Uses of P2Y12 receptors and their antagonists

Abstract

This invention relates to the field of biomedicine, and particularly to novel applications of the P2Y12 receptor and its antagonists. This invention discloses the use of the P2Y12 receptor as a target in screening GABA. A The application of P2Y12 receptor antagonists, specifically NMDA receptor anesthetics, in interventional drugs. This invention also discloses the use of the P2Y12 receptor antagonist ticagrelor in the preparation of GABA. A The use of receptor-based anesthetics in interventional drugs can significantly reduce GABA levels. A The duration of loss of consciousness induced by receptor-type anesthetics. This invention also discloses the effect of knocking out the P2Y12 receptor in microglia on GABA. A Application of general anesthesia induced by the receptor anesthetic sodium pentobarbital and the NMDA receptor anesthetic ketamine.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to novel uses of the P2Y12 receptor and its antagonists. Background Technology

[0002] General anesthesia can induce reversible loss of consciousness and pain sensation, and is widely used in modern surgical procedures and related medical examinations. There are many types of general anesthetics, each targeting different neuronal receptors or molecular targets, including opioid receptors and gamma-aminobutyric acid type A receptors (GABA). A General anesthetics, including receptor complexes such as GABA receptors, benzodiazepine receptors and GABA receptor-coupled chloride channels, N-methyl-D-aspartate (NMDA) receptors, and β2-adrenergic (β2A) receptors, exert their effects on general anesthesia. To achieve anesthesia of appropriate depth, several general anesthetics are often used in combination clinically. These anesthetics work synergistically with multiple neuronal receptors or molecular targets to produce the desired anesthetic effect. Although modern anesthesia techniques generally ensure the safety and effectiveness of the anesthesia process, a small percentage of patients still experience a series of complications due to slow recovery from anesthesia, such as permanent damage to the central nervous system and postoperative delirium, which can seriously affect the patient's prognosis.

[0003] Current technologies utilize only anesthetic arousal promoters that target opioids and benzodiazepines. Flumazenil is the only existing arousal promoter for benzodiazepine receptor-targeting anesthetics; naloxone is the only arousal promoter for opioid receptor-targeting opioids. There are no effective antagonists for other target anesthetics. Furthermore, the most commonly used general anesthetics in clinical practice primarily act on GABA. A The receptors are NMDA and NMDA receptors; therefore, finding drugs that specifically antagonize these two types of receptors will play an important role in anesthesia recovery.

[0004] Specifically, current interventions to promote rapid awakening in anesthetized patients are very limited, primarily targeting a series of receptors on neurons. For example, sedation or respiratory depression caused by opioids (which act on opioid receptors) can be treated with opioid antagonists like naloxone to promote awakening or relieve respiratory depression; benzodiazepines (which act on GABA) can also be used. A Anesthesia induced by benzodiazepine receptors (one of the components of the receptor complex) can be induced by the selective benzodiazepine receptor antagonist flumazenil. However, rapid injection of these anesthetic arousal-promoting drugs can cause side effects such as anxiety, palpitations, and fear. In addition, apart from opioids and benzodiazepines, there are no effective antagonists for other commonly used anesthetics in clinical practice.

[0005] In clinical practice, general anesthetics are often used in combination with anesthetics that target different receptors, while GABA... AGABA- and NMDA receptor anesthetics are among the most commonly used types. General anesthesia induced by these receptors often results in slow recovery and can cause severe central nervous system damage. However, there is currently a lack of interventional drugs targeting this receptor-induced general anesthesia. Therefore, the search for drugs targeting GABA is crucial. A Interventional drugs for receptor and NMDA receptor anesthetics, for those using GABA. A The restoration of normal physiological function in patients under anesthesia with GABAergic or NMDA receptor anesthetics is of significant clinical importance. Specifically, it involves identifying GABAergic receptor anesthetics. A Arousal-promoting drugs for receptor and NMDA receptor anesthetics are of great significance for reversing the state of anesthetized patients.

[0006] The P2Y12 receptor is a member of the P2 family of purine receptors, a G protein-coupled receptor composed of 342 amino acids. It is primarily expressed on platelets in the peripheral nervous system, and on microglia in the brain in the central nervous system. The natural agonist of the P2Y12 receptor is ADP. Previous studies have shown that ADP acting on P2Y12 receptors on platelets can induce platelet aggregation and activation, and acting on microglia can induce protrusion movement and migration. Currently, P2Y12 receptor antagonists are used clinically to treat thrombotic cardiovascular disease, stroke, acute pancreatitis, and other diseases. Ticagrelor, a reversible P2Y12 receptor antagonist developed by AstraZeneca, is widely used clinically. Clinical studies have shown that 12 months of ticagrelor treatment can significantly reduce the incidence of cardiovascular death / myocardial infarction / stroke without increasing the risk of bleeding. Recent studies have shown that P2Y12 receptor-mediated signaling in microglia plays an important role in regulating neuronal activity and can inhibit overactivated neurons in disease states such as cerebral ischemia and epilepsy.

[0007] The invention CN113267636A also discloses the application of P2Y12 receptor and its antagonists in the diagnosis and treatment of acute pancreatitis.

[0008] However, there are currently no reports on the association between the P2Y12 receptor and general anesthesia and anesthetic recovery. Summary of the Invention

[0009] The technical problem to be solved by the present invention is to provide a new use for the P2Y12 receptor and its antagonist.

[0010] To address the aforementioned technical problems, this invention provides the P2Y12 receptor as a target for screening GABA. A Application of receptor and NMDA receptor anesthetics in interventional drugs.

[0011] This invention also provides a P2Y12 receptor antagonist for the preparation of GABA. AApplication of receptor-type anesthetics in interventional drugs.

[0012] As an improvement to the application of this invention: the P2Y12 receptor antagonist is ticagrelor.

[0013] As a further improvement to the application of this invention: intervention with GABA A The effect of general anesthesia induced by receptor-type anesthetics.

[0014] As a further improvement to the application of this invention: significantly reducing GABA A Duration of loss of consciousness induced by receptor anesthetics.

[0015] As a further improvement to the application of this invention: GABA A The receptor-type anesthetic is sodium pentobarbital.

[0016] In summary, this invention provides information on the P2Y12 receptor and its antagonist in the intervention of GABA. A Application in the effects of general anesthesia induced by receptor-type anesthetics.

[0017] This invention also provides a method for knocking out the P2Y12 receptor in microglia to inhibit GABA. A Application of general anesthesia induced by the receptor anesthetic sodium pentobarbital and the NMDA receptor anesthetic ketamine.

[0018] This invention utilizes ticagrelor, a clinically widely used reversible P2Y12R antagonist, or knockout of the p2y12r gene in microglia to significantly alleviate GABA. A General anesthesia induced by receptor-specific anesthetics is characterized by a shortened duration of loss of consciousness and a reduced depth of anesthesia in mice. Therefore, this invention provides a method for intervening in GABA. A The general anesthesia induced by receptor-type anesthetics provides a new target, and this invention was completed based on this.

[0019] Through extensive and in-depth research, the inventors unexpectedly discovered for the first time that the microglia-specific P2Y12 receptor in the brain plays an important target and regulatory role in the maintenance of general anesthesia. Specifically, P2Y12R is specifically expressed in microglia in the brain. Using the clinically widely used reversible P2Y12R antagonist ticagrelor or knocking out the p2y12r gene in microglia can significantly alleviate GABA. A General anesthesia induced by receptor-specific anesthetics was characterized by a shortened loss of consciousness time and a reduced depth of anesthesia in experimental mice. This lays the foundation for further development of GABA. A This laid the foundation for interventional drugs using receptor-based anesthetics.

[0020] This invention is the first to discover that antagonism or knockout of the P2Y12 receptor can significantly alleviate GABA.A The depth of anesthesia induced by receptor-type anesthetics can be reduced, and the duration of loss of consciousness induced by anesthesia can be shortened.

[0021] This invention employs ticagrelor, a clinically widely used antagonist targeting the P2Y12 receptor. Experiments in this invention demonstrate that ticagrelor can reduce GABA levels. A The depth of anesthesia induced by receptor-type anesthetics is increased, and the time of loss of consciousness induced by anesthesia is shortened. Ticagrelor can be injected intracranially approximately 30 minutes before the administration of the anesthetic. Attached Figure Description

[0022] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0023] Figure 1 The P2Y12 receptor antagonist ticagrelor can significantly shorten the duration of general anesthesia induced by sodium pentobarbital;

[0024] Figure 1 middle:

[0025] A is a flowchart of the experiment, including the time of lateral ventricle cannula implantation, cannula drug administration time, and anesthesia record time points;

[0026] B. Intraventricular injection of the P2Y12 receptor antagonist ticagrelor did not affect the time it took for mice to enter anesthesia.

[0027] In C, intraventricular injection of the P2Y12 receptor antagonist ticagrelor significantly shortened the duration of general anesthesia induced by sodium pentobarbital.

[0028] Figure 2 Knockout of the microglia-specific P2Y12 receptor can significantly shorten GABA. A The duration of general anesthesia induced by receptor and NMDA receptor anesthetics;

[0029] Figure 2 middle:

[0030] A is a flowchart of the experiment, including the time points of tamoxifen injection to induce gene knockout in mice in the control group and microglia P2Y12 receptor conditional knockout group, and anesthesia records.

[0031] B indicates that knocking out the P2Y12 receptor in microglia does not affect the time to onset of anesthesia induced by pentobarbital sodium, but significantly shortens the duration of general anesthesia induced by pentobarbital sodium.

[0032] C indicates that knocking out the P2Y12 receptor in microglia does not affect the time to onset of ketamine-induced anesthesia, but significantly shortens the duration of ketamine-induced general anesthesia. Detailed Implementation

[0033] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0034] Example 1: The P2Y12R receptor antagonist ticagrelor was used to target GABA. A Effects of receptor anesthetics

[0035] (1) Laboratory animals

[0036] Twelve eight-week-old SPF-grade male C57bl / 6j mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. and housed in an SPF-grade environment.

[0037] (2) Lateral ventricle cannula implantation (routine experimental method)

[0038] All mice administered intraventricularly were 8-week-old male C57BL6 / j rats. All mice were anesthetized via intraperitoneal injection of a mixture of ketamine (100 mg / kg) and toluenethiazide (10 mg / kg) according to their body weight. After anesthesia, the mice's eyes were covered with ointment, the hair above the skull was shaved, and the skull was disinfected with iodine. The mice were then fixed to a stereotaxic apparatus; the scalp was cut with surgical scissors, and the skull surface was cleaned with medical cotton swabs. A cannula was then implanted at the location of the lateral ventricle based on brain mapping. The cannula placement coordinates were (AP, -0.45 mm; ML, 1.0 mm; DV, -2.0 mm; i.e., stereotaxic coordinates of the brain). To prevent cannula obstruction, a cannula obturator was inserted into the cannula, which was then fixed with dental cement. After the cement hardened, the mice were removed and allowed to recover for 7 days. On the day of administration via cannula, remove the occluder from the cannula 30 minutes before the experiment, and insert a polyethylene tube connected to a Hamilton microsyringe into the cannula. Manually control the microsyringe to inject the agonist or antagonist for 2 minutes. After the injection is completed, stop the injection for at least 1 minute to allow the drug to fully enter and diffuse.

[0039] (3) Statistics on anesthetic injection and anesthesia duration

[0040] Animals with implanted cannulas were divided into a control group and a P2Y12R antagonist group, with 6 mice in each group. On the day of the experiment, the weight of each animal was measured. Thirty minutes before the injection of the anesthetic, the control group was injected with 2 μL of normal saline, and the P2Y12R antagonist group was injected with ticagrelor (80 μg ticagrelor / kg body weight, dissolved in 2 μL of normal saline). GABA was then injected intraperitoneally according to the animal's weight. AThe recipient anesthetic sodium pentobarbital (100 mg sodium pentobarbital / kg body weight, dissolved in 0.2 ml physiological saline) was administered to the animals under a video recorder. The specific time when each mouse lost its righting reflex was then examined, and the specific time when each mouse regained its righting reflex was recorded by video. The duration of loss of consciousness for each mouse under general anesthesia was then calculated.

[0041] (4) Statistical methods

[0042] Data are expressed as mean ± standard error (SEM), and statistical significance analysis was performed using Graphpad Prism. Unpaired student's t-tests were used to compare two groups. For comparisons of more than two groups, one-way ANOVA and Bonferroni's test were used to calculate statistical differences among groups. In all statistical results, no significant difference was indicated by the ns notation; p < 0.05 was considered significant; * indicated p < 0.05; ** indicated p < 0.01; and *** indicated p < 0.001.

[0043] (5) Experimental Results

[0044] Eight-week-old C57bl / 6j mice were given an intraventricular injection of either the P2Y12 receptor antagonist ticagrelor or saline. Thirty minutes later, GABA was administered intraperitoneally according to the weight of each mouse. A The recipient was given sodium pentobarbital (100 mg / kg) as an anesthetic, and the anesthesia process was recorded using a video recorder. Figure 1 A). During the anesthesia induction phase, the time point at which each mouse lost its righting reflex (LORR, i.e., entered general anesthesia) was manually checked; and the time point at which the righting reflex recovered (RORR, i.e., awakened from general anesthesia) was determined by video. The durations of LORR and RORR were calculated and analyzed.

[0045] Experiments have shown that the use of the P2Y12 receptor antagonist ticagrelor can significantly shorten the duration of RORR (return of remission) in general anesthesia induced by pentobarbital sodium, while there is no statistically significant difference in LORR (lost return of remission) upon entering general anesthesia. Figure 1 (BC) indicates that administering the P2Y12 receptor antagonist ticagrelor to the brain can shorten the GABAergic response. A Duration of general anesthesia induced by the receptor anesthetic sodium pentobarbital.

[0046] Example 2: Effects of P2Y12 receptor knockout on GABA in microglia A Effects of NMDA receptor anesthetics

[0047] (1) Laboratory animals

[0048] Eight-week-old male P2y12r f / f (Control group) and Cx3cr1 CreER / + ::P2y12r f / f Mice in the (microglia P2Y12 receptor conditional knockout group) were donated to the Peng Jiyun research group at Nanchang University. At eight weeks of age, the animals were gavaged with tamoxifen (100 mg tamoxifen / kg body weight, dissolved in 0.2 ml physiological saline) every 48 hours for eight consecutive weeks to ensure effective knockout of the P2Y12 receptor in microglia. After two weeks of normal rearing, subsequent experiments were conducted to ensure specific knockout of the P2Y12 receptor only in microglia.

[0049] Cx3cr1 CreER / + ::P2y12r f / f , namely CX3CR1 in "Microglial P2Y12 Receptor Regulates Seizure-Induced Neurogenesis and Immature Neuronal Projections" CreER / + :P2Y12 fl / fl .

[0050] (2) Statistics on anesthetic injection and anesthesia duration

[0051] Animals were divided into a control group (n=8) and a P2Y12 receptor conditional knockout group (n=10). On the day of the experiment, each animal was weighed, and GABA was injected intraperitoneally according to its weight. A The mice were given either the receptor anesthetic sodium pentobarbital (100 mg sodium pentobarbital / kg body weight, dissolved in 0.2 ml of physiological saline) or the NMDA receptor anesthetic ketamine (100 mg ketamine / kg body weight, dissolved in 0.2 ml of physiological saline). The animals were placed under a video recorder. The specific time when each mouse lost its righting reflex was then examined, and the specific time when each mouse regained its righting reflex was recorded by video. The duration of loss of consciousness for each mouse under general anesthesia was calculated.

[0052] (3) Statistical methods

[0053] Data are expressed as mean ± standard error (SEM) and statistical significance analysis was performed using Graphpad Prism. Unpaired student's t-tests were used to compare the two groups. In all statistical results, no significant difference was indicated by the ns notation; p < 0.05 was considered significant, * indicated p < 0.05, ** indicated p < 0.01, and *** indicated p < 0.001.

[0054] (4) Experimental Results

[0055] Give 8-week-old P2y12r f / f (Control group) and Cx3cr1 CreER / + ::P2y12r f / f (Microglia P2Y12 receptor conditional knockout group) The p2Y12r gene knockout was induced by tamoxifen gavage. After the final induction, both groups of animals were fed normally for two weeks. Because normal peripheral monocytes and macrophages are rapidly renewed, the renewed peripheral monocytes and macrophages after two weeks of feeding express the P2Y12 receptor. However, the normal renewal rate of microglia in the brain is extremely slow; therefore, the P2Y12 receptor in microglia was still knocked out, thus achieving microglia-specific P2Y12 receptor knockout. Figure 2 A).

[0056] On the day of anesthesia recording, the weight of each mouse was recorded, and then GABA was injected intraperitoneally according to the weight of each mouse. A The recipient was given sodium pentobarbital (100 mg / kg) as an anesthetic, and the anesthesia process was recorded using a video recorder. Figure 2 B). During the anesthesia induction phase, the time point at which each mouse lost the righting reflex (LORR, i.e., entered general anesthesia) was manually examined; and the time point at which the righting reflex recovered (RORR, i.e., awakening from general anesthesia) was determined via video. The durations of LORR and RORR were calculated and analyzed. It was found that specific knockout of the P2Y12 receptor in microglia significantly shortened the RORR duration of sodium pentobarbital-induced general anesthesia, while there was no statistically significant difference in the LORR at the point of entering general anesthesia. Figure 2 B).

[0057] Three days after the sodium pentobarbital anesthesia experiment was completed (when the sodium pentobarbital in the mice was basically metabolized and cleared), the weight of each mouse was recorded again. Then, based on the weight of each mouse, the NMDA receptor anesthetic ketamine (100 mg / kg) was injected intraperitoneally, and the anesthesia process was recorded under a video recorder. Figure 2C). During the anesthesia induction phase, the time point at which each mouse lost the righting reflex (LORR, i.e., entered general anesthesia) was manually examined; and the time point at which the righting reflex (RORR, i.e., awakening from general anesthesia) was determined via video. The durations of LORR and RORR were calculated and analyzed. It was found that specific knockout of the P2Y12 receptor in microglia significantly shortened the RORR duration of ketamine-induced general anesthesia, while there was no statistically significant difference in LORR upon entering general anesthesia.

[0058] These experimental results demonstrate that specifically knocking out the P2Y12 receptor in microglia of the brain can shorten the GABAergic reaction time. A Duration of general anesthesia induced by the receptor anesthetic sodium pentobarbital and the NMDA receptor anesthetic ketamine.

[0059] Finally, it should be noted that the above examples are merely some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the disclosure of the present invention should be considered within the scope of protection of the present invention.

Claims

1. P2Y12 receptor antagonists in the preparation of GABA A The application of receptor-type anesthetics in interventional drugs is characterized by: The P2Y12 receptor antagonist is ticagrelor, GABA. A The receptor-type anesthetic is sodium pentobarbital; interventional drugs are used to significantly reduce GABA. A Duration of loss of consciousness induced by receptor anesthetics.

2. The application according to claim 1, characterized in that: Intervention in GABA A The effect of general anesthesia induced by receptor-type anesthetics.

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