Application of Hspa5 inhibitor in preparation of medicine for preventing or treating anxiety-related diseases

Abstract

The invention discloses application of an Hspa5 inhibitor in preparation of a medicine for preventing or treating anxiety disorder. An anxiety mouse model is constructed through chronic constraint stress (CRS), and in combination with medial amygdala kernel (MeA) transcriptome sequencing and qPCR verification, it is found that the endoplasmic reticulum molecular chaperone Hspa5 is remarkably up-regulated in the anxiety state. Furthermore, an Hspa5 specific inhibitor HA15 is locally injected into a MeA brain region, so that the anxiety-like behavior induced by the CRS is remarkably improved, and the exploration time of an open field experiment central region, the exploration time of an open arm of an elevated cross labyrinth and the exploration time of a bright box of a bright-dark box experiment are prolonged. The invention discloses the function of Hspa5 as a novel anti-anxiety target for the first time, and provides a direct experimental basis and a transformation direction for developing a novel anti-anxiety drug which is non-monoamine and targets an endoplasmic reticulum homeostasis.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and neuroscience, specifically to the novel use of an inhibitor of the endoplasmic reticulum molecular chaperone Hspa5 (also known as BiP) in the preparation of drugs for the prevention or treatment of anxiety and stress-related mental disorders. Background Technology

[0002] Anxiety disorders are among the most prevalent mental illnesses worldwide. Clinically, patients with anxiety disorders exhibit persistent tension, excessive worry, and fear, often accompanied by severe autonomic dysfunction. Chronic stress is a major risk factor for inducing and maintaining anxiety symptoms. Currently used anti-anxiety medications (such as selective serotonin reuptake inhibitors (SSRIs) and benzodiazepines) generally suffer from slow onset of action, poor specificity, easy development of drug tolerance, and significant side effects (such as drowsiness and addiction). Therefore, there is an urgent need to find new targets for anti-anxiety drugs with novel mechanisms of action.

[0003] The medial amygdala (MeA) is a key brain region integrating olfactory processing and neuroendocrine regulation, playing a crucial role in handling innate threats and regulating the hypothalamus-pituitary-adrenal (HPA) axis. However, the molecular pathological reprogramming mechanisms of the MeA region under chronic stress are not fully understood.

[0004] Endoplasmic reticulum (ER) homeostasis imbalance is associated with a variety of neuropsychiatric disorders. High anxiety states are often accompanied by persistent neuronal firing and elevated glucocorticoid levels, which may exceed the ER's protein folding capacity, leading to chronic activation of the unfolded protein response (UPR). Hspa5 (Heat Shock Protein Family A (Hsp70) Member 5, also known as BiP) is a core molecular chaperone for the ER UPR. However, whether Hspa5 directly participates in the regulation of anxiety-like behaviors induced by chronic stress in the MeA, and whether targeted inhibition of Hspa5 can produce an anti-anxiety effect, has never been elucidated in the current technology. Summary of the Invention

[0005] The technical problem this invention aims to solve is to provide a novel mechanism for anxiolytic drug targets and to offer new uses for Hspa5 inhibitors (especially the small molecule compound HA15) in the preparation of anxiolytic drugs. Specifically, this invention aims to address the problems of slow onset of action, poor specificity, and significant side effects of existing anxiolytic drugs (such as selective serotonin reuptake inhibitors and benzodiazepines) by targeting the endoplasmic reticulum molecular chaperone Hspa5 in the medial amygdala (MeA) region to achieve rapid and effective anxiolytic effects.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] Application of Hspa5 inhibitors in the preparation of drugs for the prevention or treatment of anxiety-related disorders.

[0008] Preferably, the Hspa5 inhibitor is selected from one or more of small molecule compounds, RNA interference molecules, antisense oligonucleotides (ASO), gene editing systems, or specific neutralizing antibodies.

[0009] Preferably, the small molecule compound is HA15 or a pharmaceutically acceptable salt, ester, solvate, or prodrug thereof. HA15 is an inhibitor that specifically targets Hspa5 / BiP.

[0010] Preferably, the RNA interference molecule is siRNA or shRNA.

[0011] Preferably, the anxiety-related disorder is one or more of generalized anxiety disorder, chronic stress-induced anxiety-like state, and other stress-related mental disorders accompanied by excessive fear and avoidance behavior.

[0012] Preferably, the drug targets the central nervous system. More preferably, the central nervous system is the medial amygdala (MeA) region. The MeA is a key brain region integrating olfactory processing and neuroendocrine regulation, playing a crucial role in processing innate threats and regulating the hypothalamus-pituitary-adrenal (HPA) axis.

[0013] Preferably, the dosage form of the drug is selected from one or more of the following: injection, oral preparation, nasal spray, or implantable sustained-release preparation.

[0014] Preferably, when the drug is a local injection, the effective concentration of HA15 in the working solution is 10 μM-50 μM. More preferably, the effective concentration of HA15 in the working solution is 20 μM. More preferably, the HA15 working solution is prepared by dissolving HA15 in dimethyl sulfoxide (DMSO) to prepare a stock solution, and diluting it with phosphate buffered saline (PBS) to the working solution concentration before use.

[0015] Preferably, when the drug is a local injectable agent, it is injected into the bilateral MeA regions via stereotactic surgery.

[0016] Preferably, the drug produces an anxiolytic effect within 0.5 hours after administration.

[0017] Preferably, the drug can significantly increase the time that anxiety model animals spend in the central area of ​​an open field experiment, the exploration time in the open arm of an elevated cross maze, and / or the time spent in the light box of a light-dark box experiment.

[0018] A pharmaceutical composition for the prevention or treatment of anxiety-related disorders, comprising a therapeutically effective amount of an Hspa5 inhibitor and a pharmaceutically acceptable carrier.

[0019] Preferably, the carrier is selected from one or more of water for injection, physiological saline, phosphate buffer, vegetable oil, liposomes, microspheres, gel matrix or sustained-release material.

[0020] Preferably, the Hspa5 inhibitor alleviates anxiety-like behavior by restoring endoplasmic reticulum homeostasis in the medial amygdala (MeA) region. Specifically, it exerts an anti-anxiety effect by inhibiting the expression or activity of the endoplasmic reticulum molecular chaperone Hspa5, reducing endoplasmic reticulum stress, and restoring the homeostasis of the unfolded protein response (UPR).

[0021] Beneficial effects:

[0022] (1) This invention provides a new target and mechanism for anti-anxiety: Based on omics data and functional verification, this invention reveals for the first time the causal regulatory role of the endoplasmic reticulum molecular chaperone Hspa5 in anxiety-like behavior induced by chronic stress, providing a novel, non-monoamine endoplasmic reticulum homeostasis intervention target for the development of anti-anxiety drugs.

[0023] (2) This invention realizes the "repurposing of old drugs": it clarifies that the small molecule compound HA15 (previously used mainly in anti-tumor and endoplasmic reticulum stress research) has significant anti-anxiety efficacy. Experiments have confirmed that even at low doses (such as local injection of 20 μM), it can exert a rapid anti-anxiety effect (effective within 0.5 hours after administration), which broadens the clinical indications of this compound and shortens the research and development cycle of new anti-anxiety drugs.

[0024] (3) This invention achieves precise anatomical intervention: it confirms that Hspa5 inhibition targeting the medial amygdala (MeA) can effectively alleviate anxiety. Given the close connection between the MeA and the olfactory pathway, this invention provides a theoretical basis for the future development of brain region-targeted drugs. Attached Figure Description

[0025] Figure 1 The study aimed to upregulate the expression level of Hspa5 in the medial amygdala (MeA) of mice under chronic restraint stress (CRS); where A is the experimental flowchart; and B is the result of qPCR detection of the relative expression level of Hspa5 mRNA in MeA.

[0026] Figure 2Local injection of the Hspa5 inhibitor HA15 into MeA significantly alleviated CRS-induced anxiety-like behavior; AC represents the trajectory heatmap and central region exploration statistics of the open field experiment (OFT); DF represents the trajectory heatmap and open box exploration statistics of the light-dark box experiment (LDT); and GI represents the trajectory heatmap and open arm exploration statistics of the elevated cross maze (EPM). Detailed Implementation

[0027] The present invention will be further described in detail below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0028] Example 1: Construction of a chronic restraint stress (CRS) mouse model and discovery of the Hspa5 target

[0029] Animal model construction:

[0030] Male C57BL / 6J mice aged 10-12 weeks were selected and subjected to restraint stress in 50mL centrifuge tubes with ventilation holes at 14:00 every day for 5 hours daily for 10 consecutive days to establish a CRS anxiety model. Control group mice remained in their original cages and were fasted and deprived of water for the same period. All animal experiments were approved by the Animal Ethics Committee of Southeast University and strictly conducted in accordance with relevant ethical guidelines.

[0031] Target discovery and validation:

[0032] After a 10-day stress period, total RNA was extracted from the MeA region of mice for transcriptome sequencing (RNA-seq). Bioinformatics analysis showed that the endoplasmic reticulum protein processing pathway was significantly enriched, and Hspa5 was significantly upregulated as a core node. Subsequently, quantitative real-time PCR (qPCR) independently verified that the mRNA expression level of Hspa5 in the MeA region of CRS group mice was significantly higher than that in the control group (P<0.05).

[0033] Example 2: Pharmacodynamic validation of the anti-anxiety effect of Hspa5-specific inhibitor HA15

[0034] Drug preparation and administration:

[0035] HA15 was dissolved in DMSO to prepare a 20 mM stock solution, which was then diluted with sterile PBS to a final concentration of 20 μM (DMSO final concentration was 0.1% (v / v)) before use. Catheters were implanted bilaterally in the MeA of mice via stereotactic surgery. After 10 days of CRS modeling, mice were randomly divided into a CRS+Vehicle group and a CRS+HA15 group. 1 μL of the drug or solvent was slowly injected into each MeA via a dual-needle injection system.

[0036] Behavioral assessment:

[0037] Behavioral tests were conducted 0.5 hours after administration.

[0038] Open Field Experiment (OFT):

[0039] Compared with the CRS+Vehicle group, the CRS+HA15 group mice had significantly increased number of times they entered the central region and the time they stayed in the central region (P<0.05).

[0040] Light-dark box experiment (LDT):

[0041] HA15 treatment significantly increased the number of times mice entered the open box and the time they stayed in the open box (P<0.05).

[0042] Elevated Cross Maze (EPM):

[0043] The number of times mice entered the open arm and the time they stayed in the open arm were significantly higher in the HA15-treated group than in the solvent control group (P<0.05).

[0044] Conclusion: The in vivo pharmacological experiments in Example 2 fully demonstrate that inhibiting Hspa5 can effectively and significantly alleviate anxiety-like behaviors induced by chronic stress, and Hspa5 inhibitors (such as HA15) have great potential to prepare anti-anxiety drugs.

[0045] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of Hspa5 inhibitors in the preparation of drugs for the prevention or treatment of anxiety-related diseases.

2. The application according to claim 1, characterized in that, The Hspa5 inhibitor is selected from one or more of small molecule compounds, RNA interference molecules, antisense oligonucleotides, gene editing systems, or specific neutralizing antibodies; the small molecule compound is HA15 or its pharmaceutically acceptable salt, ester, solvate, or prodrug; the RNA interference molecule is siRNA or shRNA.

3. The application according to claim 1, characterized in that, The anxiety-related disorders mentioned are one or more of the following: generalized anxiety disorder, chronic stress-induced anxiety-like state, and other stress-related mental disorders accompanied by excessive fear and avoidance behaviors.

4. The application according to claim 1, characterized in that, The drug targets the central nervous system.

5. The application according to claim 4, characterized in that, The central nervous system is the medial amygdala region.

6. The application according to claim 1, characterized in that, The dosage form of the drug is selected from one or more of the following: injection, oral preparation, nasal spray, or implantable sustained-release preparation.

7. The application according to claim 6, characterized in that, When the drug is a local injection, the effective concentration of HA15 in the working solution is 10 μM-50 μM; the drug produces an anxiolytic effect within 0.5 hours after administration.

8. The application according to claim 7, characterized in that, The effective concentration of HA15 in the working solution is 20 μM.

9. The application according to claim 1, characterized in that, The drug significantly increased the time spent by anxiety model animals in the central area of ​​the open field experiment, the exploration time in the open arm of the elevated cross maze, and / or the time spent in the light box of the light-dark box experiment.

10. A pharmaceutical composition for the prevention or treatment of anxiety-related disorders, characterized in that, It contains a therapeutically effective amount of an Hspa5 inhibitor and a pharmaceutically acceptable carrier.

Images