel medicine for sepsis diagnosis and treatment.

Description

Inhibitor for treating sepsis and application thereof

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to an inhibitor for treating sepsis and application thereof.

Background

According to statistics, the incidence rate of sepsis reaches 535 cases per 100000 persons, the hospitalization mortality rate accounts for 25-30%, and the sepsis is an important reason for the high incidence rate and the high mortality rate in the current intensive care unit. It is an excessive inflammatory reaction of the immune system of the body to infection after releasing inflammatory mediators, resulting in abnormal physiological, pathological and biochemical syndromes, most patients are accompanied by the abnormality of blood coagulation function, the function of systemic blood coagulation substances is activated and is accompanied by the risk of massive hemorrhage and multi-organ failure, and meanwhile, because the increase of the reaction of the blood coagulation substances is combined with the damage of an anti-coagulation mechanism, the disseminated intravascular coagulation and the vascular embolism also occur in some patients. Exogenous pathogenic bacteria relate to various cell functions and cytokine expression changes in the process from the occurrence of inflammatory reaction to the occurrence of sepsis by exciting organisms, the process presents the characteristic of waterfall-like cascade reaction, and the sepsis is difficult to predict and rapid in occurrence and development, and the specific treatment method is lacked, so that the sepsis is very difficult to diagnose and treat and has poorer prognosis.

Although the level of clinical treatment and pathogenesis of sepsis is well understood, reducing nosocomial mortality has relied primarily on early antibiotic therapy and multi-organ support therapy, and molecular-mechanism targeted therapies for sepsis immunity and infection remain lacking. Sepsis patients often die of secondary infections after immunosuppression because of early overactive inflammatory responses leading to immune dysfunction, early pathogen invasion activating innate immunity, activated innate immune cells providing pathogen antigens to lymphocytes and activating cellular immunity. Immune cells release large amounts of cytokines to trigger pro-and anti-inflammatory immune responses, leading to immunoregulatory disturbances, organ dysfunction or failure, and even death. Therefore, the regulation of early immune dysfunction and the prevention and treatment of immunosuppression caused by sepsis are important research points.

Disclosure of Invention

The present invention is directed to solving, at least to some extent, one of the technical problems in the related art. Therefore, the main object of the present invention is to provide an inhibitor for treating sepsis and the use thereof.

The purpose of the invention is realized by the following technical scheme:

in a first aspect: an inhibitor for the treatment of sepsis, said inhibitor being capable of effecting treatment of sepsis by reducing cytokine release from circulating immune cells.

Preferably, wherein the inhibitor is CP-673451, the CP-673451 has the formula:

preferably, wherein the sepsis comprises early sepsis, severe sepsis and septic shock.

The research concept of the invention is as follows: the present inventors have found that increased expression of B and T lymphocyte negative co-stimulatory factor (BTLA) on immune cells during sepsis in patients is considered to be one of the major factors responsible for sepsis-induced immune cell dysfunction; the pathophysiological mechanism of early sepsis is multiple organ damage caused by a "cytokine storm". Therefore, we hypothesized that promoting expression of BTLA in immune cells would accelerate immune cell dysfunction during sepsis, thereby attenuating the development of "cytokine storm" and exploring therapeutic drugs for sepsis. Therefore, the team of the application utilizes a cell experiment to carry out high-throughput screening on the drugs for up-regulating BTLA expression by a commercial kinase inhibitor drug library, and carries out verification on protein and mRNA expression levels in an animal model to determine that the effect of up-regulating BTLA expression by a PDGFR signal pathway inhibitor (CP-673451) is most obvious; the inventor uses the PDGFR pathway inhibitor medicine for treating CLP sepsis mice, and proves that the functions of the important organs of the CLP sepsis mice are protected and the death rate is reduced after the medicine is used for treating the CLP sepsis mice. Further exploring and discovering a mechanism of protective action of a PDGFR pathway inhibitor in sepsis, after the PDGFR pathway inhibitor is treated by using a medicament, the PDGFR pathway inhibitor can inhibit the release of chemotactic factors CXCL13, CCL1, CCL2 and CCL7 in CLP sepsis mice in vivo, reduce the chemotaxis of B and T lymphocytes in peripheral blood and important organs, further reduce the secretion of an anti-inflammatory factor IL-10 generated by the lymphocytes, and inhibit excessive anti-inflammatory activation; and meanwhile, the generation of cytokines such as IL-1 beta and the like by medullary cells is reduced, so that the activation of T cells and B cells is inhibited, the early-stage excessive immune inflammatory reaction is relieved, the sepsis is relieved, and the aim of treating the sepsis is fulfilled.

In a second aspect: application of CP-673451 in preparing medicine for treating sepsis is provided.

Preferably, the dosage form of the medicament is orally administered.

Preferably, wherein the effective dose of CP-673451 is 20 mg/kg.

Compared with the prior art, the invention has at least the following advantages:

the invention discovers for the first time that CP-673451(PDGFR inhibitor) can effectively treat sepsis, especially early sepsis;

the invention discovers the treatment effect and mechanism of CP-673451 for adjusting early-stage excessive immune inflammatory response in sepsis for the first time, and proves that CP-673451 is a medicine for effectively treating sepsis, can reduce the organ failure degree and death rate of sepsis, and can be used as a novel medicine for sepsis diagnosis and treatment.

Drawings

In order to more clearly illustrate the embodiments of the present invention, reference will now be made briefly to the embodiments or to the accompanying drawings that are needed in the description of the prior art.

FIG. 1 is a heatmap of the results of drug-induced or inhibitory BTLA expression;

FIG. 2 is a graph of flow cytometric analysis of BTLA expression in spleen immune cells;

FIG. 3 is a graph of the proportion of BTLA-expressing lymphocytes;

FIG. 4 is the expression of BTLA-mRNA levels in spleen tissue;

FIG. 5 is a survival curve for the treatment of sepsis with the inhibitor;

FIG. 6 is a graph showing that the inhibitor (CP-673451) reduces sepsis mortality by protecting lung tissue function;

FIG. 7 is a qualitative analysis of serum cytokine array of septic mice;

FIG. 8 shows the result of quantitative analysis of serum cytokine on LC chip in sepsis mice;

figure 9 is qPCR validation of cytokines in cells and tissues.

Detailed Description

In order to make the technical means, the original characteristics, the achieved purposes and the effects of the invention easily understood, the invention is further described below with reference to the specific embodiments and the attached drawings, but the following embodiments are only the preferred embodiments of the invention, and not all embodiments.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The invention carries out screening on a large number of sepsis treatment medicines at the early stage, finds that CP-673451 can reduce the death rate of sepsis by reducing the immune media such as cytokine and chemotactic factor generated and released by circulating immune cells, and can be used as an effective sepsis treatment medicine. The method specifically comprises the following steps:

first, screening identifies that inhibitors of the bioactive compound PDGFR upregulate BTLA expression. Increased expression of B and T lymphocyte negative co-stimulatory factor (BTLA) on immune cells during sepsis is believed to be one of the major factors contributing to sepsis-induced immune cell dysfunction. The pathophysiological mechanism of early sepsis is multiple organ damage caused by a "cytokine storm". Therefore, we hypothesized that promoting expression of BTLA in immune cells would accelerate immune cell dysfunction during sepsis, thereby attenuating the development of "cytokine storm" and exploring therapeutic drugs for sepsis. Therefore, a high-selectivity kinase inhibitor commercial drug library is screened, the expression of BTLA in RAW264.7 macrophages is detected through intracellular Western blot, and a part of targeted selective inhibitors promote or reduce the expression of BTLA in the macrophages.

For more intuitive and accurate comparison, we converted the results of the 20 drugs that promoted and decreased BTLA expression most significantly in RAW264.7 cells into a heat map for presentation, the specific results are shown in fig. 1, which can be seen in fig. 1, where the top 10 drugs that promoted BTLA upregulation included: CP-673451 (platelet-derived growth factor receptor inhibitor, PDGFR), SGI-7079 (histone methyltransferase inhibitor), PTC-209HBr (B cell-specific Moroney mouse leukemia virus insertion site 1, BMI-1 inhibitor), PR-619 (dihydroquinamide acylase inhibitor, DUBs), oxcarbazepine (sodium channel inhibitor), GW2580 (colony stimulating factor-1 receptor inhibitor, CSF-1R), AZD3463 (anaplastic lymphoma kinase inhibitor, ALK), prasurey ester (dihydrofolate reductase inhibitor, DHFR), LDC000067 (cyclin-dependent kinase 9, inhibitor of CDK 9), DDR1-IN-1 (inhibitor of discoid domain receptor 1, DDR 1), the drug that upregulates the most expressed BTLA is the PDGFR inhibitor CP-673451, the PDGFR inhibitor CP-673451 was therefore selected for testing in the examples described below.

The sepsis animal model in this application was made and dosed as follows:

male C57BL/6 mice (8 weeks old, 22. + -.1 g) were surgically induced sepsis:

1) isoflurane at 3% and 2% concentrations was used to induce and maintain anesthesia, respectively;

2) the incision is made along the median ventral line of the mouse, and the size of the incision is about 1 cm. Clamping the external part of the cecum, and fastening the part 1cm away from the distal end of the cecum by using a 4.0# silk thread;

3) penetrating the cecum in the middle of the far end of the ligature cecum by using a 16-gauge syringe needle, storing the cecum back to the abdomen, and suturing layer by layer;

4) subcutaneously injecting 1ml of compound sodium chloride injection into a mouse, wherein the mouse does not take antibiotics after cecal ligation and puncture;

5) mice were randomized into Control (CLP) and treatment (CLP + CP-673451) groups: the mice of the CLP + CP-673451 group received the same amount of phosphate buffer (pH 7.2-7.4) as the experimental drug (20mg/kg once daily) administered orally 2, 24, 48 and 72 hours after the establishment of the CLP (cecal ligation and perforation) sepsis model mice, respectively.

Example 1: effect of CP-673451 on BTLA expression in sepsis model mice

In order to verify the influence of CP-673451 on the BTLA expression of a sepsis model mouse, male C57BL/6 mice (8 weeks old, 22 +/-1 g) are utilized to construct a mouse caecal ligation and perforation sepsis model (the caecal ligation and perforation sepsis model mouse is constructed according to the method for manufacturing the sepsis animal model), the spleen of the mouse on the 1 st day after CLP molding is taken for flow cytometry analysis, and the method has the advantages ofAs shown in FIGS. 2 and 3, it can be seen that BTLA expression level in spleen immune cells of mice after CP-673451 treatment is higher than that in the control group; and CP-673451 mainly up-regulates CD4 in mouse spleen ⁺ 、CD8 ⁺ Expression level of BTLA in T and B lymphocytes, at CD4 ⁺ The promotion effect of T lymphocyte (P0.02) and B lymphocyte (P0.001) is most obvious and has statistical significance.

In addition, BTLA-mRNA fluorescent quantitative PCR detection is carried out on the CLP group and the CLP + CP-673451 treatment group, as shown in FIG. 4, the expression of BTLA-mRNA is consistent with the expression trend of BTLA protein, CP-673451 also increases the increase of BTLA messenger ribonucleic acid (mRNA) expression level in spleen cells of mice 1 and 3 days after CLP modeling, and the difference has statistical significance.

Example 2: effect of CP-673451 on survival in sepsis model mice

To verify the effect of CP-673451 treatment on 7-day survival in sepsis model mice. After constructing a mouse Caecal Ligation and Perforation (CLP) model, observing and recording the survival rate of the mouse; the results are shown in fig. 5, and the treatment groups using CP-673451 (oral CP-673451 treatment was given 2, 24, 48, and 72 hours after CLP molding) increased the survival rate of mice compared to the control group, and the difference was statistically significant (n-25; P-0.015), indicating that CP-673451 effectively increased the survival rate of mice.

Example 3: effect of CP-673451 on important organs of mouse model for sepsis

In order to verify the influence of CP-673451 on important organs of sepsis mice, the application team respectively carries out CP-673451 oral treatment 2, 24, 48 and 72 hours after CLP modeling, wherein the lung tissues of the mice on the 1 st day after CLP modeling are taken to be stained with hematoxylin and eosin to observe the pathological changes of the lung tissues, and the result is shown in figure 6, and the lung tissues of the mice in a normal control group have complete alveoli, no inflammatory cell infiltration and no thickening of the alveoli intervals; the lung tissue of the sepsis group has a large amount of inflammatory cell infiltration, the pulmonary alveoli are seriously damaged, and the lung interval is obviously thickened; CP-673451 is used for treating lung tissue with local small amount of inflammatory infiltration, no obvious alveolar damage and local small amount of alveolar space thickening. Compared with the CLP group, the lung tissue treated by CP-673451 has complete alveolar structure, and the infiltration degree of inflammatory cells and alveolar septal thickening are reduced, which shows that CP-673451 can effectively treat lung injury caused by sepsis.

Example 4: effect of CP-673451 on cytokine levels in sepsis model mice

The production and release of cytokines is critical for the participation of lymphocytes in immune responses. It was found that lymphocytes during sepsis are important cells involved in the sepsis "cytokine storm". The severity of sepsis is associated with a "cytokine storm", so it is reasonable to control the "cytokine storm" to reduce the host's own damage caused by the anti-infective response; BTLA plays a role in regulating lymphocyte activity during sepsis. Therefore, to verify that BTLA modulates lymphocyte activity by modulating cytokine release. We first observed the effect of CP-673451 on the level of mouse cytokines in sepsis model by using mouse cytokine array kit, which can detect the expression of 111 mouse cytokines at the same time. Firstly, collecting plasma of a normal control group, a CLP group and a CLP + CP-673451 group, wherein samples 24 hours and 72 hours after molding are taken from the CLP group and the CLP + CP-673451 group; then carrying out experimental detection according to the operation steps of the mouse cytokine array kit; in addition, after the CLP group and the CLP + CP-673451 group are modeled by adopting the liquid-phase chip kit, the change of the cell factors in the blood plasma is detected more comprehensively within 24 hours, and experimental operation is carried out according to the operation steps of the liquid-phase chip kit; finally, CCL1, CCL2, CCL7, CCL17, CCL27, CXCL11, IL-1 beta, IL-2, IL-4, IL-6, IL-10, TNF-alpha and CXCL13 factors are screened out by combining with the detection experiment results of a cytokine array and a cytokine liquid chip, and the mRNA expression change of the factors is verified by adopting fluorescent quantitative PCR. Firstly, constructing an LPS (stimulated LPS) RAW264.7 cell inflammation model, stimulating RAW264.7 cells by adopting 200ng/ul LPS, collecting cell samples at 8, 16 and 24 hours of stimulation, extracting RNA (ribonucleic acid), and carrying out fluorescent quantitative PCR (polymerase chain reaction) detection on the factors; then, a CLP mouse model is constructed, materials are taken 24 hours after model building, liver tissues and spleen are dissociated, RNA is extracted, and the fluorescent quantitative PCR detection of the factors is carried out.

The qualitative detection result of the cytokine array kit shows that the release of CXCL13, Ang-2, CCL11, CXCL1, IL-6 and IL-1ra is increased after sepsis, but is obviously reduced by about 28.5 +/-3.59 percent after CP-673451 treatment; in contrast, release of IGFBP-5, CXCL5 and IGFBP-2 was reduced by about 32.4% + -2.69% following treatment with CP-673451, with CXCL13 factor changing most significantly following mouse sepsis and treatment with CP-673451 (FIG. 7); the results of liquid chip cytokine detection experiments show that CCL1, CCL2, CCL7, CCL17, CCL27, CXCL11, IL-1 beta, IL-2, IL-4, IL-6, IL-10 and TNF-alpha factors in the CLP group are remarkably increased at 24 hours of sepsis, and CCL1, CCL2, CCL7, CCL17, CCL27, CXCL11, IL-1 beta, IL-2, IL-4, IL-6, IL-10 and TNF-alpha factors in the CP-673451 treatment group are remarkably reduced at 24 hours of administration treatment (figure 8); verification of cytokine m-RNA expression after fluorescent quantitative PCR with dosing therapy (fig. 9A, B) shows: CXCL13, CCL1, CCL2, CCL7, IL-1. beta. and IL-10 are statistically different. Through a cytokine array, a cytokine liquid phase chip and fluorescent quantitative PCR, the application of CP-673451 after sepsis is proved by protein expression and mRNA expression, the treatment is realized by up-regulating BTLA, inhibiting cytokines CXCL13, CCL1, CCL2, CCL7, IL-1 beta and IL-10 to regulate lymphocyte activity and inflammatory reaction, and the purposes of inhibiting the storm of an organism inflammatory factor and reducing organ injury are achieved.

In conclusion, the invention discovers and verifies a sepsis therapeutic drug CP-673451, and the drug reduces chemotaxis of B and T lymphocytes in circulating peripheral blood and important organs by inhibiting release of cytokines IL-10 and IL-1 beta and chemokines CXCL13, CCL1, CCL2 and CCL7 from circulating immunocytes, protects functions of important organs by inhibiting excessive inflammatory reaction of sepsis, and reduces death rate of sepsis. Therefore, CP-673451 is an effective sepsis therapeutic drug, and has important clinical application prospect and value in the treatment of sepsis patients.

The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; such modifications and substitutions do not depart from the spirit and scope of the present invention, and they should be construed as being included in the following claims and description.

Claims (6)

1. An inhibitor for the treatment of sepsis, wherein the inhibitor is capable of effecting treatment of sepsis by reducing cytokine release from circulating immune cells.

2. The inhibitor for the treatment of sepsis according to claim 1, wherein the inhibitor is CP-673451.

3. The inhibitor for use in the treatment of sepsis according to claim 2, wherein the sepsis comprises early sepsis, severe sepsis and septic shock.

Application of CP-673451 in preparing medicine for treating sepsis.

5. The use of claim 4, wherein the medicament is in a dosage form for oral administration.

6. The use of claim 4, wherein the effective amount of CP-673451 is 20 mg/kg.

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