New use of inhibitor of f-box protein 48 and drug for treating ischemic cerebrovascular disease

Abstract

The application discloses a new use of an inhibitor of F-box protein 48 and a drug for treating ischemic cerebrovascular diseases, relates to the field of biological medicines, and discloses that F-box protein 48 has a key role in neuron damage in ischemic cerebral stroke, and the inhibitor of Fbxo48 can prevent or treat ischemic cerebral stroke, and the application provides a new use of the inhibitor of F-box protein 48, and provides a new target and strategy for the treatment of ischemic cerebral stroke.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and more specifically, to novel uses of inhibitors of F-box protein 48 and drugs for treating ischemic cerebrovascular diseases. Background Technology

[0002] Vascular cognitive impairment is a clinical syndrome of stroke or subclinical vascular brain injury caused by cerebrovascular lesions and their risk factors. It involves damage to at least one cognitive domain and ranges from mild cognitive impairment to dementia, including varying degrees of cognitive impairment caused by mixed pathologies such as Alzheimer's disease. Stroke, also known as apoplexy, is a common neurological disease, often caused by sudden rupture or blockage of blood vessels in the brain, preventing blood flow and resulting in brain tissue damage. It is characterized by high incidence, high mortality, and high disability rates. In recent years, due to increased life and work pressures, the incidence of stroke has been rising annually and is trending towards younger ages. Stroke includes ischemic stroke and hemorrhagic stroke, with ischemic stroke having a higher incidence, accounting for approximately 75% to 90% of all strokes. Ischemic stroke usually occurs at rest and manifests as slurred speech, unclear pronunciation, facial drooping, difficulty swallowing, hemiplegia, and sensory disturbances. In severe cases, it can lead to urinary and fecal incontinence. Hemorrhagic stroke usually occurs during activity and manifests as headache, nausea, projectile vomiting, hemiplegia, and sensory disturbances. In severe cases, it can lead to altered consciousness and coma.

[0003] Currently, the exact etiology and pathological process of this disease remain unclear. The brain injury mechanism caused by ischemic stroke is complex, and it is currently believed to be related to a cascade of neurological damage, mainly including glutamate excitatory poisoning, oxidative stress damage, and immune inflammatory damage. However, because there is no unified explanation for the mechanism of ischemic stroke, the direction for radical cure of stroke is unclear, and existing drug treatments have problems such as poor treatment effects and poor prognosis. There is a lack of drugs that can treat or improve stroke. At the same time, stroke is an important cause of vascular cognitive impairment, so the intervention and treatment of stroke is also an important issue in addressing vascular cognitive impairment.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The purpose of this invention is to provide new uses for inhibitors of F-box protein 48 and drugs for treating ischemic cerebrovascular diseases.

[0006] This invention is implemented as follows:

[0007] In a first aspect, embodiments of the present invention provide the use of FBox protein 48 inhibitors in the preparation of medicaments for the prevention, treatment or adjuvant treatment of stroke.

[0008] Secondly, embodiments of the present invention provide a method for screening drugs for the prevention, treatment or adjunctive treatment of stroke, comprising: using FBox protein 48 inhibitors as candidate drugs to screen for drugs that can prevent, treat or adjunctive treat stroke.

[0009] Thirdly, embodiments of the present invention provide a drug for the prevention, treatment or adjunctive treatment of stroke, the active ingredients of which include: FBox protein 48 inhibitors and other drugs for the prevention or treatment of stroke.

[0010] The present invention has the following beneficial effects:

[0011] This invention has discovered that F-Box protein 48 (Fbxo48) plays a key role in neuronal damage in ischemic stroke, and that inhibitors of Fbxo48 can prevent or treat ischemic stroke. This invention provides a new use for F-Box protein 48 inhibitors, offering new targets and strategies for the treatment of ischemic stroke. Attached Figure Description

[0012] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0013] Figure 1 The effect of adding or not adding BC1618 on nerve cells in a glucose-oxygen deprivation-reperfusion model (HT22 cell model);

[0014] Figure 2 The effect of adding or not adding BC1618 on neurons in a mouse MCAO model; where A is the result of TTC staining, with white representing the infarct area; B is the statistics of the infarct area; C is the neurological score after 0 hr and 24 hr of cerebral ischemia-reperfusion.

[0015] Figure 3 For histochemical staining; tissue sections and Nissler staining were performed on mouse brain tissue after cerebral ischemia-reperfusion to mark the morphology of neurons. Detailed Implementation

[0016] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0017] In a first aspect, embodiments of the present invention provide the use of FBox protein 48 inhibitors in the preparation of medicaments for the prevention, treatment or adjuvant treatment of stroke.

[0018] F-Box protein 48, also known as FBXO48 or Fbxo48, participates in SCF-dependent proteasome ubiquitin-dependent protein catabolism. It is part of the SCF ubiquitin ligase complex.

[0019] In some embodiments, the stroke includes ischemic stroke.

[0020] In some embodiments, the prevention, treatment or adjunctive treatment of stroke includes: prevention, treatment or adjunctive treatment of neuronal cell damage in ischemic stroke.

[0021] In some embodiments, the prevention, treatment or adjunctive treatment of stroke includes: prevention, treatment or adjunctive treatment of cerebral infarction in ischemic stroke.

[0022] In some embodiments, the prevention, treatment, or adjunctive treatment of ischemic stroke includes inhibiting the infarction of ischemic stroke or reducing the infarct volume of ischemic stroke.

[0023] Validated results show that FBXO48 inhibitors can significantly improve neurological scores and reduce infarct volume after ischemia-reperfusion, indicating that FBXO48 is a key protein in MCAO-induced ischemic brain injury.

[0024] In some embodiments, the FBox protein 48 inhibitor includes: a reagent that inhibits the activity and / or expression level of FBox protein 48.

[0025] The FBox protein 48 inhibitor can inhibit the activity of FBox protein 48, causing it to lose its normal biological function; it can also inhibit the expression of the FBox protein 48 gene, such as inhibiting transcription and / or inhibiting translation. As long as FBox protein 48 is the target, it falls within the scope of protection of this application.

[0026] In some embodiments, the expression level includes protein expression level and / or gene expression level. Reagents that inhibit expression levels may include gene editing reagents.

[0027] In some embodiments, the FBox protein 48 inhibitor includes BC1618.

[0028] It should be noted that the use of Fbxo48 inhibitors other than BC1618 for the treatment of ischemic stroke is also within the scope of protection of this invention.

[0029] On the other hand, embodiments of the present invention provide a method for screening drugs for the prevention, treatment or adjunctive treatment of stroke, which includes: using FBox protein 48 as a target, using FBox protein 48 inhibitors as candidate drugs, and screening for drugs that can prevent, treat or adjunctive treat stroke.

[0030] In some embodiments, the FBox protein 48 inhibitor includes: a reagent that inhibits the activity and / or expression level of FBox protein 48.

[0031] In some embodiments, the expression level includes protein expression level and / or gene expression level. Reagents that inhibit expression levels may include gene editing reagents.

[0032] In some embodiments, the FBox protein 48 inhibitor includes BC1618.

[0033] BC1618 is an orally active Fbxo48 inhibitor that stimulates Ampk signaling (preventing the degradation of activated pAmpkα by the Fbxo48-mediated proteasome). BC1618 promotes mitochondrial division, autophagy, and increases hepatic insulin sensitivity.

[0034] In addition, embodiments of the present invention also provide a drug for the prevention, treatment or adjunctive treatment of stroke, the active ingredients of which include: FBox protein 48 inhibitors and other drugs for the prevention or treatment of stroke.

[0035] The terms "medicine" and "medicinal composition" used herein are interchangeable. The medicines described include, in addition to the active ingredients of this invention, pharmaceutically acceptable carriers.

[0036] Optionally, the carrier includes, but is not limited to: diluents, buffers, suspensions, emulsions, granules, encapsulating agents, excipients, fillers, binders, sprays, transdermal absorbents, humectants, disintegrants, absorption promoters, surfactants, colorants, flavoring agents, and adsorbent carriers.

[0037] In this article, “treatment” includes preventing or alleviating a condition, slowing the onset or development of a condition, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, reducing or stopping symptoms associated with a condition, producing a complete or partial reversal of a condition, curing a condition, or a combination of the above.

[0038] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0039] Example 1

[0040] In vitro, the HT22 neuronal cell line was used to simulate the pathological process of ischemic stroke through a glucose-oxygen deprivation-reperfusion experiment to explore the protective effect of FBXO48 inhibition against neuronal damage during this process.

[0041] Experimental procedure:

[0042] Cells were seeded at a 50% density in 96-well plates. After 24 hours of seeding, the medium was replaced with sugar-free medium, and the plates were placed in a tri-gas incubator under 5% CO2 and 95% nitrogen for 2 hours to deprive the cells of glucose and oxygen. Then, the plates were replaced with normal medium and cultured under normal conditions for 18 hours, which constitutes reperfusion. Cell viability was assessed using the MTT assay after reperfusion.

[0043] Both the experimental and control groups underwent pretreatment. In the experimental group, BC1618 was added to pre-warmed medium to a final concentration of 2 μM. The control group received an equal volume of DMSO. Four hours before switching to the sugar-free medium, the pre-prepared experimental and control media were replaced, and the treatment lasted for 4 hours. After glucose-oxygen deprivation, the culture was reperfused with the pre-prepared experimental and control media for 18 hours.

[0044] Experimental results:

[0045] The results showed that the use of BC1618 significantly reduced cell death in the HT22 neuronal cell line after glucose-oxygen deprivation-reperfusion. Figure 1 ).

[0046] Example 2

[0047] To verify the key role of FBXO48 in neuronal damage in ischemic stroke.

[0048] Experimental methods:

[0049] In this embodiment, the FBXO48 inhibitor BC1618 was administered intranasally to inhibit FBXO48 activity in mouse brain tissue, while the control group underwent solvent control treatment. The specific steps are as follows:

[0050] 5 mg of BC1618 powder was dissolved in 60 μL of DMSO, followed by the addition of 100 μL of Tween-80, 200 μL of PEG-400, and 640 μL of physiological saline, resulting in a final BC1618 concentration of 5 mg / mL. Each mouse was administered 20 μL of the solution daily. The control group received the same amount of solvent. After three consecutive days of administration, these rats underwent MCAO surgery to artificially occlude the middle cerebral artery. The occlusion was performed for 60 minutes, followed by deembolization and reperfusion for 18 hours. Neurological scoring was performed, and then intact brain tissue was surgically harvested, flash-frozen at -80°C for 4 minutes, and then evenly sliced ​​into five sections, which were stained in preheated TTC staining solution. Five replicates were performed.

[0051] Experimental results:

[0052] The results showed that mice with inhibited FBXO48 activity were significantly protected against MCAO-induced functional impairment. Figure 2 (A), cerebral infarction volume measured by TTC staining ( Figure 2 (A, B) and neurological function scores ( Figure 2 (C) shows MCAO-induced functional impairment. These results demonstrate the crucial role of FBXO48 in neuronal death in ischemic stroke.

[0053] Example 3

[0054] We verified that FBXO48 inhibitors can rescue neuronal damage in mice after cerebral ischemia-reperfusion.

[0055] Experimental methods:

[0056] Paraffin sections of the mouse brain tissue mentioned in Example 2 were prepared and Nissler staining was performed to determine the state of neuronal cells. The results are shown in [Figure 1]. Figure 3 .

[0057] Experimental results:

[0058] The results showed that neurons in mice after cerebral ischemia-reperfusion injury were significantly shrunken and morphologically abnormal, demonstrating severe neuronal damage following the procedure. The use of BC1618 could alleviate this neuronal abnormality and salvage neuronal damage in mice after cerebral ischemia-reperfusion injury. Figure 3 ).

[0059] The above data fully demonstrate that FBXO48 is a key protein in MCAO-induced ischemic brain injury and a therapeutic target for ischemic stroke.

[0060] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The use of an FBox protein 48 inhibitor in the preparation of a drug for the prevention, treatment or adjuvant treatment of stroke, wherein the FBox protein 48 inhibitor is BC1618.

2. The application according to claim 1, characterized in that, The stroke includes ischemic stroke.

3. The application according to claim 1, characterized in that, The prevention, treatment, or adjunctive treatment of stroke includes: prevention, treatment, or adjunctive treatment of neuronal cell damage in ischemic stroke.

4. The application according to claim 1, characterized in that, The prevention, treatment or adjunctive treatment of stroke includes: prevention, treatment or adjunctive treatment of cerebral infarction in ischemic stroke.

5. The application according to claim 1, characterized in that, The prevention, treatment, or adjunctive treatment of ischemic stroke includes inhibiting or reducing the volume of ischemic stroke infarction.

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