Use of anidulafungin in preventing and treating neurodegenerative diseases

Abstract

The present invention relates to the use of a compound (i.e., anidulafungin) in preventing and treating neurodegenerative diseases, in particular, in preventing and treating Alzheimer's disease. The inventors creatively discovered that anidulafungin can effectively inhibit Aβ aggregation and reduce the neurotoxicity of Aβ oligomers, thereby effectively preventing and treating Aβ aggregation-related nervous system diseases (e.g., Alzheimer's disease); and the use of the known drug for the treatment of Alzheimer's disease can ensure the safety of medication and reduce the risk of possible adverse reactions.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to the use of anidulafungin in preventing and treating neurodegenerative diseases, in particular Alzheimer's disease. Background Art

[0002] Alzheimer's disease (AD) seriously affects the quality of life of patients and their families. With the development of an aging society, it has become one of the diseases that seriously endangers the health of the elderly in China and even the world, and is a major social problem that needs to be urgently addressed.

[0003] The pathological characteristics of AD include extracellular amyloid beta (Aβ) deposition, intracellular Tau protein aggregation, neurofibrillary tangles, and neuronal loss. The etiology of AD has always been shrouded in mystery. Among many hypotheses, the Aβ cascade hypothesis is the most recognized, and the formation of amyloid plaques in the brain has become the first suspect in the pathogenesis of AD. Amyloid precursor protein (APP) is cleaved to form Aβ, which aggregates to form oligomers. Its solubility deteriorates and can be deposited into plaques. Plaques can cause neurofibrillary tangles, neuronal loss, etc., but the related pathogenesis is still unclear.

[0004] Currently, the AD treatment drugs approved by the U.S. Food and Drug Administration (FDA) mainly include cholinesterase inhibitors, NMDA receptor antagonists and Aβ monoclonal antibodies. Existing clinical evidence shows that cholinesterase inhibitors and NMDA receptor antagonists have a certain degree of improvement in the cognitive level and daily living ability of AD patients, but cannot prevent or reverse the progression of AD. Although Aβ monoclonal antibodies can clear Aβ in the patient's brain, they have side effects such as cerebral hemorrhage and cerebral edema, and are expensive. Therefore, there is currently a lack of disease-modifying drugs that can effectively prevent or reverse the course of AD.

[0005] Although the pathogenesis of AD is complex and diverse, the aggregation and deposition of Aβ in the brain still plays a vital role in the development of AD. Aβ can aggravate the severity of brain pathological changes and symptoms of AD patients by inducing neuroinflammation, oxidative stress, neuronal death, and abnormal energy metabolism.

[0006] Therefore, developing new Aβ inhibitors is one of the important ways to treat AD. Summary of the invention

[0007] Purpose of the Invention

[0008] In view of the problems or needs in the prior art, the object of the present invention is to provide a compound for preventing and treating Aβ aggregation-related nervous system diseases (such as neurodegenerative diseases); in particular, the neurodegenerative disease is Alzheimer's disease, and the drug contains at least anidulafungin.

[0009] It is currently known that anidulafungin is an echinocandin antifungal drug that can inhibit the synthesis of 1,3-β-D-glucan, an important component of the fungal cell wall, and is active against a variety of Candida and some Aspergillus in vitro.

[0010] The present inventors have discovered for the first time that anidulafungin can inhibit Aβ aggregation, and thus can prevent and treat Aβ aggregation-related nervous system diseases.

[0011] Solution

[0012] In order to achieve the above object, the present invention provides the following technical solutions:

[0013] In a first aspect, the present invention provides a use of a compound in the preparation of a medicament for treating and / or preventing a nervous system disease associated with Aβ aggregation, wherein the compound has a structure as shown in the following formula I:

[0014]

[0015] The compound shown in formula I is anidulafungin.

[0016] Without being bound by any theory, it is understood that the compounds preferably treat and / or prevent Aβ aggregation-related neurological diseases by inhibiting Aβ aggregation.

[0017] Without being bound by any theory, it is understood that the compounds preferably treat and / or prevent the Aβ aggregation-related neurological diseases by reducing the neurotoxicity of Aβ oligomers.

[0018] In certain embodiments, the compound preferably treats and / or prevents the Aβ aggregation-related neurological diseases by inhibiting Aβ aggregation and reducing the neurotoxicity of Aβ oligomers.

[0019] In certain embodiments, the treating comprises administering an effective amount of the drug to a patient in need thereof.

[0020] In certain embodiments, the treatment or prevention further comprises monoclonal antibody therapy. The monoclonal antibody therapy may be, for example, administering an effective amount of Aβ monoclonal antibody to a patient in need thereof. The drug may be administered before, after or simultaneously with the monoclonal antibody therapy.

[0021] In certain embodiments, the concentration of the compound in the drug is 0.25 to 10 μM, such as 0.25 μM, 0.5 μM, 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM, 4 μM, 4.5 μM, 5 μM, 5.5 μM, 6 μM, 6.5 μM, 7 μM, 7.5 μM, 8 μM, 8.5 μM, 9 μM, 9.5 μM or 10 μM, or any range therebetween. In certain embodiments, the concentration of the compound in the drug is 0.5 to 1 μM. In a specific embodiment, the concentration of the compound in the drug is 1 μM.

[0022] In certain embodiments, the Aβ aggregation-related neurological disease is a neurodegenerative disease, such as Alzheimer's disease.

[0023] In certain embodiments, the drug is administered by one or more of the following: oral administration, injection, implantation, spraying and / or inhalation. Exemplarily, the drug is administered by parenteral, subcutaneous, intraperitoneal, intravenous, intralesional, intracerebral, intraarterial interstitial infusion and / or implantation of a delivery device.

[0024] In certain embodiments, the dosage form of the drug is one or more selected from the following: injection, oral solution, powder, tablet, granule, capsule, syrup, decoction, sustained-release preparation, enteric solvent, aerosol or suspension.

[0025] In certain embodiments, the medicament comprises a preventively and / or therapeutically effective amount of the compound, and a pharmaceutically acceptable carrier and / or excipient.

[0026] In a second aspect, the present invention provides a use of a pharmaceutical composition comprising a compound having a structure as shown in Formula I below in the preparation of a drug for treating and / or preventing Aβ aggregation-related nervous system diseases:

[0027]

[0028] In certain embodiments, the pharmaceutical composition further comprises one or more of a cholinesterase inhibitor, an NMDA receptor antagonist, and an Aβ monoclonal antibody.

[0029] In certain embodiments, the pharmaceutical composition further comprises an Aβ monoclonal antibody.

[0030] Without being bound by any theory, it is understood that the compounds preferably treat and / or prevent Aβ aggregation-related neurological diseases by inhibiting Aβ aggregation.

[0031] Without being bound by any theory, it is understood that the compounds preferably treat and / or prevent the Aβ aggregation-related neurological diseases by reducing the neurotoxicity of Aβ oligomers.

[0032] In certain embodiments, the compound preferably treats and / or prevents the Aβ aggregation-related neurological diseases by inhibiting Aβ aggregation and reducing the neurotoxicity of Aβ oligomers.

[0033] In certain embodiments, the treating comprises administering an effective amount of the drug to a patient in need thereof.

[0034] In certain embodiments, the treatment or prevention further comprises monoclonal antibody therapy. The monoclonal antibody therapy may be, for example, administering an effective amount of Aβ monoclonal antibody to a patient in need thereof. The compound may be administered before, after or simultaneously with the monoclonal antibody therapy.

[0035] In certain embodiments, the concentration of the compound in the drug is 0.25 to 10 μM, such as 0.25 μM, 0.5 μM, 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM, 4 μM, 4.5 μM, 5 μM, 5.5 μM, 6 μM, 6.5 μM, 7 μM, 7.5 μM, 8 μM, 8.5 μM, 9 μM, 9.5 μM or 10 μM, or any range therebetween. In certain embodiments, the concentration of the compound in the drug is 0.5 to 1 μM. In a specific embodiment, the concentration of the compound in the drug is 1 μM.

[0036] In certain embodiments, the Aβ aggregation-related neurological disease is a neurodegenerative disease, such as Alzheimer's disease.

[0037] In certain embodiments, the drug is administered by one or more of the following routes: oral administration, injection, implantation, spraying and / or inhalation.

[0038] In certain embodiments, the dosage form of the drug is one or more selected from the following: injection, oral solution, powder, tablet, granule, capsule, syrup, decoction, sustained-release preparation, enteric solvent, aerosol or suspension.

[0039] In certain embodiments, the medicament comprises a preventively and / or therapeutically effective amount of the compound, and a pharmaceutically acceptable carrier and / or excipient.

[0040] In a third aspect, the present invention provides a method for treating and / or preventing Aβ aggregation-related nervous system diseases, the method comprising administering a compound having a structure as shown in the following formula I or a drug containing the same to a patient in need:

[0041]

[0042] Without being bound by any theory, it is understood that the compounds preferably treat and / or prevent Aβ aggregation-related neurological diseases by inhibiting Aβ aggregation.

[0043] Without being bound by any theory, it is understood that the compounds preferably treat and / or prevent the Aβ aggregation-related neurological diseases by reducing the neurotoxicity of Aβ oligomers.

[0044] In certain embodiments, the compound preferably treats and / or prevents the Aβ aggregation-related neurological diseases by inhibiting Aβ aggregation and reducing the neurotoxicity of Aβ oligomers.

[0045] In certain embodiments, the treatment comprises administering an effective amount of the compound or the drug to a patient in need thereof, for example, by common administration such as oral administration, injection, implantation, spraying and / or inhalation.

[0046] In certain embodiments, the treatment or prevention further comprises monoclonal antibody therapy. The monoclonal antibody therapy may be, for example, administering an effective amount of Aβ monoclonal antibody to a patient in need thereof. The compound may be administered before, after or simultaneously with the monoclonal antibody therapy.

[0047] In certain embodiments, the concentration of the compound in the drug is 0.25 to 10 μM, such as 0.25 μM, 0.5 μM, 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM, 4 μM, 4.5 μM, 5 μM, 5.5 μM, 6 μM, 6.5 μM, 7 μM, 7.5 μM, 8 μM, 8.5 μM, 9 μM, 9.5 μM or 10 μM, or any range therebetween. In certain embodiments, the concentration of the compound in the drug is 0.5 to 1 μM. In a specific embodiment, the concentration of the compound in the drug is 1 μM.

[0048] In certain embodiments, the Aβ aggregation-related neurological disease is a neurodegenerative disease, such as Alzheimer's disease.

[0049] In certain embodiments, the drug is administered by one or more of the following routes: oral administration, injection, implantation, spraying and / or inhalation.

[0050] In certain embodiments, the dosage form of the drug is one or more selected from the following: injection, oral solution, powder, tablet, granule, capsule, syrup, decoction, sustained-release preparation, enteric solvent, aerosol or suspension.

[0051] In certain embodiments, the medicament comprises a preventively and / or therapeutically effective amount of the compound, and a pharmaceutically acceptable carrier and / or excipient.

[0052] Beneficial Effects

[0053] First, the present invention uses existing marketed antimicrobial drugs to inhibit Aβ aggregation, which means that the association between Aβ aggregation and neurodegenerative diseases can be addressed with drugs that are already in clinical use. This approach reduces the time and resource investment required for clinical trials and safety assessments in the development of new drugs.

[0054] Secondly, compared with monoclonal antibodies, the use of existing marketed antimicrobial drugs has lower costs for inhibiting Aβ aggregation. Monoclonal antibodies are usually complex protein drugs prepared by biotechnology, and their production and purification processes are cumbersome and expensive. Existing marketed antimicrobial drugs have been verified in large-scale production and have relatively low production costs, which will help improve patient accessibility and reduce the overall cost of treatment.

[0055] In addition, the present invention uses existing marketed antibacterial drugs to inhibit Aβ aggregation, which is expected to avoid the possible side effects and adverse reactions of monoclonal antibody drugs. Monoclonal antibody drugs usually need to be administered by injection or intravenous infusion, which may lead to a series of adverse symptoms and potential immune reactions. In contrast, existing marketed antibacterial drugs have been widely used, and the information on their administration routes and safety is more sufficient, thereby reducing the risk of possible adverse reactions. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] One or more embodiments are exemplarily described by the pictures in the accompanying drawings, and these exemplary descriptions do not constitute limitations on the embodiments. The special word "exemplary" here means "used as an example, embodiment or illustrative". Any embodiment described as "exemplary" here is not necessarily interpreted as being superior or better than other embodiments.

[0057] Figure 1The figure shows the inhibitory effect of anidulafungin on Aβ42 aggregation; wherein, Figure A shows an in vitro aggregation experiment to detect the primary nucleation step (Primary nucleation) of Aβ42 aggregation inhibited by anidulafungin, wherein the horizontal axis is the reaction time, and the vertical axis is the total amount of Aβ42 fibers generated by the reaction, and each point in the figure represents the total amount of Aβ42 fibers generated at that time point in the presence of the corresponding concentration of anidulafungin; the curve represents the Aβ42 aggregation kinetic curve calculated and fitted by the Amylofit platform based on the data; Figure B shows the effect of anidulafungin on the reaction rate constant Kn of the primary nucleation step of Aβ42 aggregation, wherein the horizontal axis is the anidulafungin concentration, and the vertical axis is the ratio of the reaction rate constant (kn') of the primary nucleation step of Aβ42 aggregation under different anidulafungin concentrations to the reaction rate constant (Kn) of the primary nucleation step in the absence of anidulafungin.

[0058] Figure 2 The effect of anidulafungin on Aβ42 aggregation and fiber formation detected by Dot Blot experiment is shown; the figure shows the dot blot after 0h, 6h, 12h, and 30h treatment with 5μM Aβ42 in the presence and absence of anidulafungin; ANI represents anidulafungin.

[0059] Figure 3 The figure shows the effect of anidulafungin on the aggregation and fiber formation of Aβ42 observed by atomic force microscopy; wherein, Figures A, B, and C are microscope images of 0h, 6h, and 30h after treatment with 5μM Aβ42 in the absence of anidulafungin; Figures D, E, and F are microscope images of 0h, 6h, and 30h after treatment with 5μM Aβ42 in the presence of 5μM anidulafungin; in the figure, ANI represents anidulafungin.

[0060] Figure 4 :Figure A shows the toxic effect of Aβ oligomers on BV2 cells; the horizontal axis shows the concentration of Aβ oligomers, and the vertical axis shows the cell activity; Figure B shows the safety evaluation of anidulafungin on BV2 cells; the horizontal axis shows the concentration of anidulafungin, and the vertical axis shows the cell activity; Figure C shows the effect of anidulafungin on the cytotoxicity of BV2 cells induced by Aβ oligomers; the horizontal axis shows the concentration of Aβ oligomers or anidulafungin, and the vertical axis shows the cell activity; ****: P<0.0001 compared with the control group; ***: P<0.001 compared with the control group; ##: P<0.01 compared with the 5μM Aβ42 oligomer modeling group; all data are expressed as mean ± standard error, and differences among multiple groups were detected by one-way analysis of variance, and multiple comparisons were performed by Tukey correction. The differences were significant when P<0.05. AβO: Aβ42 oligomers; ANI: anidulafungin. DETAILED DESCRIPTION

[0061] Unless explicitly stated otherwise, throughout the specification and claims, the term “comprise” or variations such as “include” or “comprising”, etc., will be understood to include the stated elements or components but not to exclude other elements or components.

[0062] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art.

[0063] Unless explicitly defined otherwise in other parts of this document, technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs.

[0064] Neurodegenerative diseases are caused by the loss of neurons and / or their myelin sheaths, which progress over time and lead to functional impairment.

[0065] Alzheimer's disease (AD) is a primary degenerative brain disease that occurs in the elderly and early elderly. It refers to a persistent disorder of higher nervous function activities, that is, disorders of memory, thinking, analysis and judgment, visual-spatial recognition, and emotions in the absence of consciousness disorders. In the early stages of the disease, it can manifest as a decline in cognitive function, mainly memory loss, and in the late stages, mental and behavioral symptoms may appear.

[0066] Aβ oligomers are a conformation-specific spatial structure generated by enzymatic processing of amyloid precursor protein. After β-secretase and γ-secretase cleave amyloid precursor protein to form β-amyloid monomers, they can form oligomers ranging from 2 to 8 to 20 to 40 depending on the degree of polymerization, and then they can be transformed into regular folded structures and assembled into amyloid fibers.

[0067] Anidulafungin (CAS NO.166663-25-8) is an antifungal drug with the following structure:

[0068]

[0069] It was approved by the U.S. Food and Drug Administration (FDA) in February 2006 for the treatment of various types of Candida infections.

[0070] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustrating the present invention and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0071] In the following examples: Aβ oligomers were purchased from Shanghai Qiangyao Biotechnology Co., Ltd.; Aβ42 polypeptide was purchased from Abcam.

[0072] Example 1 Inhibitory effect of anidulafungin on Aβ42 aggregation in vitro

[0073] 1. Treatment of Aβ42 Peptide

[0074] 1) Aβ42 polypeptide was dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) to a concentration of 1 mg / mL.

[0075] 2) After the solution is clarified, mix by ultrasonic.

[0076] 3) The solution obtained in step 2) was equally divided into ten low-binding EP tubes (100 μL / tube, each containing 0.1 mg Aβ42).

[0077] 4) After air drying, store the EP tube at -80°C to maintain stability.

[0078] 2. In vitro Aβ aggregation and drug inhibition experiments

[0079] 1) Dissolution of Aβ42

[0080] The air-dried Aβ42 prepared above was taken out from the -80°C refrigerator and dissolved in 60 mM NaOH to a concentration of 200 μM. After standing at room temperature for 20 minutes, it was sonicated in an ice water bath for 20 minutes to ensure that the peptide was completely dissolved. Afterwards, it was diluted to the working concentration required for the experiment using 20 mM phosphate buffer.

[0081] 2) Mixing Anidulafungin and Aβ42

[0082] Anidulafungin was diluted to 200mM with DMSO and then diluted to 0-20μM with PBS. At the same time, ThT working solution was added to Aβ42 diluted with phosphate buffer. Anidulafungin and Aβ42 were mixed and the mixture was distributed to each well of a 96-well ELISA plate, ensuring that the final volume of each well was 200μL. The concentrations of Aβ42 were 5μM, ThT were 20μM, and the concentrations of anidulafungin were 0, 1, 2.5, and 5μM, respectively.

[0083] 3) Fluorescence detection

[0084] Place the 96-well microplate containing the samples in a microplate reader and set the reaction temperature to 37°C. Use a 440nm excitation filter and a 480nm emission filter to measure ThT fluorescence through the top of the plate. Perform fluorescence detection every 6 minutes and repeat the measurement four times for each sample.

[0085] 4) Data analysis

[0086] The AmyloFit platform was used to analyze the effects of different concentrations of anidulafungin on the aggregation kinetics of Aβ42.

[0087] Figure 1 Figure A shows the kinetic curve of Aβ42 aggregation when 5μM Aβ42 was co-incubated with different concentrations of anidulafungin (0, 1, 2.5, 5μM). The AmyloFit platform was used to analyze the kinetic data, and the "secondary nucleation dominated" model was selected, and the primary reaction rate k n The independent variable was set to perform fitting analysis of the Aβ42 aggregation kinetic curve.

[0088] Figure 1 Figure A shows that with the increase of anidulafungin drug concentration, the time for Aβ42 to aggregate and form fibers is significantly delayed; and the experimental data are highly consistent with the curve fitted by the change of the reaction rate constant kn in the primary nucleation step (the earliest stage in Aβ42 aggregation) in the Amylofit platform, which indicates that anidulafungin inhibits Aβ42 aggregation by inhibiting the primary nucleation step.

[0089] Figure 1 Figure B shows the effect of anidulafungin on the reaction rate constant Kn of the primary nucleation step of Aβ42 aggregation, which is calculated from the kinetic curve fitted by the AmyloFit platform in Figure A. The results in Figure B show that with the increase of anidulafungin concentration, the ratio of the reaction rate constants (kn' / Kn) of the primary nucleation step of Aβ42 decreased significantly, indicating that anidulafungin can effectively inhibit the primary nucleation step of Aβ42.

[0090] In summary, Figure 1 It was shown that anidulafungin significantly inhibited the aggregation of Aβ42, and this inhibitory effect was positively correlated with the drug concentration. When the concentration was 1μM, it was able to effectively inhibit the aggregation of 5μM Aβ42, prolong the lag phase of the aggregation reaction, and delay the time required for the reaction to reach the plateau phase. When the drug concentration increased to 5μM, anidulafungin significantly prolonged the lag phase of Aβ42 aggregation from about 5 hours to 30 hours, significantly delaying the onset of the aggregation reaction. In addition, with the increase in anidulafungin concentration, the rate constant k of the primary nucleation step of Aβ42 aggregation n When the concentration of anidulafungin reached 5 μM, the kn value dropped to one ten-thousandth of that without drug treatment.

[0091] Example 2 Study on the effect of anidulafungin on Aβ42 aggregation and fiber formation by Dot Blot and Atomic Force Microscopy

[0092] 1. Aβ42 aggregation assay in vitro

[0093] The Aβ42 in vitro aggregation experiment was performed according to the steps described in Example 1.

[0094] 2. Mixing Anidulafungin and Aβ42

[0095] At different stages of the aggregation reaction, samples containing different concentrations of anidulafungin were taken out from the 96-well ELISA plate, collected in low-binding capacity EP tubes, and then stored in a -80°C refrigerator for subsequent analysis.

[0096] 3. Dot Blot Experiment

[0097] Dot blot was used to evaluate the effect of anidulafungin on the aggregation and fibril formation of Aβ42 using OC-anti-amyloid fibril antibody (Sigma-Aldrich, AB2286), which can specifically bind to mature amyloid fibrils.

[0098] The specific steps are as follows:

[0099] i. Take the Aβ42 sample out of the -80°C freezer, slowly thaw on ice, and sonicate in an ice-water bath for 2 minutes to ensure homogeneity.

[0100] ii. Cut the PVDF membrane to the appropriate size and use a 100μL pipette tip to print a circular mark on the membrane as the range for subsequent sample addition. The PVDF membrane was first activated by soaking in anhydrous methanol for 2 minutes, then soaked in TBST solution (TBS-T; 20mM Tris, 150mM NaCl, 0.05% Tween 20, pH 7.5) for 5 minutes to remove excess methanol, and finally soaked in new TBST for use.

[0101] iii. Place the absorbent filter paper soaked in TBST on top of the dry absorbent filter paper, remove the PVDF membrane from the TBST, place it on top of the wet filter paper, and start dropping the Aβ42 solution sample after the excess water is absorbed. Drop 2 μL of the sample in the center of the pre-printed circular mark. After all samples have been dropped, place the membrane in a fume hood and let it air dry for 1 hour to fix the protein on the PVDF membrane.

[0102] iv. Subsequently, the PVDF membrane was blocked with a rapid blocking solution for 20 minutes, and then rinsed twice with TBST for 5 minutes each time. The blocked PVDF membrane was placed in an antibody incubation box, and OC anti-amyloid fiber antibody diluted 1:10000 with a universal antibody diluent was added, and incubated overnight on a shaker in a 4°C refrigerator.

[0103] v. After the primary antibody incubation is completed, the PVDF membrane is rinsed with TBST for 3 times, 10 minutes each time, and then the horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG secondary antibody diluted 1:10000 in universal antibody diluent is added and incubated at room temperature for 1 hour. After the secondary antibody incubation is completed, the PVDF membrane is rinsed with TBST for 3 times, 10 minutes each time.

[0104] vi. Mix the ECL luminescent solution evenly with solution I and solution II in a ratio of 1:1 and use it. Place the PVDF membrane with protein facing up in the exposure instrument, evenly cover the prepared ECL luminescent solution on the PVDF membrane, and react in the dark for 1 minute. After turning on the machine, select the appropriate exposure time and adjust it to save the most suitable image according to the specific band display situation.

[0105] The results are as follows Figure 2 As shown, Figure 2 It was shown that anidulafungin significantly inhibited Aβ42 fiber formation. Specifically, fiber generation was observed in the 5μM Aβ42 sample without anidulafungin after 6 hours of aggregation, and the number of fibers gradually increased with time. When the aggregation time reached 30h, a large number of Aβ42 fibers were formed in the sample, and a strong OC antibody positive signal was shown. On the contrary, in the presence of 5μM anidulafungin, the Aβ42 fiber formation process was significantly delayed. Even after 30 hours of aggregation, the number of fibers detected by the OC antibody was still very limited, and the antibody positive signal intensity was extremely low.

[0106] 4. Atomic force microscopy experiment

[0107] Atomic force microscopy was used to characterize the morphology formed during the Aβ42 aggregation process in the presence or absence of anidulafungin to determine the effect of anidulafungin on the Aβ42 fibrillation process. Specifically:

[0108] i. Use high-quality V1 grade mica sheets as carriers for Aβ42 samples. In a sterile operating table, use strong adhesive transparent tape to stick and etch the mica surface to prepare a clean and flat surface for sample deposition.

[0109] ii. Take the Aβ42 sample out of the -80℃ freezer and slowly thaw it on ice. Ultrasonicate it in an ice-water bath for 2 minutes to ensure a uniform solution. In a sterile operating table, add 20 μL of different Aβ42 solution samples to the surface of the mica sheet and let it stand for 3 minutes to allow the protein molecules to fully adsorb. Subsequently, gently rinse the mica surface with enzyme-free sterile water to remove impurities that may affect imaging. After rinsing, place the mica sheet in the operating table and air-dry it overnight with a gentle airflow.

[0110] iii. The air-dried mica sheet was imaged using a Multimode 8 atomic force microscope. Scanasyst-Air silicon nitride probe was used for surface characterization, with the following parameters: triangular cantilever, resonance frequency 70kHz, elastic modulus 0.4N / m, tip radius 2nm. During the imaging process, the scanning range of the probe was set to 5μm×5μm, and the resolution was set to 512 pixels. All the AFM images obtained were analyzed in detail using the analysis software Nanoscope Analysis 1.7 that comes with the microscope.

[0111] The results are as follows Figure 3 As shown, Figure 3 It was shown that anidulafungin significantly inhibited Aβ42 fibrillation. In the absence of anidulafungin, short fibril aggregates were visible in the 5μM Aβ42 sample after 6 hours, and mature fibers were formed after 30 hours. In the presence of 5μM anidulafungin, the sample morphology did not change much after 6 hours, no obvious aggregates were observed, and a small amount of circular oligomers and short linear fibrils appeared only after 30 hours. Specifically, before the reaction started, the main components in the initial sample were Aβ42 monomers (Figure A, Figure D). In the absence of anidulafungin, after 6 hours of incubation with 5μM Aβ42, a large number of short linear fibrils were visible in the sample (Figure B). When the incubation time was extended to 30 hours, mature Aβ42 fibers were formed in the sample (Figure C). In the presence of 5μM anidulafungin, the aggregation and fiber formation process of the 5μM Aβ42 sample was significantly delayed. After 6 hours of reaction, the morphology of Aβ42 in the sample did not change much from the initial state, and no obvious aggregates were observed (Figure E). Only after 30 hours of reaction did a small amount of oligomers and short linear fibrils appear in the sample (Figure F).

[0112] Example 3 Establishment of Aβ oligomer toxicity AD cell model

[0113] This example uses the BV2 cell line to establish an Aβ oligomer-induced AD cell model. BV2 is an immortalized cell line derived from mouse microglia. The BV2 cell line used in this example was purchased from the National Biomedical Experimental Cell Resource Bank. By co-incubating BV2 cells with Aβ oligomers, the significant reduction in cell activity indicates the neurotoxic effect of Aβ oligomers, that is, a cytotoxic model was successfully established. It can be used to simulate the neurotoxic effects of Aβ oligomers, and further used to evaluate whether candidate drugs can reduce the toxic effects of Aβ oligomers and play a neuroprotective role.

[0114] Preparation of Aβ oligomers

[0115] The specific preparation method is as follows:

[0116] First, human Aβ42 monomers were dissolved in HFIP at a concentration of 1 mg / mL, allowed to stand until the solution was clear, and then freeze-dried. Subsequently, the freeze-dried transparent film-like material was dissolved in a small amount of DMSO and diluted to a concentration of 1 mg / mL with PBS. After solubilization by ultrasonic vibration, the solution was incubated at 37°C for 24 to 32 hours to promote oligomerization. The formation of Aβ oligomers was confirmed by electron microscopy, and its purity was ensured to be ≥98% using high-performance liquid chromatography.

[0117] Determine the optimal concentration for establishing AD cell model in BV2 cell line

[0118] 1) Setting up Aβ oligomer concentration gradient

[0119] Take out the Aβ oligomer powder from the -80℃ freezer, add DMSO to the bottle according to the pre-calculated volume, and mix thoroughly to ensure that the Aβ oligomer is completely dissolved. Subsequently, place the mixture in a 37℃ water bath and use ultrasound to dissolve until the solution is clear and transparent. Before use, dilute it with DMEM medium to the required concentration of working solution, and set the concentration gradient to 2.5μM, 5μM, 10μM, and 20μM for use.

[0120] 2) Cultivate BV2 cells to the logarithmic phase

[0121] Add 5 ml of fetal bovine serum and 500 μl of double antibody (50 U / ml penicillin and 50 g / ml streptomycin solution) to 44.5 mL of DMEM culture medium to prepare 50 mL of DMEM complete culture medium. At this time, the concentration of fetal bovine serum is 10%, and the concentration of double antibody is 1%. Seal with sealing film and store at 4°C. The validity period is 30 days. Inoculate BV2 cells in the above-prepared complete culture medium and culture overnight in a humidified environment of 37°C, 5% CO2 and 95% air until the cells grow to the logarithmic phase.

[0122] 3) Add gradient concentrations of Aβ oligomers to BV2 cells

[0123] The cultured BV2 cells were counted and inoculated into a 96-well plate, with 5000 BV2 cells counted per well. The BV2 cells were diluted to the required cell amount with the above-mentioned complete medium, 100 μL per well, and each column corresponded to a group (each group had 6 duplicate wells), one of which was a blank group with no cells and only medium added. The cells were cultured overnight in a humidified environment of 37°C, 5% CO2 and 95% air, and the complete medium in the wells was aspirated and discarded. Each column corresponded to a group, namely, a blank group (without cells), a negative control group (control) (containing cells, without Aβ42 oligomers), and Aβ42 oligomer groups of various concentration gradients (2.5 μM, 5 μM, 10 μM, 20 μM). Aβ42 oligomer solution of corresponding concentration was added to each concentration gradient of Aβ42 oligomer group, prepared with serum-free DMEM medium (100 μl per well), and 100 μL serum-free DMEM medium was added to the blank group and negative control group (control), respectively, and cultured in a 37°C incubator for 24 hours.

[0124] 4) Cytotoxicity experiment to determine the toxic concentration of Aβ42 oligomers required to construct AD cell model

[0125] After the culture in step 3), 10 μL of CCK-8 solution (New Saimei Biotechnology Co., Ltd.) was added to the culture solution and cultured for 2 hours. The culture was terminated and placed in an enzyme-linked immunosorbent assay instrument for 2 minutes in the dark. The absorbance at 450 nm was measured and the results were recorded to obtain the absorbance values ​​of the blank group, negative control group (control), and Aβ42 oligomer groups of each concentration gradient. The blank group was used as the reference for zeroing and the toxic effects of different concentrations of Aβ42 oligomers on BV2 cells were calculated to clarify the conditions required for the construction of the AD cell model.

[0126] Cell viability of different Aβ42 oligomer concentration gradient groups = (absorbance value of Aβ42 oligomer group - average absorbance value of blank group) / (average absorbance value of negative control group - average absorbance value of blank group)

[0127] Cell viability of negative control group (control) = (absorbance value of negative control group - average absorbance value of blank group) / (average absorbance value of negative control group - average absorbance value of blank group)

[0128] 5) Data analysis

[0129] All data are presented as mean ± SEM. One-way analysis of variance was used to detect differences among three or more groups, and Tukey's post hoc test was used for comparisons between two groups. The differences were considered significant when P < 0.05. GraphPad Prism 10 software was used to analyze data and plot graphs.

[0130] The results are as follows Figure 4 As shown in Figure A, with the increase of Aβ42 oligomer concentration, the activity of BV2 cells showed a trend of gradual decline. The results showed that 5μM Aβ42 oligomers treated BV2 cells for 24 hours can significantly produce cytotoxicity, and the viability of BV2 cells is significantly reduced, indicating that the AD cell model was successfully constructed. 5μM Aβ42 oligomers were used as the Aβ42 oligomer modeling concentration in subsequent experiments.

[0131] Example 4: Determination of the safe dose range of anidulafungin on BV2 cells

[0132] 1) Set the gradient concentration of anidulafungin

[0133] Anidulafungin was dissolved in DMSO to prepare a 50 mM storage solution, which was stored at -80°C; the storage solution was diluted with serum-free DMEM culture medium to a 100 μM intermediate solution, which was stored at -20°C; when used, the working solution was diluted with serum-free DMEM culture medium to prepare the following concentration gradient working solutions: 10 nM, 100 nM, 250 nM, 500 nM, 1 μM, 2.5 μM and 5 μM.

[0134] 2) Cultivation of BV2 cells BV2 cells were cultured according to the steps in Example 3, counted and inoculated into 96-well plates, 5000 cells per well, diluted with complete culture medium to the required cell amount, 100 μL per well, each column corresponding to a group (each group of 6 replicates), one of which was not added with cells but only with culture medium as a blank group (no cells), and cultured overnight at 37°C in a humidified environment of 5% CO2 and 95% air.

[0135] 3) Add gradient concentrations of anidulafungin to BV2 cells

[0136] The culture medium in the wells was discarded and the prepared anidulafungin working solution was added. Each group with anidulafungin solution was cultured in a 37°C incubator for 24 hours.

[0137] 4) Safety assessment

[0138] The cell activity was determined by CCK-8 method (the cytotoxicity test procedure is the same as above). Figure 4 As shown in Figure B, the results show that within the concentration range of 10nM to 2.5μM, anidulafungin has no obvious toxic effect on BV2 cells.

[0139] Example 5: Determination of the effect of anidulafungin in reducing the neurotoxicity of Aβ oligomers

[0140] 1) Anidulafungin was diluted with serum-free DMEM medium to obtain the following concentration gradient solutions: 0 nM, 500 nM, 1 μM and 2 μM.

[0141] 2) Separately, a 10 μM Aβ42 oligomer solution was prepared using serum-free DMEM medium.

[0142] 3) The above anidulafungin solution and the Aβ42 oligomer solution were mixed at a volume ratio of 1:1 to obtain mixed solutions with the following concentrations: 0 nM anidulafungin + 5 μM Aβ42, 250 nM anidulafungin + 5 μM Aβ42, 500 nM anidulafungin + 5 μM Aβ42, 1 μM anidulafungin + 5 μM Aβ42.

[0143] 4) Count the cultured BV2 cells and inoculate them into 96-well plates, 5000 cells per well, dilute with complete medium to the required cell amount, 100 μL per well, each column corresponds to a group (6 replicates per group), one column is not added with cells but only with medium as a blank group (no cells). Culture overnight at 37°C, 5% CO2 and 95% air in a humidified environment.

[0144] 5) The culture medium in the wells was discarded, and a mixed solution of anidulafungin solution and Aβ42 oligomers was added. The cytotoxicity experiment steps were the same as above.

[0145] The results are as follows Figure 4 As shown in Figure C, the results show that with the increase of anidulafungin concentration, the activity of BV2 cells showed an upward trend. The cell activity of 5μM Aβ oligomers was significantly reduced to 68.66%±2.54% (P<0.0001); while in the groups with 250nM, 500nM and 1μM anidulafungin, the cell activity recovered to 70.13%±2.85%, 74.50%±3.88%, and 93.30%±4.41%, respectively (P<0.01). This result reveals that 1μM anidulafungin can significantly reduce the toxic effects of 5μM Aβ oligomers on BV2 cells, thereby improving the survival rate of cells.

[0146] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of a compound in the preparation of a drug for treating and / or preventing Alzheimer's disease, wherein the compound has a structure as shown in the following formula I: 。 2. Use of a pharmaceutical composition comprising a compound having a structure as shown in Formula I below in the preparation of a drug for treating and / or preventing Alzheimer's disease: 。 3. The use according to claim 1 or 2, characterized in that The compounds reduce Aβ oligomer neurotoxicity.

4. The use according to claim 1 or 2, characterized in that The treatment or prevention also includes monoclonal antibody therapy.

5. The use according to claim 2, characterized in that The pharmaceutical composition further comprises one or more of a cholinesterase inhibitor, an NMDA receptor antagonist and an Aβ monoclonal antibody.

6. The use according to claim 1 or 2, wherein the drug is administered by one or more of the following methods: oral administration, injection, implantation, spraying and / or inhalation.

7. The use according to claim 6, characterized in that The drug is administered parenterally, subcutaneously, intraperitoneally, intravenously, intralesionally, intracerebrally, intraarterially by interstitial infusion and / or by implantation of a delivery device.

8. The use according to claim 6, characterized in that The dosage form of the drug is one or more selected from the following: injection, oral solution, powder, tablet, granule, capsule, syrup, decoction, sustained-release preparation, enteric solvent, aerosol or suspension.

9. The use according to claim 1 or 2, characterized in that The medicament comprises a preventive and / or therapeutically effective amount of the compound, and a pharmaceutically acceptable carrier and / or excipient.

10. The use according to claim 1 or 2, characterized in that The concentration of the compound in the drug is 0.25~10 μM.

11. The use according to claim 10, characterized in that The concentration of the compound in the drug is 0.5~1 μM.

12. The use according to claim 11, characterized in that The concentration of the compound in the drug was 1 μM.

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