阅读说明：本技术 化合物epz5676及其相关抑制剂在制备抗脑缺血疾病药物中的用途 (Application of compound EPZ5676 and related inhibitor thereof in preparation of anti-cerebral ischemic disease drugs ) 是由 朱依谆 刘新华 王静欢 于 2019-10-09 设计创作，主要内容包括：本发明属制药领域,涉及化合物EPZ5676在制药中的新用途,具体涉及化合物(C-(30)H-(42)N-8O-3)及其相关抑制剂在制备预防或治疗脑缺血疾病药物中的用途,本发明通过建立的小鼠脑缺血模型和OGD/R诱导的原代神经元细胞体外模型实验,结果表明EPZ5676通过改善脑缺血小鼠行为学症状,减少脑梗死体积,抑制炎症蛋白与凋亡蛋白表达,促进链接蛋白表达,对脑缺血疾病具有保护作用。所述的化合物EPZ5676可用于制备抗脑缺血药物,同时也为脑缺血疾病的研究提供了新的方向。(The invention belongs to the field of pharmacy, relates to a new application of a compound EPZ5676 in pharmacy, and particularly relates to a compound (C) 30 H 42 N 8 O 3 ) The invention relates to the use of a related inhibitor in the preparation of a medicament for preventing or treating cerebral ischemia diseases, and the results of the mouse cerebral ischemia model and the OGD/R-induced primary neuron cell in vitro model experiments show that EPZ5676 has the effects of reducing cerebral infarction volume, inhibiting the expression of inflammatory protein and apoptosis protein, promoting the expression of link protein and protecting cerebral ischemia diseases by improving the behavioral symptoms of the cerebral ischemia mouse. The compound EPZ5676 can be used for preparing anti-cerebral ischemia medicaments, and provides a new direction for the research of cerebral ischemia diseases.)

1. The compound EPZ5676 and the related inhibitor thereof can be used for preparing the anti-cerebral ischemia drugs; the molecular formula of EPZ5676 is as follows: c₃₀H₄₂N₈O₃。

2. Use according to claim 1, wherein EPZ5676 exerts a protective effect against cerebral ischemic diseases by inhibiting the expression of apoptosis-related proteins.

3. Use according to claim 1, wherein EPZ5676 exerts an anti-inflammatory effect in cerebral ischemic diseases by inhibiting the expression of inflammation-associated proteins.

4. Use according to claim 1, wherein EPZ5676 exerts a protective effect in cerebral ischemic diseases by promoting the expression of a linkage-related protein.

Technical Field

The invention belongs to the field of pharmacy, relates to a new application of a compound EPZ5676 in pharmacy, and particularly relates to a compound (C)₃₀H₄₂N₈O₃) And the application of the related inhibitor in the preparation of the drugs for preventing or treating cerebral ischemia diseases.

Background

The prior art discloses that cerebral ischemia (also known as cerebral apoplexy) is one of three diseases threatening human health, and has the characteristics of high morbidity, high recurrence rate, high disability rate and high death rate. Investigation shows that ischemic stroke accounts for 60-80% of cerebral stroke, however, no drug for effectively treating ischemic stroke exists in clinical intervention so far; clinical practice shows that the ischemic area is edematous after cerebral ischemia, and a large amount of inflammatory factors are secreted; since neuronal cells are the most sensitive cells, neuronal cells in the center of infarction and the surrounding areas thereof die in a large amount, how to protect neurons and reduce neuronal death becomes a major key point for the research of neural repair after cerebral ischemia in the industry.

The research discloses that the compound EPZ5676 is a potent inhibitor of DOT1L histone methyltransferase DOT1L, has a remarkable effect of resisting pulmonary fibrosis, and can be used for treating leukemia. However, no report on the role of EPZ5676 in cerebral ischemia has been found so far.

Based on the current situation of the prior art, the inventor of the application intends to provide a novel medicament for effectively preventing and treating cerebral ischemic diseases, in particular to a novel application of a compound EPZ5676 in pharmacy, and especially relates to a compound (C)₃₀H₄₂N₈O₃) And the application of the related inhibitor in the preparation of the drugs for preventing or treating cerebral ischemia diseases.

Disclosure of Invention

The invention aims to provide a novel medicament for effectively preventing and treating cerebral ischemia diseases based on the current situation of the prior art, in particular to a novel application of a compound EPZ5676 in pharmacy, and particularly relates to a compound (C)₃₀H₄₂N₈O₃) And the application of the related inhibitor in the preparation of the drugs for preventing or treating cerebral ischemia diseases.

The invention provides a new pharmacological action and application of a compound EPZ5676 in preventing and treating cerebral ischemia diseases, and through an established mouse cerebral ischemia model and an OGD/R induced primary neuron cell in vitro model experiment, the result shows that in vivo EPZ5676 can improve the behavioral symptoms of a cerebral ischemia mouse, reduce the cerebral infarction volume, inhibit the expression of inflammatory proteins and apoptosis proteins and promote the expression of interlinkage proteins; in vitro, EPZ5676 can reduce the expression of inflammatory protein and apoptosis protein, and promote the expression of interlinkage protein. Therefore, the compound EPZ5676 can be used for preparing anti-cerebral ischemia medicaments.

The molecular formula of EPZ5676 is as follows: c₃₀H₄₂N₈O₃(ii) a It is an efficient DOT1L histone methyltransferase DOT1L of an inhibitor.

According to the invention, a cerebral ischemia reperfusion injury model is prepared by adopting a C57 mouse wire-embolism method for experiments, and the behavioral injury of the mouse after cerebral ischemia is detected by Bederson score, and the result shows that EPZ5676 can improve the behavioral symptoms of the cerebral ischemia mouse; the TTC method detects the cerebral infarction volume after the cerebral ischemia of the mouse, and the result shows that EPZ5676 can reduce the cerebral infarction volume; the immunofluorescence method detects changes of an inflammatory index, an apoptosis index and a link index of a mouse after cerebral ischemia, and the result shows that EPZ5676 can reduce the expression of the inflammatory index and the apoptosis index and increase the expression of the link index; the expression of inflammatory protein, apoptosis protein and interlinkage protein after cerebral ischemia of mice is detected by a Western blot method, and the result shows that EPZ5676 can reduce the expression of the inflammatory protein and the apoptosis protein and increase the expression of the interlinkage protein.

In the invention, a cerebral ischemia model is established by inducing primary cortical neuron cells of a rat through OGD/R in vitro, an action test of EPZ5676 on neuron cells after cerebral ischemia is carried out, and an immunofluorescence result shows that EPZ5676 can obviously reduce the expression of inflammation indexes and apoptosis indexes; meanwhile, Western blot results show that EPZ5676 can obviously reduce the expression of inflammatory proteins and apoptosis proteins and increase the expression of interlinkage proteins; the experimental result proves that the compound EPZ5676 has protective effect on cerebral ischemia diseases.

The present invention provides the compound EPZ5676 (C)₃₀H₄₂N₈O₃) The invention, through the established mouse cerebral ischemia model and the experiment of the primary neuron cell in vitro model induced by OGD/R, shows that EPZ5676 has the effects of improving the behavioral symptoms of the cerebral ischemia mouse, reducing the cerebral infarction volume, inhibiting the expression of inflammatory proteins and apoptosis proteins, promoting the expression of link proteins and protecting the cerebral ischemia disease; the compound EPZ5676 can be used for preparing anti-cerebral ischemia medicaments.

Drawings

Fig. 1 is a model of induced cerebral ischemia in mice, showing that EPZ5676 significantly reduced the volume of cerebral infarction.

FIG. 2 is a mouse cerebral ischemia induction model, which shows that EPZ5676 obviously reduces the location of apoptosis index and the expression of apoptosis-related protein.

FIG. 3 is a model of induced cerebral ischemia in mice, showing that EPZ5676 significantly reduces the localization of inflammatory markers, reducing the expression of inflammation-associated proteins.

FIG. 4 is a model of induced mouse cerebral ischemia, showing that EPZ5676 significantly promotes the localization of the linkage index, promoting the expression of linkage-related proteins.

FIG. 5 is a Western blot method for detecting the apoptosis-related protein expression level of primary neuron cells damaged by OGD/R.

FIG. 6 is a Westernblot method for detecting the inflammation-associated protein expression level of OGD/R damaged primary neuronal cells.

FIG. 7 is a Westernblot method for detecting the expression level of linkage-associated proteins in primary neuronal cells injured by OGD/R.

FIG. 8 is the Immunofluorescence method for detecting the expression of apoptosis-related markers of OGD/R-injured primary neuronal cells.

FIG. 9 is an Immunofluorescence method for detecting the expression of inflammation-related markers of primary neuronal cells injured by OGD/R.

Detailed Description

Example 1EPZ5676 test for protective Effect on primary neuronal cells injured by OGD/R in vitro

Grouping: primary cultured cortical neuronal cells were randomly divided into 5 groups, i.e.: 1) a Sham group; 2) group I/R; 3) inhibitor 10 μ M group; 4) inhibitor 20 μ M group; 5) inhibitor 40. mu.M group. The inhibitor is administered for 4h in advance, then hypoxia and glucose deprivation are carried out for 2h, and in-vitro cerebral ischemia model is prepared after reoxygenation is carried out for 4 h. Collecting protein, and detecting the expression of p53, PARP1, caspase3, caspase9, Bax, Bcl-2, iNOS, COX-2, VCAM-1, ZO-1 and Claudin-1 proteins by a Westernblot method; slides were stained with immunofluorescence for tunnel, caspase3, iNOS, VCAM-1 markers. The result shows that EPZ5676 can obviously reduce the expression of apoptosis indexes and inflammation indexes; meanwhile, the expression of inflammatory protein and apoptosis protein is reduced, and the expression of link protein is increased.

The experimental result proves that EPZ5676 has obvious protective effect on an OGD/R induced in-vitro cerebral ischemia model, and can be used for preparing the medicine for resisting cerebral ischemia related diseases.

Table 1 shows that EPZ5676 obviously improves the behavioral result of a mouse cerebral ischemia reperfusion induction model,

wherein Sham is a Sham group; I/R is cerebral ischemia reperfusion group prepared by the thread suppository; inhibitor:20mg/kg/day EPZ5676, I/R and inhibitor group compared to Sham group, p<0.05；^#Inhibition group compared with I/R group, p<0.05。

Table 1, Bederson score.

Example 2 therapeutic Effect of EPZ5676 on in vivo mouse cerebral ischemia model

Grouping: male C57 mice weighing 21-24g were anesthetized by intraperitoneal injection with 1% sodium pentobarbital, fixed in supine position, and had an incision of about 2cm in the median cervical region. The subcutaneous tissue is separated in a blunt manner, the left Common Carotid Artery (CCA) is exposed, the External Carotid Artery (ECA) and the Internal Carotid Artery (ICA) are sequentially and upwards separated and exposed, the common carotid artery and the external carotid artery are respectively hung at the distal end of the common carotid artery and the external carotid artery, the common carotid artery is tied by a slipknot, the external carotid artery is tied by a deadknot, and then the common carotid artery and the external carotid artery are hung at the distal end of the internal carotid artery and are pulled by a draw hook. Then, the thread is hung at the proximal end of the external carotid artery, and a slipknot is tied. Then, a small opening is cut at the position of the Y-shaped bifurcation of the external carotid artery and the internal carotid artery by microshearing, the thread plug is guided into the external carotid artery from the incision, the external carotid artery is inserted into the common carotid artery in a proper position, then the ligation opening is tightened, the external carotid artery at one side is cut off, the stump of the external carotid artery is slightly pulled, so that the external carotid artery and the internal carotid artery are on the same straight line, the angle of the thread plug is adjusted, and the angle of the thread plug and the center of the front of the neck forms an outward included angle of 45 degrees horizontally. When the thread plug is inserted into the internal carotid artery, the thread hanging of the internal carotid artery is removed, the thread plug is guided into the cranium gently and stops when meeting the resistance, and then the thread plug is fixedly ligated. And cutting off redundant wire bolts, and only leaving a residual end with the length of about 1 mm. Removing neck assemblyAfter the artery was sutured and the bleeding was observed, the incision was closed. After 1.5h of infarction, the incision is opened, and the wire plug is pulled out to the branch of the external carotid artery and the common carotid artery. The skin is sutured, the iodophor is placed in a cage after being sterilized, the whole process is kept warm, and sufficient water and food are provided after the operation. The sham operation group was the same as the model group except that no plug was inserted. EPZ5676 in 2% DMSO + 30% PEG300+ 5% Tween 80+ ddH₂O was dissolved and mice were randomly assigned to Sham (Sham, ip.n.: 10), model (I/R, solvent control, ip.n.: 10), model + EPZ5676 (inhibitor,20mg/kg/day, n.: 10). 7 days after predose, the model was made. After 24 hours of molding, performing behavioral evaluation; TTC measures cerebral infarct volume; brain tissue was harvested, fixed in 4% paraformaldehyde, embedded as paraffin sections, and fluorescently stained for tunnel, caspase3, iNOS, VCAM-1, and ZO-1 indices. Brain tissue was collected, proteins were lysed, and expression of p53, PARP1, caspase3, caspase9, Bax, Bcl-2, iNOS, COX-2, VCAM-1, ZO-1, Claudin-1 proteins was detected by Western blot. The result shows that EPZ5676 can improve the behavioral symptoms of cerebral ischemia mice, reduce the volume of cerebral infarction, reduce the expression of inflammatory index and apoptosis index, and promote the expression of linkage index; meanwhile, the expression of inflammatory protein and apoptosis protein is inhibited, and the expression of link protein is promoted. The experimental result proves that the EPZ5676 has obvious protective effect on an in-vivo cerebral ischemia model prepared by a thrombosis method.