Use of dimethyl itaconate in the prevention and treatment of chronic stress-induced anxiety

Abstract

The application provides application of dimethyl itaconate in prevention and treatment of chronic stress-induced anxiety. The application finds that dimethyl itaconate can inhibit excessive activation of microglial cells in a stress state, improve mitochondrial structure and function abnormalities of the microglial cells, and reduce expression of a lysosome marker CD68, so as to inhibit central nervous inflammation, maintain central nervous immune homeostasis, prevent and relieve anxiety-like behavior induced by chronic stress, and effectively prevent and relieve occurrence and development of chronic stress-induced anxiety (generalized anxiety disorder).

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to the treatment of anxiety disorders, specifically the application of dimethyl itaconic acid in the prevention and treatment of anxiety disorders induced by chronic stress. Background Technology

[0002] Anxiety disorders are a common group of mental disorders, encompassing various subtypes such as generalized anxiety disorder, panic disorder, and social anxiety disorder. Their core clinical features are excessive anxiety and fear, with persistent or excessive stress being a key environmental trigger. The prevalence of anxiety disorders is rising significantly worldwide, becoming a significant public health issue. Furthermore, they often co-occur with other mental disorders such as depression, increasing the complexity of diagnosis and treatment and exacerbating the clinical burden.

[0003] Recent studies have shown that neuroimmune disorders are a core component of the stress pathological process. Microglia, as the core executors of immune surveillance in the central nervous system, undergo progressive activation under stress, manifested as increased numbers, cell hypertrophy, and upregulation of the lysosomal marker CD68. At the same time, abnormalities such as cristae breakage and matrix vacuolation appear in the ultrastructure of mitochondria. Overactivated microglia can induce neuroinflammation, thereby exacerbating anxiety-like behaviors.

[0004] Existing medications for treating anxiety disorders mainly target the neurotransmitter system, such as benzodiazepines, 5-HT1A receptor agonists, and selective serotonin reuptake inhibitors. The mechanism of action of these drugs is mostly to regulate the levels of neurotransmitters such as serotonin and gamma-aminobutyric acid in the brain to relieve anxiety symptoms. However, existing drugs have many limitations, including: (1) Single target: Existing anti-anxiety drugs mainly regulate the neurotransmitter system and fail to intervene in the core pathological link of stress-induced neuroimmune imbalance. (2) Safety issues: Some drugs have adverse reactions such as addiction, withdrawal syndrome, and cognitive impairment, and the safety and tolerability of long-term use are limited. (3) Some drugs have a slow onset of action and rarely involve the regulation of neuroimmune homeostasis, making it difficult to fundamentally improve the neuroinflammatory imbalance caused by stress.

[0005] Therefore, there is an urgent need to find a new type of drug that can regulate neuroimmune homeostasis, has high safety, and can effectively prevent and alleviate stress-induced anxiety disorders. Summary of the Invention

[0006] Based on this, the purpose of this invention is to provide the application of dimethyl itaconic acid in the prevention and treatment of chronic stress-induced anxiety disorder. Dimethyl itaconic acid can target microglia to regulate central nervous system immune homeostasis, and is safe, efficient and effective in preventing and treating generalized anxiety disorder.

[0007] To achieve the above objectives, the present invention adopts the following technical solution.

[0008] The first aspect of the invention provides the use of dimethyl itaconic acid in the preparation of medicaments for the prevention and / or treatment of generalized anxiety disorder.

[0009] In some implementations, the application includes inhibiting excessive activation of microglia.

[0010] In some implementations, the application includes improving microglial cell mitochondrial damage.

[0011] In some implementations, the application includes improving anxiety-like behaviors.

[0012] In some embodiments, the dosage form of the drug includes injections and oral preparations.

[0013] In some embodiments, the oral dosage form includes tablets, capsules, granules, powders, pills, solutions, suspensions, mixtures, and syrups.

[0014] A second aspect of the invention provides a medicament for the prevention and / or treatment of generalized anxiety disorder, the active ingredient of which includes dimethyl itaconic acid ester.

[0015] In some embodiments, the dosage form of the drug includes injections and oral preparations.

[0016] In some embodiments, the oral dosage form includes tablets, capsules, granules, powders, pills, solutions, suspensions, mixtures, and syrups.

[0017] Compared with the prior art, the present invention has the following beneficial effects.

[0018] This invention has found that dimethyl itaconic acid can inhibit central nervous system inflammation, maintain central nervous system immune homeostasis, and prevent and alleviate anxiety-like behaviors induced by chronic stress by inhibiting the excessive activation of microglia under stress, improving their mitochondrial structure and function abnormalities, and reducing the expression of the lysosomal marker CD68. This can effectively prevent and alleviate the occurrence and development of chronic stress-induced anxiety disorder (generalized anxiety disorder).

[0019] Dimethyl itaconic acid has the advantages of high safety, no obvious adverse reactions, and strong targeting in the prevention and treatment of generalized anxiety disorder. It can be used for a long time and is not prone to drug resistance, so it has broad application prospects. Attached Figure Description

[0020] Figure 1 These are experimental results related to the inhibitory effect of dimethyl itaconic acid on chronic stress-induced microglial overactivation.

[0021] Figure 2This is the experimental result related to the ameliorative effect of dimethyl itaconic acid on the abnormal activation of mitochondria in microglia induced by chronic stress.

[0022] Figure 3 These are experimental results related to the preventive effect of dimethyl itaconic acid on anxiety-like behavior induced by chronic stress. Detailed Implementation

[0023] Unless otherwise specified, the experimental methods described in the following embodiments of the present invention are generally performed under conventional conditions or as recommended by the manufacturer. All commonly used chemical reagents used in the embodiments are commercially available products.

[0024] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0025] The terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusion. For example, a process, method, apparatus, product, or device that includes a series of steps is not limited to the steps or modules listed, but may optionally include steps not listed, or may optionally include other steps inherent to such process, method, product, or device.

[0026] The term "and / or" as used in this invention describes the relationship between related objects, indicating that there can be three relationships. For example, A and / or B can represent three situations: A exists alone, A and B exist simultaneously, and B exists alone.

[0027] The following description is based on specific implementation methods.

[0028] Dimethyl itaconate (DI), CAS No.: 617-52-7, has the following structural formula: .

[0029] Example 1: Inhibitory effect of dimethyl itaconic acid on chronic stress-induced microglial overactivation 1. Experimental Methods (1) Laboratory animals Twelve 8-week-old SPF-grade male C57BL / 6J mice were randomly divided into four groups after one week of acclimatization: solvent control + control group, DI + control group, solvent control + stress group, and DI + stress group, with three mice in each group.

[0030] (2) Preparation of DI solution Accurately weigh dimethyl itaconic acid (CAS No.: 617-52-7, purity ≥98%), dissolve it in sterile PBS to prepare a solution with a concentration of 10 mg / 500 μL, shake to mix well and use for later use.

[0031] (3) Drug administration and modeling Control group: 500 μL of PBS or DI solution was injected intraperitoneally at the same time every day, without restraint stress.

[0032] Stress group: Starting at 7 PM daily, mice were restrained for 2 hours for 7 consecutive days in 3D-printed acrylonitrile-butadiene-styrene (ABS) restraint tubes. The restraint tube design should ensure unobstructed breathing and limited movement. Mice were housed individually during the modeling period to avoid interference. 500 μL of PBS or DI solution was injected intraperitoneally 30 minutes before each restraint.

[0033] Specifically as follows: Solvent control + control group: Under non-stress conditions, 500 μL of sterile PBS was injected intraperitoneally daily; DI+ control group: Under non-stress conditions, 500 μL of DI solution was injected intraperitoneally daily; solvent control + stress group: 500 μL of sterile PBS was injected intraperitoneally 30 minutes before stress, for 7 consecutive days; DI+stress group: 500 μL of DI solution was injected intraperitoneally 30 minutes before the stress, for 7 consecutive days.

[0034] (4) Sample preparation and immunofluorescence staining Twenty-four hours after the last stress response, mice were deeply anesthetized and fixed via cardiac perfusion with 4% paraformaldehyde. Brain tissue was removed and fixed in the same fixative, then sequentially transferred to 20% and 30% sucrose solutions for dehydration. After OCT embedding, brain slices containing the prefrontal cortex, 25 μm thick, were coronally excised using a cryostat. The brain slices were blocked and incubated overnight at 4°C with primary antibodies (rabbit anti-IBA1, 1:1000; rat anti-CD68, 1:600). After washing, the slices were incubated with fluorescent secondary antibodies, counterstained with DAPI, and mounted.

[0035] (5) Image acquisition and analysis The subcortical limbic region (IL) of the prefrontal cortex was observed using laser confocal microscopy. Three to five brain slices were taken from each mouse, and two to three fields of view were taken from each slice. The number of IBA1-positive cells and the fluorescence intensity of CD68 (expressed as average fluorescence density) were quantitatively analyzed using ImageJ software.

[0036] 2. Experimental Results The results are as follows Figure 1 As shown.

[0037] Compared with the PBS+ control group, the relative expression level of CD68 in microglia in the subcortical limbic region of the prefrontal cortex of mice in the PBS+ stress group was significantly increased by 1.27 times. Figure 1 A- Figure 1 B), the number of cells increased significantly (P<0.0001) Figure 1 A, Figure 1 C), and morphologically, it showed a significant decrease in the total length of microglial processes and the monitoring area (C). Figure 1 D- Figure 1 E), Sholl analysis also indicated a significant reduction in intersection points (E), Figure 1 F), exhibiting typical characteristics of overactivation.

[0038] There were no significant differences in the expression level, quantity, and morphology of CD68 in microglia between DI+ control mice and PBS+ control mice. Figure 1 A- Figure 1 F) indicates that DI has no significant effect on microglia under normal physiological conditions; Compared with the PBS + stress group, the relative expression level of CD68 in microglia of mice in the DI + stress group was not significantly increased. Figure 1 A- Figure 1 B), the quantity has returned to normal levels. Figure 1 A, Figure 1 C), there was no significant reduction in the total length of the protrusion and the monitoring area, and no significant difference in Sholl analysis ( Figure 1 D- Figure 1 (F) indicates that dimethyl itaconic acid can effectively inhibit chronic stress-induced excessive activation of microglia.

[0039] Figure 1 In the image A: Representative images of immunostaining of Iba1 (green), CD68 (red), and DAPI (blue) proteins in the mouse prefrontal cortex (mPFC) of the group shown, with a ruler length of 50 micrometers; BE: Quantitative analysis was performed on the immunofluorescence intensity of CD68 protein, the number of Iba1 cells, the total length of microglia protrusions, and the "territory range" of microglia in the prefrontal cortex of mice in the indicated groups (3 mice per group, two-way ANOVA); F: Shore analysis was performed on microglia in the prefrontal cortex of mice in the indicated groups (3 mice per group, two-way ANOVA).

[0040] Example 2: The effect of dimethyl itaconic acid in improving stress-induced abnormal mitochondrial activation in microglia. 1. Experimental Methods (1) Laboratory animals and treatment The experimental animals were grouped and treated in the same manner as in Example 1.

[0041] (2) Sample preparation for transmission electron microscopy After the final stress response, mice were deeply anesthetized and rapidly perfused with pre-cooled electron microscopy fixative (e.g., PBS solution of 2.5% glutaraldehyde + 2% paraformaldehyde) via the heart. After brain harvesting, the subcortical marginal zone (IL) tissue of the prefrontal cortex was precisely dissected on ice and rapidly cut into small pieces of approximately 1 mm³. The samples were fixed with osmium tetroxide, dehydrated with graded acetone, embedded in Epon 812 resin, and cut into 80 nm thick ultrathin sections using an ultramicrotome. Finally, the sections were double-stained with uranium acetate and lead citrate.

[0042] (3) Image acquisition and mitochondrial scoring Mitochondria in microglia were observed under a transmission electron microscope (identified by cell location, karyotype, and cytoplasmic characteristics). At least 20 mitochondria were observed from each mouse. A semi-quantitative scoring method was used in a double-blind manner, with the following scoring criteria: Ridge integrity: 0 points = intact; 1 point = minor local fracture; 2 points = extensive fracture; 3 points = ridge disappearance.

[0043] Matrix state: 0 points = normal electron density; 1 point = local cavitation or reduced electron density; 2 points = widespread cavitation.

[0044] Autophagy / abnormal characteristics: 0 points = none; 1 point = mild autophagy or slight swelling; 2 points = moderate autophagy or significant swelling; 3 points = severe autophagy or mitochondrial rupture.

[0045] The scores of each item are added together to get the total score for each mitochondria. The higher the score, the more severe the mitochondrial damage.

[0046] 2. Experimental Results like Figure 2 As shown, in the PBS+RS 7-day (stress) group, mice microglia exhibited significant cristae breakage, matrix vacuolization, and abnormal autophagosome fusion in their mitochondria. Figure 2 A), the mitochondrial score was significantly higher than that of the PBS + control group (P<0.01). Figure 2 B); In the DI+ RS 7-day (stress) group, the integrity of the mitochondrial cristae structure in microglia of mice was restored, autophagy characteristics were reduced, and the mitochondrial score was not significantly different from that of the normal control group. Figure 2 A- Figure 2 B) indicates that dimethyl itaconic acid can effectively improve stress-induced microglial mitochondrial dysfunction.

[0047] Example 3: Preventive effect of dimethyl itaconic acid on anxiety-like behavior induced by chronic stress 1. Experimental Methods (1) Laboratory animals and grouping Sixty-four 8-week-old SPF-grade male C57BL / 6J mice were randomly divided into four groups (solvent control + control group, DI + control group, solvent control + stress group, and DI + stress group), with 16 mice in each group. The experiment was conducted independently in two batches, with 8 mice in each group in each batch, to verify the reliability of the results.

[0048] (2) Preparation, administration and modeling of DI solution Same as Example 1.

[0049] (3) Behavioral testing Twelve hours after the last stress response, an open field test was conducted. Mice were gently placed in the center of a 40 cm × 40 cm × 40 cm open field box, and their movement trajectory was recorded by a video tracking system over 5 minutes. Analytical metrics included dwell time in the central area (defined as a 20 cm × 20 cm central area), number of entries, and total distance traveled.

[0050] Twenty-four hours after the open field experiment, the elevated zero maze experiment was conducted. Mice were gently placed at the junction of a closed arm and an open arm of the elevated zero maze, and their movement trajectory was recorded over 5 minutes. Analytical metrics included dwell time in the open arm, number of entries, and total distance traveled.

[0051] All data were automatically analyzed using VisuTrack animal behavior analysis software.

[0052] 2. Experimental Results Experimental results are as follows Figure 3 As shown.

[0053] Open field test: Compared with the PBS+ control group, mice in the PBS+stress group had significantly reduced time spent in the central region and number of entries (P<0.01), while the total distance traveled was not significantly different. Figure 3 A- Figure 3 B), exhibiting significant anxiety-like behavior; compared with the DI+ control group, the DI+ stress group mice showed no significant difference in the time spent in the central region and the number of entries, but were significantly higher than the PBS+ stress group (B). Figure 3 A- Figure 3 (B) Anxiety-like behaviors were significantly improved.

[0054] Elevated zero maze experiment: Compared with the PBS+ control group, mice in the PBS+stress group had a significantly reduced open arm dwell time. Figure 3 A, Figure 3 C), exhibiting anxiety-like behavior; compared with the DI+ control group, there was no significant difference in the open arm dwell time and number of entries (C). Figure 3 A, Figure 3 C), no anxiety-like behaviors were observed.

[0055] There was no significant difference in the total distance traveled by mice in each experimental group. Figure 3 B) indicates that dimethyl itaconic acid has no effect on the motor function of mice, and its effect on improving behavior is a specific anti-anxiety effect.

[0056] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments 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.

[0057] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. The use of dimethyl itaconic acid in the preparation of medicines for the prevention and / or treatment of generalized anxiety disorder.

2. The application as described in claim 1, characterized in that, The application includes inhibiting excessive activation of microglia.

3. The application as described in claim 1, characterized in that, The applications include improving mitochondrial damage in microglia.

4. The application as described in claim 1, characterized in that, The applications include improving anxiety-like behaviors.

5. The application as described in any one of claims 1 to 4, characterized in that, The dosage forms of the drug include injections and oral preparations.

6. The application as described in claim 5, characterized in that, The oral preparations include tablets, capsules, granules, powders, pills, solutions, suspensions, mixtures, and syrups.

7. A medicine for the prevention and / or treatment of generalized anxiety disorder, characterized in that, The active ingredient of the drug includes dimethyl itaconic acid.

8. The drug as described in claim 7, characterized in that, The dosage forms of the drug include injections and oral preparations.

9. The medicament as described in claim 8, characterized in that, The oral preparations include tablets, capsules, granules, powders, pills, solutions, suspensions, mixtures, and syrups.

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