Application of HDAC9 inhibitors in the preparation of drugs for treating depression

Abstract

This invention belongs to the pharmaceutical field, specifically relating to the application of HDAC9 inhibition methods such as gene knockout, non-selective inhibition, selective inhibition, and shRNA knockdown in biotechnology and medicine, namely, the application of HDCA9 inhibitors in the preparation of drugs for treating depression. A biomarker for depressive episodes is also provided, wherein HDCA9, as a histone deacetylase, regulates the excitability of hippocampal neurons and participates in depressive episodes. The application of HDCA9 inhibitors and HDCA9 shRNA in the preparation of drugs for treating depression is also discussed. The research of this invention found that inhibiting HDAC9 expression in hippocampal neurons can effectively alleviate depressive episodes.

Description

Technical Field

[0001] The invention belongs to the medical field, specifically involving the application of HDAC9 inhibition methods such as gene knockout, non-selective inhibition, selective inhibition, and shRNA knockdown in biotechnology and medicine, especially in the treatment of depression. Background Technology

[0002] Depression is a common mood disorder characterized by low mood, slowed thinking, and reduced speech and movement. It severely disrupts patients' lives and work, placing a heavy burden on families and society; approximately 15% of depression patients die by suicide. According to the World Health Organization's recent Global Health Assessment report, depression is listed as the leading cause of disability worldwide and a major contributor to the global disease burden. Currently, treatment is primarily psychotherapy, with antidepressants used for moderate to severe depression. Traditional antidepressants, including tricyclic antidepressants and monoamine oxidase inhibitors, generally require 4–6 weeks to significantly improve symptoms. Furthermore, due to limited understanding of the pathophysiology of depression and its unclear pathogenesis, drug treatment for depression suffers from long treatment cycles, a large number of treatment-resistant cases, and a high relapse rate; currently, approximately 50% of patients taking medication still experience recurrent episodes. Therefore, there is an urgent need to find new therapeutic targets for depression.

[0003] In recent years, the application of epigenetics in disease has attracted increasing attention. Histone acetylation is involved in various mental illnesses, including depression. Broad-spectrum histone deacetylase inhibitors (HDACi), such as sodium butyrate, have been shown to alleviate depressive episodes in animal experiments. However, most HDAC inhibitors suffer from low selectivity for specific HDAC subtypes, resulting in problems such as low selectivity and numerous adverse reactions, which limits their clinical application. Therefore, identifying which HDAC subtypes in the brain are altered in relation to the occurrence of depression is a pressing scientific question that urgently needs to be addressed. This may also form the basis for the future development of selective HDAC inhibitors and precision treatment of depression. As an important member of the class II HDAC family, there are currently no reports of inhibiting HDAC9 for the prevention and treatment of depression. Summary of the Invention

[0004] The purpose of this invention is to provide a new direction for the treatment of depression by inhibiting HDAC9, and the application of related technologies in the preparation of drugs for the prevention and treatment of depression.

[0005] To achieve the above objectives, the present invention provides a biomarker for depressive episodes and its application in the preparation of drugs for treating depression by inhibiting HDAC9.

[0006] A biomarker for depressive episodes, wherein HDAC9 is a histone deacetylase that regulates hippocampal neuronal excitability and is involved in depressive episodes.

[0007] Application of HDAC9 inhibitors and HDAC9 shRNA in the preparation of drugs for treating depression.

[0008] Application of HDAC9 inhibitors and HDAC9 shRNA in the preparation of drugs that alleviate the onset of neuropsychiatric disorders by regulating the excitability of hippocampal neurons.

[0009] Application of HDAC9 inhibitors and HDAC9 shRNA in the preparation of drugs that alleviate the onset of neuropsychiatric disorders by regulating the development of dendritic spines in hippocampal neurons.

[0010] The neuropsychiatric disorder mentioned above is depression.

[0011] As described in the application, the expression of HDAC9 in the hippocampus of patients with major depressive episodes was significantly higher than that in healthy subjects.

[0012] As described in the application, the expression of HDAC9 in the hippocampus of mice with chronic restraint stress depression and mice with chronic unpredictable stress depression was significantly increased compared with that in normal mice.

[0013] As described in the application, normal mouse hippocampal neurons overexpressing HDAC9 exhibited significant depressive behavior.

[0014] As described in the application, overexpression of HDAC9 in normal mouse hippocampal neurons significantly reduced dendritic spine density, accompanied by a decrease in the frequency and amplitude of spontaneous excitatory postsynaptic potentials.

[0015] The application of HDAC9 inhibition as an antidepressant treatment.

[0016] As described in the application, specific knockout of HDAC9 in mouse hippocampal neurons can alleviate depressive episodes induced by chronic restraint stress.

[0017] As described in the application, HDAC9 shRNA injection into the hippocampus of mice can alleviate depressive episodes induced by chronic restraint stress.

[0018] As described in the application, injection of the non-selective HDAC9 inhibitor TMP269 into the hippocampus of mice can alleviate depressive episodes induced by chronic restraint stress.

[0019] Compared with the prior art, the present invention has the following advantages:

[0020] The HDAC9 in this invention was derived from hippocampal samples from clinical patients experiencing major depressive episodes. Furthermore, HDAC inhibitors have been proven effective in treating depressive episodes; therefore, using HDAC9 as a therapeutic target for depression has clinical applicability. Additionally, this study found that inhibiting HDAC9 expression in hippocampal neurons can effectively alleviate depressive episodes, suggesting the feasibility of HDAC9 as a therapeutic target for antidepressants.

[0021] Further research in this invention revealed that HDAC9 primarily exerts its effects by regulating dendritic spine development, thereby influencing neuronal synaptic transmission. This finding aligns with traditional hypotheses regarding the mechanisms of depressive episodes, enhancing the feasibility of HDAC9 as a target for antidepressant therapy. Furthermore, unlike HDAC inhibition, HDAC9 inhibition, due to its clearly defined target and pharmacological mechanism, offers improved safety. These data indicate that HDAC9 inhibition is a highly promising antidepressant treatment approach for development and application. Attached Figure Description

[0022] Figure 1 A shows the HDAC9 content in the hippocampus of patients with major depressive episodes and healthy subjects. Figure 1 B shows the process of constructing a mouse model of chronic restraint stress depression. Figure 1 CE showed depressive behavior in chronic restraint stress (CRS) mice in forced swimming (FST), tail suspension test (TST), sucrose preference (SPT), and open field test. Figure 1 F shows the HDAC9 content in the hippocampus of chronically restrained mice and control mice as detected by Western blotting. Figure 1 GI showed depressive behavior in chronic unpredictable stress (CUMS) mice in forced swimming (FST), tail suspension test (TST), sucrose preference (SPT), and open field test. Figure 1 J shows the HDAC9 content in the hippocampus of mice under chronic unpredictable stress and control mice as detected by Western blotting.

[0023] Figure 2 A shows the injection and behavioral process of HDAC9 overexpressing virus. Figure 2 B shows the changes in HDAC9 expression in the hippocampus after administration of an HDAC9-overexpressing virus. Figure 2 CE showed that overexpression of HDAC9 in normal mouse hippocampal neurons resulted in depressive behavior in forced swimming (FST), tail suspension test (TST), sucrose preference (SPT), and open field test.

[0024] Figure 3 A shows a typical Golgi staining pattern of hippocampal neurons in HDAC9-overexpressing mice. Figure 3BE showed the dendritic spine density of HDAC9-overexpressing mouse hippocampal neurons using imageJ software, including short and thick, mushroom-shaped, slender, and total dendritic spine density. Figure 3 FG displays the amplitude and frequency of spontaneous excitatory postsynaptic potentials (sEPSCs) in HDAC9-overexpressing mouse hippocampal neurons recorded by electrophysiological methods.

[0025] Figure 4 A shows the process of constructing a HDAC9-specific knockout mouse for hippocampal neurons. Figure 4 BC shows the mRNA and protein levels of HDAC9 in the hippocampus of HDAC9 CKO mice. Figure 4 DF showed that HDAC9 CKO mice exhibited depressive behavior in the forced swimming (FST), tail suspension test (TST), sucrose preference (SPT), and open field test after experiencing chronic restraint stress.

[0026] Figure 5 A shows the injection and behavioral process of HDAC9 shRNA virus. Figure 5 B shows the changes in HDAC9 expression in the hippocampus after administration of HDAC9 shRNA virus. Figure 5 CE showed that HDAC9 shRNA virus mice exhibited depressive behavior in the forced swimming (FST), tail suspension test (TST), sucrose preference (SPT), and open field test after experiencing chronic restraint stress.

[0027] Figure 6 AB studies showed that mice administered the non-selective HDAC9 inhibitor TMP-269 and the HDAC inhibitor JNJ-26481585, which has no inhibitory effect on HDAC9, exhibited depressive behavior in the forced swimming (FST), tail suspension test (TST), and sucrose preference (SPT) tests. Detailed Implementation

[0028] The following description, in conjunction with the accompanying drawings, uses embodiments of the present invention to further illustrate the substantive content of the invention, but does not limit the invention thereto. Example 1

[0029] Detection of HDAC9 levels in blood samples from patients with clinical major depressive episodes.

[0030] This invention utilizes an ELISA method to detect HDAC9 levels in patient blood samples. First, plasma samples were collected from patients with major depressive episodes meeting the diagnostic criteria of the International Classification of Diseases, 11th Revision (ICD 11) (Hamilton Depression Rating Scale 17 (HAMD17) score ≥17). Exclusion criteria included: individuals diagnosed with a neuropsychiatric disorder in the past or recently; individuals with severe chronic diseases of the heart, liver, kidneys, or brain; pregnant women or those planning to become pregnant; patients with leukopenia, anemia, or thrombocytopenia; individuals who have received any tested medication or medication / procedure within the past six months (i.e., those participating in another test); and individuals with allergies or other conditions that prevent them from completing the test. Next, plasma samples were collected from healthy subjects matched for age, sex, and race with the patients with major depressive episodes. All patients and healthy controls were 15-60 years old and of Asian descent (see Table 1 for details). Finally, the HDAC9 levels in plasma samples from 12 patients with major depressive episodes and 12 healthy subjects were detected using a human HDAC9 ELISA kit. The results showed that the HDAC9 levels in the plasma of patients with major depressive episodes were significantly elevated. Figure 1 A).

[0031] Example 2

[0032] Detection of HDAC9 content in the hippocampus of chronically stressed mice.

[0033] (1) Construction of chronic restraint stress (CRS) mice: Before the formal experiment, mice were placed in a behavioral room for 1 week to adapt, with a 12-hour day-night cycle and free access to water and food. After initial screening based on body weight, sucrose preference, and open field test, mice with similar behavioral scores were selected and randomly divided into two groups: a normal control (NC) group and a CRS group. CRS group mice were placed in restraint tubes made from 50 ml centrifuge tubes (the centrifuge tubes were pre-heated to create air holes), dispersed throughout the tube, with a small hole in the center of the cap through which the mouse's tail was exposed to the air. The mice were restrained for 21 consecutive days, 4 hours a day. CRS group mice were fasted and deprived of water during the restraint period, while NC group mice had free access to water and food. The flowchart is shown below. Figure 1 B.

[0034] (2) Construction of chronic unpredictable stress (CUMS) mice: Mice were repeatedly subjected to unpredictable mild stress, including day / night light cycles, 45° tilted cages, restraint, foot electrical stimulation, cold environment, cold water swimming, low-intensity strobe lighting, food and water deprivation, and damp bedding. All stressors were randomly administered throughout the stress period, which lasted for 5 weeks.

[0035] (3) Validation by behavioral studies of depression:

[0036] Forced swimming (FST): Mice were placed in a cylindrical glass tank 25 cm high and 10 cm in diameter, with a water depth of 15 cm and a water temperature of 25°C. The mice swam in the tank for 6 minutes, and the cumulative immobility time of the mice in the following 5 minutes was recorded.

[0037] Tail suspension test (TST): The mouse's tail was fixed and its head was suspended about 30 cm above the ground. The suspension time was 5 minutes, and the time the mouse remained still was recorded.

[0038] Sugar Water Preference Test (SPT): Before the experiment, mice were trained to adapt to sugary drinking water. Two water bottles were placed in each cage, one filled with 1% sucrose solution and the other with pure water, for two consecutive days. The mice were then fasted and deprived of water for 12 hours. At the start of the experiment, each cage was placed with one bottle of 1% sucrose solution and one bottle of pure water. After 1 hour, the weights of the sucrose solution and pure water were measured, and the sugar water preference percentage for each mouse was calculated as: sugar water intake / (sugar water intake + pure water intake).

[0039] Open field test (OFT): The mouse open field reaction chamber is 28 cm high and 70 cm long at the bottom. The mouse is placed in the center of the bottom surface of the chamber, and video is taken over 30 minutes to record the mouse's movement trajectory and total distance traveled.

[0040] The results showed that the immobility time of CRS mice and CUMS mice was significantly increased in forced swimming and tail suspension tests. Figure 1 C, 1G), while their sugar water preference rate decreased significantly ( Figure 1 The presence of D and 1H indicates that they exhibit a clear depressive phenotype. Furthermore, their performance in the open field test was not significantly different from that of the control group. Figure 1 E, 1I), indicating that their exercise volume was not affected.

[0041] (4) This invention utilizes Western blot to detect HDAC9 content: hippocampal tissues from CRS mice, CUMS mice, and normal mice were isolated, and total protein was extracted using RIPA lysis buffer. The HDAC9 protein content was then further detected using Western blot. The results showed that the HDAC9 content in the hippocampus of both CRS and CUMS mice was significantly increased. Figure 1 F, 1J). Example 3

[0042] Depressive behavioral manifestations in mice with HDAC9 overexpression in the hippocampus

[0043] Adeno-associated virus carrying HDAC9 was injected into the hippocampus of mice using a mouse stereotaxic instrument. Figure 2 A), 3 weeks later, Western blot analysis of HDAC9 levels in the hippocampus revealed a significant increase, indicating successful viral infection. Figure 2 B). Next, forced swimming, tail suspension test, sucrose preference test, and open field test were used to examine the depressive behavioral manifestations in mice. The results showed that HDAC9 overexpressing mice also exhibited significant depressive-like behaviors. Figure 2 CE). Example 4

[0044] Changes in neuronal excitability after HDAC9 overexpression in mouse hippocampus

[0045] This invention utilizes Golgi staining to detect the number of dendritic spines in hippocampal neurons. Mice were anesthetized, and their brains were rapidly removed and fixed in a fixative for over 48 hours. The mouse brain tissue was then cut into 2-3 mm thick sections. The brain tissue was gently rinsed several times with physiological saline and placed in 45 ml round-bottom EP tubes. Golgi staining solution (G1069, Servicebio) was added to completely submerge the brain tissue, which was then placed in a cool, ventilated place away from light for 14 days. The tissue was then rinsed three times with distilled water, submerged in 80% glacial acetic acid overnight, and washed with distilled water after softening. The tissue was then placed in 30% sucrose solution. The tissue was cut into 100 μm sections using a vibratory microtome, adhered to gelatin slides, and dried overnight in the dark. The dried tissue sections were treated with concentrated ammonia for 15 minutes, washed with distilled water for 1 minute, treated with acidic dura mater fixative for 15 minutes, washed with distilled water for 3 minutes, dried, and fixed with glycerol gelatin. Finally, panoramic multi-slice scanning using a digital slide scanner was used to obtain a panoramic image of the brain tissue. Morphological analysis was performed on the apical dendrites of neurons. For each group, at least three dendritic segments were randomly selected from each neuron, and at least five neurons from each mouse were analyzed. Figure 3 A). The results showed that the density of short dendritic spines, mushroom-shaped dendritic spines, and total dendritic spines was significantly reduced in the hippocampal neurons of HDAC9-overexpressing mice. Figure 3 BE).

[0046] Furthermore, this invention utilizes patch-clamp technique to record the amplitude and frequency of spontaneous excitatory postsynaptic potentials (sEPSCs) in hippocampal neurons of HDAC9-overexpressing mice. The results showed that compared to control mice, the amplitude of sEPSCs in the hippocampal neurons of HDAC9-overexpressing mice remained unchanged, while the frequency was significantly reduced, suggesting a significant decrease in neuronal excitability. Figure 3 FG). Example 5

[0047] Depressive phenotype in HDAC9 conditional knockout mice

[0048] This invention utilizes CRE-LOXP technology to integrate HDAC9 flox / flox Neuron-specific HDAC9 knockout mice were obtained by crossing genetically engineered mice with CaMKIIα-Cre tool mice. Figure 1 Further analysis using RT-PCR and Western blotting revealed a significant decrease in both HDAC9 mRNA and protein levels in the hippocampus of HDAC9CKO mice, indicating successful knockout mouse construction (Figure BC). Further testing using forced swimming, tail suspension test, sucrose preference test, and open field test revealed that the behavioral characteristics of HDAC9 CKO mice were similar to those of the control group. Figure 4 DF). Furthermore, we found that the depressive phenotype of HDAC9 CKO mice did not change after undergoing CRS ( ). Figure 4 (DF), suggesting that HDAC9 CKO can alleviate depressive behaviors induced by CRS. Example 6

[0049] Depressive phenotype in HDAC9 shRNA mice

[0050] Adeno-associated virus carrying HDAC9 shRNA was injected into the hippocampus of mice using a mouse stereotaxic instrument. Figure 5 A), after 3 weeks, Western blot analysis of HDAC9 levels in the hippocampus revealed a significant decrease, indicating successful viral infection. Figure 5 B). Next, after viral infection mice underwent CRS, their depressive behavioral manifestations were assessed using forced swimming, tail suspension test, sucrose preference test, and open field test. The results showed that, compared with normal mice undergoing CRS, HDAC9shRNA mice exhibited significantly reduced depressive phenotypes after CRS. Figure 5 (CE), suggesting that HDAC9 knockdown can alleviate CRS-induced depressive episodes. Example 7

[0051] Effects of HDAC9 inhibitors on depressive behavior

[0052] This invention selects the broad-spectrum HDAC inhibitors TMP269 and JNJ-26481585, where TMP269 has inhibitory activity against HDAC9, while JNJ-26481585 has almost no inhibitory activity against HDAC9. Normal mice undergoing critical respiratory syndrome (CRS) were intraperitoneally injected daily with both inhibitors at a dose of 15 mg / kg. After the CRS ended, the depressive behavioral manifestations of each group of mice were assessed using forced swimming, tail suspension test, sucrose preference test, and open field test. The results showed that concurrent administration of TMP269 during CRS could alleviate the depressive phenotype in mice in the later stages. Figure 6 AB), while administering JNJ-26481585 concurrently with CRS did not significantly alter depressive behavioral patterns ( ). Figure 6 (AB), suggesting that inhibiting HDAC9 can alleviate depressive episodes.

Claims

1. Application of HDAC9 inhibitor TMP269 in the preparation of drugs for treating depression.

2. The application of the HDAC9 inhibitor TMP269 in the preparation of a drug that alleviates the onset of neuropsychiatric disorders by regulating the excitability of hippocampal neurons, wherein the neuropsychiatric disorder is depression.

3. The use of the HDAC9 inhibitor TMP269 in the preparation of a drug that alleviates the onset of neuropsychiatric disorders by regulating the development of dendritic spines in hippocampal neurons, wherein the neuropsychiatric disorder is depression.

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