Application of N-acetylneuraminic acid in the preparation of drugs for treating methamphetamine addiction

Abstract

This application relates to the field of biomedical technology, and in particular to the application of N-acetylneuraminic acid in the preparation of drugs for treating methamphetamine addiction. This application is the first to discover the key role of N-acetylneuraminic acid (Neu5Ac) in the treatment of methamphetamine addiction, which can significantly reduce the CPP score in methamphetamine-addicted mice, demonstrating a good therapeutic effect on methamphetamine addiction. Furthermore, Neu5Ac can be synthesized endogenously, exhibiting advantages such as good biocompatibility and low toxicity.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, and in particular to the application of N-acetylneuraminic acid in the preparation of drugs for treating methamphetamine addiction. Background Technology

[0002] Methamphetamine addiction is a chronic, highly relapsing, and complex brain disorder characterized by a progressive development from impulsive drug-seeking triggered by initial use to compulsive drug-seeking, leading to strong physical and psychological dependence. Current treatments for methamphetamine addiction primarily employ psychosocial therapy (such as cognitive behavioral therapy) and pharmacological interventions (such as methylphenidate and bupropion). However, the available drug options for treating methamphetamine addiction are limited, and the overall efficacy of interventions is unsatisfactory. Furthermore, many of these drugs are exogenous synthetic compounds with poor biocompatibility and a high risk of toxic side effects. Therefore, the search for safe and effective drugs to treat methamphetamine addiction is of great significance to global public health and human health.

[0003] N-acetylneuraminic acid (Neu5Ac) is the major form of sialic acid, a naturally occurring nine-carbon amino monosaccharide with the chemical formula C5. 11 H 19 Neu5Ac (NO9, CAS No. 131-48-6) plays a crucial role in neuronal differentiation, growth, and regeneration, supporting synaptic transmission, maintaining normal cellular function, and enhancing learning and memory. It is abundant in human breast milk and has become an essential nutrient for brain development, influencing learning and memory functions. In the vascular system, Neu5Ac regulates endothelial integrity and participates in the pathogenesis of atherosclerosis. However, the role of Neu5Ac in drug addiction, particularly methamphetamine addiction, has not been reported. Summary of the Invention

[0004] Based on this, one or more embodiments of this application provide the use of N-acetylneuraminic acid in the preparation of medicaments for treating methamphetamine addiction. N-acetylneuraminic acid can be synthesized endogenously, has good biocompatibility, few toxic side effects, and has a good therapeutic effect on methamphetamine addiction.

[0005] Furthermore, the drug can reduce the CPP score in methamphetamine-induced mice.

[0006] Furthermore, the drug comprises a therapeutically effective amount of the N-acetylneuraminic acid.

[0007] In some embodiments, the drug includes pharmaceutically acceptable excipients.

[0008] Furthermore, the pharmaceutically acceptable excipients include one or more of the following: diluents, wetting agents, binders, disintegrants, lubricants, flavoring agents, solvents, solubilizers, cosolvents, emulsifiers, antioxidants, preservatives, and pH adjusters.

[0009] In some embodiments, the dosage form of the drug is a capsule, tablet, pill, powder, granule, emulsion, solution, suspension, syrup, or tincture.

[0010] In some embodiments, the routes of administration of the drug include oral administration, intraperitoneal injection, intravenous injection, and intracerebral injection.

[0011] In some embodiments, the drug comprises the N-acetylneuraminic acid and a solvent; optionally, the concentration of the N-acetylneuraminic acid is 10 mg / mL to 30 mg / mL.

[0012] In some embodiments, the subject of the drug is a mammal.

[0013] Furthermore, the subjects of the drug are humans or mice.

[0014] This application is the first to discover the crucial role of N-acetylneuraminic acid (Neu5Ac) in the treatment of methamphetamine addiction, which can reduce the CPP score in methamphetamine-induced mice and has a good therapeutic effect on methamphetamine addiction. Furthermore, Neu5Ac can be synthesized endogenously, exhibiting advantages such as good biocompatibility and low toxicity. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the specific embodiments of this application or the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0016] Figure 1 These are the behavioral test results for the mice. Figure 1 In the figure, a represents the exploration time (in seconds) of the methamphetamine model group (MC group) mice in the drug-related compartment (L) and non-drug-related compartment (R) of the conditioned place preference (CPP) device at baseline. Figure 1 In the figure, b represents the exploration time (in seconds) of the methamphetamine model group (MC group) mice in the drug-related compartment (L) and non-drug-related compartment (R) of the conditioned position preference (CPP) device during the pre-test phase of the CPP experiment. "**" indicates extremely significant differences between groups (P<0.01). Figure 1In the middle, c represents the preference scores (in seconds) of mice in the methamphetamine model group (MC group, blue dot) and mice in the methamphetamine plus Neu5Ac treatment group (N group, green dot) after the CPP experiment; the horizontal axis represents the experimental group, "MC" represents the model group, and "N" represents the intervention group; the vertical axis represents the preference score (seconds), and "*" represents a significant difference between groups (P<0.05).

[0017] Figure 2 The test results are for samples from different groups of mice. Figure 2 In the middle column, 'a' represents the Neu5Ac content (in ng / L) in the striatum of mice in the control group (CC group, black column), the methamphetamine model group (MC group, green column), and the methamphetamine plus Neu5Ac treatment group (N group, red column); the horizontal axis represents the experimental group; the vertical axis represents the Neu5Ac level in the striatum (ng / L); "*" indicates a significant difference between groups (P<0.05), and "**" indicates an extremely significant difference between groups (P<0.01). Figure 2 In the middle column, b represents the Neu5Ac content (unit: ng / L) in the whole brain tissue of mice in the control group (CC group, black column), the methamphetamine model group (MC group, green column), and the methamphetamine plus Neu5Ac treatment group (N group, red column); the horizontal axis represents the experimental group; the vertical axis represents the whole brain Neu5Ac level (ng / L); the error bar represents the standard error; "*" represents significant difference between groups (P<0.05), and "**" represents extremely significant difference between groups (P<0.01).

[0018] Figure 3 The results of Western blot analysis of striatal dopamine D2 receptor (D2R) protein in control mice (CC group, left band), methamphetamine model mice (MC group, middle band), and methamphetamine plus Neu5Ac treatment mice (N group, right band) are shown. The band labeled with D2R is the target D2R protein band, and the band labeled with β-actin is the β-actin internal reference protein band.

[0019] Figure 4 The results show the quantitative analysis of striatal D2R protein expression in control mice (CC group, blue column), methamphetamine model mice (MC group, green column), and methamphetamine plus Neu5Ac treatment mice (N group, red column); the horizontal axis represents the experimental group; the vertical axis represents the relative expression level of D2R protein (corrected with β-actin as an internal reference); "**" indicates extremely significant differences between groups (P<0.01).

[0020] Figure 5Immunofluorescence staining results of striatal dopamine D2 receptor (D2R) in control mice (CC group, left column), methamphetamine model mice (MC group, middle column), and methamphetamine plus Neu5Ac treatment mice (N group, right column); the blue fluorescence in the DAPI row represents cell nuclear staining, used to mark cell location; the red fluorescence in the D2R row represents the specific signal of D2R protein; the Merge row is an overlay image of DAPI and D2R fluorescence; the scale bar represents the length unit of the field of view; the scale bar is 50 μm.

[0021] Figure 6 The results show the quantitative analysis of striatal D2R immunofluorescence intensity in control mice (CC group, black column), methamphetamine model mice (MC group, green column), and methamphetamine plus Neu5Ac treatment mice (N group, red column); the horizontal axis represents the experimental group; the vertical axis represents the mean fluorescence intensity (MFI) of D2R; "*" indicates significant difference between groups (P<0.05), and "**" indicates extremely significant difference between groups (P<0.01). Detailed Implementation

[0022] The present application is further described below with reference to embodiments and examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the application. Furthermore, it should be understood that after reading the teachings of this application, those skilled in the art can make various alterations or modifications to this application, and these equivalent forms also fall within the protection scope of the appended claims.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the specification of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of this application.

[0024] In this application, "pharmaceutically acceptable" means those ligands, materials, compositions, and / or dosage forms that are suitable for administration to patients within the bounds of reasonable medical judgment and that are commensurate with a reasonable benefit / risk ratio.

[0025] In this application, "medicine" includes any agent, compound, composition, or mixture that provides physiological and / or pharmacological effects in vivo or in vitro, and often provides beneficial effects. The scope of the physiological and / or pharmacological effects produced by the "medicine" in vivo is not particularly limited; it may have systemic effects or only local effects. The activity of the "medicine" is not particularly limited; it may be an active substance that can interact with other substances or an inert substance that does not interact with other substances.

[0026] In this application, "one or more" means any one, two or more of the listed items.

[0027] In this application, "further" is used to describe the purpose and indicate differences in content, but should not be construed as a limitation on the scope of protection of this application.

[0028] Unless otherwise specified, the percentage content mentioned in this application refers to mass percentage for solid-liquid mixtures and solid-phase-solid mixtures, and volume percentage for liquid-phase-liquid mixtures.

[0029] In this application, the abbreviations are as follows: MA refers to methamphetamine; Neu5Ac refers to N-acetylneuraminic acid; D2R refers to dopamine receptor D2; GNE enzyme refers to UDP-N-acetylglucosamine 2-epimerase / N-acetylmannosamine kinase (the rate-limiting enzyme in Neu5Ac biosynthesis); liver-brain axis refers to the physiological mechanism by which liver metabolites act on the brain through blood circulation, affecting nerve function; CPP score refers to conditioned position preference (CPP) score, a behavioral experimental method for assessing drug addiction and reward effects, which calculates preference scores by recording the time animals spend in drug-related and non-drug-related environments; LC-MS / MS refers to liquid chromatography-tandem mass spectrometry (LC-MS / MS); ELISA refers to enzyme-linked immunosorbent assay; Western spectroscopy is also used. blotting refers to the Western blotting method for protein immunoblotting; immunofluorescence refers to the detection of the expression and distribution of specific proteins in tissues or cells using fluorescently labeled antibodies; transmission electron microscopy (TEM) refers to transmission electron microscopy; Golgi staining refers to the Golgi staining method; other abbreviations not listed can be referred to the general definitions in the field of neurobiology.

[0030] Application method:

[0031] There are no particular restrictions on the dosage form and method of administration of the drug in this application.

[0032] Representative methods of administration include, but are not limited to, oral and injection.

[0033] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following components: (a) fillers or compatibilizers, such as starch, lactose, sucrose, glucose, mannitol, and silica; (b) binders, such as hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and gum arabic; (c) humectants, such as glycerin; (d) disintegrants, such as agar, calcium carbonate, potato starch or cassava starch, alginate, certain complex silicates, and sodium carbonate; (e) slowing agents, such as paraffin; (f) absorption accelerators, such as quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glyceryl monostearate; (h) adsorbents, such as kaolin; and (i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium dodecyl sulfate, or mixtures thereof. In capsules, tablets, and pills, the dosage form may also contain a buffer. Solid dosage forms such as tablets, sugar pills, capsules, pills, and granules can be prepared using coatings and shells, such as casings and other materials known in the art. They may contain opacifying agents, and the release of the active compound or compound in such compositions may be delayed in a portion of the digestive tract. Examples of encapsulating components that may be used are polymeric substances and waxes. If necessary, the active compound may also be formed into microcapsules with one or more of the excipients described above.

[0034] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or tinctures. In addition to the active compound, liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers, and emulsifiers, specifically, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butanediol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil, or mixtures thereof. Besides these inert diluents, the composition may also contain adjuvants such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, and fragrances. For example, suspensions may contain suspending agents, specifically, for example, ethoxylated isooctadecyl alcohol, polyoxyethylene sorbitol and dehydrated sorbitan esters, microcrystalline cellulose, aluminum methoxide, and agar, or mixtures thereof.

[0035] Liquid dosage forms for injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions, or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous or non-aqueous carriers, diluents, solvents, or excipients include water, ethanol, polyols, and suitable mixtures thereof.

[0036] The following are some specific examples.

[0037] For experimental parameters not specified in the following specific embodiments, please refer to the guidelines provided in this application, or consult experimental manuals or other known experimental methods in the art, or refer to the manufacturer's recommended experimental conditions. The raw materials and reagents not specified in the following specific embodiments are all commercially available, or can be prepared by those skilled in the art using known methods.

[0038] Materials and reagents: Neu5Ac was purchased from MedChemExpress (catalog number HY-I0400), with a purity ≥98%. Methamphetamine was provided by the Sichuan Provincial Key Laboratory of Intelligent Policing, Sichuan Police Academy, with a purity of 99.9%. Sulpiride (D2R antagonist) was purchased from MedChemExpress (catalog number HY-B1019). Phosphate-buffered saline (PBS buffer, pH 7.4), water for injection, and physiological saline were purchased from Thermo Fisher Scientific.

[0039] Laboratory animals: 8-week-old male C57BL / 6J mice, weighing 22 to 25 grams, SPF grade, purchased from Chengdu Dashuo Laboratory Animal Co., Ltd. or other qualified suppliers. SPF grade animal laboratory housing, 12-hour 24-hour light cycle, room temperature 26±3 degrees Celsius, relative humidity 40% to 60%, free access to food and water.

[0040] Experimental setup: The CPP device was purchased from Jiangsu Saions Technology Co., Ltd. (Jiangsu, China). The device consists of two identical compartments (15cm×17cm×20cm) on the left (black stripe) and right (white) sides, and a central corridor (6cm×15cm×20cm). Each compartment has a separate camera at the top to collect mouse trajectories. All data are recorded using the accompanying video tracking system.

[0041] Example 1

[0042] In this embodiment, a methamphetamine addiction mouse model was established by combining conditioned place preference (CPP) test with intraperitoneal administration of methamphetamine. Neu5Ac was administered intraperitoneally as an intervention. The addiction of mice in the model group and the administration group was evaluated by CPP test.

[0043] 1.1 Animal Grouping

[0044] Eight-week-old male C57BL / 6J mice were randomly divided into a control group (CC group), a methamphetamine model group (MC group), and a methamphetamine plus Neu5Ac treatment group (N group), with 10 mice in each group.

[0045] 1.2 Conditional Place Preference (CPP) Experiment

[0046] Day 1 (Adaptation period): Mice were placed in the central corridor of the CPP device and allowed to explore freely for 10 minutes.

[0047] Day 2 (baseline period): Record the time the mice spent in the two compartments.

[0048] Days 3-9 (Conditioning Period): At 10:00 AM daily, mice in the MC and N groups were intraperitoneally injected with methamphetamine solution at a dose of 1 mg / kg / day (calculated as methamphetamine). The methamphetamine solution was prepared by dissolving methamphetamine in physiological saline to a specific concentration. The concentration needed to be calculated based on the mouse's weight, ensuring that the injection volume = mouse weight (g) × 0.005 mL. For example, for a mouse weighing 25g (0.025kg), the daily dose of methamphetamine was 0.025mg, and the injection volume = 25 × 0.005mL = 0.125mL. 0.025mg of methamphetamine should be dissolved in 0.125mL of physiological saline to prepare a solution with a concentration of 0.2mg / mL. Mice in the CC group were injected with physiological saline, with the injection volume = mouse weight (g) × 0.005mL. After injection, each group of mice was placed in a drug-specific compartment for 30 minutes. Every afternoon at 18:00, mice in each group were injected with an equal volume of physiological saline. After the injection, the mice in each group were placed in a non-drug compartment for 30 minutes.

[0049] Day 10 (Pre-CPP test): Mice explored freely for 10 minutes, and the time spent there was recorded.

[0050] Days 11 to 24 (administration): Mice in the MC and N groups were intraperitoneally injected with Neu5Ac solution at 10:00 AM daily, at a dose of 20 mg / kg / day (Neu5Ac). The Neu5Ac solution was prepared by dissolving Neu5Ac in PBS buffer to a specific concentration. The concentration needed to be calculated based on the mouse's weight, ensuring that the injection volume = mouse weight (g) × 0.005 mL. For example, for a mouse weighing 25g (0.025kg), the daily dose of Neu5Ac was 0.5 mg, and the injection volume = 25 × 0.005 mL = 0.125 mL. 0.5 mg of Neu5Ac should be dissolved in 0.125 mL of PBS buffer to prepare a solution with a concentration of 4 mg / mL. Mice in the CC group were injected with physiological saline, with an injection volume = mouse weight (g) × 0.005 mL. After injection, each group of mice was placed in a drug-specific compartment for 30 minutes. Every afternoon at 18:00, mice in each group were injected with an equal volume of physiological saline. After the injection, the mice in each group were placed in a non-drug compartment for 30 minutes.

[0051] On day 25 (post-CPP test), mice freely explored the CPP device for 10 minutes, and the dwell time was recorded. The CPP score was calculated. CPP score = dwell time in drug-related compartments - dwell time in non-drug-related compartments.

[0052] 1.3 CPP Scoring Results

[0053] Figure 1 These are the behavioral test results for the mice. Figure 1 In the figure, 'a' represents the exploration time (in seconds) of the methamphetamine model group (MC group) mice in the drug-related compartment (L) and non-drug-related compartment (R) of the conditioned place preference (CPP) device at baseline. The "ns" marked in the figure indicates that there is no statistically significant difference in the exploration time of mice in the L and R compartments within the same group, proving that there is no significant difference in the time mice spend in the left and right compartments at baseline. Figure 1 In the figure, b represents the exploration time (in seconds) of the methamphetamine model group (MC group) mice in the drug-related compartment (L) and non-drug-related compartment (R) of the conditioned position preference (CPP) device during the pre-test phase of the CPP experiment. "*" indicates significant differences between groups (P<0.05). This figure demonstrates that the mice spent significantly more time in the drug-related compartment (R) during the pre-test phase. Figure 1 In the middle, c represents the preference scores (in seconds) of mice in the methamphetamine model group (MC group, blue dot) and the methamphetamine plus Neu5Ac treatment group (N group, green dot) after the CPP experiment; the horizontal axis represents the experimental group, "MC" represents the model group, and "N" represents the intervention group; the vertical axis represents the preference score (seconds); "**" indicates that the difference between the groups is extremely significant (P<0.01), proving that compared with the MC group, the N group mice spent significantly less time in the right box.

[0054] Figure 1 The experimental results showed that, compared with the MC group, the CPP score of mice in the N group was significantly reduced, indicating that Neu5Ac can effectively alleviate methamphetamine addiction behavior.

[0055] 1.4 Sample Collection and Testing

[0056] After the Conditional Place Preference (CPP) test scores were completed, mice in each group were anesthetized with 4% sodium pentobarbital (50 mg / kg, intraperitoneal injection); 1-2 mL of blood was collected from the apex of the heart, centrifuged at 3000 rpm for 15 min, and the serum was separated and stored at -80℃; liver, striatum, and brain tissue were rapidly removed, flash-frozen in liquid nitrogen, and stored at -80℃; the above samples were then tested using the following methods:

[0057] 1.4.1 The levels of Neu5Ac in serum, liver, and striatum were detected by liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0058] 1.4.1.1 Preparation of striatal and liver samples

[0059] (1) All extraction reagents should be pre-cooled at -20℃ before use.

[0060] (2) Take an appropriate amount of sample and add 400 μL of pre-cooled (4℃) methanol-water solution (v / v=4:1), which contains 0.1% formic acid and the internal standard succinic acid-2,2,3,3-d4.

[0061] (3) Add two small steel balls to the sample and place it in a -20°C freezer for 2 minutes. The sample pan used for homogenization should be pre-cooled at -20°C to ensure that the temperature does not get too high during homogenization. Then, put the sample into a grinder and grind it at a frequency of 60 Hz for 2 minutes.

[0062] (4) After grinding, the sample was vortexed for 1 minute, followed by sonication in an ice-water bath for 10 minutes. Then, it was centrifuged at 4°C and 12,000 rpm for 10 minutes, and then 300 μL of the supernatant was evaporated.

[0063] (5) After evaporation, add 200 μL of pre-cooled (4°C) methanol-water solution (v / v=4:1, also containing 0.1% formic acid and internal standard succinic acid-2,2,3,3-d4) to the residue again.

[0064] Repeat steps (4)-(5) to perform vortex mixing, ice-water bath sonication, and centrifugation on the sample. Then, take 200 μL of the supernatant and evaporate it to dryness.

[0065] (6) After evaporation, the sample was reconstituted with 200 μL of a water-acetonitrile mixture (v / v=95:5, containing L-2-chlorophenylalanine). Then, the sample was vortexed for 30 seconds and subjected to sonication in an ice-water bath for 5 minutes.

[0066] (7) Centrifuge at 4°C and 13000 rpm for 5 minutes. Use a syringe to draw 200 μL of the supernatant and filter it through a 0.22 μm organic phase pinhole filter. Finally, transfer the filtered solution to a brown vial for LC-MS / MS analysis.

[0067] (8) The quality control sample is made by mixing the extracts of all samples in equal volumes.

[0068] 1.4.1.2 Preparation of serum samples

[0069] (1) All extraction reagents were pre-cooled at -20°C before use.

[0070] (2) Take an appropriate amount of sample and then add 320 μL of pre-cooled (4°C) methanol-acetonitrile mixed solution (v / v=2:1), which contains 0.1% formic acid, 0.1 mM BHT, and internal standards succinic acid-2,2,3,3-d4 and C-17.

[0071] (3) Vortex the mixed sample for 1 minute, then sonicate it in an ice-water bath for 10 minutes. After that, centrifuge it at 4°C and 12,000 rpm for 10 minutes. After centrifugation, take 300 μL of the supernatant and evaporate it to dryness.

[0072] (4) After evaporation, the solution was reconstituted with 200 μL of a water-acetonitrile mixture (v / v = 95:5) containing L-2-chlorophenylalanine. Then, the mixture was vortexed for 30 seconds and sonicated in an ice-water bath for 5 minutes.

[0073] (5) After ultrasonic treatment, the sample was centrifuged at 4°C and 13,000 rpm for 5 minutes. Then, 200 μL of the supernatant was drawn up using a syringe and filtered through a 0.22 μm organic phase pinhole filter. The filtered solution was transferred to a brown vial for LC-MS / MS analysis.

[0074] (6) The quality control sample is prepared by mixing the extracts of all samples in equal volumes.

[0075] 1.4.1.3 Chromatography-Mass Spectrometry (LC-MS / MS) Detection Method

[0076] Chromatographic conditions: injection volume was 5 μL; flow rate was 0.35 mL / min; mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was acetonitrile; gradient elution was used, and the gradient elution program is shown in Table 1.

[0077] Table 1 Gradient elution procedure

[0078]

[0079] Mass spectrometry conditions: curtain gas 35 psi; collision-induced ionization parameter medium; positive ion spray voltage 4500 V; negative ion spray voltage -4500 V; ion source temperature 450 °C; column temperature 40 °C; spray gas 60 psi; auxiliary heating gas 55 psi.

[0080] 1.4.2 The level of Neu5Ac in brain tissue was detected by enzyme-linked immunosorbent assay (ELISA).

[0081] 1.4.2.1 Preparation of brain tissue samples

[0082] Pre-cool the PBS solution, then use this solution to wash the tissue block three times to thoroughly remove blood stains. Cut an appropriate amount of brain tissue, weigh it, place it in a homogenization tube, and centrifuge it using butanol:methanol:water (V / V / V=5:25:70) as the extraction solution at 3000 rpm, 4°C, and for 20 minutes. Carefully collect the supernatant after centrifugation.

[0083] 1.4.2.2 ELISA Detection Method

[0084] (1) Reagent rewarming: Ensure that all reagents have reached room temperature.

[0085] (2) Sample addition: Set up standard wells, blank wells, and sample wells on the microplate. Add diluted standard to the standard wells. Add sample dilution to the sample wells first, followed by the sample to be tested. Ensure that the sample does not come into contact with the well walls during all operations, and mix gently.

[0086] (3) Adding enzyme label: Add enzyme labeling reagent to each well except for the blank well.

[0087] (4) Incubation: After sealing the microplate with the sealing film, incubate at 37°C for 60 minutes.

[0088] (5) Preparation of washing solution: Dilute the concentrated washing solution with distilled water to the required multiple and set aside.

[0089] (6) Washing: Remove the sealing film, discard the liquid in the well, and wash repeatedly with washing solution to remove unbound reagents and impurities.

[0090] (7) Color reaction: Add color reagent A and B to each well, mix gently, and develop color for 15 minutes at 37°C in the dark to allow the enzyme to catalyze the substrate to produce a color change.

[0091] (8) Termination of reaction: Add stop solution to stop the colorimetric reaction.

[0092] (9) Measurement: Using blank wells as a reference, the absorbance of each well was measured at a wavelength of 450 nm.

[0093] (10) Calculation: Plot a standard curve based on the concentration and corresponding absorbance value of the standard. By measuring the absorbance value of the sample, find the corresponding concentration on the standard curve, and take into account the dilution factor to obtain the actual concentration of the sample.

[0094] 1.4.3 Western blotting was used to detect striatal D2R protein expression.

[0095] 1.4.3.1 Preparation of striatal samples

[0096] (1) Pre-cool the PBS solution, then use this solution to wash the tissue block three times to thoroughly remove blood stains. Cut the tissue into several small pieces using surgical scissors, weigh them, and place them in a homogenization tube. Add two 3 mm homogenization beads to the homogenization tube and add lysis buffer equivalent to ten times the tissue volume. Two minutes before use, add protease inhibitor to the lysis buffer and set the homogenization program.

[0097] (2) After homogenization, remove the homogenizing tube from the machine and transfer the homogenate to a centrifuge tube. Place the centrifuge tube on ice and continue adding lysis buffer, then let it stand for 30 minutes. To ensure complete tissue lysis, shake the centrifuge tube on a shaker every 5 minutes.

[0098] (3) Set the centrifuge parameters to 12000 rpm and 4℃, and centrifuge the homogenate tube for 10 minutes. After centrifugation, collect the supernatant for subsequent experiments.

[0099] (4) Follow the instructions of the BCA protein quantitative detection kit. The process includes preparing protein standard stock solution, preparing protein standard working solution, plotting standard curve (ELISA method), preparing test samples, preparing BCA colorimetric working solution, sample detection, and data calculation.

[0100] (5) Add 5 times the volume of reduced protein loading buffer to the protein solution at a ratio of 4:1, then place the test tube in the instrument and heat the protein for 15 minutes to denature the protein.

[0101] 1.4.3.2 SDS-PAGE electrophoretic separation

[0102] Prepare separating and stacking gels of varying concentrations based on the molecular weight of the target protein. Smaller proteins require higher-concentration separating gels, while larger proteins require lower-concentration ones. Pour the prepared separating gel between glass plates, add distilled water to seal the top, and let it stand for 30 minutes to solidify. Then, pour out the distilled water, fill the stacking gel completely (avoiding air bubbles), and insert the comb at an angle. After the gel has solidified, fix the glass plates in the electrophoresis tank, pour in the electrophoresis buffer, remove the comb, and add protein samples to each lane according to the calculated concentration and volume, ready for electrophoresis.

[0103] At the start of electrophoresis, set the voltage to 70V. Once the proteins have entered the separating gel, adjust the voltage to 110V. Stop electrophoresis when the bromophenol blue is approximately 1-2 cm from the bottom of the electrophoresis tank.

[0104] 1.4.3.3 Protein Transfer

[0105] Soak the PVDF membrane in methanol for later use. Assemble the membrane in the following order: sponge (2 sheets), filter paper (2 sheets), gel, PVDF membrane, filter paper (2 sheets), sponge (2 sheets), ensuring all air bubbles are removed. Fix the assembled membrane transfer structure in the electroporation apparatus, ensuring an ice-water bath environment. Select a constant current of 220mA for membrane transfer, with a transfer time of approximately 80 minutes.

[0106] 1.4.3.4 Western blot and detection of proteins

[0107] After transfer, the PVDF membrane was blocked with BSA for two hours. Then, the membrane was transferred to primary antibody solutions (β-actin dilution 1:1000, GAPDH dilution 1:10000, D2R dilution 1:200, α-SYN dilution 1:1000). The membrane was incubated overnight at 4°C. After primary antibody incubation, the membrane was eluted three times with TBST on a shaker for 15 minutes each time. Then, horseradish enzyme-labeled goat anti-rabbit / mouse secondary antibody (dilution 1:5000) was diluted with BSA. The membrane was incubated at room temperature on a shaker for 2 hours. After secondary antibody incubation, the membrane was eluted three more times with TBST. Then, in a dark room, ECL solutions A and B were mixed in a 1:1 ratio, and an appropriate amount of the mixture was reacted with the membrane. After the reaction was complete, residual liquid on the membrane was blotted off with filter paper. Finally, the membrane was placed in a computer for development and fixing to observe and record the experimental results.

[0108] 1.4.3.5 Gel Image Analysis

[0109] The film was scanned and archived, and the target band of the image was analyzed using ImageJ software.

[0110] 1.4.4 Immunofluorescence was used to detect the expression and distribution of D2R in the striatum.

[0111] 1.4.4.1 Preparation of striatal samples

[0112] (1) Dewaxing to water: The sections were treated with xylene I, II and III for 15 minutes each, then dehydrated with anhydrous ethanol I and II, 85% alcohol and 75% alcohol gradient for 5 minutes each time, and finally washed with distilled water.

[0113] (2) Antigen retrieval: The slides were immersed in citrate buffer (pH 6.0) and microwaved for 20 minutes for retrieval. After cooling, they were washed three times with PBS for 5 minutes each time.

[0114] (3) Blocking endogenous peroxidase: Place the slices in 3% hydrogen peroxide and incubate at room temperature in the dark for 25 minutes, then wash them three times in PBS (pH 7.4) for 5 minutes each time;

[0115] (4) Serum blocking: bovine serum albumin, at room temperature for more than 30 minutes;

[0116] (5) Add D2R primary antibody (1:100, Stata) dropwise and incubate overnight at 4°C;

[0117] (6) After washing three times with PBS, add CY3 secondary antibody (Servicebio) diluted to 1:100, incubate at 37°C for 30 minutes, and then wash three times with PBS again.

[0118] (7) Add DAPI dropwise and incubate at room temperature for 10 min; wash 3 times with PBS, 5 min each time;

[0119] (8) Use anti-fluorescence decay mounting medium to mount the slides.

[0120] 1.4.4.2 Microscopic observation

[0121] Observation and photography were performed using a laser scanning confocal microscope. Channel settings: DAPI channel, excitation light ~405nm, emission light collection range 430-470nm (blue), used to display all cell nuclei and define cell and tissue structures; CY3 channel, excitation light 550nm, emission light collection range 570-620nm (red), used to display the localization and expression intensity of D2R protein.

[0122] 1.4.5 Observation of the ultrastructure of striatal synapses using transmission electron microscopy

[0123] Transmission electron microscopy revealed that methamphetamine exposure reduced the number of striatal synapses from 28.5±3.2 per field of view to 15.3±2.7 (P<0.001), and the number of synaptic vesicles from 45.7±5.8 per synapse to 22.3±4.2 (P<0.001). After Neu5Ac treatment, these numbers recovered to 25.7±3.1 and 40.8±5.5, respectively (P<0.01).

[0124] 1.4.6 Dendritic spine density was observed using Golgi staining.

[0125] Golgi staining showed that the dendritic spine density decreased from 18.5±2.3 per 50 μm in the control group to 9.7±1.8 (P<0.001), and recovered to 16.8±2.2 per 50 μm after Neu5Ac treatment (P<0.01). These results indicate that Neu5Ac can effectively improve synaptic structure and function and restore synaptic plasticity.

[0126] 1.5 Sample Test Results

[0127] Figure 2 The results are from striatal samples of different groups of mice. Figure 2In the middle column, 'a' represents the Neu5Ac content (ng / L) in the striatum of mice in the control group (CC group, black column), the methamphetamine model group (MC group, green column), and the methamphetamine plus Neu5Ac treatment group (N group, red column); the horizontal axis represents the experimental group; the vertical axis represents the striatum Neu5Ac level (ng / L); "*" indicates a significant difference between groups (P<0.05), and "**" indicates an extremely significant difference between groups (P<0.01); the striatum Neu5Ac level in the MC group was significantly lower than that in the CC group, suggesting that methamphetamine can inhibit the synthesis of endogenous Neu5Ac or accelerate its consumption; the striatum Neu5Ac level in the N group was significantly higher than that in the MC group, proving that exogenous supplementation of Neu5Ac can effectively increase the Neu5Ac concentration in the striatum; Figure 2 In the middle column (b), the Neu5Ac content (in ng / L) in the whole brain tissue of mice in the control group (CC group, black column), the methamphetamine model group (MC group, green column), and the methamphetamine plus Neu5Ac treatment group (N group, red column) is represented. The horizontal axis represents the experimental group; the vertical axis represents the whole brain Neu5Ac level (ng / L); the error bar represents the standard error; "*" indicates a significant difference between groups (P<0.05), and "**" indicates an extremely significant difference between groups (P<0.01). The whole brain Neu5Ac level in the MC group was significantly lower than that in the CC group, reflecting the extensive effect of methamphetamine on Neu5Ac metabolism in the central nervous system; the whole brain Neu5Ac level in the N group was significantly higher than that in the MC group, confirming that Neu5Ac can cross the blood-brain barrier and increase the Neu5Ac level in the central nervous system.

[0128] Figure 3 This data represents the Western blot results of striatal dopamine D2 receptor (D2R) protein in control mice (CC group, left band), methamphetamine model mice (MC group, middle band), and methamphetamine plus Neu5Ac treatment mice (N group, right band). The D2R-labeled band represents the target D2R protein, and the β-actin-labeled band represents the β-actin internal reference protein. The D2R protein band in the MC group was significantly weaker than that in the CC group, suggesting that methamphetamine addiction can downregulate striatal D2R expression. The D2R protein band in the N group was significantly brighter than that in the MC group, indicating that Neu5Ac can repair the abnormal expression of D2R.

[0129] Figure 4The results show the quantitative analysis of striatal D2R protein expression in control mice (CC group, blue column), methamphetamine model mice (MC group, green column), and methamphetamine plus Neu5Ac treatment mice (N group, red column). The horizontal axis represents the experimental group; the vertical axis represents the relative expression level of D2R protein (corrected with β-actin as an internal reference); "**" indicates extremely significant differences between groups (P<0.01). The relative expression level of D2R protein in the MC group was significantly lower than that in the CC group, further verifying the inhibitory effect of methamphetamine on D2R; the expression level of D2R protein in the N group was significantly upregulated compared with the MC group, confirming that Neu5Ac can reverse the downregulation of D2R expression and repair the dopamine signaling pathway.

[0130] Figure 5 Immunofluorescence staining results of striatal dopamine D2 receptor (D2R) in control mice (CC group, left column), methamphetamine model mice (MC group, middle column), and methamphetamine plus Neu5Ac treatment mice (N group, right column); the DAPI row (blue fluorescence) represents nuclear staining, used to mark cell location; the D2R row (red fluorescence) represents the specific signal of D2R protein; the Merge row (overlay image of DAPI and D2R fluorescence) represents the superimposed image of DAPI and D2R fluorescence; the scale bar represents the length unit of the field of view; the scale bar is 50 μm. The red fluorescence signal of D2R in the MC group was almost absent, reflecting that methamphetamine addiction leads to severe abnormalities in the expression and distribution of striatal D2R; the red fluorescence signal of D2R in the N group was significantly restored compared with the MC group, suggesting that Neu5Ac can improve the cellular expression and distribution of D2R.

[0131] Figure 6 The table shows the quantitative analysis results (D2RMFI) of striatal D2R immunofluorescence intensity in control mice (CC group, black column), methamphetamine model mice (MC group, green column), and methamphetamine plus Neu5Ac treatment mice (N group, red column). The horizontal axis represents the experimental group; the vertical axis represents the mean fluorescence intensity (MFI) of D2R. "*" indicates significant difference between groups (P<0.05), and "**" indicates extremely significant difference between groups (P<0.01). The D2R fluorescence intensity in the MC group was significantly lower than that in the CC group, further demonstrating the inhibitory effect of methamphetamine on D2R expression. The D2R fluorescence intensity in the N group significantly increased compared to the MC group, confirming that Neu5Ac can repair the expression and function of D2R at the cellular level, thereby improving the abnormality of the dopamine neural circuit.

[0132] The technical features of the above-described embodiments and examples can be combined in any suitable manner. For the sake of brevity, not all possible combinations of the technical features in the above-described embodiments and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0133] The embodiments described above merely illustrate several implementation methods of this application and should not be construed as limiting the scope of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Furthermore, it should be understood that after reading the above teachings, those skilled in the art can make various alterations or modifications to this application, and the equivalent forms obtained also fall within the protection scope of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the protection scope of the appended claims. Therefore, the protection scope of this patent application should be determined by the appended claims, and the specification can be used to interpret the content of the claims.

Claims

1. Application of N-acetylneuraminic acid in the preparation of drugs for treating methamphetamine addiction.

2. The application according to claim 1, characterized in that, The drug can reduce the CPP score in methamphetamine-induced mice.

3. The application according to claim 1 or 2, characterized in that, The drug comprises a therapeutically effective amount of the N-acetylneuraminic acid.

4. The application according to claim 1 or 2, characterized in that, The drug includes pharmaceutically acceptable excipients.

5. The application according to claim 4, characterized in that, The pharmaceutically acceptable excipients include one or more of the following: diluents, wetting agents, binders, disintegrants, lubricants, flavoring agents, solvents, solubilizers, cosolvents, emulsifiers, antioxidants, preservatives, and pH adjusters.

6. The application according to claim 1 or 2, characterized in that, The dosage form of the drug is capsule, tablet, pill, powder, granule, emulsion, solution, suspension, syrup or tincture.

7. The application according to claim 1 or 2, characterized in that, The drug can be administered orally, via intraperitoneal injection, via intravenous injection, or via intracerebral injection.

8. The application according to claim 1 or 2, characterized in that, The drug comprises the N-acetylneuraminic acid and a solvent; the concentration of the N-acetylneuraminic acid is 10 mg / mL to 30 mg / mL.

9. The application according to claim 1 or 2, characterized in that, The subjects of the drug are mammals.

10. The application according to claim 9, characterized in that, The subjects of the drug are humans or mice.

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