阅读说明：本技术 HIF-1α降解抑制剂在制备酮体水平升高的冠心病药物中的应用 (Application of HIF-1 alpha degradation inhibitor in preparing medicine for treating coronary heart disease with elevated ketone body level ) 是由 孙爱军 葛均波 马秀瑞 董震 于 2021-02-07 设计创作，主要内容包括：本发明请求保护了一种HIF-1α降解抑制剂罗沙司他在制备循环酮体水平升高的冠心病药物中的应用。以HIF-1α为靶点,临床上应用罗沙司他,抑制HIF-1α降解,升高HIF-1α水平,可改善因酮体水平升高介导的心肌缺血缺氧性损伤。临床中将罗沙司他用于缺血性心脏病的治疗,尤其用于血酮体水平升高的冠心病患者,以减少心肌细胞损伤。(The invention provides application of a HIF-1 alpha degradation inhibitor roxarstat in preparation of a coronary heart disease drug with high circulating ketone body level. By taking HIF-1 alpha as a target spot, the clinical application of the roxasistat can inhibit the degradation of the HIF-1 alpha, increase the HIF-1 alpha level and improve myocardial ischemia and anoxic injury mediated by the increase of the ketone body level. Rosemastat is used clinically in the treatment of ischemic heart disease, particularly in coronary heart disease patients with elevated levels of blood ketone bodies to reduce myocardial cell damage.)

1. An application of HIF-1 alpha degradation inhibitor in preparing medicine for treating coronary heart disease with high ketone body level is provided.

2. The use of claim 1, wherein said elevated ketone body levels are elevated circulating ketone body levels.

3. The use of claim 1 or 2, wherein the HIF-1 α degradation inhibitor is rosxastat.

4. Use according to claim 1 or 2, wherein the use comprises reducing the myocardial infarct size, improving the ejection fraction and/or increasing the cardiac output.

Technical Field

The invention relates to the technical field of medicines, in particular to application of an HIF-1 alpha degradation inhibitor in preparation of a coronary heart disease medicine with an elevated ketone body level.

Background

According to the Chinese cardiovascular disease report 2018, the death caused by cardiovascular diseases is the first of the death component ratio of Chinese residents. Myocardial ischemia (myocarpialischemia) is a pathological condition in which the heart is difficult to maintain normal work due to reduced blood perfusion of the heart, reduced oxygen supply to the heart, abnormal energy metabolism of the heart muscle, and various causes. The energy required by the heart activity is almost completely provided by aerobic metabolism, so that the blood oxygen intake rate of the cardiac muscle is high even in a resting state, and under a normal condition, the body can promote the blood supply and demand to be relatively constant through self regulation so as to ensure the normal work of the heart, but when the blood supply and demand of the cardiac muscle are unbalanced due to some reason, the myocardial ischemia is formed. Coronary heart disease is the most prominent and common cause of myocardial ischemia. Acute myocardial infarction is the most serious type of coronary heart disease, myocardial cell death is caused by ischemia and hypoxia, cardiac function is seriously damaged due to massive myocardial cell necrosis, and life-threatening events such as heart failure, malignant arrhythmia and the like occur. Therefore, active measures are taken to reduce cell damage during myocardial ischemia and hypoxia, and the center of gravity for saving endangered myocardium is the center of gravity of the current treatment.

Structural reconstruction, electrical reconstruction, metabolic reconstruction and the like occur in the myocardium in the absence of oxygen, wherein the metabolic reconstruction occurs earliest and runs through the whole process of the disease, so that intervention of the metabolic reconstruction is a potential target for prevention and treatment of the myocardial reconstruction. Under normal conditions, the myocardial energy metabolism has extremely high flexibility, and the metabolic spectrum can be adjusted in time according to the organism condition so as to deal with the change of energy requirements under various conditions; but the flexibility of substrate selection and metabolic regulation is significantly reduced in the absence of oxygen. However, the metabolic pathways of ischemic and hypoxic cardiac muscle are not clear, and few studies have been made on how to improve ischemic and hypoxic myocardial damage by intervening in metabolic regulation.

Ketone bodies are important metabolic fuel sources of almost all organisms, are mainly synthesized by the liver and are metabolized in organs such as heart, brain, kidney and the like; is a collective name of beta-hydroxybutyric acid (beta-OHB), acetoacetic acid and acetone, and the main component of the compound is beta-OHB, accounting for 78%. Under physiological conditions, the normal heart oxidizes ketone bodies in the proportion of their delivery, the heart being the organ that consumes the most ketone bodies per unit mass of myocardium; moreover, ketone bodies are more energy efficient and provide more energy than fatty acid oxidation. In 2018, JACC issued, the level of ketone bodies in blood of myocardial infarction patients is positively correlated with poor prognosis, and the fact that the metabolism of the ketone bodies has important significance under the condition of myocardial ischemia and hypoxia is suggested. The method has important significance for reducing myocardial cell injury by screening corresponding medicines by changing the metabolism of ketone bodies into targets during ischemia and hypoxia and treating coronary heart disease. Therefore, the key problem to be solved urgently is to develop safe and efficient targeted drugs by deeply researching relevant molecular action mechanisms, more effectively treat coronary heart disease and simultaneously reduce side effects.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention is realized by the following technical scheme:

use of HIF-1 alpha degradation inhibitor in preparation of medicine for treating coronary heart disease with elevated ketone body level is provided;

further, the elevated ketone body levels are elevated circulating ketone body levels;

further, the HIF-1 alpha degradation inhibitor is roxasistat;

further, the use comprises reducing myocardial infarct size, improving ejection fraction and/or increasing cardiac output.

Drawings

FIG. 1: beta-hydroxybutyrate exacerbates myocardial cell hypoxic injury by down-regulating HIF-1 alpha.

Among them, fig. 1A and 1B show: the primary myocardial cells of the mice are subjected to dose-dependent cell death after being administered with beta-hydroxybutyric acid under the anoxic condition; FIG. 1C shows that: western results show that the expression of HIF-1 alpha of hypoxic cardiac myocytes is down-regulated after the administration of beta-hydroxybutyric acid and is dose-dependent;

FIG. 2: by using HIF-1 alpha as intervention target, the damage of hypoxic myocardial cells caused by high beta-hydroxybutyric acid can be alleviated.

Wherein, FIGS. 2A and 2B show that, after administration of hypoxic stimuli and beta-hydroxybutyrate, use of rospastat inhibits HIF-1 α degradation, thereby partially reversing the inhibitory effect of beta-hydroxybutyrate on HIF-1 α; FIG. 2C: the immunofluorescence result statistical analysis chart can also reflect the experimental result;

FIG. 3: the increase of the level of the systemic ketone body aggravates the myocardial damage of the myocardial infarction mice, and the roxasistat partially reverses the effect.

Among them, fig. 3A shows that the blood ketone body levels of mice steadily increased after administration of the ketogenic diet; FIG. 3B shows that after ligation of anterior descending branch, the area of myocardial infarction was increased in the ketogenic diet compared to the normal diet, and decreased by the intervention of Rosxastat; FIG. 3C shows that after ligation of the anterior descending branch, ejection fraction was reduced in the ketogenic diet group and improved with Rosesastat intervention; FIG. 3D shows that after ligation of the anterior descending branch, cardiac output was reduced in the ketogenic diet and significantly increased after dry-treatment with Rosemastat.

Advantageous effects

The invention is proved by experiments that:

the research firstly confirms that the high-level beta-hydroxybutyric acid can aggravate the hypoxia injury of the myocardial cells by changing the metabolism of the myocardial cell ketone body into the entry point during ischemia and hypoxia, and the mechanism is mainly mediated by inhibiting HIF-1 alpha; therefore, by taking HIF-1 alpha as an intervention target, and applying the HIF-1 alpha degradation inhibitor rasagiltat to up-regulate the expression of HIF-1 alpha at high level of beta-hydroxybutyric acid, the hypoxia injury of cardiac muscle cells is reduced.

The research relates to the extraction of rat myocardial cells, and an animal model of myocardial ischemia has an early experimental foundation, the method is real and reliable, and preliminary research results prove that the use of the HIF-1 alpha degradation inhibitor roxasistat can obviously reduce myocardial damage caused by ischemia and hypoxia in the environment with the increased level of ketone bodies.

The invention fully proves that under the pathological conditions of myocardial ischemia and hypoxia, the application of the roxasistat can reverse myocardial damage caused by high beta hydroxybutyric acid mediated ketone body level increase, reduce myocardial cell death and improve cardiac function.

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.

Rosemastat, chemical name 2- (4-hydroxy-1-Methyl-7-phenyloxoquinoneine-3-carboxamid) acetic acid, CAS registry number: 808118-40-3, the molecular formula is: c₁₉H₁₆N₂O₅The structure is shown as formula I:

the compound is a novel Hypoxia Inducible Factor (HIF) -Prolyl Hydroxylase (PH) enzyme inhibitor. HIF is an important transcription factor in the body that adapts to oxygen changes, and HIF-PH enzymes promote HIF degradation under normoxic conditions; when the body is lack of oxygen, the activity of HIF-PH enzyme is inhibited, the accumulation of HIF is increased, thus inducing the corresponding gene expression and making the body adapt to the change of lack of oxygen. Under the condition that the organism is not lack of oxygen, the roxasistat inhibits the activity of HIF-PH enzyme, so that the accumulation amount of HIF is increased, and further, the corresponding physiological reaction is generated. By stabilizing HIF, inhibiting its degradation, activating the transcription of related genes, generating corresponding physiological response, increasing the concentration of erythropoietin moderately, improving the sensitivity of Erythropoietin (EPO) receptor, coordinating the generation of red blood cells, reducing the level of hepcidin, increasing the content and activity of transferrin receptor, promoting the absorption and utilization of iron, having good tolerance, being a novel medicine for treating renal anemia and having no application in cardiovascular diseases.

In the following examples, sodium beta-hydroxybutyrate was obtained from sigma; rosemastat (Roxadustat) was purchased from seleck; the rest of the drugs, reagents and instruments are commercially available.

The following examples are presented to enable one of ordinary skill in the art to more fully understand the present invention and are not intended to limit the invention in any way.

Adopting primary adult mouse myocardial cells, culturing by using a culture medium containing ketone bodies, then giving anoxic stimulation for 24 hours, collecting the cells, respectively carrying out Live/death, Western-Blot, RT-PCR, immunofluorescence and other experiments, and detecting the myocardial cell injury condition and HIF-1 alpha expression; under the condition, taking HIF-1 alpha as an intervention target, and administering an HIF-1 alpha degradation inhibitor, namely, roxasistat, to up-regulate HIF-1 alpha expression and reduce myocardial cell hypoxia injury; the experimental results are reflected in examples 1 to 3.

Example 1: increasing β -hydroxybutyrate levels results in increased hypoxic cardiomyocyte death.

As shown in fig. 1, it is clear that β -hydroxybutyrate aggravates myocardial cell hypoxic injury by inhibiting HIF-1 α, wherein fig. 1A and 1B show that: Live/Death knot shows that primary mouse myocardial cells are subjected to dosage-dependent cell Death after being given with beta-hydroxybutyric acid under the anoxic condition; FIG. 1C shows that: western results showed that HIF-1 α expression in hypoxic cardiomyocytes was down-regulated and dose-dependent following β -hydroxybutyrate administration.

Example 2: by using HIF-1 alpha as intervention target, the method can reduce the injury of hypoxic myocardial cells caused by high beta-hydroxybutyric acid.

As shown in figure 2, after the administration of hypoxia stimulation and beta-hydroxybutyrate by using primary adult mouse cardiomyocytes and human ventricular myocyte cell lines, the HIF-1 alpha degradation inhibitor roxasistat is applied to up-regulate HIF-1 alpha expression, so that the damage of the cardiomyocytes caused by beta-hydroxybutyrate under the hypoxia condition can be partially reversed; specifically, as shown in FIGS. 2A and 2B, Western Blot and immunofluorescence results show that, after hypoxia stimulation and beta-hydroxybutyrate are given, the use of rosixostat can inhibit HIF-1 alpha degradation, and further partially reverse the inhibition effect of beta-hydroxybutyrate on HIF-1 alpha; FIG. 2C is a chart of statistical analysis of immunofluorescence results, which also reflects the results of this experiment.

Example 3: the increase of the level of the systemic ketone body aggravates the myocardial damage of the myocardial infarction mice, and the roxasistat partially reverses the effect.

Animal level experiments: 60 male C57BL/6 mice with the age of 6-8 weeks are adopted, 30 male mice are given common diet, 30 male mice are given ketogenic diet to establish a hyperketonic mouse model, the mice are raised for 4 weeks and then subjected to coronary artery (anterior descending branch) ligation to establish a myocardial infarction animal model, after 1 week of myocardial infarction, cardiac ultrasonography is carried out, then cardiac samples are kept and subjected to TTC, HE and Masson staining, and the myocardial infarction condition is identified from pathology and morphology. As shown in FIG. 3, the expression of HIF-1. alpha. was detected by Western-Blot and RT-PCR experiments, and it was found that the myocardial infarction area of mice was increased and myocardial damage was aggravated, compared to the administration of normal diet mice, in which the "CD" group represents the normal diet group and the "KD" group represents the ketogenic diet group. Specifically, figure 3A shows that mice have steadily elevated levels of blood ketone bodies following administration of the ketogenic diet; FIG. 3B shows that B, after ligation of anterior descending branch, the myocardial infarction area of ketogenic diet group is increased compared with that of common diet group, and the myocardial infarction area can be reduced by adopting Rosxastat intervention; FIG. 3C shows that after ligation of the anterior descending branch, ejection fraction was reduced in the ketogenic diet group and improved with Rosesastat intervention; figure 3D shows that after ligation of the anterior descending branch, cardiac output was reduced in the ketogenic diet and increased after intervention with rosxastat.

The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.