Application of cannabidiol in preparation of methylamphetamine addiction memory intervention medicine

Abstract

The invention discloses an application of cannabidiol in preparation of a methylamphetamine addiction memory intervention medicine. The research shows that the cannabidiol can slow down the formation of the methamphetamine addiction memory, the pharmacological mechanism of the cannabidiol in slowing down the methamphetamine addiction memory is explored, and the result shows that the cannabidiol can weaken the activation of a TRPV1 channel on hippocampus blotting cells and hinder the coding of the addiction memory blotting in mouse hippocampus; according to the invention, the pharmacological mechanism of cannabidiol in slowing down methamphetamine addiction memory is explored; and a new treatment strategy can be provided for drug rehabilitation treatment of methamphetamine addiction memory.

Description

Technical Field

[0001] The present invention relates to the technical field of addiction treatment and biopharmaceuticals, and particularly to the application of cannabidiol in the preparation of drugs for intervening methamphetamine addiction memory. Background Art

[0002] According to the World Drug Report 2024, there are approximately 292 million drug users globally, and drug addiction is a global public health problem. Addiction memory plays a key role in controlling drug cravings and relapse. The pathological persistence of addiction memory leads to recurrent drug-seeking and compulsive drug use, and drug addiction is regarded as a memory-related encephalopathy. Memory engrams, as the neural substrates of memory, store memory information and mediate recall. Addiction memory depends on the encoding of memory engrams, and capturing and labeling addiction memory engrams is the gold standard for evaluating memory formation. Currently, there is a lack of safe and effective treatment methods for drug addiction. Drug addiction treatment mainly focuses on psychological intervention, exercise therapy, and alternative therapy; intervening in addiction memory is a highly potential treatment method, and studying safe and effective intervention drugs is a scientific problem that urgently needs to be solved at present.

[0003] Cannabidiol is the main non-addictive component in the plant cannabis and has considerable therapeutic potential in neuropsychiatric diseases such as epilepsy, Alzheimer's disease, Parkinson's disease, depression, and anxiety. Current clinical data show that cannabidiol has good tolerance, and few adverse reactions occur at therapeutic doses for children and adults.

[0004] Therefore, this article proposes the application of cannabidiol in the preparation of drugs for intervening methamphetamine addiction memory. Summary of the Invention

[0005] In order to solve the above technical problems, the present invention designs the application of cannabidiol in the preparation of drugs for intervening methamphetamine addiction memory

[0006] In order to achieve the above technical effects, the present invention is realized through the following technical solutions: the application of cannabidiol in the preparation of drugs for intervening methamphetamine addiction memory.

[0007] Further, the cannabidiol is used during the reconsolidation window period after the extraction of addiction memory.

[0008] Further, the reconsolidation window period is within 3 hours after the extraction of addiction memory.

[0009] Further, the drug prepared from cannabidiol is directly infused into the hippocampus or nucleus accumbens of mice through a delivery catheter.

[0010] Further, the cannabidiol acts on the TRPV1 channel.

[0011] The beneficial effects of the present invention are as follows:

[0012] By means of the visual marking and detection of methamphetamine addiction memory engrams, the present invention explores the effect of cannabidiol on methamphetamine addiction memory; in order to explore the pharmacological mechanism of cannabidiol in alleviating methamphetamine addiction memory;

[0013] The research on the visual marking of methamphetamine addiction memory engrams of the present invention shows that both intra-nucleus accumbens cannabidiol infusion and intra-hippocampus cannabidiol infusion can reduce the formation of CPP addiction memory in mice; cannabidiol intervention can hinder the encoding of addiction memory engrams in the hippocampus of mice; at the same time, cannabidiol intervention can also weaken the activation of TRPV1 channels on hippocampal engram cells; this indicates that cannabidiol can be used to prepare drugs, pharmaceuticals, etc. for methamphetamine addiction memory intervention, and the application prospect is very broad. Brief Description of the Drawings

[0014] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0015] Figure 1 It is a schematic diagram of the principle for the c-fos-Tet3G; TRE-hM4Di-FLAG transgenic mice described in Example 1 of the present invention to capture and mark methamphetamine addiction memory engrams;

[0016] Figure 2 It is a chromatogram and mass spectrum for detecting the purity of methamphetamine by LC-MS described in Example 1 of the present invention;

[0017] Figure 3 It is a chromatogram and mass spectrum for detecting the purity of cannabidiol by LC-MS described in Example 1 of the present invention;

[0018] Figure 4 It is a schematic diagram of the effect of intra-hippocampus cannabidiol infusion on mouse addiction memory, memory engram encoding, and TRPV1 channel activation described in Example 1 of the present invention;

[0019] Figure 5 It is a schematic diagram of the effect of intra-nucleus accumbens cannabidiol infusion on mouse addiction memory, memory engram encoding, and TRPV1 channel activation described in Example 1 of the present invention;

[0020] Figure 6 It is a schematic diagram of the molecular docking prediction and SPR verification of the binding of cannabidiol to the human TRPV1 channel described in Example 1 of the present invention. Detailed Embodiments

[0021] The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

[0022] Example 1

[0023] An exploration of the application of cannabidiol in the preparation of drugs for intervening methamphetamine addiction memory, the steps are as follows:

[0024] S1. Drug preparation

[0025] The preparation and administration doses of methamphetamine and cannabidiol:

[0026] ① Methamphetamine hydrochloride (purity ≥ 99%, Figure 2 as shown, A is the chromatogram and B is the mass spectrum) was obtained from the Yunnan Institute for Drug Dependence Control and Prevention, dissolved in sterile physiological saline, and the prepared methamphetamine solution was injected into mice intraperitoneally at an injection concentration of 2 mg / kg.

[0027] ② Cannabidiol (purity ≥ 98%, Figure 3 as shown, C is the chromatogram and D is the mass spectrum) was purchased from Chengdu Pulisi Biotechnology Co., Ltd., dissolved in dimethyl sulfoxide (DMSO) and resuspended in sterile physiological saline, and the final DMSO concentration was 12%. The prepared cannabidiol solution was infused into the mouse hippocampus or nucleus accumbens through a microdialysis cannula at an infusion concentration of 10 μg / 2 μl.

[0028] S2. Marking and detection of methamphetamine addiction memory engrams in mice. By controlling the intake of doxycycline (Dox), the transgenic system was turned on or off, and the memory engrams were marked by the expression of the reporter gene FLAG; that is, Dox (4 mg / ml) was given to mice for drinking during methamphetamine administration, and Dox was in a closed state at other times, so as to capture and mark methamphetamine addiction memory engrams; the specific steps are as follows:

[0029] ① Adaptation: Three days before the experiment, c-fos-Tet3G; TRE-FLAG mice were placed in the CPP device every day for adaptation, 30 minutes per mouse.

[0030] ② Pre-detection: The mice were placed in the CPP device, the tracking system was started, and the residence time of the mice in the black box and the white box was recorded and the percentage was calculated. The box with a higher percentage was defined as the natural preference box of the mouse, and the box with a lower percentage was defined as the drug-paired box of the mouse.

[0031] ③ Administration: All mice were intraperitoneally injected with normal saline in the natural preference chamber for 2 days, and the mice were restricted in the natural preference chamber for 30 minutes after each injection; after the injection, the mice to be injected with methamphetamine were given Dox (4 mg / ml) to drink, and then the mice were intraperitoneally injected with methamphetamine in the drug-paired chamber for 4 days. The control group was injected with an equal volume of normal saline, and the mice were restricted in the drug-paired chamber for 30 minutes after each injection; after the injection, the mice were changed to normal drinking water, and then all mice were intraperitoneally injected with normal saline in the natural preference chamber for 2 days, and the mice were restricted in the natural preference chamber for 30 minutes after each injection.

[0032] ④ Post-detection: The mice after administration were placed in the CPP apparatus, the tracking system was started, and the residence time of the mice in the black chamber and the white chamber was recorded. The addiction memory of the mice was evaluated by the CPP score.

[0033] ⑤ Sampling: The mice were anesthetized by inhaling isoflurane using a small animal anesthesia machine, the chest cavity was quickly opened, and the mice were perfused through the heart with normal saline; after the organs such as the liver and lungs turned white, perfusion through the heart with 4% paraformaldehyde fixative was carried out, the head was quickly cut off to take the brain, and the whole brain was immersed in 4% paraformaldehyde fixative and stored at 4 °C for standby.

[0034] ⑥ Visualization labeling of memory engrams: Mouse brain sections were prepared using a cryostat for sectioning, and memory engram cells were co-labeled with c-fos antibody and FLAG antibody. The memory engram encoding was evaluated by the number of memory engram cells, and the phosphorylation level of TRPV1 channels on the engram cells was detected.

[0035] S3. Intervention strategy of cannabidiol

[0036] ① Intervention strategy: The intervention strategy of cannabidiol is directed at the reconsolidation window period (3 hours) after the extraction of addiction memory. During this window period, the addiction memory is in an unstable state and is easily modified and updated.

[0037] ② Administration route: Cannabidiol was directly infused into the mouse brain region through a drug delivery catheter. The mice were anesthetized by inhaling isoflurane using a small animal anesthesia machine, and micro-drug delivery cannulas were implanted into the hippocampus and nucleus accumbens of the mice respectively through brain stereotaxic positioning. The mice recovered for 1 week after the surgery.

[0038] ③Operation method: Fix the syringe needle on the micro-injection pump, fill the PU tube with mineral oil, connect the two ends of the PU tube to the syringe needle tip and the injection inner tube equipped with a locking nut respectively, start the micro-injection pump, and aspirate the drug. After each methamphetamine injection, use a small animal anesthesia machine to perform isoflurane inhalation anesthesia on the mice. After disinfecting the cannula with 75% alcohol, unscrew the catheter cap, insert the injection inner tube, and install the locking nut. Start the micro-injection pump and inject cannabidiol. The methamphetamine group injects an equal amount of sterile normal saline. After the drug injection is completed, unscrew the locking nut, pull out the injection inner tube, reinstall the catheter cap, and transfer the mice to a warm place to wake up.

[0039] S4. Detection of the binding of cannabidiol to the TRPV1 channel

[0040] ①Molecular docking: Obtain the protein structure of the human TRPV1 channel from the AlphaFold protein structure database and obtain the 3D chemical structure of cannabidiol from the PubChem chemical structure database. Predict the binding pocket and interaction of cannabidiol with the human TRPV1 channel through AutoDock software, and perform visual analysis of the binding pocket and interaction through PyMOL software;

[0041] ②SPR detection: Install the CM5 chip according to the standard operating procedure of the Biacore T200TM instrument. Use 10 mM NaAc buffer solutions with different pH values (pH 4.0, 4.5, 5.0, 5.5) to pre-enrich with the TRPV1 protein diluted to 20 μg / mL respectively. When the coupling amount is the largest at pH 5.0, then select 10 mM NaAc at pH 5.0 as the buffer solution for diluting the TRPV1 protein. After loading the EDC / NHS (1:1) solution to activate the chip, then load the TRPV1 protein diluted with 10 mM NaAc (pH 5.0) (concentration of 100 μg / mL) and fix it on the chip for 1800 s. Finally, add ethanolamine to block the chip, and the final fixation amount is 13462.0 RU. Dilute cannabidiol to 10 μM with PBS buffer solution, and sequentially test the regeneration effect of the chip under 10 mM Glycine-HCl solutions with different pH values (pH 1.5, 2.0, 2.5, 3.0). Select 10 mM Glycine-HCl (pH 1.5) with high and stable binding ability as the regeneration buffer solution. Dilute cannabidiol with PBS buffer solution, and the dilution factors are 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, 0.78125, 0.390625, 0.1953123, 0 μM. And load at 30 μL / min, the binding time of cannabidiol to the protein is 180 s; natural dissociation is 300 s.

[0042] S5. Result analysis

[0043] The results were analyzed using SPSS statistical software and Biacore T200 Evaluation analysis software, and the following results were obtained:

[0044] ① As Figure 4 shown, within the time window of reconsolidation (3 hours) after each methamphetamine injection, cannabidiol was infused into the hippocampus of mice. CPP detection showed ( Figure 4 C): Administration of methamphetamine could significantly induce the formation of CPP addictive memory in mice (P < 0.001), while infusion of cannabidiol into the hippocampus could reduce the formation of CPP addictive memory in mice (P < 0.01); Visualization markers of methamphetamine addictive memory engrams showed ( Figure 4 D): Cannabidiol intervention could hinder the encoding of addictive memory engrams in the hippocampus of mice (P < 0.001); At the same time, cannabidiol intervention could also weaken the activation of TRPV1 channels on hippocampal engram cells ( Figure 4 E, P < 0.001);

[0045] Figure 4 In which, A is the experimental flow chart; B is the implantation trajectory of the drug delivery cannula; C is the conditioned place preference score; D is the visualization marker of methamphetamine addictive memory engrams in the hippocampus; E is the detection of TRPV1 channel activation in hippocampal engram cells.

[0046] ② As Figure 5 shown, within the time window of reconsolidation (3 hours) after each methamphetamine injection, cannabidiol was infused into the nucleus accumbens of mice. CPP detection showed ( Figure 5 C): Administration of methamphetamine could significantly induce the formation of CPP addictive memory in mice (P < 0.001), while infusion of cannabidiol into the nucleus accumbens could reduce the formation of CPP addictive memory in mice (P < 0.05); Visualization markers of methamphetamine addictive memory engrams showed ( Figure 5 D): Cannabidiol intervention could hinder the encoding of addictive memory engrams in the nucleus accumbens of mice (P < 0.001); At the same time, cannabidiol intervention could also weaken the activation of TRPV1 channels on nucleus accumbens engram cells ( Figure 5 E, P < 0.001);

[0047] Figure 5 In which, A is the experimental flow chart; B is the implantation trajectory of the drug delivery cannula; C is the conditioned place preference score; D is the visualization marker of methamphetamine addictive memory engrams in the nucleus accumbens; E is the detection of TRPV1 channel activation in nucleus accumbens engram cells.

[0048] ③ As Figure 6 shown, the action targets of cannabidiol were predicted and verified. Molecular docking showed ( Figure 6A): Cannabidiol can form a docking pocket with the TRPV1 channel, and the binding free energy is -7.65 ± 0.37 kcal / mol. There may be a direct binding interaction between cannabidiol and the TRPV1 channel; SPR detection shows ( Figure 6 B): Cannabidiol directly binds to the TRPV1 channel, and the KD coefficient is 88.2 μM.

[0049] Figure 6 In A, it is a molecular docking schematic diagram of cannabidiol and the human TRPV1 channel; among them, the protein structure of the TRPV1 channel is shown as a green strip, the protein residues at the binding site are marked in yellow, cannabidiol is marked in cyan, hydrogen bonds are marked as blue dotted lines, and hydrophobic interactions are marked as gray dotted lines; B is the SPR detection result diagram of cannabidiol and the human TRPV1 channel.

Claims

1. Use of cannabidiol in the preparation of a drug for intervening methamphetamine addiction memory.

2. The use of cannabidiol according to claim 1 in the preparation of a drug for intervening in methamphetamine addiction memory, characterized in that, The cannabidiol is used in the reconsolidation window period after the extraction of addiction memory.

3. The use of cannabidiol according to claim 2 in the preparation of a drug for intervening in methamphetamine addiction memory, characterized in that, The reconsolidation window period is within 3 hours after the extraction of addiction memory.

4. The use of cannabidiol according to claim 1 in the preparation of a drug for intervening methamphetamine addiction memory, characterized in that, The drug prepared from cannabidiol is directly infused into the hippocampus or nucleus accumbens of mice through a drug delivery catheter.

5. The use of cannabidiol according to claim 1 in the preparation of a drug for intervening in methamphetamine addiction memory, characterized in that, The cannabidiol acts on the TRPV1 channel.

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