Use of a trpm11-specific small molecule inhibitor ml-si3

Abstract

The application provides application of a TRPML1 specific small molecule inhibitor ML-SI3 in preparation of a drug for treating or preventing brain injury. The application has the following beneficial effects: the TRPML1 specific small molecule inhibitor ML-SI3 can significantly reduce the injury degree of neurons of a brain injury rat; the learning and memory ability of the brain injury rat is improved; the movement disorder of the brain injury rat is improved, and the brain function of a rat after cardiopulmonary resuscitation after cardiac arrest is comprehensively protected; the TRPML1 specific small molecule inhibitor ML-SI3 provides a new drug for brain function protection of a cardiac arrest patient, and has good market value and clinical application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to application of a TRPML1 specific small molecule inhibitor ML-SI3. BACKGROUND

[0002] Cardiac arrest (CA) refers to sudden stop of heart beating caused by various reasons, sudden stop of normal heart pump function and effective circulation, and severe ischemia, hypoxia and metabolic disorder of cells in the whole body. Once cardiac arrest occurs, if immediate and timely rescue and resuscitation is not provided, irreversible damage of brain and other important organs and tissues of the patient will be caused after 4-6 minutes, therefore, cardiopulmonary resuscitation (CPR) after cardiac arrest is a key step that should be taken to improve the survival probability of the patient as much as possible, and is called a cardiac arrest survival chain. CPR is an effective method for rescuing cardiac arrest. However, in the process of cardiopulmonary resuscitation, severe brain hypoxia symptoms often occur, and the brain cells of the patient will be greatly damaged, and central nervous damage in different degrees is left, which is called post-cardiac arrest brain injury (PCABI). 60%-70% of the patients whose cardiac arrest resuscitation is successful die due to post-cardiac arrest brain injury, and only about 10.2% of the patients whose cardiac arrest is survived in China keep good neurological function after resuscitation. Improving acute nervous system injury caused after cardiac arrest is a key to improve the survival and discharge rate of the patient. At present, the strategies for preventing and treating post-cardiac arrest brain injury can be divided into intervention measures aiming at reducing excitotoxicity, improving neuron metabolism, limiting mitochondrial injury and reducing neuroinflammation. Although various strategies for protecting brain function after cardiac arrest are attempted at present, no neuroprotective drug can effectively improve the survival rate or neurological function prognosis of the patient. NMDA, AMPA receptor antagonists, antioxidants, mitochondrial function protectors, anti-inflammatory drugs, immune regulation and other treatment means are still in preclinical research. In-depth understanding of the complex pathophysiological changes after post-cardiac arrest brain injury is the basis and key to the development of brain function protection agents for patients with cardiac arrest. SUMMARY

[0003] The application provides application of a TRPML1 specific small molecule inhibitor ML-SI3 in preparation of a drug for treating or preventing brain injury caused by cardiac arrest.

[0004] The first aspect of the application provides application of a TRPML1 specific small molecule inhibitor ML-SI3 in preparation of a drug for treating or preventing brain injury.

[0005] According to some embodiments of the application, the brain injury comprises brain injury caused by cardiac arrest.

[0006] According to some embodiments of the application, the TRPML1 specific small molecule inhibitor ML-SI3 has the following structure:

[0007]

[0008] According to some embodiments of the application, in the preparation of a drug for treating or preventing brain injury, the effective dose of the TRPML1 specific small molecule inhibitor ML-SI3 is 1-4 mg / kg.

[0009] In a second aspect of the application, a drug for treating brain injury caused by cardiac arrest is provided, comprising a pharmaceutically active ingredient and a pharmaceutically acceptable excipient, wherein the pharmaceutically active ingredient comprises a TRPML1 specific small molecule inhibitor ML-SI3.

[0010] According to some embodiments of the drug for treating brain injury caused by cardiac arrest, the content of the pharmaceutically active ingredient in the drug is 1-4 mg / kg.

[0011] According to some embodiments of the drug for treating brain injury caused by cardiac arrest, the content of the pharmaceutically active ingredient in the drug is 3 mg / kg.

[0012] According to some embodiments of the drug for treating brain injury caused by cardiac arrest, the dosage form of the drug comprises injection, tablet, capsule, granule, aerosol or oral liquid.

[0013] The beneficial effects of the application include that the TRPML1 specific small molecule inhibitor ML-SI3 can significantly reduce the degree of injury of neurons in brain injury rats, improve the learning and memory ability of brain injury rats, and improve the motor dysfunction of brain injury rats, and has a comprehensive protective effect on brain function of rats after cardiopulmonary resuscitation after cardiac arrest.

[0014] The TRPML1 specific small molecule inhibitor ML-SI3 provides a new drug for brain function protection of cardiac arrest patients, and has good market value and clinical application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 A preparation and administration time mode diagram for brain injury rats after cardiac arrest (CA) cardiopulmonary resuscitation (CPR) and restoration of spontaneous circulation (ROSC);

[0016] Figure 2a A brain cortex section Nissl staining diagram of brain injury rats after administration of ML-SI3;

[0017] Figure 2b This is a statistical chart of the degree of cerebral cortical damage (Nissl body ratio) in brain-injured rats after ML-SI3 administration;

[0018] Figure 3a This is an analysis diagram of the water maze test results of brain-injured rats after ML-SI3 administration;

[0019] Figure 3b This is a statistical graph showing the time rats spent in the target quadrant on the sixth day after ML-SI3 administration, used to evaluate their spatial memory ability (longer time means better memory ability);

[0020] Figure 4 This is an analysis chart of the balance beam test results in brain-injured rats after ML-SI3 administration;

[0021] Figure 5 This is an analysis diagram of the results of the rotarod test in brain-injured rats after administration of ML-SI3;

[0022] Figure 6a This is an analysis diagram of the results of the open field test in brain-injured rats after administration of ML-SI3;

[0023] Figure 6b The middle left picture shows the total distance traveled by brain-injured rats in an open field after administration of ML-SI3. Figure 6b The middle right figure shows the average movement rate statistics of brain-injured rats in an open field after ML-SI3 administration. DETAILED DESCRIPTION

[0024] The embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to be used to explain the present invention, but should not be understood as limiting the present invention.

[0025] In the present invention, the terms "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and the features of different embodiments or examples without contradiction.

[0026] The application provides the application of the TRPML1 specific small molecule inhibitor ML-SI3 in preparing a brain function protection agent after cardiac arrest. Specifically, when the inhibitor is used in preparing a drug for treating brain injury of a rat after cardiac arrest, the inhibitor can significantly reduce the injury degree of neurons of the brain injury rat, improve the learning and memory ability of the brain injury rat, reduce the movement disorder of the brain injury rat, and has a comprehensive protection effect on the brain function of the rat after cardiac arrest and cardiopulmonary resuscitation.

[0027] When the TRPML1 specific small molecule inhibitor ML-SI3 is used in treating a brain injury rat after cardiac arrest and cardiopulmonary resuscitation, the effective dose is 1-4 mg / kg, preferably 3 mg / kg.

[0028] Brain injury after cardiac arrest is the main cause of death of a patient after cardiac arrest and cardiopulmonary resuscitation. Excessive zinc and iron ions in neurons participate in the pathological process of brain injury after cardiac arrest, but the source and mechanism of these metal ions are not clear. The inventors have verified through experiments that lysosomal ion channel TRPML1 mediates zinc ion release to cause autophagy flow blockage and mitochondrial damage to cause myocardial cell damage, but the role of TRPML1 in brain injury after cardiac arrest is not clear. The inventors have found that down-regulation of TRPML1 can improve the brain injury degree of a rat after cardiopulmonary resuscitation and reduce the zinc and iron ion levels in neurons. Therefore, it is speculated that the TRPML1 channel is a key factor for excessive zinc and iron ions in neurons after cardiac arrest. This study will provide a solid theoretical basis for more accurately understanding the pathological mechanism of brain injury after cardiac arrest and provide a new target—TRPML1 for protecting brain function after cardiac arrest in clinic. The following is an experiment of the application to verify the therapeutic effect of the TRPML1 specific small molecule inhibitor ML-SI3 on a brain injury rat after cardiac arrest and cardiopulmonary resuscitation.

[0029] 1. Establishment of a CA / CPR self-circulation recovery model (ROSC) of an SD rat and intervention of ML-SI3.

[0030] The ML-SI3 intervention mode diagram of the ROSC rat / model group is shown in Figure 1 .

[0031] 1.1 Test instrument: respirator, cold light source, monitor, oxygen cylinder, heating pad;

[0032] 1.2 Surgical instruments: intravenous puncture needle (24G), tracheal catheter (14G), surgical suture (5-0, 2-0), shaving knife, cotton swab, cotton ball, gauze, medical adhesive tape, syringe, rubber band, air reservoir;

[0033] 1.3 Test materials: anesthetic 4% chloral hydrate (3-4 ml / 500 g), adrenaline (1:50 dilution, 1-2 ml at a time, additional 1-2 ml / time according to the condition of the rat), furosemide (10 mg / ml), 1 ml of the stock solution for each rat, injected during rescue (when there is no diuretic, the infusion volume should be reduced as much as possible), rocuronium (stock solution 1:10 dilution, 1 mg / ml; first dose 0.5 ml, additional 0.2 ml / time according to the condition of the rat, a total of about 1 ml for a 450 g rat; oxygen inhalation for 3 min after injection; a small amount of diaphragm muscle strength can be retained.), anticoagulant heparin (0.5 ml heparin + 500 ml normal saline), normal saline.

[0034] 1.4 Test animals: male SD rats 450-500 g are optimal. Rats weighing about 400 g can also be used. The number of rats is 24, which are randomly divided into 4 groups, with 6 rats in each group.

[0035] 1.5 Chloral hydrate 4%, onset time 2 min, anesthesia maintenance time 95 min; every 1 h injection 0.5 ml to maintain anesthesia, pay attention to keep warm, heating pad, limb fixation, door tooth fixation. Stick the testis to the lower abdomen, remove the hair near the heart end of the tail, and tie it with a rubber band. The tail is cut layer by layer, the tail artery is separated with a glass needle, the heart end is clamped, 3 cm-4 cm tail artery is inserted into 3 sutures, the heart end is clamped, the lower end of the suture (1 root ligation far end, placed in the tail end pad under the tail artery, venous indwelling needle puncture, open the blood vessel clamp, the venous indwelling needle is gently pushed in, the upper end of the suture is 2 roots) is connected with the one end of the converter, the other end of the converter is connected with the monitor, the blood pressure (normal value: 120-90 / 80 mmHg) and heart rate (normal value: 250-600 times / min) are monitored.

[0036] 1.6 Tracheal intubation, straighten the tongue, and remove oral secretions with a cotton swab. Align the cold light source with the throat of the rat, observe the epiglottis site, and insert the tracheal catheter (tracheal catheter tip upward) at the moment of epiglottis opening. Pull out the tube core. After successful intubation, inhale pure oxygen for 5 min, and then inject muscle relaxant when the blood pressure is stable. The breathing machine (parameter setting: RR 50-75 times / min, tidal volume 4-5 ml) is connected with the oxygen storage bag filled with oxygen at one end, and the tracheal catheter at the other end, and the airway pressure is monitored (normal range before muscle relaxation 8-12 cmH2O, airway pressure <20 cmH2O after muscle relaxation, possible lung fluid or small airway obstruction, appropriate increase in frequency and reduction in oxygen flow to ensure minute ventilation. Airway pressure is too small, which may indicate that the catheter has come out).

[0037] 1.7 Induce cardiac arrest, give muscle relaxant, inject rocuronium 0.5 mg / time (two slow administrations to avoid excessive blood pressure fluctuation), and clamp the tracheal catheter when spontaneous breathing disappears.

[0038] 1.8 Rescue, first should observe the rat heart beat, combined with mean arterial pressure dropped to 40mmHg, try to start timing: 35s-60s, start rescue when the heart fibrillation no ejection fraction and MAP close to 30mmHg: airway management: open airway, cardiopulmonary resuscitation: index finger, middle finger in the lower half of the sternum for cardiac compression (200 times / min). Fluid management: epinephrine (1-2ml), 1min after no spontaneous circulation (systolic blood pressure 65-90mmHg), repeat injection of epinephrine. Sodium bicarbonate (1ml), normal saline (0.5-1ml / time). Cardiopulmonary resuscitation more than 5-10min still no spontaneous circulation, then give up rescue.

[0039] 1.9 extubation, when the pharyngeal reflex recovery, 5min blood pressure can be maintained by inhaling air, that is, the tracheal tube can be removed. Ligation of blood vessels, suture incision.

[0040] The rats were divided as follows:

[0041] Sham group: the control rats in the sham operation group were given normal saline intraperitoneally;

[0042] ML-SI3 group: the control rats in the sham operation group were given ML-SI3 (3mg / kg) intraperitoneally;

[0043] ROSC group: modeling group; SD rats were given normal saline intraperitoneally after CA / CPR self-circulation recovery model;

[0044] ROSC+ML-SI3 group: modeling group combined with ML-SI3 administration; SD rats were given ML-SI3 intraperitoneally after CA / CPR self-circulation recovery model.

[0045] 2. Effect of TRPML1 specific small molecule inhibitor ML-SI3 administration on the degree of damage to damaged rat neurons

[0046] 2.1 Measurement method:

[0047] After modeling for one day, the control group (Sham), modeling group (ROSC), modeling combined with ML-SI3 group (ROSC+ML-SI3), control combined with ML-SI3 (Sham+ML-SI3) rats were anesthetized, and the left ventricle was punctured and perfused with PBS buffer.

[0048] The whole brain was collected, fixed in 4% paraformaldehyde at room temperature for 24 hours, then embedded in paraffin, cut into 3 pm thick sections with a microtome, and deparaffinated in xylene. After 30 minutes, the sections were transferred to 100%, 95%, 90%, 80%, and 70% ethanol solutions, respectively, for 5 minutes each, and finally to distilled water for 5 minutes. The deparaffinized sections were stained with 1% thionine. The staining solution was left to stand for 10 minutes, and then washed with distilled water. After staining, the sections were dehydrated in ethanol and then transferred to xylene for 5 minutes, and finally sealed with neutral glue for storage. Under a microscope, the Nissl bodies appeared dark, indicating damaged neurons, and the results are shown in Figs. 1A-1D. Figure 2a and Figure 2b

[0049] As can be seen from Figure 2a : Compared with the model group (ROSC; lower left panel), the ML-SI3 administration group (ROSC+ML-SI3; lower right panel) significantly reduced the number of dark blue Nissl bodies in the cerebral cortex tissue of the model rats, indicating that ML-SI3 administration can significantly reduce the degree of neuronal damage in brain-injured rats. The sham operation group (Sham+Vehicle; upper left panel) and the sham operation+ML-SI3 group (Sham+ML-SI3; upper right panel) are negative controls for the model rats, and the Nissl body staining of the rat cerebral cortex tissue is negative. The scale of the staining picture is 50 microns. Figure 2b The proportion of damaged neurons (Nissl staining positive) in the cortical neurons of rats in each group.

[0050] 3. Effect of ML-SI3 administration on the learning and memory ability of brain-injured rats

[0051] 3.1 Measurement method

[0052] After ROSC for five days, the control group (Sham), model group (ROSC), model combined with ML-SI3 group (ROSC+ML-SI3), and control combined with ML-SI3 group (Sham+ML-SI3) were subjected to the Morris water maze test on the sixth day. All data were statistically analyzed and plotted using Graphpad 7.0 software.

[0053] Acquired training

[0054] (1) The rat was placed head-first into the water, and the starting position was randomly selected from one of the four directions: east, west, south, and north. The time (s) taken by the animal to find the underwater platform was recorded. If this time exceeded 60 s in the first few training sessions, the animal was guided to the platform. The animal was allowed to stay on the platform for 10 s.

[0055] ​(2) Remove the animal and dry it. If necessary, put the rat under a 150W incandescent lamp for 5 min and put it back into the cage. Train the animal 4 times a day, with an interval of 15-20 min between two training sessions, for 5 consecutive days.

[0056] Probe training

[0057] The second day after the last acquisition training, remove the platform and start the 60s probe training. Put the animal into the water from the opposite quadrant of the original platform quadrant. Record the time spent in the target quadrant and the number of entries into the target quadrant as the index of spatial memory.

[0058] Place training

[0059] Determine the working memory of the animal. The second day after the probe training, start the 4-day place training. Put the platform in the opposite quadrant of the original platform quadrant, and the method is the same as the acquisition training. Train 4 times a day. Record the time to find the platform, the swimming distance, and the swimming speed each time.

[0060] Place probe training

[0061] The second day after the last place training. The method is similar to the above-mentioned probe training. Record the time spent in the target quadrant and the number of entries into the target quadrant within 60s, and the results are shown in Figure 3a and Figure 3b .

[0062] From Figure 3a and Figure 3b , it can be seen that: the water maze trajectory chart shows that compared with the model group rats (ROSC), the ML-SI3 administration group rats (ROSC+ML-SI3) can find the target quadrant faster, and stay in the target quadrant for a longer time within 60 seconds. For comparison of the specific time of each group of rats staying in the target quadrant within 60 seconds, see the statistical chart. The water maze test shows that ML-SI3 administration can significantly improve the learning and memory ability of brain injury rats.

[0063] 4. The effect of ML-SI3 administration on the motor disorder of brain injury rats

[0064] 4.1 Measurement method

[0065] After ROSC for two days, on the third day, rats in the control group (Sham), the model group (ROSC), the model combined with ML-SI3 group (ROSC+ML-SI3), and the control combined with ML-SI3 group (Sham+ML-SI3) were tested on the balance beam. All data were statistically analyzed and statistical charts were made using Graphpad 7.0 software.

[0066] Balance beam training

[0067] Before formal testing, rats are trained on the balance beam. First, rats are familiarized with the experimental environment by allowing them to spend some time in the laboratory. Next, rats are gradually trained on the balance beam, starting with a wider beam and allowing them to walk multiple times until they become proficient. Then, the diameter of the beam is gradually reduced to allow rats to adapt to different diameters. The training time can be determined based on the rats' adaptation level.

[0068] Formal operation

[0069] Rats are placed at the starting end of the balance beam. Timing begins, and the time it takes for the animal to walk to the other end of the beam (passing time) is observed. The balance beam times of rats in each group are statistically analyzed, and the results are shown in Figure 4

[0070] As can be seen from Figure 4 , compared with the model group (ROSC), ML-SI3 administration (ROSC+ML-SI3) can significantly reduce the balance beam time of the model rats, indicating that ML-SI3 administration can significantly reduce the motor impairment of brain injury rats.

[0071] 5. Effect of ML-SI3 administration on motor impairment in brain injury rats.

[0072] Two days after ROSC, the control group (Sham), model group (ROSC), model combined with ML-SI3 group (ROSC+ML-SI3), and control combined with ML-SI3 (Sham+ML-SI3) rats are tested on the rotating rod. All data are statistically analyzed and plotted using Graphpad 7.0 software.

[0073] 5.1 Measurement method

[0074] The rat rotating rod experiment procedure is as follows:

[0075] a. Place the rat on the rotating rod, start the rotating rod, and gradually increase the speed;

[0076] b. Observe the behavior of the rat, record the time of continuous rotation and the speed of the rotating rod;

[0077] c. When the rat falls or loses balance, record the falling time and the speed of the rotating rod;

[0078] d. Perform multiple tests to determine the rat's endurance and motor ability indicators;

[0079] e. Statistically analyze the falling time of rats in each group;

[0080] The results are shown in Figure 5 .

[0081] From​Figure 5 As can be seen: compared with the modeling group (ROSC), ML-SI3 administration (ROSC+ML-SI3) can significantly prolong the time of rats falling on the rotating rod, indicating that ML-SI3 administration can significantly alleviate the motor dysfunction of brain injury rats.

[0082] 6. The effect of ML-SI3 administration on the motor dysfunction of brain injury rats.

[0083] 6.1 Measurement method

[0084] Two days after ROSC, the control group (Sham), modeling group (ROSC), modeling combined with ML-SI3 group (ROSC+ML-SI3), and control combined with ML-SI3 (Sham+ML-SI3) rats were tested in the open field on the third day. All data were statistically analyzed and graphed using Graphpad 7.0 software.

[0085] Experimental preparation

[0086] Experimental device: The size of the open field device used for rats is usually 100cm×100cm×40cm.

[0087] Environmental conditions: The laboratory should be kept quiet, and the lighting conditions should be controlled at a moderate level to reduce the impact of external factors on animal behavior.

[0088] Adaptation period: Before the experiment, rats should be adapted to the laboratory environment for at least 30 minutes to reduce stress reactions to the new environment.

[0089] Experimental steps

[0090] Place the animals: At the beginning of the experiment, gently place the rats in the center or a corner of the open field device.

[0091] Behavior observation: Start recording the behavior of rats in the open field. The observation time is usually 5 to 10 minutes, which is the standard length in most studies.

[0092] Behavioral indicators: Record the behavior of rats, including the time spent in the center and edge of the open field, movement distance, cleaning behavior, standing behavior, etc.

[0093] Data recording: Use cameras and motion tracking software to record and analyze the behavior of rats to improve the accuracy and objectivity of the data.

[0094] Data analysis

[0095] Behavior analysis: According to the video and motion tracking software data, analyze the behavior patterns of rats, including movement trajectory, stay area, etc.

[0096] Statistical processing: data were analyzed using One-way ANOVA to draw scientific conclusions, and the results are shown in Figure 6a and Figure 6b

[0097] From Figure 6a and Figure 6b It can be seen that: compared with the modeling group (ROSC), ML-SI3 administration (ROSC+ML-SI3) can significantly increase the total distance and speed of rats in the open field, and these behavioral changes indicate that ML-SI3 administration can significantly reduce the motor dysfunction of brain injury rats.

[0098] The inventors believe that the TRPML1 specific small molecule inhibitor ML-SI3 can significantly improve the application of brain injury function in rats after cardiopulmonary resuscitation after cardiac arrest, reduce the degree of neuronal injury in brain injury rats, improve the learning and memory ability of brain injury rats, and reduce the motor dysfunction of brain injury rats.

[0099] The above experimental results prove that: the TRPML1 small molecule inhibitor ML-SI3 has a comprehensive protective effect on the brain function of rats after cardiopulmonary resuscitation after cardiac arrest. The application of the TRPML1 specific small molecule inhibitor ML-SI3 described in the present application in the preparation of a drug for treating or preventing brain injury provides a new drug for brain function protection for cardiac arrest patients, and has good market value and clinical application prospect.

[0100] Although the above embodiments have been shown and described, it can be understood that the above embodiments are exemplary and cannot be understood as limiting the present application, and the changes, modifications, replacements and variations of the above embodiments made by those of ordinary skill in the art are within the protection scope of the present application.​

Claims

1. Use of a TRPML1-specific small molecule inhibitor, ML-SI3, as an active ingredient in the preparation of a drug for treating or preventing brain damage caused by cardiac arrest; The structural formula of the TRPML1-specific small molecule inhibitor ML-SI3 is shown below: 。 2. The use according to claim 1, characterized in that The content of the active pharmaceutical ingredient in the medicine is 1-4 mg / kg.

3. The use according to claim 1, characterized in that The content of the active ingredient in the medicine is 3 mg / kg.

4. The use according to claim 1, characterized in that The dosage form of the drug is selected from injection, tablet, capsule, granule, aerosol or oral liquid.

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