Application of sulforaphane in preparation of medicine for abstinging amphetamine addiction

Abstract

The invention discloses an application of sulforaphane in preparation of a medicine for abstinging amphetamine addiction, and relates to the technical field of biological medicines. The invention also provides an application of sulforaphane in preparation of a product for relieving the reduction of the activity level of neuronal cells. A methylamphetamine drug use disorder model is utilized to prove that the sulforaphane has the effect of relieving malaise, anxiety and other adverse mental symptoms caused by drug withdrawal, and the pharmacological action rule of the sulforaphane in treatment is clarified; therefore, a theoretical basis is provided for preparation of drugs for withdrawal of methamphetamine addiction and clinical withdrawal of drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to the use of sulforaphane in the preparation of a drug for treating methamphetamine addiction. Background Art

[0002] Methamphetamine (METH), commonly known as "ice", is a highly addictive new amphetamine-type psychostimulant. Its abuse has become a serious public health problem worldwide.

[0003] Currently, psychological cognitive training is commonly used clinically to improve patients' psychiatric symptoms in response to negative emotions caused by drug abuse. This includes response inhibition control training, comprehensive intervention training across multiple cognitive dimensions, and mindfulness therapy combined with goal management training. However, these therapies have significant limitations in clinical application, and when addicts have low subjective initiative, it is often difficult to achieve good rehabilitation results. Currently, there are no effective pharmacological treatments for abnormal psychiatric symptoms during methamphetamine withdrawal, and the development and utilization of therapeutic drugs is a major gap in current drug research. Therefore, the search for drugs to improve drug-induced cognitive dysfunction is of great significance for drug addiction treatment. Summary of the Invention

[0004] The present invention aims to provide the use of sulforaphane in the preparation of a methamphetamine addiction treatment drug to address the problems of the prior art. The present invention has found that sulforaphane can alleviate adverse mental symptoms such as general fatigue and anxiety caused by drug withdrawal, thereby providing a theoretical basis for the preparation of methamphetamine addiction treatment drugs and for clinical drug withdrawal.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides the use of sulforaphane in preparing a medicament for quitting methamphetamine addiction.

[0007] Furthermore, the drug has the effect of alleviating adverse mental symptoms that occur during the withdrawal process of methamphetamine addicts.

[0008] Furthermore, the adverse mental symptoms include general fatigue or anxiety.

[0009] The present invention also provides the use of sulforaphane in preparing a product for alleviating decreased neuronal cell activity levels caused by methamphetamine.

[0010] The present invention also provides a drug for quitting methamphetamine addiction, wherein the active ingredient includes sulforaphane.

[0011] Furthermore, the medicine also includes pharmaceutically acceptable excipients.

[0012] Furthermore, the dosage form of the drug includes tablets, powders, granules, capsules, oral solutions or injections.

[0013] The present invention discloses the following technical effects:

[0014] The present invention uses a methamphetamine drug use disorder model to demonstrate that sulforaphane can alleviate mental symptoms such as general fatigue and anxiety caused by drug withdrawal, and clarifies the pharmacological action of sulforaphane in treatment, thereby providing a theoretical basis for the preparation of drugs for quitting methamphetamine addiction and clinical drug withdrawal. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0016] Figure 1 is the structural formula of sulforaphane;

[0017] Figure 2 Schematic diagram for constructing an animal model of methamphetamine drug use disorder;

[0018] Figure 3 This is a schematic diagram of the animal experiment process for the effect of sulforaphane on methamphetamine withdrawal symptoms;

[0019] Figure 4Figure 3 is the result of open field test and elevated plus maze test; A is the activity trajectory of representative experimental animals in open field test; B is the statistical result of the average moving distance of mice in the open field box (Averaged Moved Distance of Total Observation); C is the statistical result of the average movement speed of mice in the open field box (Averaged Velocity of Total Observation); D is the statistical result of the duration of mice in the central zone of the open field box (Duration in Central Zone); E is the statistical result of the number of mice crossing the central zone of the open field box (Number of Central Zone Crossing); F is the statistical result of the latency of mice entering the central zone from the periphery (Latency of Surrounding to Center); G is the activity trajectory of representative experimental animals in elevated plus maze test; H is the statistical result of the number of mice entering the open arm and the closed arm (Open / Closed Armi entries); I is the statistical result of the proportion of mice entering the open arm (Open Arm entry percentage); J is the proportion of mice entering the open arm (Open Arm time K is the statistical result of the time mice stay in the open arm and closed arm (Open / Closed Arm time);

[0020] Figure 5 The results of molecular biology experiments verify that sulforaphane can alleviate the withdrawal effects of addictive drugs; A shows the trend of changes in the levels of apoptosis markers Bcl2 and Bax in the prefrontal cortex of mice in the three groups; B shows the statistical results of protein expression grayscale values by Western Blot in the three groups of mice; C shows the statistical results of Caspase-3 activity detection in the three groups of mice; D shows the trend of changes in the phosphorylation levels of PI3K / AKT signaling pathway proteins in the prefrontal cortex of the three groups of mice; E shows the statistical results of the grayscale values of PI3K phosphorylation levels; F shows the statistical results of the grayscale values of AKT phosphorylation levels; G shows the changes in the mRNA expression levels of AKT1 in the prefrontal cortex of the three groups of mice; H shows the trend of changes in the phosphorylation levels of PI3K / AKT signaling pathway proteins detected by immunohistochemistry; I shows the statistical results of the positive expression of PI3K phosphorylated proteins; and J shows the statistical results of the positive expression of AKT phosphorylated proteins.

[0021] Figure 6Results of a cell experiment detecting the effect of sulforaphane on the viability of HT22 cells induced by methamphetamine; A shows the effects of different concentrations and treatment time periods of methamphetamine on HT22 cell viability; B shows the effects of different concentrations and treatment time periods of sulforaphane on HT22 cell viability; C shows the changes in HT22 cell viability 12 hours after the addition of methamphetamine (1 mmol / L) after 40 minutes of pre-treatment with different concentrations of sulforaphane; D shows the changes in HT22 cell viability 12 hours after the addition of different concentrations of sulforaphane after 40 minutes of methamphetamine (1 mmol / L); E shows the changes in HT22 cell viability 24 hours after the addition of methamphetamine (1 mmol / L) after 40 minutes of pre-treatment with different concentrations of sulforaphane; F shows the changes in HT22 cell viability 24 hours after the addition of different concentrations of sulforaphane after 40 minutes of methamphetamine (1 mmol / L). DETAILED DESCRIPTION

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0024] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0025] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0026] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0027] Terminology Notes:

[0028] Sulforaphane (SFN) referred to in the present invention, also known as sulforaphane, is a natural isothiocyanate derived from cruciferous plants with a molecular formula of C6H 11 S2NO, structural formula is Figure 1 shown.

[0029] Example 1

[0030] 1. Preparation of sulforaphane and methamphetamine

[0031] In vivo methamphetamine solution preparation method: take 20 mg of methamphetamine hydrochloride (molar mass 185.70 g / mol, purity >99.99%) and dissolve it in 20 mL of normal saline to a final concentration of 1 mg / mL. After filtering, store at 4°C until use.

[0032] In vitro methamphetamine solution preparation method: take 20 mg of methamphetamine hydrochloride (molar mass 185.70 g / mol, purity >99.99%) and dissolve it in 1.077 mL PBS to a final concentration of 100 mM. After filtering, place it at 4°C for use.

[0033] In vivo sulforaphane preparation: Dissolve 20 mg of sulforaphane (molar mass 177.28 g / mol) from one vial in 20 ml of saline containing 1% dimethyl sulfoxide (DMSO) to a final concentration of 1 mg / mL. Filter and store at -20°C.

[0034] In vitro sulforaphane preparation: Dissolve 20 mg of sulforaphane (177.28 g / mol) in 11.3 mL of saline containing 1% DMSO to a final concentration of 10 mM. Filter and store at -20°C.

[0035] 2. Animal Experiments

[0036] 2.1 Model Construction

[0037] C57 / BL6J mice were used to establish a methamphetamine use disorder model and a sulforaphane neuroprotection model (the methamphetamine use disorder model construction process and animal experiment process are as follows: Figure 2 and Figure 3 The model establishment method is as follows:

[0038] Pre-test period (1 day): On the first day of the experiment, all experimental mice were first allowed to acclimate in a behavioral laboratory with appropriate temperature and humidity for 30 minutes. Then, the mice were placed in a test box for 30 minutes to acclimate. After the end, both groups of mice were given an intraperitoneal injection of 0.9% saline, and the autonomous activity of the experimental mice in the test box was recorded for 1 hour.

[0039] Formation period (2-15 days): Over the following 2-15 days, mice in the control group were intraperitoneally injected with 1% DMSO in saline at a volume of 10 mL / kg, followed by an intraperitoneal injection of saline at a volume of 10 mL / kg 40 minutes later. Mice in the methamphetamine use disorder model group were still intraperitoneally injected with 1% DMSO in saline at a volume of 10 mL / kg, followed by an intraperitoneal injection of methamphetamine at a concentration of 10 mg / kg 40 minutes later. Mice in the sulforaphane nerve injury protection model group were intraperitoneally injected with sulforaphane at a volume of 10 mL / kg, followed by an intraperitoneal injection of methamphetamine at a concentration of 10 mg / kg 40 minutes later.

[0040] Conversion period (Days 16-17): During Days 16-17, the three groups of mice were placed in their cages in the animal room without any treatment. On Day 17, the mice were treated as in the pre-test period, and their locomotor activity was recorded within 1 hour after intraperitoneal injection of 0.9% saline to assess the withdrawal effect.

[0041] Testing period (18-31 days): During the period of 18-31 days, the three groups of mice were tested for motor ability and anxiety behavior using the open field test and elevated plus maze test to evaluate the neuroprotective ability of sulforaphane.

[0042] Open-field test method: The open-field box consisted of a 60 cm × 60 cm × 50 cm blue cube. Mice were transferred to the behavioral laboratory for 2 hours of acclimatization before the experiment began. At the beginning of the experiment, mice were placed at the bottom of the open-field box and allowed to move freely for 6 minutes. TopScan 3.00 animal behavior analysis software was used to record and analyze the average distance moved (Average Moved Distance of Total Observation), average velocity (Average Velocity of Total Observation), duration in the central zone (Duration in Central Zone), number of central zone crossings (Number of Central Zone Crossings), and latency of surrounding areas entering the central zone (Latency of Surrounding to Center). After each mouse was measured, the open-field box and its surroundings were disinfected with 75% alcohol to ensure that no residual mouse odor would affect the next set of experiments.

[0043] Elevated Plus Maze Method: The elevated plus maze consists of a cross-shaped apparatus consisting of two closed arms (35 cm long × 5 cm wide × 15 cm high) and two similar open arms, located approximately 55 cm above the ground. At the start of the experiment, mice were placed in the center of the apparatus, facing the same open arm, and allowed to freely explore for 5 minutes. The number of open and closed arm entries, the time spent in the open and closed arms, the percentage of open arm entries, and the percentage of time spent in the open arms were recorded to assess anxiety-like behavior. After each mouse was tested, the apparatus was thoroughly cleaned with 75% alcohol to ensure that no residual mouse odor could affect the next set of experiments.

[0044] like Figure 4As shown, compared with control mice, the average moved distance (Average Moved Distance of Total Observation), average velocity (Average Velocity of Total Observation), duration in the central zone (Duration in Central Zone), and number of central zone crossings in the open field box were all reduced in the methamphetamine use disorder model group. However, these indicators were increased in the sulforaphane neuroprotection group compared with the methamphetamine use disorder group, indicating that sulforaphane can improve the reduced motor ability during methamphetamine withdrawal. Compared with control mice, the latency of the peripheral to central zone in the methamphetamine use disorder model group was significantly prolonged, and this parameter was reduced under the neuroprotective effect of sulforaphane, indicating that sulforaphane can enhance the exploratory ability of mice during withdrawal. Through the elevated plus maze experiment, it was found that the percentage of times mice entered the open arm (Open Arm percentage) and the percentage of time they entered the open arm (Open Arm time percentage) were significantly reduced during the withdrawal period, while sulforaphane can increase the number and time mice entered the open arm, indicating that sulforaphane can improve the anxiety of mice during the withdrawal period and enhance their desire to explore.

[0045] Test results on experimental animals show that sulforaphane has the pharmacological effect of alleviating anxiety during the withdrawal period of addictive drugs.

[0046] 2.2 Tissue testing

[0047] The prefrontal cortex of the experimental animal model was sampled and subjected to protein and mRNA extraction and tissue sectioning. Western blotting and immunofluorescence techniques were used to detect neuronal apoptosis and the expression levels of apoptosis-related markers and signaling pathway proteins.

[0048] Method for extracting protein from experimental animal tissues: Pre-chill EP tubes at -80°C. Add 1 mL of the prepared RIPA lysis buffer mixture (RIPA lysis buffer: protease inhibitor = 99:1) per 100 mg of tissue. Add two steel balls to each EP tube and place in a tissue grinder. Grind at 60 Hz for 1 minute, followed by 30-second rests. After three cycles, place on ice and lyse for 30 minutes. Centrifuge at 12,000 rpm for 10 minutes at 4°C. After centrifugation, aspirate the supernatant into a fresh 1.5 mL EP tube and store at -80°C or proceed with protein quantification and denaturation.

[0049] Immunohistochemical staining: Mouse brain tissue was fixed with 4% formaldehyde for 24 hours. The tissue was then washed three times with 1× PBS on a shaker for 20 minutes each. After washing, the tissue was dehydrated with a gradient of ethanol and cleared with xylene. The cleared brain tissue was paraffin-impregnated, embedded side down, and placed flat in an embedding rack at an angle. The rack was then completely soaked in paraffin solution. After the block cooled, it was trimmed and sectioned at a thickness of 4 μm. Tissue sections were deparaffinized and rehydrated. Antigen retrieval was then performed by slowly heating 0.01 M (pH 6.0) citrate buffer to 95–98°C. The slides were then immersed in the solution for 2 minutes and cooled to room temperature. The sections were washed with 1× PBS and blocked with a blocking reagent containing 3% goat serum at 37°C for 30 minutes. The sections were washed with 1× PBS and incubated with the appropriately diluted primary antibody at 4°C overnight on a shaker. The sections were washed with 1× PBS and incubated with the appropriately diluted secondary antibody at 37°C for 1.5 hours. The sections were labeled with horseradish peroxidase in the dark for 30 min at room temperature, washed, incubated with diaminobenzidine, sealed with neutral resin, and air-dried before being photographed and observed under a microscope.

[0050] like Figure 5 As shown, the Bcl2 / Bax ratio, a marker of apoptosis, was significantly decreased in the methamphetamine use disorder group, while caspase-3 activity and TUNEL expression levels were significantly increased in the methamphetamine use disorder group, indicating increased apoptosis in the prefrontal cortex neurons of the mice. However, the sulforaphane neuroprotection group showed reduced apoptosis compared to the withdrawal group, suggesting that sulforaphane can mitigate methamphetamine-induced apoptosis in the prefrontal cortex neurons. The PI3K / AKT signaling pathway is a key regulatory molecule in the nervous system. The results from the three experimental groups showed that sulforaphane could increase the reduced phosphorylation levels of PI3K and AKT molecules caused by methamphetamine, suggesting that sulforaphane exerts its neuroprotective effects by activating the PI3K / AKT signaling pathway.

[0051] Testing has shown that sulforaphane can attenuate methamphetamine-induced neuronal apoptosis in the prefrontal cortex, which is believed to be the molecular mechanism by which sulforaphane alleviates anxiety levels during methamphetamine withdrawal.

[0052] 3. Cell Experiment

[0053] The HT22 cell poisoning model was induced by methamphetamine, and the HT22 cell neuroprotection model was induced by sulforaphane. The model establishment method is as follows:

[0054] Well-grown HT22 cells were seeded in 12-well plates (1×10 6When the HT22 cells reached a density of approximately 80%, the culture medium was removed from the wells and the cells were washed once with PBS. The cells were divided into the control group, the methamphetamine group, and the pre-protection and treatment groups with varying concentrations of sulforaphane. Cell viability was assessed using the Cell Counting Kit-8.

[0055] Cell Counting Kit-8 assay for HT22 cell viability: 5,000 cells in good growth condition were plated in a 96-well plate. 100 μL of culture medium was added to each well and different interventions were performed. The culture medium was then discarded and the Cell Counting Kit-8 (CCK-8) reagent was diluted with culture medium at a ratio of 10:100. The mixture was thoroughly mixed and 100 μL of the diluted CCK-8 solution was added to each well. Incubate at 37°C in the dark for 1 hour. The absorbance at 450 nm was measured using a microplate reader. The results were calculated using the following formula:

[0056] Cell viability (%) = [OD 模型组 -OD 空白 / OD 对照组 -OD 空白 ]×100%.

[0057] like Figure 6 As shown, methamphetamine can reduce HT22 cell viability, while sulforaphane can enhance HT22 cell viability within 24 hours at concentrations of 2-10 μM. When the two drugs were mixed and treated with cells, it was found that when sulforaphane was added first to pre-protect HT22 cells and then methamphetamine was added 40 minutes later, neuronal viability was enhanced at a concentration of 2-5 μM, demonstrating the best neuroprotective effect. However, when methamphetamine was first added to the cells and then sulforaphane was added, sulforaphane had no significant therapeutic effect.

[0058] HT22 cell experiments verified that sulforaphane has the effect of reversing the decrease in HT22 cell viability caused by methamphetamine.

[0059] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. Use of sulforaphane in the preparation of a drug for treating methamphetamine addiction.

2. The use according to claim 1, characterized in that The medicine has the effect of alleviating adverse mental symptoms occurring in methamphetamine addicts during the withdrawal process.

3. The use according to claim 1, characterized in that The adverse mental symptoms include general fatigue or anxiety.

4. The use of sulforaphane in preparing a product for alleviating the reduction of neuronal cell activity level, characterized in that: The decreased neuronal cell viability level is caused by methamphetamine.

5. A drug for quitting methamphetamine addiction, characterized in that: Active ingredients include sulforaphane.

6. The drug according to claim 5, characterized in that The drug also includes pharmaceutically acceptable excipients.

7. The drug according to claim 6, characterized in that The dosage forms of the drug include tablets, powders, granules, capsules, oral solutions or injections.

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