Use of IMRC-exo in preparation of drug for alleviating multiple organ injury after resuscitation from cardiac arrest and drug

Abstract

Use of IMRC-Exo in the preparation of a drug for alleviating multiple organ injury after resuscitation from cardiac arrest and a drug. Through research on the efficacy of the use of IMRC-Exo in the treatment of multiple organ injury after resuscitation from cardiac arrest, it has been found that IMRC-Exo can effectively alleviate multiple organ injury after resuscitation from cardiac arrest. After administering a pharmaceutical dose of IMRC-Exo to rat and pig models resuscitated from cardiac arrest, compared to a conventional cardiac arrest resuscitation model group, the IMRC-Exo group shows statistically significant improvements in effects regarding cardiac function, neurological function, cardiac and brain injury markers, as well as regarding the analysis of pro-inflammatory factor levels in cardiac and brain organ tissues and the analysis of the degree of cell apoptosis in cardiac and brain organ tissues, providing a new idea for the research and development of therapeutic drugs for multiple organ injury after resuscitation from cardiac arrest.

Description

Application of IMRC-Exo in the preparation of drugs to alleviate multi-organ injury after cardiac arrest resuscitation and related drugs

[0001] Cross-references to related applications

[0002] This application claims priority to Chinese Patent Application No. 202410558225.2, filed on May 8, 2024, entitled "Application of IMRC-Exo in the Preparation of a Drug for Alleviating Multi-Organ Injury After Cardiac Arrest Resuscitation and the Drug", the entire contents of which are incorporated herein by reference. Technical Field

[0003] This application relates to the field of cell drug formulation technology, and in particular to the application of IMRC-Exo in the preparation of drugs to alleviate multi-organ damage after cardiac arrest resuscitation and the drug thereof. Background Technology

[0004] Cardiac arrest remains a major health problem with high incidence and mortality rates worldwide. Reports indicate that the annual incidence of cardiac arrest in Europe and the United States is 86.5 per 100,000 and 88.8 per 100,000, respectively, with hospital survival rates of only 8.0% and 9.3%. Surveys in my country show that the number of people experiencing cardiac arrest annually is close to 97.1 per 100,000, with a hospital survival rate as low as 1.15%. In 2021, the National Center for Disease Control and Prevention issued a statement indicating that cardiac arrest will become a significant factor hindering the achievement of life expectancy as part of the "Healthy China 2030" initiative. Currently, improving treatment strategies for cardiac arrest and continuously enhancing the clinical outcomes of these patients has become a crucial task in this field.

[0005] Studies show that post-cardiac arrest syndrome (PCOS) is a significant cause of death in patients who have successfully resuscitated after cardiac arrest. It stems from systemic ischemia-reperfusion injury caused by cardiac arrest resuscitation, clinically manifesting as multiple organ system dysfunction. Currently, international guidelines recommending therapeutic hypothermia and other methods are insufficient to significantly improve the clinical treatment outcomes of PCOS. Furthermore, some laboratory-proven treatments have not yet been successfully translated into clinical applications. Therefore, there is an urgent need to explore new, effective, and clinically feasible methods for the efficient treatment of PCOS following cardiac arrest resuscitation.

[0006] In view of the above, this application is hereby submitted.

[0007] Summary of the Invention

[0008] Explanation of abbreviations used in this application:

[0009] MSC: Mesenchymal stem cell (MSC);

[0010] IMRC: Immunity and matrix regulatory cells (IMRC) are formed by inducing differentiation of human embryonic stem cells as seed cells, which have unlimited stable expansion and pluripotent differentiation capabilities.

[0011] Exo: exosome (Exosome, Exo);

[0012] IMRC-Exo: refers to exosomes derived from human embryonic stem cell-derived immune and matrix regulatory cells (hESC-IMRC-Exo) formed by directed differentiation of human embryonic stem cells.

[0013] The purpose of this application is to confirm the efficacy of IMRC-Exo in the treatment of multi-organ injury after cardiac arrest resuscitation in rats and pigs, and to explore the research and clinical translation of IMRC-Exo exosome drugs based on this, which will be beneficial to the development of novel and highly effective drugs for multi-organ protection after cardiac arrest resuscitation, and has important scientific research significance and clinical application prospects.

[0014] In order to achieve the above-mentioned objectives of this application, the following technical solution is adopted:

[0015] This application provides the use of IMRC-Exo in the preparation of drugs to alleviate multi-organ injury after cardiac arrest resuscitation.

[0016] Optionally, the application is the administration of a pharmaceutical dose of IMRC-Exo.

[0017] Optionally, the IMRC-Exo is obtained by secretion from IMRC cells with cell surface markers CD105, CD73, and CD90 all >95%, and immunogenicity-related protein expression achieving HLA-DR <5% and HLA-E and HLA-G >50%.

[0018] Optionally, the administration method is intravenous administration at a concentration of 5 × 10⁻⁶. 10 IMRC-Exo particles / mL injection.

[0019] Optionally, the pharmaceutically effective dose of the IMRC-Exo is 2.5 × 10⁻⁶. 10 ~2.5×10 11 IMRC-Exo particles / kg.

[0020] Optionally, the multi-organ injury following cardiac arrest resuscitation includes:

[0021] Cardiac dysfunction after cardiac arrest resuscitation, neurological dysfunction after cardiac arrest resuscitation, myocardial injury after cardiac arrest resuscitation, brain injury after cardiac arrest resuscitation, renal function injury after cardiac arrest resuscitation, and intestinal function injury after cardiac arrest resuscitation.

[0022] This application provides a medicament for reducing multi-organ damage after cardiac arrest resuscitation, wherein the active ingredient of the medicament comprises IMRC-Exo and pharmaceutically acceptable excipients.

[0023] Optionally, the content of IMRC-Exo in the drug is 1×10⁻⁶. 10 ~1×10 13 One particle.

[0024] Optionally, the pharmaceutically acceptable excipients include one or more of diluents, binders, wetting agents, disintegrants, lubricants, solubilizers, pH adjusters, and osmotic pressure adjusters.

[0025] Optionally, the dosage form of the drug is an injection;

[0026] Preferably, the route of administration of the injection includes one of intravenous injection, intraperitoneal injection, intramuscular injection or subcutaneous injection, with intravenous injection being the preferred method.

[0027] This application also provides a method for reducing multi-organ damage after cardiac arrest resuscitation, characterized by administering a pharmaceutically effective dose of IMRC-Exo to the subject.

[0028] Optionally, the IMRC-Exo is obtained by secretion from IMRC cells with cell surface markers CD105, CD73, and CD90 all >95%, and immunogenicity-related protein expression achieving HLA-DR <5% and HLA-E and HLA-G >50%.

[0029] Optionally, the administration is carried out intravenously at a concentration of 5 × 10⁻⁶. 10 IMRC-Exo particles / mL.

[0030] Optionally, the pharmaceutically effective dose of the IMRC-Exo is 2.5 × 10⁻⁶. 10 ~2.5×10 11 IMRC-Exo particles / kg.

[0031] Optionally, the multi-organ injury after cardiac arrest resuscitation includes: cardiac dysfunction after cardiac arrest resuscitation, neurological dysfunction after cardiac arrest resuscitation, myocardial injury after cardiac arrest resuscitation, brain injury after cardiac arrest resuscitation, renal function injury after cardiac arrest resuscitation, and intestinal function injury after cardiac arrest resuscitation.

[0032] Optionally, the subject is a mammal.

[0033] Optionally, the subjects are selected from humans, rats, or pigs.

[0034] Compared with the prior art, the beneficial effects of this application are as follows:

[0035] This application discloses the application of IMRC-Exo in the preparation of drugs to alleviate multi-organ injury after cardiac arrest resuscitation. This application confirms the efficacy of IMRC-Exo in the treatment of multi-organ injury after cardiac arrest resuscitation in rats and pigs through research, and explores the research and development and clinical translation of IMRC-Exo exosome drugs based on this. This has important scientific research significance for the development of novel and highly effective drugs for multi-organ protection after cardiac arrest resuscitation.

[0036] This application provides a medicament for alleviating multi-organ injury after cardiac arrest resuscitation, wherein the active ingredient of the medicament comprises IMRC-Exo and pharmaceutically acceptable excipients. Experiments have shown that IMRC-Exo at pharmaceutically effective doses can effectively alleviate multi-organ injury after cardiac arrest resuscitation. Attached Figure Description

[0037] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0038] Figure 1a shows the changes in stroke volume (SV) of pigs in the Sham, CPR, and CPR+IMRC-Exo groups provided in Example 2 of the present invention;

[0039] Figure 1b shows the changes in total ejection fraction (GEF) in pigs in the Sham, CPR, and CPR+IMRC-Exo groups provided in Example 2 of the present invention.

[0040] Figure 1c shows the changes in porcine cardiac troponin (cTnI) in the Sham, CPR, and CPR+IMRC-Exo groups provided in Example 2 of the present invention.

[0041] Figure 2a shows the changes in neuron-specific enolase (NSE) in the Sham, CPR, and CPR+IMRC-Exo groups provided in Example 2 of the present invention.

[0042] Figure 2b shows the changes in the neurological deficit score (NDS) of pigs in the Sham, CPR, and CPR+IMRC-Exo groups provided in Example 2 of the present invention;

[0043] Figure 2c shows the changes in the brain function performance grading (CPC) of pigs in the Sham, CPR, and CPR+IMRC-Exo groups provided in Example 2 of the present invention;

[0044] Figure 3a shows the changes in creatinine (Cr) in pigs in the Sham, CPR, and CPR+IMRC-Exo groups provided in Example 2 of the present invention;

[0045] Figure 3b shows the changes in porcine intestinal fatty acid-binding protein (IFABP) in the Sham, CPR, and CPR+IMRC-Exo groups provided in Example 2 of the present invention.

[0046] Figure 4a is a staining diagram of the degree of apoptosis in multiple organ tissues of pigs in the Sham, CPR and CPR+IMRC-Exo groups 24 h after resuscitation provided in Example 2 of the present invention.

[0047] Figure 4b is a bar chart showing the degree of apoptosis in multiple organ tissues of pigs in the Sham, CPR and CPR+IMRC-Exo groups 24 h after resuscitation, as provided in Example 2 of the present invention.

[0048] Figure 5a is a graph showing the content analysis of tumor necrosis factor-a (TNF-a) in multiple organ tissues of pigs in the Sham, CPR and CPR+IMRC-Exo groups 24 h after resuscitation provided in Example 2 of the present invention.

[0049] Figure 5b is an analysis of the interleukin-1β (IL-1β) content in multiple organ tissues of pigs in the Sham, CPR, and CPR+IMRC-Exo groups 24 h after resuscitation, as provided in Example 2 of this invention.

[0050] Figure 6 is an analysis of the baseline status and modeling outcome of rats in the Sham, CPR, and CPR+IMRC-Exo groups provided in Example 3 of the present invention;

[0051] Figure 7 shows the changes in cardiac and cerebral injury markers and neurological function in rats in the Sham, CPR, and CPR+IMRC-Exo groups provided in Example 3 of the present invention.

[0052] Figure 8 is an analysis of the degree of cell apoptosis in the heart and brain organ tissues of rats in the Sham, CPR and CPR+IMRC-Exo groups 24 h after modeling, as provided in Example 3 of the present invention.

[0053] Figure 9 is an analysis of the content of pro-inflammatory factors in the heart and brain tissues of rats in the Sham, CPR and CPR+IMRC-Exo groups 24 h after modeling, as provided in Example 3 of the present invention. Detailed Implementation

[0054] The technical solutions of this application will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0055] According to one aspect of this application, the use of an IMRC-Exo in the preparation of a drug to reduce multi-organ injury after cardiac arrest resuscitation.

[0056] This application discloses the application of IMRC-Exo in the preparation of drugs to alleviate multi-organ injury after cardiac arrest resuscitation. This application confirms the effectiveness of IMRC-Exo in the treatment of multi-organ injury after cardiac arrest resuscitation in rats and pigs through research, and explores the research and development and clinical translation of IMRC-Exo exosome drugs based on this. This has important scientific research significance for the development of novel and highly effective drugs for multi-organ protection after cardiac arrest resuscitation.

[0057] It should be noted that recent studies have shown that mesenchymal stem cells (MSCs) possess various biological effects, including anti-inflammatory, antioxidant, and anti-apoptotic properties. Furthermore, research has found that human embryonic stem cells, with their unlimited stable expansion and pluripotent differentiation capacity, used as seed cells to induce differentiation into immune and matrix regulatory cells (IMRCs), exhibit superior cell quality, immune regulation, and damage repair capabilities compared to MSCs derived from traditional adult tissues such as bone marrow, umbilical cord, and adipose tissue, thus becoming a more advantageous stem cell therapy approach. In addition, exosomes (Exo), as important mediators for MSC therapy of various acute diseases, possess the various biological effects of MSCs while being easier to obtain, store, and safer, thus becoming a good alternative to MSC therapy. However, there are currently no reported studies on the application of IMRC-Exo in the preparation of drugs to alleviate multi-organ injury after cardiac arrest resuscitation.

[0058] In a preferred embodiment of this application, the application includes administering a pharmaceutical dose of IMRC-Exo, wherein:

[0059] The administration method is to administer an equal volume of IMRC-Exo dissolved in solvent via intravenous injection;

[0060] The pharmaceutically effective dose of the IMRC-Exo is 2.5 × 10⁻⁶. 10 -2.5×10 11 IMRC-Exo particles / kg.

[0061] In a preferred embodiment of this application, the IMRC-Exo is obtained by secretion from IMRC cells with cell surface markers CD105, CD73 and CD90 all >95% and immunogenicity-related protein expression achieving HLA-DR <5% and HLA-E and HLA-G >50%.

[0062] Preferably, the preparation process of the IMRC-Exo is as follows:

[0063] 1) Human embryonic stem cells are used to induce their differentiation into IMRCs with a purity >95%. By controlling the expression of immunogenicity-related proteins such as CD105, CD73 and CD90 to be >95%, HLA-DR <5% and HLA-E and HLA-G >50%, standardized, low-immunogenic IMRCs are obtained.

[0064] 2) Extract IMRC-derived Exo using methods such as tangential flow, ultrafiltration, ultracentrifugation, chromatography, and immunoaffinity. Then, obtain standard-quality IMRC-Exo through purification, identification, counting, expression of specific membrane proteins, and morphological observation.

[0065] In a preferred embodiment of this application, the multi-organ injury following cardiac arrest resuscitation includes:

[0066] Cardiac dysfunction after cardiac arrest resuscitation, neurological dysfunction after cardiac arrest resuscitation, myocardial injury after cardiac arrest resuscitation, brain injury after cardiac arrest resuscitation, renal function injury after cardiac arrest resuscitation, and intestinal function injury after cardiac arrest resuscitation.

[0067] According to one aspect of this application, a medicament for reducing multi-organ damage after cardiac arrest resuscitation, the active component of the medicament comprising IMRC-Exo and pharmaceutically acceptable excipients.

[0068] This application provides a medicament for alleviating multi-organ injury after cardiac arrest resuscitation, wherein the active ingredient of the medicament comprises IMRC-Exo and pharmaceutically acceptable excipients. Experiments have shown that IMRC-Exo at pharmaceutically effective doses can effectively alleviate multi-organ injury after cardiac arrest resuscitation.

[0069] In a preferred embodiment of this application, the content of IMRC-Exo in the drug is 1×10⁻⁶. 10 ~1×10 13 One particle.

[0070] Preferably, the content of IMRC-Exo in each 20 ml of the drug is 1 × 10⁻⁶. 12 IMRC-Exo particles;

[0071] It should be noted that the drug in this application must contain a pharmaceutically effective dose of IMRC-Exo particles, wherein the pharmaceutically effective dose of IMRC-Exo is 2.5 × 10⁻⁶. 10 ~2.5×10 11 IMRC-Exo particles / kg. For example, for a pig weighing 35-40kg, the intravenous administration dose is 20-200ml at a concentration of 5×10⁻⁶. 10 The formulation of IMRC-Exo particles / mL; for rats weighing 350-400g, the intravenous administration dose is 0.2-2ml at a concentration of 5×10⁻⁶. 10 IMRC-Exo particle / mL formulation.

[0072] In the preferred embodiments described above, the pharmaceutically acceptable excipients include one or more of the following: diluents, binders, wetting agents, disintegrants, lubricants, solubilizers, pH adjusters, and osmotic pressure adjusters.

[0073] In a preferred embodiment of this application, the dosage form of the drug is an injection;

[0074] Preferably, the route of administration of the injection includes one of intravenous injection, intraperitoneal injection, intramuscular injection or subcutaneous injection, with intravenous injection being the preferred method.

[0075] The technical solution of this application will be further described below with reference to the embodiments.

[0076] Example 1

[0077] (a) Animal preparation:

[0078] 1. Fast for 12 hours the night before the experiment, but water is allowed.

[0079] 2. On the morning of the experiment, anesthesia was induced by intramuscular injection of telastatin / zoprazepam 5 mg / kg and thiamethoxam 1 mg / kg, followed by general anesthesia by intravenous bolus injection of propofol 2 mg / kg and maintenance of anesthesia by intravenous infusion of propofol 4 mg / kg / h. At the same time, analgesia was administered by intravenous bolus injection of butorphanol 8 μg / kg and intravenous infusion of 3 μg / kg / h.

[0080] 3. Intubate via endotracheal tube and connect the end-expiratory carbon dioxide partial pressure (ETCO2) monitoring device to the ventilator. The ventilation parameters of the ventilator are set to volume control mode, tidal volume 10 ml / kg, and oxygen concentration 21%. The initial ETCO2 is maintained within the normal range of 35-40 mmHg by adjusting the respiratory rate.

[0081] 4. Under direct vision, incise the skin, dissect the subcutaneous fascia and muscles, and expose the bilateral femoral arteries and veins. Insert the arterial end catheter of a pulse-indicating continuous cardiac output (PiCCO) monitor into the left femoral artery, and insert pressure monitoring catheters into the right femoral artery and vein, respectively, connecting them to the thoracic aorta and right atrium. Additionally, under direct vision, incise the skin, dissect the aforementioned tissues, and expose the right internal and external jugular veins, inserting ventricular fibrillation induction electrodes and the venous end catheters of the PiCCO monitor, respectively.

[0082] 5. Use a temperature-controlled blanket to maintain a normal body temperature of around 38°C throughout the process.

[0083] (II) Model Establishment:

[0084] 1. Conditions for the cardiac arrest resuscitation model: 12 minutes of cardiac arrest + 6 minutes of cardiopulmonary resuscitation.

[0085] 2. Method for inducing cardiac arrest: A ventricular fibrillation induction electrode was inserted through the right ventricle, connected to a power source, and a 1mA AC current was applied to induce ventricular fibrillation. After successful induction, the patient was observed without intervention for 12 minutes.

[0086] 3. Methods of cardiopulmonary resuscitation:

[0087] 1) Mechanical chest compressions, with parameters of 5cm depth and 100 compressions / minute;

[0088] 2) Ventilator-assisted ventilation, with parameters of volume control mode, tidal volume 7 ml / kg, oxygen concentration 100%, and respiratory rate 10 breaths / min;

[0089] 3) Adrenaline: 20 μg / kg intravenously 2 minutes into cardiopulmonary resuscitation, and then repeated every 3 minutes thereafter;

[0090] 4) Defibrillation: Defibrillate once with 150J after 6 minutes of cardiopulmonary resuscitation;

[0091] 5) If spontaneous circulation is not restored, immediately restart cardiopulmonary resuscitation (CPR) for 2 minutes, then defibrillate once. Repeat this cycle until resuscitation is successful or after a maximum of 5 resuscitation attempts, declare resuscitation failure.

[0092] 4. Post-resuscitation monitoring methods: 1) Continue anesthesia monitoring; 2) Restart mechanical ventilation with the same parameters as before, i.e., volume control mode, tidal volume 10ml / kg, oxygen concentration 21%, and respiratory rate restored to the pre-modeling state; 3) Continuous monitoring for 6 hours.

[0093] 5. Post-resuscitation observation method: After the animal monitoring is completed, disinfect and suture all incisions, wean the animal off the ventilator and remove the endotracheal tube, and transfer the animal to the pig pen for continued observation for 18 hours.

[0094] Example 2

[0095] (I) Randomization and Intervention in Animals:

[0096] Experimental grouping: 18 healthy male white pigs from China, weighing 35-40kg, were randomly divided into 3 groups: sham surgery group, cardiopulmonary resuscitation (CPR) group, and CPR+IMRC-Exo group, with 6 pigs in each group.

[0097] Intervention measures:

[0098] 1) Sham group: No porcine cardiac arrest resuscitation model was established, and the same amount of solvent was administered to the same groups within the same time period.

[0099] 2) CPR group: A porcine cardiac arrest resuscitation model was established, and 5 minutes after successful resuscitation, the same amount of solvent was administered to the group at the same time as other groups.

[0100] 3) CPR + IMRC-Exo group: A porcine cardiac arrest resuscitation model was established, and 5 minutes after successful resuscitation, an equal volume of IMRC-Exo prepared with solvent was administered via the internal jugular vein, with a total particle count of 1 × 10⁻⁶. 12 indivual.

[0101] Note: All three solvents were 20ml of normal saline, which was infused via internal jugular vein pump within 30 minutes.

[0102] (II) Observation Indicators:

[0103] 1. Before modeling, record the weight, heart rate, blood pressure, ETCO2, and other values ​​of the three groups of animals, as well as the results of arterial blood gas analysis.

[0104] 2. During cardiopulmonary resuscitation, the changes in aortic blood pressure and right atrial pressure were continuously monitored, and the coronary perfusion pressure was calculated by the difference between the two. The cardiopulmonary resuscitation duration, adrenaline dosage, number of defibrillation attempts, and resuscitation success rate of the three groups of animals were also recorded.

[0105] 3. Before modeling and at 1h, 2h, 4h and 6h after resuscitation, use a PiCCO monitor to regularly assess changes in cardiac function indicators such as stroke volume (SV) and total ejection fraction (GEF).

[0106] 4. Before modeling and at 1h, 2h, 4h, 6h, and 24h after recovery, collect 2ml of venous blood samples, centrifuge to obtain plasma, and freeze at -80℃. Detect the plasma using enzyme-linked immunosorbent assay (ELISA) at selected times.

[0107] Myocardial injury marker - cardiac troponin I (cTnI);

[0108] Brain injury marker - neuron-specific enolase (NSE);

[0109] Kidney injury marker - creatinine (Cr);

[0110] Serum levels of intestinal injury marker - intestinal fatty acid-binding protein (iFABP).

[0111] 5. At 24 hours after resuscitation, the neurological function status of the three groups of animals was assessed using two methods: the Neurological Deficit Scale (NDS) and the Brain Function Performance Scale (CPC).

[0112] 6. Once the animals have completed the collection of all the above samples and data within 24 hours after resuscitation, they are immediately euthanized. Tissues from the left ventricular apex, cerebral cortex, hippocampus, upper pole of the right kidney, and terminal ileum are rapidly obtained. Some of these tissues are fixed, embedded, and sectioned to prepare pathological specimens. The apoptosis index of organs and tissues such as the heart, brain, kidney, and intestine is then detected using the TUNEL assay at a later date.

[0113] In addition, some fresh tissues were frozen in a -80°C cryogenic freezer, and the levels of pro-inflammatory factors, tumor necrosis factor-a (TNF-α) and interleukin-1β (IL-1β), in various organs and tissues were detected by enzyme-linked immunosorbent assay at selected times.

[0114] (III) Research Results:

[0115] 1. Baseline status of the three groups of animals before modeling:

[0116] Before modeling, there were no statistically significant differences among the three groups of animals in terms of basic physiological indicators such as body weight, heart rate, mean arterial pressure, ETCO2, pH value, PO2, PCO2, and lactate (all P>0.05). See Table 1.

[0117] Table 1. Basic Information of the Three Groups of Animals

[0118] Note: ETCO2, partial pressure of carbon dioxide at end-tidal; PO2, partial pressure of oxygen; PCO2, partial pressure of carbon dioxide; Sham, sham surgery; CPR, cardiopulmonary resuscitation; IMRC-Exo, exosomes derived from immune and matrix regulatory cells.

[0119] 2. The effects and outcomes of cardiopulmonary resuscitation during the modeling process in the two groups of animals:

[0120] During the modeling process, the CPR group and the CPR+IMRC-Exo group established experimental animal models using parameters of 12 minutes of cardiac arrest and 6 minutes of cardiopulmonary resuscitation.

[0121] Data showed that during cardiopulmonary resuscitation (CPR), the coronary perfusion pressure of the two groups of animals maintained the same trend and remained at a generally consistent level, with no statistically significant differences between the groups (all P>0.05). Consequently, there were no statistically significant differences between the two groups in terms of CPR duration, adrenaline dosage, number of defibrillation attempts, and resuscitation success rate (all P>0.05). See Table 2.

[0122] Table 2. Cardiopulmonary resuscitation effects and outcomes in the two groups of animals.

[0123] Note: CPR stands for cardiopulmonary resuscitation; IMRC-Exo stands for exosomes derived from immune and stromal regulatory cells. Data from animals that failed resuscitation in the CPR group and the CPR+IMRC-Exo group were used only for the analysis of cardiopulmonary resuscitation efficacy and outcome, and no further data were used for other indicator analyses.

[0124] 3. Changes in cardiac function indicators and myocardial injury markers before and after modeling in the three groups of animals:

[0125] Figures 1a to 1c show the changes in cardiac function indicators and myocardial injury markers in the Sham, CPR, and CPR+IMRC-Exo groups provided in this embodiment, where:

[0126] Figure 1a shows the changes in stroke volume (SV) of animals in the Sham, CPR, and CPR+IMRC-Exo groups provided in this embodiment;

[0127] Figure 1b shows the changes in total ejection fraction (GEF) in the Sham, CPR, and CPR+IMRC-Exo groups provided in this embodiment.

[0128] Figure 1c shows the changes in cardiac troponin (cTnI) in the Sham, CPR, and CPR+IMRC-Exo groups provided in this embodiment.

[0129] In Figures 1a-1c: BL, baseline; Sham, sham surgery; CPR, cardiopulmonary resuscitation; IMRC-Exo, exosomes derived from immune and stromal regulatory cells; as shown in Figures 1a-1c, compared with the Sham group, the CPR group and the CPR+IMRC-Exo group... * P<0.05; Compared with the CPR group, the CPR+IMRC-Exo group # P<0.05.

[0130] Before modeling, there were no statistically significant differences in the baseline levels of cardiac function indicators SV and GEF, and the myocardial injury marker cTnI among the three groups of animals (all P>0.05). After resuscitation, compared with the Sham group, the CPR group and the CPR+IMRC-Exo group showed significantly lower SV and GEF values, and the differences between the groups were statistically significant (all P<0.05).

[0131] However, the SV values ​​at 1 h and 2 h after resuscitation and the GEF values ​​at each time point after resuscitation in the CPR+IMRC-Exo group were significantly higher than those in the CPR group, and the differences between the groups were statistically significant (all P < 0.05).

[0132] In addition, compared with the Sham group, the serum levels of cTnI in the CPR group and the CPR+IMRC-Exo group were significantly increased at all time points after animal resuscitation, and the differences between the groups were statistically significant (all P<0.05).

[0133] However, the serum cTnI levels in the CPR+IMRC-Exo group were significantly lower than those in the CPR group 2 hours after resuscitation, and the differences between the groups were statistically significant (all P < 0.05). See Figures 1a to 1c for details.

[0134] 4. Changes in brain injury markers and neurological function indicators before and after modeling in the three groups of animals:

[0135] Figures 2a to 2c show the changes in brain injury markers and neurological function indicators in the Sham, CPR, and CPR+IMRC-Exo groups provided in this embodiment, where:

[0136] Figure 2a shows the changes in neuron-specific enolase (NSE) in the Sham, CPR, and CPR+IMRC-Exo groups provided in this embodiment.

[0137] Figure 2b shows the changes in neurological deficit scores (NDS) of the Sham, CPR, and CPR+IMRC-Exo groups provided in this embodiment;

[0138] Figure 2c shows the changes in brain function performance grading (CPC) of animals in the Sham, CPR, and CPR+IMRC-Exo groups provided in this embodiment;

[0139] In Figures 2a-2c: BL, baseline; Sham, sham surgery; CPR, cardiopulmonary resuscitation; IMRC-Exo, exosomes derived from immune and stromal regulatory cells; as shown in Figures 2a-2c, compared with the Sham group, the CPR group and the CPR+IMRC-Exo group... * P<0.05; Compared with the CPR group, the CPR+IMRC-Exo group # P<0.05.

[0140] Before modeling, there were no statistically significant differences in serum levels of the brain injury marker NSE among the three groups of animals (all P>0.05).

[0141] After resuscitation, compared with the Sham group, the serum NSE levels of animals in the CPR group and the CPR+IMRC-Exo group were significantly increased at all time points, and the differences between the groups were statistically significant (all P < 0.05). However, the serum NSE levels of animals in the CPR+IMRC-Exo group 4 hours after resuscitation were significantly lower than those in the CPR group, and the differences between the groups were statistically significant (all P < 0.05).

[0142] In addition, compared with the Sham group, the neurological function indicators NDS and CPC scores of animals in the CPR group and the CPR+IMRC-Exo group were significantly increased at 24h after resuscitation, and the differences between the groups were statistically significant (all P<0.05).

[0143] However, the NDS and CPC scores of animals in the CPR+IMRC-Exo group at 24 hours after resuscitation were significantly lower than those in the CPR group, and the differences between the groups were statistically significant (all P < 0.05). See Figures 2a to 2c for details.

[0144] 5. Changes in renal and intestinal injury markers before and after modeling in three groups of animals

[0145] Figures 3a and 3b show the changes in renal and intestinal injury markers in the Sham, CPR, and CPR+IMRC-Exo groups provided in this embodiment, where:

[0146] Figure 3a shows the changes in creatinine (Cr) in the Sham, CPR, and CPR+IMRC-Exo groups provided in this embodiment;

[0147] Figure 3b shows the changes in intestinal fatty acid-binding protein (IFABP) in the Sham, CPR, and CPR+IMRC-Exo groups provided in this embodiment;

[0148] In Figures 3a and 3b: BL, baseline; Sham, sham surgery; CPR, cardiopulmonary resuscitation; IMRC-Exo, exosomes derived from immune and stromal regulatory cells; as shown in Figures 3a and 3b, compared with the Sham group, the CPR group and the CPR+IMRC-Exo group... * P<0.05; Compared with the CPR group, the CPR+IMRC-Exo group # P<0.05.

[0149] As shown in Figures 3a and 3b, before modeling, there were no statistically significant differences in serum levels of Cr and IFABP, markers of kidney and intestinal injury, among the three groups (all P > 0.05). After resuscitation, compared with the Sham group, the serum levels of Cr and IFABP in the CPR group were significantly higher at all time points, and the serum levels of Cr and IFABP in the CPR+IMRC-Exo group were significantly higher at all time points and 2 hours after resuscitation, with statistically significant differences among the groups (all P < 0.05). However, the serum levels of Cr and IFABP in the CPR+IMRC-Exo group 2 hours after resuscitation were significantly lower than those in the CPR group, with statistically significant differences among the groups (all P < 0.05).

[0150] 6. Analysis of the degree of apoptosis in multiple organ tissues of three groups of animals 24 hours after resuscitation

[0151] Figures 4a and 4b are analysis diagrams of the degree of cell apoptosis in multiple organ tissues of the Sham, CPR, and CPR+IMRC-Exo groups 24 hours after resuscitation provided in this embodiment.

[0152] Figure 4a shows the staining of apoptosis levels in multiple organ tissues of the Sham, CPR, and CPR+IMRC-Exo groups 24 h after resuscitation provided in this embodiment.

[0153] Figure 4b is a bar chart showing the degree of apoptosis in multiple organ tissues of the Sham, CPR and CPR+IMRC-Exo groups 24 h after resuscitation provided in this embodiment.

[0154] In Figures 4a and 4b: Sham, sham surgery; CPR, cardiopulmonary resuscitation; IMRC-Exo, exosomes derived from immune and stromal regulatory cells; as shown in Figures 4a and 4b, compared with the Sham group, the CPR group and the CPR+IMRC-Exo group... * P<0.05; Comparison between the CPR+IMRC-Exo group and the CPR group, # P<0.05.

[0155] As shown in Figures 4a and 4b, euthanasia was performed on all animals 24 hours after resuscitation. Tissue samples from the left ventricular apex, cerebral cortex, hippocampus, upper pole of the right kidney, and terminal ileum were then rapidly harvested for pathological analysis. The results showed that compared to the Sham group, the CPR group and the CPR+IMRC-Exo group had significantly increased apoptosis indices in the heart, brain, kidney, and intestines, with statistically significant differences between groups (all P < 0.05). However, the apoptosis indices in the aforementioned organs and tissues of the CPR+IMRC-Exo group were significantly lower than those of the CPR group, with statistically significant differences between groups (all P < 0.05).

[0156] 7. Analysis of pro-inflammatory factor content in multiple organ tissues of three groups of animals 24 hours after resuscitation

[0157] Figures 5a and 5b are analysis diagrams of the content of pro-inflammatory factors in multiple organ tissues of animals in the Sham, CPR, and CPR+IMRC-Exo groups 24 hours after resuscitation provided in this embodiment.

[0158] Figure 5a is a graph showing the content analysis of tumor necrosis factor-a (TNF-a) in multiple organ tissues of the Sham, CPR and CPR+IMRC-Exo groups 24 h after resuscitation provided in this embodiment.

[0159] Figure 5b is a graph showing the content analysis of interleukin-1β (IL-1β) in multiple organ tissues of the Sham, CPR, and CPR+IMRC-Exo groups 24 h after resuscitation provided in this embodiment.

[0160] In Figures 5a and 5b: Sham, sham surgery; CPR, cardiopulmonary resuscitation; IMRC-Exo, exosomes derived from immune and stromal regulatory cells; as shown in Figures 5a and 5b, compared with the Sham group, the CPR group and the CPR+IMRC-Exo group... * P<0.05; Comparison between the CPR+IMRC-Exo group and the CPR group, # P<0.05.

[0161] As shown in Figures 5a and 5b, 24 hours after resuscitation, the animals were euthanized as before, and samples of organs and tissues such as the heart, brain, kidney, and intestine were obtained for analysis of pro-inflammatory factor content. The results showed that compared with the Sham group, the CPR group and the CPR+IMRC-Exo group had significantly increased levels of pro-inflammatory factors TNF-α and IL-1β in organs and tissues such as the heart, brain, kidney, and intestine, with statistically significant differences between groups (all P < 0.05). However, the levels of pro-inflammatory factors in the aforementioned organs and tissues of the CPR+IMRC-Exo group were significantly lower than those in the CPR group, with statistically significant differences between groups (all P < 0.05).

[0162] Example 3: Evaluation of the efficacy of IMRC-Exo in reducing cardiac and cerebrovascular damage after cardiac arrest resuscitation in rats

[0163] 1. Animal source: 15 male SD rats aged 10-12 weeks and weighing 350-400g were obtained.

[0164] 2. Experimental model establishment:

[0165] (1) Model conditions: 6 minutes of cardiac arrest + 6 minutes of cardiopulmonary resuscitation.

[0166] (2) Methods for inducing cardiac arrest: ventricular fibrillation-type cardiac arrest was induced by continuous percutaneous epicardial electrical stimulation for 3 minutes, followed by observation for 3 minutes without intervention.

[0167] (3) Methods of cardiopulmonary resuscitation: 1) Use manual chest compressions and mechanical ventilation. The chest compression rate is about 200 times / min, and the compression depth should be controlled to maintain the diastolic pressure of the aorta >20mmHg. The mechanical ventilation parameters are set as follows: tidal volume 0.60ml / 100g, frequency 100 times / min, oxygen concentration 100%, and the whole duration is 6min; 2) At the beginning of cardiopulmonary resuscitation, administer 20μg / kg of adrenaline intravenously, and repeat once every 2min thereafter; 3) At 6min of cardiopulmonary resuscitation, administer a biphasic wave defibrillation with an energy of 4J once, and quickly determine whether the rat has restored spontaneous circulation; 4) If spontaneous circulation has not been restored, immediately restart chest compressions and mechanical ventilation for 2min and then defibrillate once. Repeat this cycle ≤3 times until resuscitation is successful or declared unsuccessful.

[0168] (4) Post-resuscitation monitoring: After successful cardiopulmonary resuscitation, continue anesthesia monitoring and restart mechanical ventilation. The parameters of the latter are set as follows: tidal volume 0.60ml / 100g, frequency 100 breaths / min, oxygen concentration 21%, and duration 4h.

[0169] (5) Post-resuscitation observation: After 4 hours of monitoring, the catheter was removed, the wound was sutured, and the animal was weaned off the ventilator and extubated. The animal was then returned to its enclosure for further observation for 20 hours.

[0170] 3. Experimental grouping and processing methods:

[0171] (1) Sham group: No cardiac arrest resuscitation model was established. 0.2 ml of normal saline was injected via the tail vein. A total of 5 animals were included.

[0172] (2) Cardiopulmonary resuscitation (CPR) group: A cardiac arrest resuscitation model was established, and 0.2 ml of normal saline was injected via the tail vein after successful resuscitation. A total of 5 animals were included.

[0173] (3) CPR+IMRC-Exo group: A cardiac arrest resuscitation model was established, and 2.5×10⁻⁶ mmol / L was injected via the tail vein after successful resuscitation. 10 IMRC-Exo particles / kg, 0.2ml exosome preparation, 5 in total.

[0174] 4. Observation indicators:

[0175] (1) Before modeling, record the weight, heart rate, blood pressure and other values ​​of each group of animals.

[0176] (2) During CPR, the duration of CPR, the amount of adrenaline used, and the number of defibrillations were recorded for each group of animals.

[0177] (3) Before modeling and at 1h, 4h and 24h after recovery, 1ml of venous blood samples were collected, and serum was obtained by centrifugation and frozen in a -80℃ deep low temperature freezer. The serum levels of myocardial injury marker - cardiac troponin I (cTnI), brain injury marker - neuron-specific enolase (NSE) and S100β protein (S100β) were detected by enzyme-linked immunosorbent assay (ELISA) at selected times.

[0178] (4) At 24 hours after resuscitation, the neurological function status of each group of animals was assessed using two neurological function scoring methods.

[0179] (5) 24 hours after resuscitation, once the animal has completed all the above sample and data collection, it will be euthanized immediately and tissues from the left ventricular apex, cerebral cortex, hippocampus and other parts will be quickly obtained. Some of these tissues will be fixed, embedded and sectioned to make pathological specimens. The apoptosis index of the heart and brain organs will be detected by the in situ terminal labeling method (TUNEL) at a later date.

[0180] (6) In addition, some fresh tissues were frozen in a -80°C deep low temperature freezer, and the contents of pro-inflammatory factors - tumor necrosis factor-a (TNF-a) and interleukin-1β (IL-1β) in the heart and brain tissues were detected by ELISA at selected times.

[0181] 5. Experimental Results:

[0182] (1) Baseline status and modeling outcome of rats in each group:

[0183] Before modeling, there were no statistically significant differences in baseline physiological indicators such as body weight, heart rate, and mean arterial pressure among the three groups of rats (all P>0.05). During modeling, the CPR group and the CPR+IMRC-Exo group established rat experimental models using parameters of 6 minutes of cardiac arrest and 6 minutes of cardiopulmonary resuscitation. There were no statistically significant differences in modeling outcome indicators such as CPR duration, adrenaline dosage, and number of defibrillation attempts between the two groups (all P>0.05), as shown in Figure 6.

[0184] (2) Changes in cardiac and cerebral injury markers and neurological function in rats before and after modeling:

[0185] Before modeling, there were no statistically significant differences in the baseline levels of myocardial injury marker cTnI, and brain injury markers NSE and S100β among the three groups of rats (all P>0.05). After modeling, compared with the Sham group, the serum levels of cTnI, NSE, and S100β in the CPR group and the CPR+IMRC-Exo group were significantly increased at all time points, with statistically significant differences among the groups (all P<0.05). However, the serum levels of cTnI and S100β, as well as the serum level of NSE 4 h after modeling, in the CPR+IMRC-Exo group were significantly lower than those in the CPR group at all time points after modeling, with statistically significant differences among the groups (all P<0.05). In addition, compared with the Sham group, the NFS-1 score and NFS-2 score of the CPR group and the CPR+IMRC-Exo group were significantly increased and decreased at 24 h after modeling, with statistically significant differences among the groups (all P<0.05). However, the NFS-1 and NFS-2 scores of rats in the CPR+IMRC-Exo group were significantly better than those in the CPR group 24 h after modeling, and the differences between the groups were statistically significant (all P < 0.05), see Figure 7.

[0186] (3) Analysis of the degree of apoptosis in the heart and brain tissues of rats in each group 24 hours after modeling

[0187] Twenty-four hours after modeling, rats in each group were euthanized, and tissues from the left ventricular apex, cerebral cortex, and hippocampus were rapidly obtained for pathological analysis. Results showed that compared with the Sham group, the apoptosis index of the heart and brain tissues of rats in the CPR group and the CPR+IMRC-Exo group was significantly increased, with statistically significant differences between groups (all P < 0.05). However, the apoptosis index of the heart and brain tissues of rats in the CPR+IMRC-Exo group was significantly lower than that in the CPR group, with statistically significant differences between groups (all P < 0.05), as shown in Figure 8.

[0188] (4) Analysis of pro-inflammatory factor content in heart and brain tissues of rats in each group 24 hours after modeling

[0189] Twenty-four hours after modeling, rats were sacrificed as before, and samples of heart and brain tissue were collected for analysis of pro-inflammatory factor content. Results showed that compared with the Sham group, the CPR group and the CPR+IMRC-Exo group had significantly increased levels of pro-inflammatory factors TNF-α and IL-1β in the heart and brain tissues of rats, with statistically significant differences between groups (all P < 0.05). However, the levels of pro-inflammatory factors in the heart and brain tissues of rats in the CPR+IMRC-Exo group were significantly lower than those in the CPR group, with statistically significant differences between groups (all P < 0.05), see Figure 9.

[0190] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application. Industrial applicability

[0191] The application provides the use of IMRC-Exo in the preparation of drugs to alleviate multi-organ injury after cardiac arrest resuscitation. This application confirms the efficacy of IMRC-Exo in the treatment of multi-organ injury after cardiac arrest resuscitation in rats and pigs through research, and explores the research and development and clinical translation of IMRC-Exo exosome drugs based on this. It has important scientific research significance for the development of novel and highly effective drugs for the protection of the heart and brain organs after cardiac arrest resuscitation.

[0192] This application provides a medicament for alleviating multi-organ injury after cardiac arrest resuscitation, wherein the active ingredient of the medicament comprises IMRC-Exo and pharmaceutically acceptable excipients. Experiments have shown that IMRC-Exo at pharmaceutically effective doses can effectively alleviate multi-organ injury after cardiac arrest resuscitation.

Claims

1. Application of IMRC-Exo in the preparation of drugs to reduce multi-organ injury after cardiac arrest resuscitation.

2. The application according to claim 1, characterized in that, The application is the administration of a pharmaceutically effective dose of IMRC-Exo.

3. The application according to claim 1, characterized in that, The IMRC-Exo was obtained by secretion from IMRC cells with cell surface markers CD105, CD73, and CD90 all >95%, and immunogenicity-related protein expression achieving HLA-DR <5% and HLA-E and HLA-G >50%.

4. The application according to claim 2, characterized in that, The administration method is intravenous, and the formulation concentration is 5 × 10⁻⁶. 10 IMRC-Exo particles / mL.

5. The application according to claim 1, characterized in that, The pharmaceutically effective dose of the IMRC-Exo is 2.5 × 10⁻⁶. 10 ~2.5×10 11 IMRC-Exo particles / kg.

6. The application according to claim 1, characterized in that, The multiple organ injury following cardiac arrest resuscitation includes: Cardiac dysfunction after cardiac arrest resuscitation, neurological dysfunction after cardiac arrest resuscitation, myocardial injury after cardiac arrest resuscitation, brain injury after cardiac arrest resuscitation, renal function injury after cardiac arrest resuscitation, and intestinal function injury after cardiac arrest resuscitation.

7. A drug for reducing multi-organ damage after cardiac arrest resuscitation, characterized in that, The active ingredient of the drug includes IMRC-Exo and pharmaceutically acceptable excipients.

8. The medicament for reducing multi-organ injury after cardiac arrest resuscitation according to claim 7, characterized in that, The content of IMRC-Exo in the drug is 1×10 10 ~1×10 13 One particle.

9. The medicament for reducing multi-organ injury after cardiac arrest resuscitation according to claim 7, characterized in that, The pharmaceutically acceptable excipients include one or more of the following: diluents, binders, wetting agents, disintegrants, lubricants, solubilizers, pH adjusters, and osmotic pressure adjusters.

10. The medicament for reducing multi-organ injury after cardiac arrest resuscitation according to claim 7, characterized in that, The drug is in the form of an injection; Preferably, the route of administration of the injection includes one of intravenous injection, intraperitoneal injection, intramuscular injection or subcutaneous injection, with intravenous injection being the preferred method.

11. A method for mitigating multi-organ injury after cardiac arrest resuscitation, characterized in that, Administer a pharmaceutically effective dose of IMRC-Exo to the subjects.

12. The method according to claim 11, characterized in that, The IMRC-Exo was obtained by secretion from IMRC cells with cell surface markers CD105, CD73, and CD90 all >95%, and immunogenicity-related protein expression achieving HLA-DR <5% and HLA-E and HLA-G >50%.

13. The method according to claim 11, characterized in that, The administration method is intravenous infusion at a concentration of 5 × 10⁻⁶. 10 IMRC-Exo particles / mL.

14. The method according to claim 11, characterized in that, The pharmaceutically effective dose of the IMRC-Exo is 2.5 × 10⁻⁶. 10 ~2.5×10 11 IMRC-Exo particles / kg.

15. The method according to claim 11, characterized in that, The multiple organ injury following cardiac arrest resuscitation includes: Cardiac dysfunction after cardiac arrest resuscitation, neurological dysfunction after cardiac arrest resuscitation, myocardial injury after cardiac arrest resuscitation, brain injury after cardiac arrest resuscitation, renal function injury after cardiac arrest resuscitation, and intestinal function injury after cardiac arrest resuscitation.

16. The method according to claim 11, characterized in that, The subjects were mammals.

17. The method according to claim 16, characterized in that, The subjects were selected from humans, rats, or pigs.

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