Use of ponatinib for the preparation of a medicament for the prevention and / or treatment of alzheimer's disease

Abstract

The application relates to the technical field of biological medicine, and provides an application of ponatinib in preparation of a drug for preventing and / or treating Alzheimer's disease. As an effective specific MAVS inhibitor, the ponatinib can effectively inhibit overactivation of microglia cells in a disease state by inhibiting activation of a MAVS signal path, and further relieve a neuroinflammatory reaction in the brain. The ponatinib can effectively improve memory and behavior of AD mice, protect brain lesions, and effectively treat Alzheimer's disease.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the use of ponatinib in the preparation of drugs for the prevention and / or treatment of Alzheimer's disease. Background Technology

[0002] Alzheimer's disease (AD) is a neurodegenerative disease with insidious onset and progressive development. It primarily affects the cognitive function of older adults and is the most common type of dementia, accounting for approximately 60%-70% of dementia cases. The disease is characterized by progressive cognitive impairment and behavioral disturbances, and mainly occurs in the elderly or pre-elderly population.

[0003] Ponatinib is a third-generation tyrosine kinase inhibitor (TKI) primarily used to treat chronic myeloid leukemia (CML) and Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL), especially in patients resistant to or intolerant of previous treatments. It inhibits cancer cell proliferation by targeting the BCR-ABL1 protein (including the T315I mutant), demonstrating significant efficacy in refractory leukemia. However, there are no reports of ponatinib being used in the development of drugs for treating Alzheimer's disease. Summary of the Invention

[0004] In view of this, the present invention proposes the use of ponatinib in the preparation of drugs for the prevention and / or treatment of Alzheimer's disease.

[0005] The ponatinib described in this invention has the chemical name 3-(2-(imidazo[1,2-b]pyridazin-3-yl)ethynyl)-4-methyl-N-(4-((4-methylpiperazin-1-yl)methyl)-3-(trifluoromethyl)phenyl)benzamide; molecular formula: C 29 H 27 F3N 60 CAS No.: 943319-70-8, its structural formula is shown in formula (I):

[0006]

[0007] The technical solution of the present invention is achieved as follows: The present invention provides the use of ponatinib in the preparation of drugs for the prevention and / or treatment of Alzheimer's disease.

[0008] Based on the above technical solutions, preferably, the administration method in the application is subcutaneous injection, intravenous injection, intramuscular injection, or oral administration.

[0009] Based on the above technical solutions, preferably, the drug for preventing and / or treating Alzheimer's disease is a drug that targets and inhibits MAVS.

[0010] This invention is the first to discover that ponatinib can be used to treat Alzheimer's disease, and the first to identify ponatinib as an effective and specific MAVS inhibitor. By inhibiting the activation of the MAVS signaling pathway, it can effectively suppress the excessive activation of microglia in the disease state, thereby alleviating the neuroinflammatory response in the brain. Ponatinib can effectively improve memory and behavior in AD mice, protect against brain lesions, and effectively treat Alzheimer's disease. Attached Figure Description

[0011] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0012] Figure 1 This is a schematic diagram of an experiment involving mice treated with ponatinib.

[0013] Figure 2 The results of the open field experiment are shown in the diagram. The left diagram shows the walking distance and dwell time of the mouse in the central region, and the right diagram shows a representative heatmap of the movement trajectory; n=10.

[0014] Figure 3 The results of the Y-maze experiment are shown in the left figure, which represents the spontaneous alternation rate, and the right figure shows the representative trajectories of the mouse in different arms; n=10.

[0015] Figure 4 The results of the new object recognition experiment are shown in the figure. The left figure shows the preference index, the number of times the new object was explored, and the exploration time, respectively. The right figure shows a schematic diagram of the representative trajectory; n=10.

[0016] Figure 5 Immunofluorescence analysis results of IBA-1 in the cerebral cortex (CTX) and hippocampus (HIP). Representative immunofluorescence images of the microglial cell marker IBA-1 (red) and nuclear staining (DAPI, blue), as well as quantitative analysis of IBA-1 fluorescence intensity; scale bar, 100 μm; n=10.

[0017] Figure 6 Immunofluorescence analysis results of β-amyloid (Aβ) in the cerebral cortex (CTX) and hippocampus (HIP). Representative immunofluorescence images of Aβ (red) and nuclear staining (DAPI, blue), and quantitative analysis of Aβ fluorescence intensity; scale bar, 100 μm; n=10.

[0018] Figure 7 Image showing the results of Golgi staining analysis of dendritic spines in neurons of the cerebral cortex (CTX) and hippocampus (HIP). Representative microscopic images with magnified insets and quantitative analysis of dendritic spine density (per 10 μm); scale bar, 10 μm; n=10.

[0019] Figure 8 The relative mRNA level of Ifnb in WT or MAVS knockout peritoneal macrophages (PM) pretreated with ponatinib and transfected with dsRNA was detected by RT-qPCR. N=3 biological replicates; data are mean ± SEM. Detailed Implementation

[0020] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0021] This invention provides the use of ponatinib in the preparation of drugs for the prevention and / or treatment of Alzheimer's disease, administered via subcutaneous injection, intravenous injection, intramuscular injection, or oral administration. MAVS is the target of ponatinib; by inhibiting the activation of the MAVS signaling pathway, ponatinib can effectively suppress the excessive activation of microglia in the disease state, thereby alleviating the neuroinflammatory response in the brain. Simultaneously, inhibiting the MAVS pathway can effectively reduce the abnormal accumulation of amyloid protein in the pathological process of AD.

[0022] The present invention will be described in detail below with reference to embodiments.

[0023] Example 1

[0024] 1. Experimental Materials 1.1 Animals Animal model: 5×FAD mice (20 weeks old).

[0025] 1.2 Reagents Ponatinib: Purchased from Shanghai Taoshu Biotechnology Co., Ltd., product number, CAS No.: 943319-70-8.

[0026] 2. Experimental Methods 2.1 Animal Models and Ethical Statements This study used 5×FAD mice (catalog number 006554), wild-type mice, and MAVS knockout mice (MAVS). - / - All mice (product number 008684) were purchased from Shulaibao Biotechnology. All mice were housed in a specific pathogen-free (SPF) animal housing at an ambient temperature of 22±2℃ and a humidity of 50±10%, with a 12-hour light-dark cycle. The mice had free access to food and water.

[0027] 2.2 Drug Intervention Experiment Seven-month-old male 5×FAD mice were randomly divided into two groups of 10 mice each: Solvent control group (Ctrl group): Daily intraperitoneal injection of solvent (0.9% saline containing 0.1% dimethyl sulfoxide DMSO), with a single injection volume of 200 μL; In the treatment group, ponatinib (10 mg / kg dissolved in the above solvent) was administered intraperitoneally every 3 days for a total of 7 times.

[0028] During all stages of the assay, the laboratory personnel were unaware of the mouse groupings.

[0029] After the complete drug administration process was completed in both the solvent control group and the drug administration group, the open field test (OFT), novel object recognition (NOR) test, and Y-maze test were performed sequentially. Mice were then sacrificed and brain tissue was collected for subsequent analysis. (See the overall experimental protocol for details.) Figure 1 ).

[0030] 2.3 Behavioral testing All behavioral experiments were conducted sequentially: Open Field Experiment (OFT) → Y-maze Spontaneous Alternation Experiment → New Object Recognition Experiment (NOR), with an interval of no less than 24 hours between adjacent experiments. The experiments were conducted in a quiet, low-light environment (light intensity < 30 lux).

[0031] After mice were transferred to the testing room, they were allowed to acclimatize for at least 1 hour before testing began. After each mouse was tested, the experimental chamber was thoroughly wiped with 70% ethanol to eliminate odor interference. The entire experimental process was recorded using a digital camera. Data analysts blinded the mice by genotype and treatment group, and quantified various behavioral indicators using ANY-maze animal behavior analysis software.

[0032] 2.3.1 Open Field Experiment (OFT) A rectangular open-field test chamber (50 cm long × 50 cm wide × 40 cm high, with a light intensity of approximately 20 lux) was used to assess the spontaneous activity and anxiety-like behavior of mice. Mice were placed in the center of the chamber and allowed to explore freely for 5 minutes. The total distance traveled and the time spent in the central area (defined as the area within 25% of the center of the chamber's bottom surface) were recorded, reflecting the level of spontaneous activity and anxiety, respectively. Simultaneously, the peripheral approach-avoidance index was calculated as: (time spent in the peripheral area ÷ total test time) × 100%.

[0033] 2.3.2 Spontaneous Alternation Experiment in the Y-Maze Spatial working memory was assessed using a symmetrical Y-maze, which consisted of three arms of equal length (each arm measuring 35 cm × 8 cm × 15 cm, with an angle of 120° between arms). Mice were placed at the end of any arm as the starting point and allowed to explore freely for 5 minutes. Each entry into an arm was defined as the mouse's paws completely entering the arm, and the total number of entry attempts was recorded. Each instance of spontaneous alternation (e.g., ABC, BCA) involved entering three different arms consecutively. The spontaneous alternation rate was calculated as: [Number of spontaneous alternation attempts ÷ (Total number of entry attempts - 2)] × 100%. Data from mice with fewer than 8 entry attempts were not included in the statistical analysis.

[0034] 2.3.3 Novel Object Recognition Experiment (NOR) Object recognition and memorization are performed in three stages within a square box (50 cm × 50 cm): Adaptation period: Mice were placed in an empty box and allowed to move freely for 5 minutes; Familiarization training period: Two identical objects are placed in the box, and the mouse explores freely for 10 minutes; Testing period: After 24 hours, one of the familiar objects was replaced with a new object that was different in shape and texture but similar in height, and the mice were allowed to explore freely for 5 minutes.

[0035] A mouse's nose tip being ≤2 cm from an object is considered exploratory behavior.

[0036] The preference index is calculated as follows: (exploration time of new object ÷ total exploration time of new object + familiar object) × 100%. The order of object placement was rotated during the experiment to eliminate interference from the mice's innate object preferences.

[0037] 2.4 Tissue Collection and Sample Processing After all behavioral tests were completed, mice were anesthetized with isoflurane (induction concentration 5%, maintenance concentration 2%), and then perfused with pre-cooled sterile PBS (20–30 mL) via the heart until the perfusion fluid flowing from the right atrium was clear. The cerebral cortex (CTX) and hippocampus (HIP) were rapidly separated on ice.

[0038] Molecular testing samples: tissues are rapidly frozen in liquid nitrogen and stored at -80°C for long-term preservation.

[0039] Histopathological (immunofluorescence staining) samples: After PBS perfusion, the heart was further perfused with 4% paraformaldehyde (PFA, pH 7.4) prepared with 0.1 M PBS. Intact brain tissue was isolated and fixed with 4% paraformaldehyde at 4°C for 24 h. The tissue was then dehydrated sequentially with 15% and 30% sucrose PBS solutions at 4°C until it completely settled. The tissue was then embedded in cryopreservation medium (OCT, Sakura cryopreservation gel), and 30 μm thick brain slices were cut using a Leica CM3050S cryostat. The free brain slices were stored in cryoprotectant at -20°C protected from light for later use.

[0040] Histopathological (Golgi staining) samples: Animals were euthanized under anesthesia and the brain was quickly removed, avoiding cardiac perfusion (or only briefly rinsing with physiological saline). The tissue was trimmed to 10 mm. 3 Cut the blood into small pieces, rinse with PBS or distilled water to remove blood, and set aside.

[0041] 2.4.1 Immunofluorescence staining and confocal imaging The free brain slices were washed three times with PBS for 5 min each time; permeabilized with PBS containing 0.3% Triton X-100 at room temperature for 20 min; and blocked with 5% donkey normal serum (NDS) + 0.1% Triton X-100 PBS blocking solution at room temperature for 1 h.

[0042] Brain slices were incubated overnight at 4°C with primary antibody dilution buffer. After washing three times with PBS, the corresponding species' Alexa Fluor secondary antibody (1:500 dilution, Thermo Fisher Scientific) was added and incubated at room temperature in the dark for 1 h. Nuclear staining was performed with DAPI (1 μg / mL, Sigma-Aldrich) for 10 min. After thorough washing with PBS, the brain slices were mounted on anti-detachment slides (SuperFrost Plus, Thermo Fisher Scientific) and mounted with Fluoromount-G mounting medium.

[0043] Aβ amyloid plaque staining procedure: After the secondary antibody incubation, 1% thioflavone S (ThS) was prepared with 50% ethanol and incubated in the dark for 8 min. The plaques were then washed twice with 50% ethanol and once with PBS to mark dense amyloid plaques.

[0044] Images were acquired using an Olympus FV3000 laser scanning confocal microscope, with uniform imaging parameters for all samples. Three brain slices containing the cerebral cortex and hippocampus were taken from each mouse. Three non-overlapping fields of view were randomly selected from each slice. The average fluorescence intensity of MAVS, IBA-1, and GFAP, as well as the number and total area of ​​Aβ plaques, were quantified using ImageJ / FIJI software with uniform thresholds.

[0045] 2.4.2 Golgi staining (Golgi-Cox method) The reagents used were the FD Rapid GolgiStain kit (PK401, FD NeuroTechnologies, Inc.).

[0046] Immersion in dye solution: Mix equal volumes of solution A (potassium dichromate / potassium chromate) and solution B (mercuric chloride / silver nitrate), let stand for 24 hours, take the supernatant, immerse the tissue in it, and incubate at room temperature in the dark for 10-14 days (during which time the solution is changed by gentle shaking every 2-3 days); then transfer to solution C and treat at room temperature or 4°C in the dark for 3-7 days to harden the tissue.

[0047] Section preparation: Remove the tissue and wipe it dry. Use a vibratory microtome (no embedding required) or OCT embedding followed by freezing to cut 100-200μm thick sections. Place them on gelatin-coated glass slides and dry at room temperature in the dark for 24-72 hours.

[0048] Development reaction: Prepare the developing solution (usually solution D + solution E + water = 1:1:2), immerse the dried slices in the solution at room temperature for 10-15 minutes until the neurons turn brownish-black, and rinse twice with distilled water.

[0049] Dehydration and mounting: The slides are sequentially dehydrated with 50%, 75%, 95%, and 100% ethanol (4 min each), cleared with xylene (2-3 times), and finally mounted with neutral resin or Permount. Store in the dark.

[0050] Images were acquired and the third-order dendrites were selected for quantitative analysis. The number of dendritic spines was calculated using ImageJ software.

[0051] 2.5 Molecular docking To further explore the target, molecular docking was performed in this embodiment, and the three-dimensional structure of the MAVS CARD domain was predicted using AlphaFold 3. The three-dimensional structures of 1615 FDA-approved small molecule drugs were downloaded from the ZINC 20 database. Molecular docking was performed using AutoDock v1.2.3, with the entire surface of the MAVS CARD domain set as the docking region. The 12 compounds with the highest docking scores were selected for molecular dynamics simulations. Molecular dynamics simulations of the force field of AMBER99SB-ILDN were performed using GROMACS (v2021.3) software. The ligand-protein complex was placed in a TIP3P water tank, and Na was added. + and Cl -To neutralize the charge, the system was first subjected to energy minimization, followed by 100 ps NVT and 100 ps NPT equilibrations, and finally 100 ns molecular dynamics simulations. The conformational stability of the complex was evaluated by calculating the root mean square deviation (RMSD) of the ligand's heavy atoms. The binding free energy was calculated using the MM / GBSA method.

[0052] 2.6 Wild-type and MAVS knockout PM cell isolation stimulation Peritoneal macrophage (PM) isolation method: 8-10 week old mice (mouse source as described above) were dislocated at the cervical spine and immersed in 75% ethanol. One side of the peritoneum was exposed, and 6-8 ml of 1×PBS was injected into the peritoneal cavity using a 10 ml syringe to wash the cavity. Peritoneal fluid was collected and centrifuged at 1500 rpm for 5 min. The supernatant was discarded, and the cell pellets were resuspended in DMEM containing 10% FBS and 1% penicillin-streptomycin, and cultured at 37℃ in a humidified atmosphere of 5% CO2. After 24 h, the cell culture medium was changed, and the cells were washed once with PBS to remove non-adherent cells before proceeding with subsequent experiments. PM cells were transfected with Poly(I:C) (2 μg / 10⁶ cells) for 12 h. Subsequently, cell samples were lysed using Trizol reagent (Invitrogen), and tissue samples were homogenized using a tissue homogenizer. Total RNA was extracted using chloroform and isopropanol, and cDNA was synthesized using HiScript II Q RT SuperMix (Vazyme, China, R222-01). Real-time quantitative PCR was performed using ArtiCanCEO SYBR qPCR Mix (Tsingke, China, TSE401), with specific primers serving as the cDNA template. The relative expression levels of mRNA were calculated using the 2-ΔΔCt method, and the expression levels of target gene mRNA in all experimental samples were normalized to those in the reference sample.

[0053] 3. Experimental Results Behavioral testing results showed that mice in the ponatinib-treated group exhibited significant functional improvements across all tested behavioral dimensions. In the open field test, compared to the solvent control group, the treated mice spent a significantly longer time in the central region of the experimental setup, suggesting that ponatinib can effectively alleviate anxiety-like behavioral abnormalities associated with AD model mice (experimental results are shown in [link to results]). Figure 2 The results of the Y-maze spontaneous alternation experiment showed that the accuracy rate of spontaneous alternation in the drug-treated group was significantly increased. This directly reflects that the spatial working memory ability of AD model mice was significantly improved after drug intervention (experimental results are shown in...). Figure 3In the novel object recognition experiment, the mice in the drug-treated group showed a significantly higher preference index for novel objects and more active autonomous exploration of these objects, indicating that the impaired recognition and memory function in AD model mice was effectively restored after treatment with ponatinib (experimental results are shown in [link to results]). Figure 4 The above series of behavioral results collectively confirm that systemic administration of ponatinib can effectively improve cognitive deficits in AD model mice and alleviate their emotion-related behavioral abnormalities.

[0054] Neuropathological analysis, echoing the behavioral improvement results, showed that all core pathological endpoints related to Alzheimer's disease (AD) exhibited a consistent trend of improvement after ponatinib administration. Immunofluorescence staining revealed that the fluorescence intensity and protein expression level of IBA-1, a microglia-specific marker in the cerebral cortex and hippocampus of mice in the treatment group were significantly downregulated (see results). Figure 5 These results suggest that ponatinib can effectively inhibit the excessive activation of microglia in the disease state by suppressing the activation of the MAVS signaling pathway, thereby alleviating the neuroinflammatory response in the brain. Meanwhile, compared with the solvent control group, the number of β-amyloid (Aβ) plaques and the overall deposition level of Aβ protein in the brains of mice treated with ponatinib were significantly reduced (see results). Figure 6 This indicates that inhibiting the MAVS pathway can effectively reduce the abnormal accumulation of amyloid protein in the pathological process of AD.

[0055] Golgi staining results further confirmed the neuroprotective effect of the drug: the density of dendritic spines in the cerebral cortex and hippocampus of mice in the drug-treated group was significantly higher than that in the control group (see results). Figure 7 This indicates that ponatinib treatment has a clear protective effect on synaptic structure and synaptic plasticity in the brain affected by Alzheimer's disease. In conclusion, ponatinib effectively improves memory and behavior in Alzheimer's mice, protects against brain lesions, and is an effective treatment for Alzheimer's disease.

[0056] Molecular docking experiments showed that MAVS is the target of ponatinib, and the final target verification was achieved by using MAVS knockout cells: ponatinib effectively inhibited the poly(I:C)-mediated interferon response in WT cells, but had no effect on MAVS-deficient cells. Figure 8 This application is the first to identify ponatinib as an effective specific MAVS inhibitor, demonstrating that ponatinib, as an effective specific MAVS inhibitor, inhibits neuroinflammation and treats AD.

[0057] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The use of ponatinib in the preparation of drugs for the prevention and / or treatment of Alzheimer's disease.

2. The application as described in claim 1, characterized in that: The administration methods used in this application include subcutaneous injection, intravenous injection, intramuscular injection, or oral administration.

3. The application as described in claim 1, characterized in that: The drugs mentioned for the prevention and / or treatment of Alzheimer's disease are drugs that target and inhibit MAVS.

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