Application of minocycline hydrochloride in preparation of anti-anxiety drugs
Anti-anxiety drugs prepared by using minocycline hydrochloride can effectively inhibit microglia activity, solve the dependence and side effects of existing drugs, and provide a safe and reliable treatment plan for anxiety disorders.
Patent Information
- Application Number
- CN202510601501.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-12
- Publication Date
- 2025-07-08
AI Technical Summary
Existing anti-anxiety drugs such as benzodiazepines are highly dependent and have limited efficacy, while anti-depressants have great side effects and lack safe, reliable and effective anti-anxiety drugs.
Minocycline hydrochloride is used as an active ingredient to prepare it into tablets, capsules or injections to inhibit microglia activity and reduce anxiety symptoms.
Minocycline hydrochloride can significantly reduce anxiety-like behavior in anxious rats, inhibit microglia activity, and improve anxiety symptoms, especially alcohol-induced anxiety disorder.
Smart Images
Figure CN120267683A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of new medical uses of minocycline hydrochloride, and particularly relates to the use of minocycline hydrochloride in the preparation of antianxiety drugs. Background Art
[0002] Anxiety is an emotional disorder characterized by excessive worry, nervousness, fear, and physical symptoms such as palpitations, sweating, and insomnia. According to statistics from the World Health Organization, 4% of the world's population currently suffers from anxiety disorders. As of 2019, 301 million people worldwide suffer from anxiety disorders, making them the most common of all mental disorders. [1] Anxiety is closely related to physical health and is affected by physical health. Although it does not directly lead to physical diseases, long-term anxiety may lead to health problems such as hypertension and cardiovascular disease. At present, anxiety is treated with psychological intervention as the main method, supplemented by antidepressants or benzodiazepines. Psychological intervention is based on the principles of cognitive behavioral therapy, which is limited to exposure therapy that allows people to learn how to face their fears. The course of treatment is relatively long. [2] Benzodiazepines have historically been used to treat anxiety disorders, but are generally not recommended because of their high potential for dependence and limited long-term effectiveness. [3] Antidepressants can also be used to treat anxiety disorders in adults, but they have significant side effects, such as nausea, diarrhea, dizziness, drowsiness, headache, insomnia, dry mouth, constipation, blurred vision, etc. Therefore, it is urgent to develop new anti-anxiety drugs that are effective, safe and reliable. [4] It is of great significance for the research and development of anti-anxiety drugs to find lead compounds with good selectivity, high efficacy, clear targets, low toxicity and side effects on the body, low price and anti-anxiety effects from various compounds.
[0003] Microglia are resident immune cells in the central nervous system that mediate the occurrence and development of neuroinflammation and participate in anxiety-related mechanisms [5-6] Microglia are rapidly activated after being exposed to harmful stimuli (e.g., alcohol). Activated microglia are generally divided into M1 and M2 types based on the expression of specific markers and the secretion of pro-inflammatory or anti-inflammatory cytokines. [7] . Microglia M1 produce pro-inflammatory mediators, such as tumor necrosis factor α, interleukin (IL, such as IL-1β, IL-6) and nitric oxide synthase, which affect the vitality and function of neurons and aggravate neuronal damage. Microglia M2 produce anti-inflammatory mediators, such as arginase-1 and IL-10, which play an important role in neuronal development and neuroprotection. [8] Imbalance in polarization or repolarization of resident microglia is mainly associated with inflammation, infection, and autoimmune diseases [9]Therefore, compounds that inhibit microglial activity may have an anti-anxiety effect.
[0004] Minocycline hydrochloride (Mino) is a tetracycline antibiotic that can easily cross the blood-brain barrier. It is a microglial inhibitor and has powerful anti-microglial activation and anti-inflammatory properties. [10-11] Minocycline hydrochloride has been shown to protect neurons in various neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, cerebral ischemia, and spinal cord injury. [12-13] Minocycline hydrochloride strongly inhibits the activity of microglia, making it potentially useful for the preparation of drugs for the prevention and treatment of anxiety. However, minocycline hydrochloride is not currently used in the treatment of anxiety.
[0005] References:
[0006] [1] GBD Results Tool. In: Global Health Data Exchange [website]. Seattle: Institute for Health Metrics and Evaluation; 2019 (https: / / vizhub.healthdata.org / gbd-results?params=gbd-api-2019-permalink / 716f37e05d94046d6a06c1194a8eb0c9, accessed 5 September 2023).
[0007] [2] Alonso J, Liu Z, Evans-Lacko S, et al. Treatment gap for anxiety disorders is global: results of the World Mental Health Surveys in 21 countries. Depress Anxiety. 2018;35(3):195–208.
[0008] [3] Sake FT, Wong K, Bartlett DJ, Saini B. Benzodiazepine use risk: Understanding patient specific risk perceptions and medication beliefs. Res Social Adm Pharm. 2019;15(11):1317-1325.
[0009] [4] Griebel G, Holmes A. 50 years of hurdles and hope in anxiolytic drug discovery. Nat Rev Drug Discov. 2013 Sep; 12(9): 667 - 687.
[0010] [5] Duarte JM, Gaspar R, Caetano L, et al. Region - specific control of microglia by adenosine A(2A) receptors: uncoupling anxiety and associated cognitive deficits in female rats. Glia. Jan 2019; 67(1): 182 - 192.
[0011] [6] Schmidtner AK, Slattery DA, Glasner J, et al. Minocycline alters behavior, microglia and the gut microbiome in a trait - anxiety - dependent manner. Transl Psychiatry. Sep 13 2019; 9(1): 223.
[0012] [7] Nelson JC, Greengrove E, Nwachukwu KN, Grifasi IR, Marshall SA. Repetitive binge - like consumption based on the Drinking - in - the - Dark model alters the microglial population in the mouse hippocampus. J Integr Neurosci. Dec 30 2021; 20(4): 933 - 943.
[0013] [8] Tang Y, Le W. Differential Roles of M1 and M2 Microglia in Neurodegenerative Diseases. Mol Neurobiol. Mar 2016; 53(2): 1181 - 1194.
[0014] [9]Guo Y, Gao J, Ye P, et al. Comparison of atrial fibrillation in CKD and non-CKD populations: A cross-sectional analysis from the Kailuan study. Int J Cardiol. Feb 15 2019; 277: 125-129.
[0015]
[10] BLOCK M L, ZECCA L, HONG J S. Microglia-mediated neurotoxicity: uncovering the molecular mechanisms[J]. Nat Rev Neurosci, 2007, 8(1): 57-69.
[0016]
[11] MOLLER T, BARD F, BHATTACHARYA A, et al. Critical data-based re-evaluation of minocycline as a putative specific microglia inhibitor[J]. Glia, 2016, 64(10): 1788-1794.
[0017]
[12] BUDNI J, GARCEZ M L, DE MEDEIROS J, et al. The Anti-Inflammatory Role of Minocycline in Alzheimer s Disease[J]. Curr Alzheimer Res, 2016, 13(12): 1319-1329.
[0018]
[13] CHEN Z, JALABI W, SHPARGEL K B, et al. Lipopolysaccharide-induced microglial activation and neuroprotection against experimental brain injury is independent of hematogenous TLR4[J]. J Neurosci, 2012, 32(34): 11706-11715. Summary of the Invention
[0019] The object of the present invention is to provide a new medical use of minocycline hydrochloride, that is, the application of minocycline hydrochloride in the preparation of anti - anxiety drugs.
[0020] In order to achieve the above object, the present invention adopts the following technical solution: the application of minocycline hydrochloride in the preparation of anti - anxiety drugs, wherein the molecular formula of minocycline hydrochloride is C 23 H 28 CLN3O7, and the chemical structural formula is:
[0021]
[0022] The application of minocycline hydrochloride described in the present invention in the preparation of anti - alcohol - induced anxiety drugs.
[0023] The application of minocycline hydrochloride described in the present invention in the preparation of anti - anxiety drugs, using minocycline hydrochloride as the active ingredient, and preparing a pharmaceutically acceptable preparation with excipients and auxiliary materials. The dosage form of this preparation is tablet, capsule or injection.
[0024] The present invention has the following advantages and beneficial effects: Minocycline hydrochloride described in the present invention can effectively inhibit anxiety, and there are statistical differences in the behaviors of anxiety - treated rats and control rats. Minocycline hydrochloride with a dosage of 30 mg / kg can significantly reduce the anxiety - like behaviors of rats, further indicating that minocycline hydrochloride can effectively inhibit anxiety, especially alcohol - induced anxiety disorder, and is expected to be developed into an effective drug ingredient for preventing or treating anxiety disorder. Description of the Drawings
[0025] Figure 1 It is a statistical chart of the trajectories and analysis data of four groups of rats, namely Water + Veh group, EtOH + Veh group, Water + Mino group, and EtOH + Mino group, observed in the elevated plus - maze experiment. A. Representative diagram of the trajectories of the four groups of rats in the elevated plus - maze experiment; B. Time (s) of the four groups of rats moving in the open arms; C. Number of times the four groups of rats entered the open arms; *p < 0.05, where the concentration of minocycline hydrochloride used is 7.5 mg / mL.
[0026] Figure 2 It is a fluorescence representative diagram and data statistical chart of microglial markers of four groups of rats, namely Water + Veh group, EtOH + Veh group, Water + Mino group, and EtOH + Mino group, measured by immunofluorescence method. A. Representative immunofluorescence image; B. Average fluorescence intensity of Iba1; *p < 0.05, **p < 0.01, and the concentration of minocycline hydrochloride used is 7.5 mg / mL.
[0027] Figure 3It is the representative diagram and data statistical chart of neuron dendritic spines in the mPFC of rats in the Water+Veh group, EtOH+Veh group, Water+Mino group, and EtOH+Mino group measured by the Golgi staining method. A. Representative images of Golgi staining of the four groups of rats; B. Dendritic spine density of mPFC neurons (n / 10μm); *p<0.05, and the concentration of minocycline hydrochloride used is 7.5mg / mL.
[0028] Figure 4 It is the representative fluorescence diagram and data statistical chart of inflammatory factor markers of rats in the Water+Veh group, EtOH+Veh group, Water+Mino group, and EtOH+Mino group measured by immunofluorescence method. A. Representative immunofluorescence images; B. Mean fluorescence intensity of IL-1β; **p<0.01, and the concentration of minocycline hydrochloride used is 7.5mg / mL. Specific implementation mode
[0029] The above content of the present invention will be further described in detail through the following examples. However, it should not be understood that the scope of the above theme of the present invention is limited to the following examples. Any technology implemented based on the above content of the present invention belongs to the scope of the present invention.
[0030] Example 1
[0031] Observation of the effect of minocycline hydrochloride on alcohol-induced anxiety by the elevated plus maze test
[0032] Experimental materials: Male SD rats were purchased from Beijing Sparf Biotechnology Co., Ltd. 0.9wt% sodium chloride injection was purchased from Shandong Qidu Pharmaceutical Co., Ltd. Absolute ethanol was purchased from Tianjin Damao Chemical Reagent Factory. 75Vol% alcohol was purchased from Tianjin De'en Chemical Reagent Company. Minocycline hydrochloride was purchased from MedChemExpress LLC (HY-17412). PBS was purchased from Wuhan Sevier Biotechnology Co., Ltd.
[0033] Absolute ethanol was diluted with tap water to a 20% (v / v) alcohol solution, and minocycline hydrochloride was diluted with PBS to a concentration of 7.5mg / mL.
[0034] Experimental protocol: An intermittent alcohol exposure model was established. Thirty-six Sprague-Dawley rats were divided into four groups: Water+Veh group, EtOH+Veh group, Water+Mino group, and EtOH+Mino group. After one week of acclimation to the rearing environment, the rats were intragastrically administered an alcohol solution (5 mL / kg, i.g.) / Water (5 mL / kg, i.g.) intermittently for 14 days (administered for two days and then rested for two days, with a total of 8 administrations), and then abstained from alcohol for 14 days. During this period, minocycline hydrochloride (4 mL / kg, i.p.) / PBS (4 mL / kg, i.p.) was administered for treatment. After abstinence, the elevated plus-maze test was conducted. The experimental apparatus consisted of two open arms measuring 45 cm × 10 cm, two closed arms measuring 50 cm × 10 cm, and a central platform at the connection of the open arms and the closed arms with a size of 10 cm × 10 cm. The closed arms had baffles 30 cm high, and the entire plus-maze was 50 cm above the ground. Before the experiment, the lights were adjusted to provide uniform illumination. The rats were placed in the test chamber for 1 h to adapt to the test environment. At the start of the experiment, the rats were gently taken out of the cage and placed in the central area of the maze facing the open arms, and their behaviors were monitored for 15 min. After the experiment ended, the animal feces were removed, the maze was sprayed with 75Vol% alcohol, and wiped with paper towels to avoid the excreta and odor of the previous experimental rat affecting subsequent experiments. The data were analyzed and statistical graphs were drawn.
[0035] Experimental results: Compared with the Water+Veh group, the rats in the EtOH+Veh group had a reduced movement time in the open arms, a reduced number of entries into the open arms, and showed anxiety-like behaviors. Compared with the EtOH+Veh group, the EtOH+Mino group had an increased movement time in the open arms, an increased number of entries into the open arms, and the anxiety-like behaviors were improved ( Figure 1 ).
[0036] Example 2
[0037] Determination of microglial markers in the mPFC of rats by immunofluorescence
[0038] Experimental materials: Male Sprague-Dawley rats were purchased from Beijing Spearfright Biotechnology Co., Ltd. 0.9 wt% sodium chloride injection was purchased from Shandong Qidu Pharmaceutical Co., Ltd. Absolute ethanol was purchased from Tianjin Damao Chemical Reagent Factory. Minocycline hydrochloride was purchased from MedChemExpress LLC (HY-17412). PBS was purchased from Wuhan Sevier Biotechnology Co., Ltd. Paraformaldehyde was purchased from Tianjin Damao Chemical Reagent Factory. Bovine serum albumin was purchased from Beijing Solarbio Science & Technology Co., Ltd. Goat serum was purchased from Wuhan Sevier Biotechnology Co., Ltd. Triton-X-100 was purchased from MP Biomedicals, USA. 30 wt% H2O2 was purchased from Tianjin Damao Chemical Reagent Factory. 0.01 M sodium citrate buffer (pH 6.0) was purchased from Wuhan Sevier Biotechnology Co., Ltd. DAPI Fluoromount-G was purchased from Shanghai Beyotime Biotechnology Co., Ltd. Tween-20 was purchased from Shanghai Beyotime Biotechnology Co., Ltd. Iba1 / AIF-1(E404W) Rabbit mAb was purchased from Cell Signaling Technology, Inc (#17198). Cy3-labeled Goat Anti-Rabbit IgG (H+L) was purchased from Shanghai Beyotime Biotechnology Co., Ltd.
[0039] Absolute ethanol was diluted with tap water to a 20% (v / v) alcohol solution. Minocycline hydrochloride was diluted with PBS to a concentration of 7.5 mg / mL.
[0040] Experimental protocol: Rewarming: Sections were taken out from the -80°C refrigerator and rewarmed at room temperature for about 30 min until the slides were dry. Hydration: The slides were placed in the diluted PBS and hydrated on a shaker at room temperature for 5 min. High-pressure antigen retrieval: According to the antibody instructions, the corresponding acidic or basic antigen retrieval solution was selected, boiled for 5 min, then the heat was turned off and cooled for 30 min. The sections were taken out and cooled to room temperature, and washed 3 times with diluted PBS for 5 min each. Membrane permeabilization: Blocking endogenous catalase: 3 wt% H2O2 solution was added dropwise and incubated at room temperature for 10 min to block endogenous catalase, then washed 3 times with diluted PBS for 5 min each. Blocking non-specific antigens: 5 wt% goat serum was added dropwise and blocked in an incubator at 37°C for 1 h. Incubation with primary antibody (Iba1 / AIF-1(E404W) Rabbit mAb): Add the primary antibody diluted proportionally and incubate overnight in a refrigerator at 4°C; Rewarming: After overnight incubation, place the sections in an oven at 37°C for 1 h of rewarming, and wash 3 times with PBS-T solution for 5 min each; Incubate the secondary antibody (Cy3-labeled Goat Anti-Rabbit IgG(H+L)): Add the fluorescent secondary antibody, place it in a humid box and incubate in the dark at 37°C for 1 h, and wash 3 times with PBS-T for 5 min each; Mounting: Add DAPI Fluoromount-G mounting medium for mounting and store in the dark at 4°C. Observe and take pictures using a fluorescence microscope, and perform statistical analysis on the target molecules using ImageJ software.
[0041] Experimental results: Compared with the Water+Veh group, the average fluorescence intensity of Iba1 in rats in the EtOH+Veh group increased; compared with the EtOH+Veh group, the average fluorescence intensity of Iba1 in the EtOH+Mino group decreased ( Figure 2 ).
[0042] Example 3
[0043] Determination of neuronal dendritic spines in the mPFC of rats by Golgi staining method
[0044] Experimental materials: Male SD rats were purchased from Beijing Spebef Biotechnology Co., Ltd., 0.9 wt% sodium chloride injection was purchased from Shandong Qidu Pharmaceutical Co., Ltd., absolute ethanol was purchased from Tianjin Damao Chemical Reagent Factory, minocycline hydrochloride was purchased from MedChemExpress LLC (HY-17412), and the Golgi kit was purchased from FD NeuroTechnologies, Inc (#PK401).
[0045] Dilute absolute ethanol with tap water to a 20% (v / v) alcohol solution, and dilute minocycline hydrochloride with PBS to a concentration of 7.5 mg / mL.
[0046] Experimental protocol: Staining: Rats were anesthetized and their brains were removed. The blood on the brain surface was washed with physiological saline, and the tissue was immersed in a mixed solution composed of 2 mL of solution A (5% potassium dichromate solution) and 2 mL of solution B (5% mercuric chloride solution) for 24 h. The mixed solution was replaced, and the tissue was soaked in the dark for 2 weeks. Then it was transferred to solution C (5% potassium chromate solution), and solution C was changed after 24 h, and the tissue was soaked for 3 days; Slicing: At -22 °C, the mPFC part was sliced into 100-μm-thick slices with a cryostat microtome and air-dried in the dark; Chromogenic reaction: The slices were soaked in double-distilled water twice, 4 min each time. Subsequently, the slices were soaked in a solution prepared by mixing solution D (ammonia water solution), solution E (Kodak fixative solution) and double-distilled water in a ratio of 1:1:2 for 10 min, rinsed with distilled water 2 times / 4 min, dehydrated successively with gradient ethanol, cleared with xylene, and then sealed for use. Photographs were taken with an optical microscope, and the target molecules were statistically analyzed using Image J software.
[0047] Experimental results: Compared with the Water+Veh group, the dendritic spine density of neurons in the EtOH+Veh group decreased; compared with the EtOH+Veh group, the dendritic spine density of neurons in the EtOH+Mino group increased ( Figure 3 ).
[0048] Example 4
[0049] Determination of inflammatory molecular markers in the mPFC of rats by immunofluorescence
[0050] Experimental materials: Male SD rats were purchased from Beijing Spearf Bio-Technology Co., Ltd. 0.9 wt% sodium chloride injection was purchased from Shandong Qidu Pharmaceutical Co., Ltd. Absolute ethanol was purchased from Tianjin Damao Chemical Reagent Factory. Minocycline hydrochloride was purchased from MedChemExpress LLC (HY-17412). PBS was purchased from Wuhan Sevier Biotechnology Co., Ltd. Paraformaldehyde was purchased from Tianjin Damao Chemical Reagent Factory. Bovine serum albumin was purchased from Beijing Solarbio Science & Technology Co., Ltd. Goat serum was purchased from Wuhan Sevier Biotechnology Co., Ltd. Triton-X-100 was purchased from MP Biomedicals, USA. 30 wt% H2O2 was purchased from Tianjin Damao Chemical Reagent Factory. 0.01 M sodium citrate buffer (pH 6.0) was purchased from Wuhan Sevier Biotechnology Co., Ltd. DAPI Fluoromount-G was purchased from Shanghai Beyotime Biotechnology Co., Ltd. Tween-20 was purchased from Shanghai Beyotime Biotechnology Co., Ltd. Anti-IL-1beta was purchased from Abcam (EPR16805-15). Cy3-labeled Goat Anti-Rabbit IgG (H+L) was purchased from Shanghai Beyotime Biotechnology Co., Ltd.
[0051] Absolute ethanol was diluted with tap water to an alcohol solution with a concentration of 20% (v / v), and minocycline hydrochloride was diluted with PBS to a concentration of 7.5 mg / mL.
[0052] Experimental protocol: Rewarming: Sections were taken out from the -80°C refrigerator and rewarmed at room temperature for about 30 min until the slides were dry; Hydration: The slides were placed in the diluted PBS and hydrated on a shaker at room temperature for 5 min; High-pressure antigen retrieval: According to the antibody instructions, the corresponding acidic or alkaline antigen retrieval solution was selected, repaired for 5 min after boiling, then the fire was turned off and cooled for 30 min. The sections were taken out and cooled to room temperature, and washed 3 times with PBS for 5 min each; Membrane disruption: Blocking endogenous catalase: 3 wt% H2O2 was added dropwise and incubated at room temperature for 10 min to block endogenous catalase, and then washed 3 times with diluted PBS for 5 min each; Blocking non-specific antigens: 5 wt% goat serum was added dropwise and blocked in an incubator at 37°C for 1 h; Incubating the primary antibody (Anti-IL-1beta): The primary antibody diluted in proportion was added and incubated overnight in a 4°C refrigerator; Rewarming: After overnight incubation, the sections were placed in an oven at 37°C for 1 h and washed 3 times with PBS-T (Phosphate Buffered Saline with Tween-20) solution for 5 min each; Incubating the secondary antibody (Cy3-labeled Goat Anti-Rabbit IgG(H+L)): The fluorescent secondary antibody was added and incubated in a wet box at 37°C in the dark for 1 h, and washed 3 times with PBS-T for 5 min each; Mounting: DAPI Fluoromount-G mounting medium was added for mounting and stored in the dark at 4°C. Observation and photography were performed using a fluorescence microscope, and statistical analysis of the target molecules was performed using ImageJ software.
[0053] Experimental results: Compared with the Water+Veh group, the average fluorescence intensity of IL-1β in the EtOH+Veh group of rats increased; compared with the EtOH+Veh group, the average fluorescence intensity of IL-1β in the EtOH+Mino group decreased ( Figure 4 ).
[0054] The above shows and describes the basic principles, main features and advantages of the present invention. Without departing from the spirit and scope of the present invention, there are various changes and improvements to the present invention, and these changes and improvements all fall within the scope of the present invention claimed.
Claims
1. Use of minocycline hydrochloride in the preparation of an anti-anxiety drug, wherein the molecular formula of minocycline hydrochloride is C 23 H 28 CLN3O7, and the chemical structural formula is:
2. The application according to claim 1, characterized in that: Use of minocycline hydrochloride in the preparation of anti-alcohol-induced anxiety drugs.
3. The application according to claim 1 or 2, characterized in that: Using minocycline hydrochloride as the active ingredient, and formulating it with excipients and auxiliary materials into a pharmaceutically acceptable preparation.
4. The application according to claim 3, characterized in that: The dosage form of the preparation is tablet, capsule or injection.