Application of adenosine receptor stimulant in preparation of medicine for improving multiple organ injuries

Abstract

The invention discloses application of an adenosine receptor stimulant in preparation of a medicine for improving multiple organ injuries, and belongs to the technical field of medicines. In-vivo and in-vitro experiments prove that the adenosine receptor stimulant can improve multiple organ injuries, especially multiple organ injuries caused by sepsis and syndromes after cardiac arrest. Further, the adenosine receptor stimulant is proved to be capable of improving the organ function recovery level of a patient with sepsis multi-organ function impairment; the clinical prognosis of the patient with the post-cardiac arrest syndrome is improved, especially the brain and heart function recovery is improved, and the survival rate of the patient with the multi-organ function impairment is improved. Meanwhile, starting from the core ATP of human body energy metabolism, the purpose of improving multiple organ injuries is achieved by regulating adenosine and adenosine receptors, and the method has a protection effect on the heart, brain, lung and the like of the important organs of the human body, and is safe and free of toxic and side effects. Therefore, the application way of the invention can increase treatment means and strategies, and further promote continuous improvement of the treatment technology.

Description

[0001] The present invention claims priority to the prior application with the patent name: Application of Adenosine and Its Receptor in Cardiopulmonary Cerebral Resuscitation and Multiple Organ Injury, patent application number: 202510319533.4, filing date: March 18, 2025. TECHNICAL FIELD

[0002] The present invention relates to the technical field of medicine, and specifically relates to application of an adenosine receptor agonist in preparation of a medicine for improving multiple organ injury. BACKGROUND

[0003] Adenosine (ADO) is widely distributed in various tissues and organs of the human body, and regulates various important physiological and pathological processes of the human body by acting on adenosine receptors (ARs). The ARs family includes four subtypes, namely A1R, A2AR, A2BR and A3R, and adenosine participates in the regulation of inflammatory response, microcirculation and cell metabolism by binding to them.

[0004] Multiple organ dysfunction caused by sepsis and post-cardiac arrest syndrome are two major problems that seriously threaten human health. There are more than 18 million severe sepsis cases worldwide each year, and the survival rate of patients with cardiac arrest who have successfully undergone cardiopulmonary resuscitation is only about 10%, which is a clinical problem that has not been solved in the field of critical care today.

[0005] However, the current treatment still has problems such as limited methods and means, and poor efficacy, the main reason being the lack of specific treatment methods and drugs. For example, for patients who have undergone cardiopulmonary resuscitation after cardiac arrest, successful cardiopulmonary resuscitation is often only the first step in successful treatment, and the recovery of important organ functions such as heart, brain, lung and kidney is crucial to the recovery and prognosis of patients. However, the current treatment methods and drugs are extremely limited, so most patients who have successfully undergone resuscitation are in a deep coma, paralysis, lifelong hemodialysis, etc., which seriously affects the survival period and quality of life.

[0006] In view of this, the present invention is proposed. SUMMARY

[0007] The purpose of the present invention is to provide application of an adenosine receptor agonist in preparation of a medicine for improving multiple organ injury, which can improve multiple organ dysfunction caused by sepsis and post-cardiac arrest syndrome using an adenosine receptor agonist.

[0008] The present invention is implemented as follows: The present invention provides application of an adenosine receptor agonist in preparation of a medicine for improving multiple organ injury.

[0009] In some embodiments, the multiple organ injury is caused by post-cardiac arrest syndrome.

[0010] In some embodiments, the multiple organ injury comprises heart injury, kidney injury, and liver injury in post cardiac arrest syndrome.

[0011] In some embodiments, the adenosine receptor agonist is used to improve the success rate of cardiopulmonary resuscitation.

[0012] In some embodiments, the adenosine receptor agonist is used to reduce the frequency of ventricular fibrillation.

[0013] In some embodiments, the multiple organ injury is caused by sepsis.

[0014] In some embodiments, the multiple organ injury comprises liver injury, lung injury, kidney injury, heart injury, and brain injury caused by sepsis.

[0015] In some embodiments, the adenosine receptor comprises A1 receptor, 2A receptor, 2B receptor, and A3 receptor.

[0016] In some embodiments, the adenosine receptor agonist comprises CCPA, Capadenoson, Tecadenoson, Neladenoson, Binodenoson, Regadenoson, CGS 21680, Evodenoson, MRE 0094, LUF5834, BAY60-6583, MRS3558, A3AR agonist 4, Piclidenoson, 2-Chloro-3-deazaadenosine, and inosine.

[0017] In some embodiments, the drug further comprises a pharmaceutically acceptable excipient.

[0018] The present application has the following beneficial effects: The present application proves through in vivo and in vitro experiments that the adenosine receptor agonist can improve multiple organ injury, especially multiple organ injury caused by sepsis and post cardiac arrest syndrome. It is further proved that the adenosine receptor agonist can improve the recovery level of organ function of patients with sepsis multiple organ injury; improve the clinical prognosis of cardiopulmonary brain resuscitation patients, especially the recovery of brain and heart function, and increase the survival rate of multiple organ injury patients. At the same time, starting from the core ATP of human energy metabolism, the present application achieves the purpose of improving multiple organ injury by regulating adenosine and adenosine receptors. This method has protective effect on important organs of the human body such as heart, brain, and lung, and is safe and has no side effects. Therefore, the application of the present application can increase the treatment means and strategy, and further promote the continuous improvement of treatment technology.

[0019] The present application is supported by the National Natural Science Foundation Youth Fund, Fund No. 82202424, ADORA1 through GBP1 inhibits pyroptosis in the role and mechanism of sepsis-related acute kidney injury. Attached Figure Description

[0020] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 The results of RNA pathway enrichment in blood samples from sepsis patients (A) and changes in the expression of different subtypes of adenosine receptors (B). Figure 2 This document presents the technical route for the LPS-induced sepsis mouse model in Example 1 and a comparison of the body weight of mice in each group. In example a, the technical route uses an LPS dose of 20 mg / kg; in example b, the technical route uses an LPS dose of 15 mg / kg; and in example c, the body weight comparison results of mice in each group. Figure 3 This section compares the survival rate and adenosine concentration of mice in each group in Example 1; where a represents the experimental results of survival rate, and b represents the LC values ​​of kidney tissue from the control group and the model group mice. MS / MS chromatogram, c represents the adenosine concentration in the kidney tissue of mice in the control group and the model group; Figure 4 The results of serum creatinine and blood urea nitrogen detection for each group of mice in Example 1 are shown, where a is the serum creatinine detection result and b is the blood urea nitrogen detection result. Figure 5 HE staining results of kidney tissue from each group of mice in Example 1; Figure 6 The results of immunofluorescence staining of kidney tissue from each group of mice in Example 1; Figure 7 The results are the RNA sequencing results of mouse kidney tissues in each group of Example 1; Figure 8 The results of Western blotting of mouse kidney tissues in each group in Example 1 are shown. Figure 9 The results of multicolor immunofluorescence of mouse kidneys in each group in Example 1 are shown. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0023] Energy is required for all life activities, and the main source of human energy is food. In pathological conditions, especially for patients with severe sepsis or post-cardiac arrest syndrome, the body is in a state of high stress, and the energy is consumed in large quantities. In this case, the extreme hunger for energy is an important pathophysiological change. ATP is the most direct source of energy for the human body, and as the "energy currency" of intracellular energy transfer, it stores and transfers chemical energy. How to evaluate and supplement energy from the perspective of pathophysiology has become a difficult problem that needs to be solved in clinical practice.

[0024] Adenosine is a core metabolic product of ATP and an important signal molecule in life activities. Adenosine mainly participates in the regulation of inflammation, vasodilation, cardiac rhythm, tumor immunity and other physiological and pathological processes by combining with specific G protein-coupled receptors on the cell membrane. However, the inventors of the present application found in the research process that adenosine and adenosine receptors can affect multiple organ damage caused by various diseases or symptoms, including but not limited to: sepsis, septic shock, coronavirus pneumonia, systemic lupus erythematosus, rheumatoid arthritis, heavy metal poisoning, acute pancreatitis (severe), malignant tumors, and cardiovascular and cerebrovascular diseases. In order to prove the above point, the inventors verified two of the diseases: sepsis and post-cardiac arrest syndrome.

[0025] Sepsis is a systemic inflammatory response syndrome caused by infection, commonly seen in patients with severe trauma or infectious diseases. The causes of the disease include infection caused by bacteria, fungi, viruses and parasites, leading to imbalance of inflammatory response and immune regulation in the body. Multiple organ damage occurs in the early stage of sepsis, and further development can cause severe sepsis, septic shock, multiple organ dysfunction syndrome (such as lung, kidney, liver, heart, brain and other organ damage) and even death.

[0026] Post-cardiac arrest syndrome is an abnormal pathophysiological state that occurs after the restoration of spontaneous circulation after cardiac arrest. It is the most common complication after the restoration of spontaneous circulation and one of the important reasons for the low survival rate of patients with cardiac arrest. After cardiac arrest, the body will experience severe ischemia, hypoxia and acidosis, directly damaging the tissues and cells of the whole body, causing severe changes in various aspects of the body. In addition, with the restoration of effective spontaneous circulation, various tissues and organs will experience ischemia-reperfusion "second hit", and in the process of reperfusion, the body will activate and release a large amount of oxygen free radicals, inflammatory mediators and other harmful substances, further aggravating the extensive damage to the organs of the whole body, such as brain damage, myocardial dysfunction, acute kidney injury, lung injury, intestinal injury and liver injury.

[0027] Sepsis and post cardiac arrest syndrome are two diseases caused by completely different causes, and are two representative diseases causing multiple organ dysfunction syndrome. The present application proves through in vivo and in vitro experiments that adenosine receptor agonists can improve multiple organ injury.

[0028] Further, the multiple organ injury is caused by sepsis, and the injury includes liver injury, lung injury, kidney injury, heart injury and brain injury caused by sepsis.

[0029] Further, the multiple organ injury belongs to post cardiac arrest syndrome, that is, multiple organ injury caused after cardiopulmonary cerebral resuscitation, and the injury includes heart injury, kidney injury and liver injury caused by ischemia-reperfusion.

[0030] It has been verified that the adenosine receptor agonist is used to improve the success rate of cardiopulmonary resuscitation, reduce the frequency of ventricular fibrillation and recover organ function.

[0031] In the present application, the adenosine receptor includes but is not limited to A1 receptor (A1), 2A receptor (A2A), 2B receptor (A2B) and A3 receptor (A3). Under stress conditions such as ischemia, inflammation and hypoxia, the level of adenosine rises sharply, and as a danger signal, it coordinates a series of protective and adaptive responses by activating the four receptors.

[0032] In the present application, the adenosine receptor agonist includes but is not limited to CCPA, Capadenoson, Tecadenoson, Neladenoson, Binodenoson, Regadenoson, CGS 21680, Evodenoson, MRE 0094, LUF5834, BAY60-6583, MRS3558, A3AR agonist 4, Piclidenoson, 2-Chloro-3-deazaadenosine and inosine. In addition, it can also be other conventional adenosine receptor agonists in the art, as long as it can promote the corresponding receptor to exert its specific physiological function.

[0033] In some embodiments, the adenosine receptor agonist used includes CCPA and Capadenoson. Among them, CCPA directly and strongly activates adenosine A1 receptor, and can simulate the effect of endogenous adenosine. Capadenoson selectively activates the A1 receptor of the heart, which can slow down the heart rate, reduce the oxygen consumption of the myocardium, and has a protective effect on the heart, thereby relieving angina symptoms and controlling the ventricular rate during atrial fibrillation.

[0034] When the above adenosine receptor agonist is prepared as a drug for improving multiple organ injury, the drug further comprises a pharmaceutically acceptable excipient.

[0035] Further, the excipient includes at least one of a filler, a binder, a disintegrant, and a lubricant. The filler includes at least one of dextrin, lactose, microcrystalline cellulose, starch, and mannitol; the binder includes at least one of hydroxypropyl cellulose, hydroxypropyl methyl cellulose, povidone, carboxymethyl cellulose, and corn starch; the disintegrant includes at least one of croscarmellose sodium, crospovidone, and sodium carboxymethyl starch; and the lubricant includes at least one of magnesium stearate, talc, polyethylene glycol, and microfine silica gel.

[0036] Further, the dosage form of the drug includes tablets, pills, powders, suspensions, granules, capsules, suppositories, injections, or sprays. The above-mentioned dosage forms can be combined to achieve intravenous, oral, anal, and other routes of administration.

[0037] The features and performances of the present application are further described in detail below in combination with examples.

[0038] In the previous research, the inventors first detected the RNA pathway enrichment and expression changes of different pressure types of adenosine receptors in blood samples of sepsis patients and normal persons, and the results are shown in Figure 1 As can be seen from the figure, the RNA pathway enrichment results of the blood samples of the sepsis patients and the normal control group show that the adenosine metabolism pathway changes significantly and shows a significant downward trend; the four receptors of adenosine all have different degrees of expression changes, among which ADORA1 is significantly reduced, and ADOR2A and ADOR2B are significantly increased. On this basis, the following research was carried out: The ADORA1 activator in the following examples is CCPA, and the ADORA inhibitor is DPCPX. Example 1 This example verifies the influence of adenosine receptor agonists on multiple organ injury caused by sepsis, which uses three sepsis mouse models: LPS-induced sepsis mouse model, adenosine receptor knockout gene mouse model, and mouse model constructed by synchronous cecal ligation and puncture.

[0039] 1. The method of the LPS-induced sepsis mouse model is referred to a, b in the reference Figure 2 , and is specifically as follows: 6-8 weeks old male wild C57 mice are used, 10 mice per group, and the animal model is established by using the method of 2 times of intraperitoneal injection of LPS.

[0040] At the same time, a blank control group (i.e. mice without treatment), a control group (an equal amount of sterile water for injection), a CCPA experimental group (1 mg / kg CCPA is given at 5.5 h of modeling), and a DPCPX experimental group (1 mg / kg CCPA is given at 5.5 h of modeling) are set.

[0041] At the 6th hour of modeling, a lethal dose of LPS (20 mg / kg) was intraperitoneally injected into each group of mice, and the mortality rate was observed for 7 days to draw a survival curve.

[0042] Figure 2 c is the weight comparison result of each group of mice, from which it can be seen that there is no significant difference in the weight of mice in the model group, the control group, the CCPA experimental group and the DPCPX experimental group. The body weight of mice in the blank control group was significantly higher than that in the model group.

[0043] The results are shown in a of Figure 3 From which it can be seen that except for the blank control group, the survival rate of mice in the CCPA experimental group is the highest, proving that ADORA1 activators can improve the survival rate of LPS-induced sepsis mice.

[0044] Figure 3 b of is the LC-MS / MS chromatogram of the kidney tissue of the control group and the model group of mice, MS / MS chromatogram, Figure 3 c is the adenosine concentration of the kidney tissue of the control group and the model group of mice. From which it can be seen that the adenosine concentration of the kidney of the model group of mice is significantly higher than that of the control group.

[0045] 2. In vivo exploration of the protective effect of ADORs on organ function Referring to the construction method described above, 6-8 week old wild type (WT) C57 mice were randomly divided into groups and sepsis was established using LPS (10 mg / kg) and CLP. The control group was given an equal dose of solvent, the CCPA experimental group was given CCPA at the 5.5th hour of modeling, and the DPCPX experimental group was given CCPA at the 5.5th hour of modeling. Sampling was performed at 48 hours and 24 hours after modeling, respectively.

[0046] The lungs were taken for fixation to further determine the degree of inflammatory damage. A portion of the kidney tissue was fixed with light microscope and electron microscope fixing solution for subsequent HE, PAS, electron microscope, and multi-color immunofluorescence pathological analysis, especially under electron microscope to observe pyroptosis bodies near the cell membrane, cytoplasmic membrane rupture, and other pyroptosis manifestations. The remaining kidney tissue was divided into tubes and placed in a -80°C freezer for kidney homogenate to detect adenosine, inflammatory factors, and the expression of N-GSDMD and IL-1β by qPCR and Western Blotting.

[0047] Blood was collected from the heart and centrifuged to detect liver, kidney, and brain function, as well as inflammatory factors such as IL-6, IL-1β, and TNF-α. Histopathological fixation of each tissue was performed to further clarify the degree of inflammatory damage. A portion of the tissue was fixed using light and electron microscopy fixatives for subsequent pathological analysis including HE, PAS, electron microscopy, and multicolor immunofluorescence. The remaining tissues were placed in cryovials and stored at -80°C. Tissue homogenates were used to detect inflammatory factors using PCR and Western blotting.

[0048] like Figure 4 As shown in the figure, a and b are the serum creatinine and blood urea nitrogen test results of each group, respectively. It can be seen that compared with the blank control group, the serum creatinine and blood urea nitrogen levels in the model group and the control group are significantly increased, while the levels in the CCPA experimental group are significantly decreased, and the levels in the DPCPX experimental group are significantly increased compared with the model group and the control group.

[0049] Figure 5 HE staining results for kidney tissue. Figure 6 The images show the immunofluorescence staining results of kidney tissue. As can be seen from the figures, after LPS induction, the kidney tissue exhibited tubular vacuolar degeneration and necrosis, erythrocyte casts, and glomerular atrophy, indicating kidney damage in the septic mouse model. However, administration of CCPA significantly improved the degree of tubular degeneration and glomerular damage.

[0050] Figure 7 The results of RNA sequencing of mouse kidney tissue show that pyroptosis and inflammatory damage markers were significantly enriched after ADORA1 activation. In particular, the core pyroptosis marker Caspase11 showed the most significant change.

[0051] Figure 8 The results of Western blotting of kidney tissue show that the expression level of Caspase-11 in the CCPA experimental group was significantly lower than that in the model group.

[0052] Figure 9 The results of multicolor immunofluorescence of mouse kidneys showed that GBP1 expression was significantly increased in septic mice, and tissue damage was improved after activation of ADORs.

[0053] 5. In vitro investigation of the protective effects of ADORs on organ function Six hours after LPS transfection, agonists (10 μM, 5 μM, 2, 5 μM) and inhibitors (2 μM, 4 μM, 6 μM) of various adenosine receptors were added. Samples were taken 48 hours later. The release ratio of LHD in the cell supernatant was detected by the kit, and the expression of inflammatory factors such as IL-1β and TNF-α in the culture medium was detected by ELISA. Supernatant proteins were extracted and Western blotting was performed.

[0054] Figure 8 For Western Blotting detection results, after homogenization of kidney tissue, the expression of caspase11 and GSDMD, the key indicators of pyroptosis in each group, was detected, and the results showed that the activation of adenosine receptor could significantly reduce its expression and improve inflammatory damage. Example 2 This example verifies the influence of adenosine receptor agonists on the multiple organ damage caused by cardiopulmonary cerebral resuscitation after cardiac arrest, as follows: 1. Preparation of cardiac arrest model: Wistar rats were intraperitoneally injected with pentobarbital 35 mg / kg, and after satisfactory anesthesia, they were fixed on their backs, the trachea was cut open and intubated, and the electrocardiogram was monitored by standard limb leads. The right carotid artery was isolated and connected to a multi-lead electrocardiogram monitor to monitor blood pressure. The inguinal region was exposed for venous puncture with a trocar needle. The animal respirator was connected, with a breathing frequency of 60 times / min, a tidal volume of 6 ml / kg, and an oxygen concentration of 100%. After stabilization for 10 min, the tracheal cannula was clamped at the end of expiration for 5 min, and resuscitation was started. The diagnostic criteria for cardiac arrest were disappearance of arterial blood pressure or heart rate <40 times / min. Cardiac massage was performed at a frequency of 160 times / min, with a compression depth of 1 / 3 of the rat's thoracic diameter, and mechanical ventilation was also performed. On this basis, the conventional resuscitation group was given epinephrine 0.2 mg / kg every 5 min, and the other three groups were given different doses of adenosine receptor agonists and inhibitors in addition to epinephrine. When ventricular fibrillation occurred, lidocaine 1.5 mg / kg was given. The electrocardiogram and carotid artery blood pressure were continuously detected and recorded. If resuscitation was ineffective after 15 min, the rat was sacrificed and the heart, brain, lung, and blood were taken. (The criteria for successful resuscitation were HR≥180 times / min and SBP≥80 mmHg), which lasted at least 5 min. The tissues were treated, and the heart was taken for blood testing to detect liver and kidney function using a biochemical analyzer, and the tissues were fixed in fixative for testing.

[0055] 2. Experimental procedure: 30 male SD rats weighing (280±20) g were randomly divided into 3 groups: a, conventional group, intravenous injection of epinephrine 0.2 mg / kg each time, n=10; b, adenosine receptor inhibitor group, in addition to epinephrine 0.2 mg / kg, adenosine receptor inhibitor 1 mg / kg was given intravenously each time, n=10; c, adenosine receptor agonist group, in addition to epinephrine 0.2 mg / kg, adenosine agonist 1 mg / kg was given intravenously each time, n=10.

[0056] Table 1 Survival of rats during resuscitation, number of cases of ventricular fibrillation, and drug dosage

[0057] Note: a, the conventional group, each intravenous injection of adrenaline 0.2 mg / kg, n=10; b, adenosine receptor inhibitor group, in addition to the conventional given adrenaline.2 mg / kg, each intravenous administration of adenosine receptor inhibitor 1 mg / kg, n=10; c, adenosine receptor agonist group.

[0058] Adenosine receptor agonist and inhibitor intervention, detection of each group of heart, kidney, liver and other organ damage, table 1 shows that the results of the adenosine receptor can significantly improve the success rate of cardiopulmonary resuscitation, reduce the frequency of ventricular fibrillation and improve liver and kidney function.

[0059] The above only for the preferred embodiments of the present application and is not intended to limit the present application, for those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. within the spirit and principles of the present application, should be included in the scope of protection of the present application.

Claims

1. Application of adenosine receptor agonists in the preparation of drugs to improve multi-organ damage.

2. The application according to claim 1, characterized in that, The multiple organ damage was caused by post-cardiac arrest syndrome.

3. The application according to claim 2, characterized in that, The multi-organ injury includes cardiac, kidney, and liver damage caused by post-cardiac arrest syndrome.

4. The application according to claim 3, characterized in that, The adenosine receptor agonist is used to improve the success rate of cardiopulmonary resuscitation.

5. The application according to claim 3, characterized in that, The adenosine receptor agonist is used to reduce the frequency of ventricular fibrillation.

6. The application according to claim 1, characterized in that, The multiple organ damage was caused by sepsis.

7. The application according to claim 6, characterized in that, The multi-organ damage includes liver damage, lung damage, kidney damage, heart damage, and brain damage caused by sepsis.

8. The application according to any one of claims 1 to 7, characterized in that, The adenosine receptors include A1 receptors, 2A receptors, 2B receptors, and A3 receptors.

9. The application according to claim 8, characterized in that, The adenosine receptor agonists include CCPA, Capadenoson, Tecadenoson, Neladenoson, Binodenoson, Regadenoson, CGS 21680, Evodenoson, MRE 0094, LUF5834, BAY 60-6583, MRS3558, A3AR agonist 4, Piclidenoson, 2-Chloro-3-deazaadenosine, and inosine.

10. The application according to claim 9, characterized in that, The drug also includes pharmaceutically acceptable excipients.

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