WO2022027237A1

WO2022027237A1 - Application of quizartinib as necroptosis inhibitor

Info

Publication number: WO2022027237A1
Application number: PCT/CN2020/106841
Authority: WO
Inventors: 查运红; 李敏; 何小燕; 魏钧; 王君; 余雅婕; 胡昕倩; 牟彦; 祝国锋; 赵海龙; 黄明; 张宏冰
Original assignee: 宜昌市第一人民医院（三峡大学人民医院）
Priority date: 2020-08-04
Filing date: 2020-08-04
Publication date: 2022-02-10
Also published as: CN112076198B; CN112076198A

Abstract

An application of quizartinib as a necroptosis inhibitor. An application of quizartinib as an RIPK1-dependent cell necroptosis inhibitor. An application of quizartinib as a cell necroptosis initiation marker phosphorylation inhibitor, the cell necroptosis initiation marker comprising an S166 site of RIPK1 and an S345 site of MLKL. An application of quizartinib as a necroptosis inhibitor in diseases characterized by abnormal activation of necroptosis, the diseases characterized by abnormal activation of necroptosis comprising COVID-19, pneumonia, amyotrophic lateral sclerosis, multiple sclerosis, inflammatory bowel disease, Crohn'S disease, ischemia-reperfusion injury, systemic inflammatory response syndrome, sepsis, cerebral arterial thrombosis, hemorrhagic stroke, psoriasis, rheumatoid arthritis and the like. A new method for treating related diseases characterized by abnormal activation of necroptosis is provided for existing treatment bottlenecks.

Description

Use of quizatinib as a programmed necrosis inhibitor

technical field

The invention relates to the application of quizatinib as a programmed necrosis inhibitor, and belongs to the technical field of medicine.

Background technique

[Corrected 12.04.2021 according to Rule 26]
At present, the diagnosis and treatment of new coronavirus pneumonia are carried out in accordance with the diagnosis and treatment plan of the "New Coronavirus Pneumonia Diagnosis and Treatment Plan" (trial version 7).

The incidence of new coronavirus pneumonia patients turning into severe patients is 30%, and the death rate is 15%. Severe cases mostly develop dyspnea after 1 week, and severe cases rapidly progress to acute respiratory distress syndrome (SARS) and sepsis shock. Prevention and treatment of sepsis is a difficult point in the treatment of new coronavirus pneumonia, and it is also a key link in determining the survival and prognosis of patients. Studies have shown that the levels of pro-inflammatory cytokines in patients with severe new coronary pneumonia have significantly increased, opening the "inflammatory storm" mode, which is considered to be an important factor in the development of severe and critically ill patients with new coronary pneumonia. Cytokines were significantly elevated in both ICU and non-ICU patients, and some of these cytokines were more concentrated in critically ill ICU patients. Compared with ordinary patients with novel coronavirus pneumonia, the plasma levels of inflammatory cytokines IL-2, IL-7, IL-10, GSCF, IP10, MCP1, MIP1A and TNFα were higher in severe patients. Prevention and treatment of sepsis in patients with novel coronavirus pneumonia and inhibition of inflammatory cytokine storm are expected to improve the survival prognosis of patients with novel coronavirus pneumonia.

Recent studies have found that receptor interaction protein kinase 1 (RIPK1)-dependent programmed cell necrosis (Necroptosis) is an important mechanism for the occurrence and development of sepsis and the imbalance of pro-inflammatory factors. Programmed cell necrosis can lead to cell membrane rupture, and due to the release of cell contents, programmed cell necrosis can cause a large number of inflammatory cell infiltration in the body and induce a severe inflammatory response. Studies have shown that programmed cell necrosis, as a new type of programmed cell death, plays an important role in the pathophysiological process of viral infections, immune diseases, sepsis and other diseases. Activation of RIPK1 promotes the production of cytokines IL-6, IFN-g, MCP-1, IL-10, GSCF, and TNFα, which is consistent with a key feature of elevated proinflammatory cytokines in severe COVID-19 patients.

"Inflammatory storm" is an important mechanism for the transformation of patients with new coronary pneumonia from mild to severe and critical. The activation of RIPK1 is a key target to open the "inflammatory storm". By inhibiting programmed necrosis and inhibiting excessive systemic inflammatory response, RIPK1 inhibitors are expected to prevent and treat sepsis in patients with novel coronavirus pneumonia, inhibit inflammatory cytokine storm, and improve the survival prognosis of patients with novel coronavirus pneumonia.

Review published on July 15, 2020 (Paper title: Receptor-interacting protein kinase1 (RIPK1) as a therapeutic target; Journal title: Nature Reviews Drug Discovery (Nature Reviews: Drug Discovery); Authors: Lauren Mifflin, Dimitry Ofengeim and Junying Yuan) further summarizes human diseases related to abnormal activation of RIPK1 that promotes the occurrence and development of diseases, including inflammatory bowel disease (Crohn's disease, ulcerative colitis), psoriasis, inflammatory bowel disease (UCD), Rheumatoid arthritis, multiple sclerosis, and systemic lupus erythematosus. Research and clinical data from multiple different research groups have shown that amyotrophic lateral sclerosis, frontotemporal dementia, Alzheimer's disease, and Parkinson's disease are closely related to the activation and programmed necrosis of RIPK1. Our data suggest that activation of RIPK1 may play an important role in the pathogenesis of both Huntington's disease and Duchenne muscular dystrophy.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is to provide an application of quizatinib as a programmed necrosis inhibitor.

In order to solve the above-mentioned technical problems, the technical scheme adopted in the present invention is:

Use of quizatinib as a programmed necrosis inhibitor.

Use of quizatinib as an inhibitor of RIPK1-dependent programmed necrosis.

Application of quizatinib as an inhibitor of phosphorylation of a marker for the initiation of programmed necrosis.

The necroptosis initiation markers include the S166 site of RIPK1 and the S345 site of MLKL.

Use of quizatinib as a programmed necrosis inhibitor in diseases characterized by abnormal activation of programmed necrosis.

Diseases characterized by abnormal activation of programmed necrosis include COVID-19, pneumonia, amyotrophic lateral sclerosis, multiple sclerosis, inflammatory bowel disease, Crohn's disease, ischemia-reperfusion injury, systemic inflammatory response syndrome, pus Toxicosis, ischemic stroke, hemorrhagic stroke, psoriasis, rheumatoid arthritis, systemic lupus erythematosus, Alzheimer's disease, Parkinson's disease, frontotemporal dementia, Huntington's disease, and Duchenne muscle Malnutrition.

Use of quizatinib as a hypothermic regulator in patients with systemic inflammatory response syndrome.

Use of quizatinib as a hypothermic regulator in septic patients.

Use of quizatinib as a pneumonia inhibitor.

Use of quizatinib as a novel coronavirus pneumonia inhibitor.

Necroptosis is a receptor-mediated, caspase-independent, highly regulated cell death process with typical necrotic morphological characteristics. After cells are stimulated with TNF-α (tumor necrosis factorα), RIPK1 (receptor-interacting Protein kinase 1) interacts with RIPK3 (receptor-interacting protein kinase 3) to phosphorylate downstream MLKL (mixed-lineage kinase domain-like pseudokinase) to form "Necrosome", which induces programmed necrosis. This process was rescued by the RIPK1 kinase-specific inhibitor Necrostatin-1 (Nec-1). RIPK1 plays an important regulatory role in this pathway. The drug TNFα+SM-164+zVAD (TSZ for short) can effectively induce the occurrence of programmed necrosis. Studies have shown that the antitumor drug Quizartinib (AC220) can inhibit TSZ-induced programmed necrosis, and further verified at the molecular level that quizartinib rescues programmed necrosis by inhibiting RIPK1 activity.

"Inflammatory storm" occurs in chemotherapy, H1N1 infection, and new coronavirus pneumonia. At present, there is no effective treatment plan for similar diseases in clinical practice. Generally, hormone therapy, antibiotic or antiviral therapy, and symptomatic and supportive therapy are used. It is now clear that quizatinib (AC220) can significantly improve the survival rate of TNFα-induced SIRS mice, improve their hypothermic state, and significantly reduce lung inflammation in mice.

The present invention has the following beneficial effects:

(1) The present invention proposes a new method for treating related diseases characterized by abnormal activation of programmed necrosis, aiming at the existing treatment bottleneck.

(2) The present invention administers a sufficient amount of quizatinib (AC220) orally, thereby inhibiting the abnormal activation of programmed necrosis, relieving the clinical symptoms of such diseases, improving the prognosis of patients, and improving the quality of life of patients.

(3) In the present invention, quizatinib (AC220) can significantly inhibit RIPK1-dependent apoptosis and programmed necrosis. In the mouse model of TNFα-induced systemic inflammatory response syndrome, quizatinib (AC220) can significantly reduce the mortality of TNFα-induced systemic inflammatory response syndrome mice, and can alleviate the hypothermic state of mice, significantly Attenuates inflammatory cell infiltration in the lungs of mice, thereby exerting a therapeutic effect on systemic inflammatory response syndrome. Thus revealing the new application of quizatinib in the treatment of new coronary pneumonia.

Description of drawings

Fig. 1 is the accompanying drawing that quizatinib inhibits the programmed cell necrosis induced by T/S/Z in the present invention;

Fig. 2 is the accompanying drawing of the safety and effectiveness of quizatinib in different cell lines in the present invention;

Fig. 3 is the accompanying drawing that quizatinib can obviously inhibit the activation of programmed necrosis in the present invention;

Fig. 4 is the accompanying drawing that quizatinib significantly improves the survival rate and body temperature of SIRS mice in the present invention;

Fig. 5 is a drawing showing that quizatinib in the present invention significantly reduces lung inflammation in SIRS mice.

detailed description

The present invention will be further described below in conjunction with the accompanying drawings.

This example discloses the application of quizatinib as a programmed necrosis inhibitor.

Use of quizatinib as an inhibitor of RIPK1-dependent programmed necrosis.

Use of quizatinib as a hypothermic regulator in septic patients.

Use of quizatinib as a pneumonia inhibitor.

Use of quizatinib as a novel coronavirus pneumonia inhibitor.

In this example, in mouse hippocampal neuron cells (HT22) and human colon cancer cells (HT-29), DMSO, 200nM SM-164+20μM zVAD(S/Z), 200nM SM-164+20μM zVAD+10μM Nec -1(S/Z/N), 200nM SM-164+20μM zVAD+500nM quizatinib (AC220)(S/Z/Q) pretreated cells for 30min, HT22 added 10ng/ml mTNF-α, HT- 29 The programmed necrosis of cells was induced by adding 10ng/ml hTNF-α, and the number of surviving cells was detected by ATP assay.

As shown in Figure 1, the results showed that quizatinib (AC220) could inhibit the occurrence of programmed cell necrosis induced by T/S/Z (TNF-α/SM-164/zVAD). The specific results are shown in Table 1.

Table 1 Results of rescue of programmed necrosis with quizatinib in different cell lines

Note: T: TNFα; S: SM164; Z: zVAD; N: Nec-1; Q: Quizartinib. In human and murine cell lines, the quizatinib group was compared with the model group (****P<0.0001).

As shown in Figure 2, the safety and efficacy of quizatinib (AC220) in different cell lines. In Figure 2, A. HT22 cells were treated with different concentrations of quizatinib (AC220), and the number of viable cells was detected by ATP assay. B. HT-29 cells were treated with different concentrations of quizatinib (AC220), and the number of viable cells was detected by ATP assay. C. In HT22 cells, pre-treated cells with different concentrations of quizatinib (AC220) + 200nM SM-164 + 20μM zVAD for 30min, then add 10ng/ml mTNF-α to induce programmed necrosis, and use ATP assay to detect the number of viable cells. D. In HT-29 cells, pre-treated cells with different concentrations of quizatinib (AC220) + 200nM SM-164 + 20μM zVAD for 30min, then add 10ng/ml hTNF-α to induce programmed necrosis, and use ATP assay to detect viable cells number.

Results: The EC ₅₀ of quizatinib (AC220) in HT22 and HT-29 were 155.5nM and <31.25nM, respectively, and the toxicity was low ( _HT22IC50 : 62.796μM, HT- _29IC50 : 212.738μM). The specific results are shown in the table. 2. Table 3.

Table 2 Comparison of cell viability after treatment with different concentrations of quizatinib

Table 3 Comparison of cell viability of different concentrations of quizatinib in inhibiting programmed necrosis

Note: All cells were treated with TNFα/SM164/zVAD to induce programmed necrosis.

As shown in Figure 3, in this example, Western blotting experiments were used to confirm that quizatinib can significantly inhibit RIPK1 (S166) in mouse fibroblasts (L929), mouse retinal ganglion cells (RGC5), and HT22 cells. The phosphorylation of the site and the phosphorylation of the MLKL (S345) site, the phosphorylation of these two sites is the sign of the initiation of programmed cell necrosis.

Figure 3 shows that quizatinib (AC220) can significantly inhibit the activation of programmed necrosis. Three cell lines, L929, HT22, and RGC5 were treated with T/S/Z to induce programmed necrosis of cells. It could be clearly observed that the addition of quizatinib could significantly rescue the programmed cell necrosis induced by T/S/Z at 4 hours. . Cell samples were collected at 0, 2, and 4 hours, and after protein extraction, Western blotting was performed. The markers of programmed necrosis activation, p-RIPK1 (S166) and p-MLKL (S345), could be significantly inhibited, suggesting that quizatinib Programmed necrosis can be inhibited at the cellular level and cell death can be rescued.

As shown in Figure 4, the RIPK1-dependent cell death pathway is involved in TNF-α-induced systemic inflammatory response syndrome (SIRS). The SIRS mouse model was successfully established by injecting 5 μg of TNFα into the tail vein of 8-week-old C57BL/6 mice. Quizatinib (AC220) can significantly improve the hypothermic state of mice (****P<0.0001) and improve the survival rate of mice (***P<0.001). The specific results are shown in Table 4 and Table 5.

Figure 4 shows that quizatinib (AC220) can significantly improve the survival rate and body temperature of SIRS mice. In a mouse model of systemic inflammatory response syndrome characterized by abnormal activation of programmed necrosis, quizatinib can significantly improve the hypothermic state of mice (****P<0.0001) and improve the survival rate of mice (*** P<0.001).

Table 4 Comparison of the number of surviving mice with different treatments in the mTNF-α-induced SIRS mouse model

Table 5 Comparison of body temperature of mice with different treatments in mTNF-α-induced SIRS mouse model (Mean±SD)

As shown in Figure 5, the lung inflammation in mice was significantly reduced after administration. Provided for the treatment of related diseases characterized by abnormal activation of programmed necrosis (such as pneumonia, new coronary pneumonia, amyotrophic lateral sclerosis, multiple sclerosis, inflammatory bowel disease, ischemia-reperfusion injury, systemic inflammatory response syndrome, etc.) New Direction.

Figure 5 shows that quizatinib (AC220) can significantly reduce lung inflammation in SIRS mice. In a mouse model of systemic inflammatory response syndrome characterized by abnormal activation of programmed necrosis, quizatinib was applied. Compared with mice in the quizatinib-treated group (using T/S/Z model), the Quizatinib can significantly reduce lung inflammation in mice and reduce inflammatory cell infiltration in the lungs of mice, suggesting that quizatinib can treat new coronary pneumonia.

Currently in clinical diagnosis and treatment, the present invention can be used as a new treatment method for related diseases characterized by abnormal activation of programmed necrosis. Related diseases characterized by abnormal activation of programmed necrosis (such as amyotrophic lateral sclerosis, multiple sclerosis, inflammatory bowel disease, ischemia-reperfusion injury, systemic inflammatory response syndrome, etc.), clinical treatment still has not achieved good results , The purpose of the present invention is to propose a new method for treating related diseases characterized by abnormal activation of programmed necrosis, aiming at the existing treatment bottleneck.

In order to achieve the above objects, the present invention is useful in related diseases (such as amyotrophic lateral sclerosis, multiple sclerosis, inflammatory bowel disease, ischemia-reperfusion injury, systemic inflammatory response syndrome, etc.) characterized by abnormal activation of programmed necrosis. In the clinical treatment of patients with patients, a sufficient amount of quizatinib (AC220) is administered orally to inhibit the abnormal activation of programmed necrosis, relieve the clinical symptoms of these diseases, and improve the prognosis and quality of life of patients.

The above is only the preferred embodiment of the present invention, it should be pointed out: for those skilled in the art, under the premise of not departing from the principle of the present invention, several improvements and modifications can also be made, and these improvements and modifications are also It should be regarded as the protection scope of the present invention.

Claims

Use of quizatinib as a programmed necrosis inhibitor.
The use according to claim 1, characterized in that, the use of quizatinib as an inhibitor of RIPK1-dependent programmed cell necrosis.
The application according to claim 1, characterized in that, the application of quizatinib as a phosphorylation inhibitor of a programmed cell necrosis initiation marker.
The use according to claim 3, characterized in that the necroptosis initiation marker comprises the S166 site of RIPK1 and the S345 site of MLKL.
The use according to claim 1, characterized in that the use of quizatinib as a programmed necrosis inhibitor in a disease characterized by abnormal activation of programmed necrosis.
The application according to claim 5, wherein the diseases characterized by abnormal activation of programmed necrosis include new coronary pneumonia, pneumonia, amyotrophic lateral sclerosis, multiple sclerosis, inflammatory bowel disease, Crohn's disease, Systemic inflammatory response syndrome, sepsis, ischemic stroke, hemorrhagic stroke, psoriasis, rheumatoid arthritis, systemic lupus erythematosus, Alzheimer's disease, Parkinson's disease, frontotemporal dementia , Huntington's disease, and Duchenne muscular dystrophy.
The application according to claim 6, wherein the application of quizatinib as a hypothermic regulator for patients with systemic inflammatory response syndrome.
The application according to claim 6, characterized in that, the application of quizatinib as a hypothermic regulator in sepsis patients.
The application according to claim 6, wherein the application of quizatinib as a pneumonia inhibitor.
The application according to claim 6, wherein the application of quizatinib as a novel coronavirus pneumonia inhibitor.