Animal model of acetic acid intervention of methamphetamine addiction, preparation and preparation method and application

Abstract

The application discloses an acetic acid intervention methamphetamine addiction animal model, a preparation and a preparation method and application. The application proves that acetic acid as a key metabolic product of the intestinal tract can directly regulate the function of an addiction-related intestinal-brain axis and inhibit addiction-related reward behavior. In combination with the characteristics of safety in source, clear effect and easy administration, acetic acid is used to intervene C57 mice through a drinking water mode and a methamphetamine addiction model is constructed. The results show that, compared with a control group, the conditional position preference score of the acetic acid intervention group of mice is significantly reduced, and addiction-related behavior is obviously weakened, and there is no obvious toxic side effect. The acetic acid preparation prepared by the application has the effects of preventing and / or weakening methamphetamine addiction, has the advantages of safety, non-toxicity, wide source, simple administration and no secondary addiction risk, and the like, and provides a safe and effective new treatment strategy for intervention of methamphetamine addiction.

Description

Technical Field

[0001] This invention relates to the field of methamphetamine (METH) addiction prevention and treatment technology, specifically to an animal model, formulation, preparation method and application of acetic acid intervention for methamphetamine addiction, particularly to a methamphetamine addiction model constructed by intervening in C57 mice through drinking water with acetic acid, the preparation of an acetic acid formulation with the effect of preventing and reducing methamphetamine addiction, and the use of this formulation to achieve a safe, simple, addiction-free method of addiction intervention without the need for complex equipment. Background Technology

[0002] Methamphetamine (METH), also known as crystal meth, is a potent neurostimulant belonging to the amphetamine family of stimulants. It selectively acts on the central nervous system above the brainstem, and is highly likely to cause psychological dependence and neurotoxicity. It has become one of the most harmful drugs in the world today.

[0003] Currently, the main methods for treating METH addiction include:

[0004] (1) Traditional drug treatment: This is divided into two categories: replacement therapy and non-replacement therapy. Replacement therapy commonly uses drugs such as methadone and buprenorphine hydrochloride sublingual tablets. Its core advantage is that it can quickly reduce withdrawal symptoms and help addicts smoothly get through the detoxification period. However, methadone itself is addictive and must be administered in strictly managed drug rehabilitation medical institutions. It may also cause adverse reactions such as dry mouth, nausea, vomiting, and dizziness. Overdose can also lead to symptoms of poisoning such as respiratory depression and coma. Although buprenorphine hydrochloride has a lower risk of respiratory depression, it still has adverse reactions such as drowsiness, dizziness, and hypotension. It is not suitable for patients with respiratory diseases, severe liver disease, or pregnant women. Non-replacement therapy is represented by clonidine and lofexidine. It can reduce mild to moderate withdrawal symptoms, but its effect on controlling withdrawal symptoms is weaker than that of replacement therapy drugs. It is also prone to adverse reactions such as dry mouth, fatigue, and orthostatic hypotension. After long-term use, sudden discontinuation of the drug can also cause symptoms such as rebound hypertension and headache. The duration of drug use is limited.

[0005] (2) Psychotherapy: Primarily targeting psychological dependence and relapse prevention, core methods include cognitive behavioral therapy and motivational reinforcement therapy. These methods have no drug side effects and can help addicts correct erroneous cognitions, establish healthy behavioral patterns, and assist in reducing relapse rates. However, the treatment cycle is long, the onset of action is slow, and it requires a very high level of professional expertise from the therapist. Furthermore, the treatment effect heavily depends on the patient's cooperation, and the efficacy is extremely poor for patients who resist treatment, making large-scale application difficult.

[0006] (3) Novel peptide drug therapy: Oxytocin, for example, has been found in recent years to effectively block the reward effect of METH and inhibit addiction-related conditioned positional behavior through nasal administration. Its mechanism of action is related to the regulation of central oxytocin receptors and the paraventricular nucleus-nucleus accumbens core neural circuit in the hypothalamus. Its advantages are that it has no obvious addictive properties and high safety. However, this approach is still in the basic research stage and a mature clinical treatment plan has not yet been formed. Key parameters such as dosage and treatment cycle still need further verification.

[0007] (4) Neural circuit modulation therapy: Interventions based on dopaminergic neural circuit mechanisms modulate two key pathways, the mesocortical and mesolimbic pathways, through pharmacological or optogenetic methods. Strengthening the mesocortical pathway (acting as a brake) and weakening the mesolimbic pathway (acting as an accelerator) can reduce addicts' craving for METH. Studies have also found that experiences of social victory can inhibit drug-seeking behavior by reshaping this neural circuit. This method provides a new theoretical perspective for addiction treatment, but currently relies on invasive techniques (such as optogenetics) and is difficult to apply directly in clinical practice. The development of non-invasive interventions is still in the exploratory stage.

[0008] While the above-mentioned treatment methods have some effect in METH addiction intervention, they all have significant drawbacks. For example, traditional drug treatment generally carries the risk of adverse reactions, some drugs are prone to secondary dependence, and the applicable population is limited; psychological and behavioral therapy has a long cycle, depends on the patient's cooperation, and is difficult to promote; novel peptide drugs and neural circuit modulation therapy are still in the research stage, and there are many obstacles to clinical translation.

[0009] Therefore, there is an urgent need for a safe, effective, and readily applicable mature solution for the prevention and treatment of METH addiction that is free from the risk of secondary addiction and easy to promote. Summary of the Invention

[0010] The purpose of this invention is to overcome the above-mentioned shortcomings and provide an application of acetic acid in the preparation of formulations for preventing and / or reducing methamphetamine addiction.

[0011] This invention demonstrates that acetic acid, as a key intestinal metabolite, can directly regulate addiction-related gut-brain axis function. Combining its safe source, clear effects, and ease of administration, acetic acid was used to intervene in C57 mice via drinking water to construct a methamphetamine addiction model. An acetic acid preparation with the effect of preventing and reducing methamphetamine addiction was prepared. This preparation provides a safe, simple, addiction-free method of addiction intervention that does not require complex equipment.

[0012] The technical solution adopted by the present invention to achieve the above objectives is as follows:

[0013] Acetic acid is an important short-chain fatty acid metabolite of the gut microbiota, and it is safe, non-toxic, widely available, and stable. Studies have shown that acetic acid can effectively reduce methamphetamine (METH)-induced addictive behavior by regulating the gut-brain axis, inhibiting excessive activation of the central reward circuit, and reducing neuroinflammatory responses. This invention established a METH addiction mouse model and intervened by supplementing acetic acid through drinking water. The results demonstrated that acetic acid significantly reduced conditioned place preference scores in mice, effectively inhibiting the formation and development of METH addiction, and exhibiting a clear anti-addiction effect.

[0014] A method for preparing an acetic acid intervention agent includes the following steps:

[0015] Step 1, Raw material preparation: Select food-grade sodium acetate (purity ≥99%), sterile drinking water, and 0.22μm sterile filter membrane;

[0016] Step 2, Solution preparation: Add sodium acetate to sterile drinking water at a concentration of 150 mmol / L and stir thoroughly until completely dissolved;

[0017] Step 3, sterilization filtration: The prepared sodium acetate solution is filtered through a 0.22μm sterile filter membrane for sterilization;

[0018] Step 4, aliquoting and storage: aliquot under aseptic conditions, store at 4°C, and replace with fresh solution weekly to avoid contamination and loss of activity.

[0019] A method for constructing an animal model of methamphetamine addiction using acetic acid intervention includes the following steps:

[0020] Step 1, Experimental animals: Select healthy SPF-grade 6-8w male C57BL / 6 mice, weighing 20-22g, and house them in an environment with a temperature of 22-25℃, relative humidity of 50%-60%, and 12h light / dark conditions, with free access to food and water. After 7 days of acclimatization, they will be used for experiments.

[0021] Step 2, Reagent preparation: METH (purity ≥98%), sodium acetate solution, sterile drinking water, conditioned position preference (CPP) test chamber, electronic balance, syringe, etc.;

[0022] Step 3, Experimental grouping: The animals were randomly divided into two groups of 8 each: ① Addiction control group (METH+Control): The animals drank regular sterile drinking water for 0-21 days to establish a METH addiction model; ② Acetic acid intervention group (METH+SodiumAcetate): The animals freely drank 150mmol / L sodium acetate solution for 0-21 days to establish a METH addiction model.

[0023] Step 4, Intervention method: After the adaptive feeding of each group of mice, they were allowed free access to the corresponding solution throughout the entire model establishment process;

[0024] Step 5, Addiction Model Construction: ① Adaptation Period (Day 14): Mice freely explore the CPP box for 30 minutes daily; ② Pre-testing Period (Day 15): Free exploration for 15 minutes, initial preferences recorded; ③ Training Period (Days 15-20): METH (2 mg / kg) and saline are injected intraperitoneally alternately every other day, and the mice are placed in the corresponding boxes for 30 minutes each; ④ Post-testing Period (Day 21): Free exploration for 15 minutes, CPP score calculated (drug pairing box time - saline pairing box time);

[0025] Step 6, Detection indicators: including CPP score, activity heatmap, and total distance of spontaneous activities; Step 7, Data analysis: GraphPadPrism software was used for processing. Quantitative data are expressed as mean ± standard deviation. Two-way ANOVA was used for comparisons among multiple groups, and post-hoc tests were performed using Tukey's multiple comparison test. P < 0.05 was considered statistically significant.

[0026] This invention provides the use of acetic acid in the preparation of formulations for the prevention and / or reduction of methamphetamine addiction; further, the acetic acid may be used alone or in combination with other active ingredients for the prevention, early intervention and relapse control of methamphetamine addiction.

[0027] This invention has many beneficial effects:

[0028] 1. Acetic acid is a natural intestinal metabolite with high safety, no toxic side effects, and no risk of addiction, thus avoiding the problem of secondary dependence on traditional drugs;

[0029] 2. The preparation process is simple, the cost is low, and the sources are widely available, making it suitable for large-scale industrial production;

[0030] 3. The administration method is simple, no special equipment is required for water-based intervention, compliance is good, and it is suitable for long-term prevention and intervention;

[0031] 4. Animal experiments have confirmed that the intervention has a stable effect and can significantly reduce addiction-related behaviors, providing a reliable basis for clinical translation. Attached Figure Description

[0032] Figure 1 : Flowchart of animal experiments involving the intervention of Bifidobacterium pseudosporidis in this invention.

[0033] Figure 2 : A heatmap of the conditioned position preference experiment of Bifidobacterium pseudosporidis intervention.

[0034] Figure 3 : Statistical graph of total distance of spontaneous activity in conditional position preference experiment of Bifidobacterium pseudosporidis intervention.

[0035] Figure 4Statistical graph of CPP score in the conditional position preference experiment of Bifidobacterium pseudosporidis intervention.

[0036] Figure 5 : Quantitative statistical graph of Bifidobacterium pseudosporidis in feces of mice treated with Bifidobacterium pseudosporidis using qPCR.

[0037] Figure 6 Detection of short-chain fatty acid content in mouse feces after intervention with Bifidobacterium pseudosporidis.

[0038] Figure 7 : Whole genome sequencing analysis diagram of Bifidobacterium pseudosporidis.

[0039] Figure 8 Statistical chart of short-chain fatty acid detection in Bifidobacterium pseudosporidioides culture medium.

[0040] Figure 9 Flowchart of animal experiments using acetic acid in drinking water to intervene in methamphetamine addiction.

[0041] Figure 10 : A heatmap of conditioned place preference in a mouse model of methamphetamine addiction induced by acetic acid intervention.

[0042] Figure 11 Statistical graph of total distance of spontaneous activity in conditioned place preference experiment of acetic acid-interventional methamphetamine addiction model mice.

[0043] Figure 12 Statistical graph of CPP scores in conditioned place preference test of acetic acid-interventional methamphetamine addiction model mice. Detailed Implementation

[0044] Example 1

[0045] An animal experimental method for the intervention of Bifidobacterium pseudosporidis in methamphetamine addiction, see [link to relevant documentation]. Figure 1 The experimental procedure shown includes the following steps:

[0046] Step 1, Experimental Animals and Grouping: SPF-grade healthy mice were selected and, after acclimatization, were randomly divided into two groups of eight mice each. The addiction control group (METH+PBS group) and the Bifidobacterium pseudosporidis intervention group (METH+BP group) were set up. The mice were kept in a routine environment with free access to food and water throughout the process.

[0047] Step 2, bacterial strain intervention: Mice in the METH+BP group were administered bacterial strains via gavage for 7 consecutive days. The strains administered via gavage were Bifidobacterium pseudosporidis isolated from low-addictive individuals. Mice in the METH+PBS group were administered an equal volume of sterile PBS solution via gavage at the same time. The operating conditions were kept consistent across all groups.

[0048] Step 3, addiction model construction: After the strain intervention, all mice were used to construct a methamphetamine-induced CPP addiction model, which was divided into three stages: pre-testing period, training period, and post-testing period. In these stages, behavioral baseline detection, drug alternation training, and addictive behavior detection were completed in sequence.

[0049] Step 4, Sample collection and index detection: After the behavioral experiment, mouse fecal samples were collected. The relative abundance of fecal Bifidobacterium pseudosporidis was detected by qPCR, and the content of short-chain fatty acids in the feces of mice in each group was determined by biochemical detection method. At the same time, the spontaneous activity distance of the mice was recorded and the CPP score was calculated.

[0050] Step 5, Effect Detection: See details for the results. Figure 2 , Figure 3 , Figure 4 , Figure 5 , Figure 6 Behavioral heatmap analysis ( Figure 2 The results showed no significant difference in area exploration time between the groups during the pre-test phase. During the post-test phase, the METH+BP group mice spent significantly less time in the drug pairing box than the METH+PBS group. During behavioral testing, there was no significant difference in the total distance of spontaneous activity between the two groups. Figure 3 The results indicate that BP gavage intervention does not impair motor function and spontaneous activity in mice, and has no significant acute somatic toxicity. CPP score results ( Figure 4 The results showed no significant differences in baseline position preferences among the groups during the pre-test phase. In the post-test phase, the CPP score in the METH+PBS group was significantly higher than in the pre-test phase, indicating successful modeling of the METH addiction phenotype; while the CPP score in the METH+BP group showed a slight increase, it remained significantly lower than the control group (P < 0.05). Fecal microbiota quantification results ( Figure 5 The results showed that the abundance of BP in feces was significantly upregulated in the METH+BP group compared to the METH+PBS group, confirming that this strain can effectively colonize the intestine. Fecal short-chain fatty acid detection results ( Figure 6 The results showed that the levels of acetic acid, isobutyric acid, and valeric acid in the feces of mice in the METH+BP group were significantly higher than those in the control group, with acetic acid showing the most significant upregulation. In conclusion, intervention with Bifidobacterium pseudosporidis can effectively reduce METH-induced addictive behavior and significantly upregulate the levels of short-chain fatty acids such as acetic acid in the intestines.

[0051] Example 2

[0052] A method for verifying the in vitro synthesis of short-chain fatty acids by Bifidobacterium pseudoshort, specifically including the following steps:

[0053] Step 1, strain preparation: Select a strain of Bifidobacterium pseudomicrobium isolated from low-addictive individuals, revive and activate it for later use;

[0054] Step 2, Genomic pathway analysis: Whole-genome sequencing (WGS) technology was used to annotate the short-chain fatty acid synthesis metabolic pathway of Bifidobacterium pseudomicrobium and analyze the gene functional basis of acetic acid synthesis in the strain.

[0055] Step 3, in vitro culture experiment: Set up a blank culture medium control group and a Bifidobacterium pseudobulb culture group, and culture them under the same culture conditions at constant temperature and anaerobic conditions to ensure that the culture environment is free from contamination by other bacteria;

[0056] Step 4, Effect Detection: See details for results. Figure 7 , Figure 8 Genomic pathway analysis results ( Figure 7 The results showed that the Bifidobacterium pseudosporidis genome possesses a complete acetic acid synthesis metabolic pathway, indicating the genetic basis for acetic acid synthesis. In vitro culture detection results ( Figure 8 The results showed that the acetic acid content in BP medium was significantly higher than that in the blank control group, confirming that acetic acid is the main metabolite of this strain, consistent with the genomic pathway prediction results. In conclusion, *Bifidobacterium pseudosporidis* possesses a stable acetic acid synthesis capacity, and acetic acid synthesis is its core metabolic characteristic.

[0057] Example 3

[0058] A method for preparing an acetic acid-interventional methamphetamine addiction formulation specifically includes the following steps:

[0059] Step 1, Raw material preparation: Select food-grade sodium acetate (purity ≥99%), sterile drinking water, and 0.22μm sterile filter membrane;

[0060] Step 2, Solution preparation: Add sodium acetate to sterile drinking water at a concentration of 150 mmol / L and stir thoroughly until completely dissolved;

[0061] Step 3, sterilization filtration: The prepared sodium acetate solution is filtered through a 0.22μm sterile filter membrane for sterilization;

[0062] Step 4, aliquoting and storage: aliquot under aseptic conditions, store at 4°C, and replace with fresh solution weekly to avoid contamination and loss of activity, thus obtaining the acetic acid intervention preparation.

[0063] Example 4

[0064] Animal experiments on the intervention of acetic acid in drinking water for methamphetamine addiction, see [link to relevant documentation]. Figure 9 The experimental procedure shown includes the following steps:

[0065] Step 1, Experimental grouping: 16 SPF-grade 6-8w male C57BL / 6 mice were randomly divided into 2 groups (8 mice in each group): addiction control group (METH+Control group, drinking regular sterile drinking water + methamphetamine addiction modeling) and acetic acid intervention group (METH+SodiumAcetate group, drinking 150mmol / L sodium acetate solution throughout the process + methamphetamine addiction modeling).

[0066] Step 2, water intervention: Starting from day 0 of the experiment, the two groups of mice were allowed to drink the corresponding solutions freely. Acetic acid intervention continued throughout the entire experimental period (days 0-21), and the mice had free access to food and water throughout the process.

[0067] Step 3, Addiction Model Construction (Days 14-21, CPP Phase): The experiment is divided into three phases: pre-testing, training, and post-testing. The specific steps are as follows: ① Pre-testing (Day 14): The partition of the two-box conditioned place preference test chamber is opened, and mice are allowed to explore freely for 15 minutes. The time spent by mice in the drug pairing box and the saline pairing box is recorded using a video tracking system. ② Training (Days 15-20): METH (2 mg / kg) and an equal volume of saline are injected intraperitoneally every other day, and the mice are placed in their respective boxes for 30 minutes after injection. ③ Post-testing (Day 21): The pre-testing procedure is repeated, the partition is opened, and mice are allowed to move freely for 15 minutes. The time spent by mice in the two boxes is recorded, and the CPP score (time spent in the drug pairing box - time spent in the saline pairing box) is calculated to verify the addiction model construction. After the behavioral tests, the mice are sacrificed, and samples are collected for subsequent testing.

[0068] Step 4, Effect Detection: See details for results. Figure 10 , Figure 11 , Figure 12 Behavioral heatmap analysis ( Figure 10 The results showed no significant difference in area exploration time between the groups of mice in the pre-test phase. In the post-test phase, the METH+Sodium Acetate group mice spent significantly less time in the drug pairing box than the METH+Control group. During behavioral testing, there was no significant difference in the total distance of spontaneous activity between the two groups of mice. Figure 11 The results indicate that acetic acid supplementation in drinking water does not impair motor function or spontaneous activity in mice, and has no significant acute somatic toxicity. CPP score results ( Figure 12 The results showed no significant differences in baseline position preferences among the groups during the pre-test phase. In the post-test phase, the CPP score in the METH+Control group was significantly higher than in the pre-test, indicating successful modeling of the METH addiction phenotype; while the CPP score in the METH+Sodium Acetate group showed a slight increase, it was still significantly lower than that in the control group (P = 0.0339). In conclusion, oral acetic acid can attenuate METH-induced addictive behavior.

Claims

1. An animal model of acetic acid intervention in methamphetamine addiction, characterized in that, The model is constructed through the following steps: Step 1, Experimental animals: Select healthy SPF-grade 6-8w male C57BL / 6 mice for feeding; Step 2, Preparation of experimental instruments and reagents; Step 3, Experimental Grouping: C57BL / 6 mice fed in Step 1 were randomly divided into two groups: an addiction control group and an acetic acid intervention group, with 8 mice in each group: ① Addiction Control Group: Drinked regular drinking water to establish a METH addiction model; ② Acetic Acid Intervention Group: Drinked 150 mmol / L sodium acetate solution to establish a METH addiction model; Free access to drinking water was available from 0 to 21 days. Step 4, Water intervention: Starting from day 0 of the experiment, the two groups of mice were allowed to drink the corresponding solutions freely, and the intervention continued throughout the entire experimental period; Step 5, addiction model construction: ① The first 14 days are the adaptation period: mice freely explore the CPP box for 30 minutes each day; ② Day 15 is the pre-test period: free exploration for 15 minutes, and initial preferences are recorded; ③ Days 15-20 are the training period: METH and saline are injected intraperitoneally alternately every other day, with a METH dose of 2 mg / kg, and then placed in the corresponding box for 30 minutes each; ④ Day 21 is the post-test period: free exploration for 15 minutes, and the CPP score is calculated, where the CPP score = drug pairing box stay time - saline pairing box stay time. Step 6, Intervention effect detection: Core indicator detection: CPP score, activity heat map, and spontaneous activity distance were used as the main evaluation indicators; Weight monitoring: The weight of mice was measured weekly during the experiment to assess the physiological effects of acetic acid on mice; Step 7, Statistical analysis of experimental data: GraphPadPrism software was used. Quantitative data were expressed as mean ± standard deviation. Two-way ANOVA was used for comparisons among multiple groups, and Tukey's multiple comparison test was used for post-hoc tests. P < 0.05 was considered statistically significant.

2. An application of acetic acid in the intervention of methamphetamine addiction, characterized in that, Animal models of METH addiction were established using acetic acid as described in claim 1.

3. The application as described in claim 2, characterized in that, The acetic acid is food-grade sodium acetate with a purity of ≥99%. The acetic acid plays a role in preventing and / or reducing METH addiction by supplementing short-chain fatty acids, regulating the gut-brain axis, inhibiting neuroinflammation, and weakening the activation of the central reward circuit.

4. The application as described in claim 3, wherein the prevention and / or reduction of METH addiction includes intervening in the process of METH addiction formation.

5. An acetic acid preparation for intervening in methamphetamine addiction, characterized in that, This is intended to implement the use of an acetic acid as described in any one of claims 2-4 in the intervention of methamphetamine addiction; the intervention of methamphetamine addiction includes prevention and / or reduction of METH addiction.

6. The acetic acid preparation according to claim 5, characterized in that, Acetic acid, alone or in combination with other active ingredients, is used in the preparation of oral medications, beverages, or health foods.

7. A method for preparing an acetic acid preparation for intervening in methamphetamine addiction as described in claim 5, characterized in that, include: Step 1, Raw material preparation: Select food-grade sodium acetate, sterile drinking water, and 0.22μm sterile filter membrane; Step 2, Solution preparation: Add sodium acetate to sterile drinking water at a concentration of 150 mmol / L and stir thoroughly to dissolve; Step 3, sterilization filtration: The prepared sodium acetate solution is filtered through a 0.22μm sterile filter membrane for sterilization; Step 4, aliquoting and storage: aliquot under aseptic conditions, store at 4°C, and replace with fresh solution weekly to avoid contamination and loss of activity.

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