Related application of target compound in treating alzheimer disease

Abstract

The invention discloses related application of a target compound in treatment of Alzheimer's disease, and relates to the field of biological medicine. In order to solve the problem that an existing autophagy inducer depending on an mTOR signal channel may cause side effects such as immunosuppression or metabolic disorder, the invention provides application of at least one small molecule compound of phytolaccin, 2-hydroxycinnamic acid, clevidipine butyrate or 3 ', 4', 7-trimethoxy quercetin as the autophagy inducer. The compound does not depend on an mTOR signal channel, has the blood-brain barrier penetrating capacity and can effectively induce autophagy. In caenorhabditis elegans and mouse Alzheimer's disease models, the compound shows a remarkable neuroprotective effect, accumulation of toxic protein aggregates such as beta-amyloid protein and over-phosphorylated tau in the brain can be reduced, cognitive impairment and related neuropathological characteristics are improved, and the compound can be used for preventing and treating neurological diseases. A new strategy is provided for treatment and / or prevention of Alzheimer's disease.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the application of a target compound in the treatment of Alzheimer's disease. Background Art

[0002] Neurodegenerative diseases are a complex class of disorders characterized by the progressive loss of neurons in the central nervous system. Alzheimer's disease (AD), the most common form, has become a major global public health challenge. With the increasing trend of population aging, the prevalence of AD continues to rise, placing a heavy economic and care burden on families and society. Currently, the main pathological hallmarks of AD include abnormal deposition of β-amyloid protein (Aβ) in the brain, hyperphosphorylation of tau protein, and the resulting neurofibrillary tangles. These pathological changes not only reflect the impairment of neuronal function but also indicate a critical window for early intervention.

[0003] In terms of treatment strategies, currently clinically approved drugs for the treatment of AD primarily include cholinesterase inhibitors and N-methyl-D-aspartate (NMDA) receptor antagonists, whose mechanisms of action primarily focus on improving cognitive function or alleviating neurotoxicity. However, these drugs can only alleviate symptoms to a certain extent and are not yet able to effectively delay or reverse the disease progression. Therefore, the development of new disease-modifying treatments has become an urgent need in this field.

[0004] In recent years, autophagy, a highly conserved protein degradation and quality control system within cells, has been recognized as a key mechanism for maintaining neuronal homeostasis and clearing abnormal protein aggregates. Studies have confirmed that autophagy dysfunction is closely associated with the pathological progression of neurodegenerative diseases such as AD. Activating the autophagy pathway can effectively promote the clearance of Aβ and hyperphosphorylated tau protein, improve neuronal function, and slow disease progression, suggesting that autophagy-enhancing strategies have potential application in the treatment of AD.

[0005] Currently, most known small-molecule autophagy inducers, such as rapamycin and its derivatives, achieve their effects by inhibiting the mammalian target of rapamycin (mTOR) signaling pathway. These compounds have demonstrated efficacy in various tumor models and the treatment of autoimmune diseases. However, associated adverse effects, such as immunosuppression, metabolic disturbances, and nephrotoxicity, significantly limit their long-term application in central nervous system diseases. Furthermore, due to the presence of the blood-brain barrier, most mTOR inhibitors have difficulty effectively entering the brain, further diminishing their feasibility in the treatment of AD.

[0006] Therefore, mTOR-independent autophagy enhancers have attracted widespread attention due to their potential to circumvent these limitations. However, the number of such compounds is currently limited, and there is a lack of systematic in vitro and in vivo efficacy evaluation and safety data. Furthermore, most candidate molecules still have significant deficiencies in structural diversity, mechanism of action clarity, and brain targeting capabilities. Therefore, the development of a class of mTOR-independent autophagy enhancers with novel chemical structures, clear mechanisms of action, the ability to effectively cross the blood-brain barrier, and good safety and pharmacokinetic properties is of great significance for promoting the development of AD therapeutics.

[0007] In view of this, the present invention is proposed. Summary of the Invention

[0008] In response to the technical defects of existing mTOR-dependent autophagy inducers, such as systemic toxic side effects and low blood-brain barrier penetration, the present invention provides a class of mTOR-independent small molecule compounds for use in the treatment and / or prevention of Alzheimer's disease, aiming to circumvent the above defects and achieve effective neuroprotection.

[0009] The present invention is achieved in that: An embodiment of the present invention provides use of a target compound in preparing a product for treating or preventing Alzheimer's disease, wherein the target compound comprises at least one of phytolanthin, 2-hydroxycinnamic acid, clevidipine butyrate, and 3',4',7-trimethoxyquercetin.

[0010] The present invention has the following beneficial effects: (1) Mechanistic innovation: The target compound induces autophagy through an mTOR-independent pathway, avoiding the risks of immunosuppression and metabolic disorders caused by mTOR inhibitors; (2) Delivery effectiveness: The target compound has the ability to penetrate the blood-brain barrier, significantly increasing drug exposure in the central nervous system; (3) Therapeutic Evidence: It has been demonstrated in Alzheimer's disease models (Caenorhabditis elegans and mice) that the target compound can enhance autophagy activity, reduce the accumulation of Aβ plaques and hyperphosphorylated tau protein in the brain, and improve cognitive dysfunction, providing a new approach for the effective treatment or prevention of AD patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0012] Figure 1The autophagy-inducing effect and mechanism verification of the target compound in the cell model; A is the LC3B-II and SQSTM1 / p62 protein levels in N2a cells after treatment with the target compound (WB assay); BC is the statistical analysis of the results of A; D is the GFP / RFP punctate aggregation in PC12 mRFP-EGFP-LC3B cells (indicating autophagic flux); E is the effect of the target compound on mTOR pathway proteins and their phosphorylation levels in N2a cells (WB assay); F is the DQ-Red BSA uptake and fluorescence release in PC12 cells (indicating lysosomal activity); G is the statistical analysis of the results of F; Figure 2 The target compound is shown to improve tau and Aβ pathology in AD cell models; A is the tau phosphorylation site and autophagy marker content in PC12 TauP301L cells (WB detection); BG is the statistical analysis of the results of A; H is the APP cleavage product and autophagy marker content in PC12APP KM595 / 596NL and V642F cells (WB detection); IM is the statistical analysis of the results of H; Figure 3 is the neuroprotective effect of the target compound in the nematode AD model; AG is the chemotaxis index, lifespan, and developmental phenotype of the hTau[P301L] strain of Caenorhabditis elegans treated with phytoalexin and 2-hydroxycinnamic acid; Figure 4 represents the cognitive improvement effect of the target compound in the 3×Tg-AD mouse model; AF represents the behavioral parameter of the Morris water maze after the mice were treated with phytosan and 2-hydroxycinnamic acid; GH represents the spontaneous alternation rate in the Y maze; IJ represents the memory index of the novel object recognition test; Figure 5 Verification of the target compound's pathological improvement and blood-brain barrier penetration ability in the 3×Tg-AD mouse model; AB is the immunohistochemistry of phosphorylated tau (p-Thr217) and Aβ (4G8) in the CA1 region of the mouse hippocampus; CE is the content of tau / APP pathway proteins and autophagy markers in hippocampal tissue (WB detection); F is the concentration of phytosalicylic acid and 2-hydroxycinnamic acid in the brain tissue of C57BL / 6 mice (HPLC-MS / MS detection, indicating BBB penetration ability). DETAILED DESCRIPTION

[0013] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0014] Definition of noun "Ombuin" in this article refers to the compound 3,5,3'-trihydroxy-7,4'-dimethoxyflavone (English name: Ombuin, abbreviated as Omb, CAS number: 529-40-8).

[0015] "2-Hydroxycinnamic acid" in this article refers to (E)-3-(2-hydroxyphenyl) acrylic acid (English name: 2-Hydroxycinnamic acid, abbreviated as 2-HCA, CAS number: 614-60-8).

[0016] "Clevidipine butyrate" in this article refers to 4-(2,3-dichlorophenyl)-1,4-dihydro-2,6-dimethyl-3,5-pyridinedicarboxylic acid methyl (1-butyryloxy)methyl ester (English name: Clevidipine, CAS number: 167221-71-8).

[0017] The "3',4',7-trimethoxyquercetin" in this article refers to 2-(3,4-dimethoxyphenyl)-3,5-dihydroxy-7-methoxy-4H-1-benzopyran-4-one (English name: 3',4',7-Trimethoxyquercetin, CAS number: 6068-80-0).

[0018] The term "prevention," as used herein, refers to medical strategies that proactively intervene (such as medication) to prevent the onset, progression, or worsening of a disease or health condition before it occurs or at an early stage. The core goal of prevention is to reduce the likelihood of disease development, delay its onset, and / or mitigate its negative impact on an individual's health.

[0019] The term "treatment" as used herein has a broad medical meaning, including but not limited to one or more of the following aspects: preventing the occurrence of a certain disease condition or reducing its severity; reducing the rate of occurrence or progression of the disease condition; reducing the risk of developing the disease condition; preventing or delaying the onset of clinical symptoms associated with the disease condition; alleviating or terminating the symptoms associated with the disease condition; achieving complete or partial reversal of the disease condition; achieving a cure for the disease condition; and any combination of the above-mentioned treatment methods.

[0020] Technical Solution On the one hand, an embodiment of the present invention provides a use of a target compound in preparing a product for treating or preventing Alzheimer's disease, wherein the target compound includes at least one of phytolane, 2-hydroxycinnamic acid, clevidipine butyrate and 3',4',7-trimethoxyquercetin.

[0021] In some embodiments, the target compound includes at least one of phytolanthus and 2-hydroxycinnamic acid.

[0022] In some embodiments, the target compound further comprises: at least one of clevidipine butyrate and 3',4',7-trimethoxyquercetin. In some embodiments, the treatment of Alzheimer's disease comprises one or more of the following: (1) delaying the pathological progression of Alzheimer's disease; (2) ameliorating symptoms caused by Alzheimer's disease.

[0023] In some embodiments, the improving symptoms caused by Alzheimer's disease includes improving any one or more of: cognitive dysfunction, decreased independent exploratory behavior, decreased learning ability, and memory loss.

[0024] In some embodiments, the product comprises a pharmaceutical or a food.

[0025] In some embodiments, the product is used to reduce the accumulation of beta-amyloid and / or hyperphosphorylated tau in the brain.

[0026] In some embodiments, the product exerts neuroprotective effects by enhancing autophagy.

[0027] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0028] Example 1: Verification of the Effect of Phytolansin, 2-Hydroxycinnamic Acid, Clevidipine Butyrate, and 3',4',7-Trimethoxyquercetin on Autophagy Enhancement via an mTOR-Independent Pathway In the N2a cell line, phytolane, 2-hydroxycinnamic acid, clevidipine butyrate, and 3',4',7-trimethoxyquercetin had little effect on LC3-II levels, but significantly reduced SQSTM1 / p62 protein levels ( Figure 1 AC in Figure 1). Changes in the number of autophagosomes can reflect the state of autophagic flux, and their increase may be due to increased synthesis or blocked degradation. To observe the dynamics of autophagosomes under drug stimulation, this example uses a PC12 cell line expressing a dual fluorescent protein tandem marker LC3 (mRFP-EGFP-LC3B) ( Figure 1D in Figure 3). In the control group (CTRL), unstimulated by drugs, only a small number of red fluorescent protein (RFP) punctate aggregates were observed, representing basal autophagy levels. Treatment with the autophagy enhancer Torin1 upregulated autophagic flux, with autophagosomes fusing with lysosomes to form autolysosomes and substrate degradation. Compared to RFP, which is less susceptible to quenching and degradation, green fluorescent protein (GFP) is quenched and degraded in autolysosomes, resulting in more RFP puncta. Treatment with the autophagy inhibitor Bafilomycin A1, however, resulted in numerous GFP and RFP puncta. This is because Bafilomycin A1, by increasing lysosomal pH, disrupts lysosomal degradation, hindering quenching and degradation of GFP and RFP. Consequently, numerous yellow puncta were observed (i.e., overlapping GFP and RFP signals). Treatment with the four compounds described above all showed similar results to those observed with Torin1 stimulation, demonstrating their autophagy-enhancing effects. Further investigation was conducted to examine the effects of these drugs on the mTOR (mammalian target of rapamycin) signaling pathway. The classic mTOR inhibitor Torin1 can significantly reduce the protein levels of phospho-mTOR (Ser2448), phospho-p70 S6 Kinase (Ser371), and phospho-4E-BP1 (Thr37 / 46). However, after treatment with the four compounds, the phosphorylation levels of the above proteins did not decrease significantly, suggesting that they do not enhance autophagy by inhibiting the mTOR pathway ( Figure 1 E in).

[0029] DQ-Red BSA detection is an effective method for quantitatively evaluating autophagy-related substrate turnover and lysosomal proteolytic activity. After being endocytosed by the cell, DQ-Red BSA is transported through early and late endosomes and finally delivered to the autophagosome and fused with the lysosome. In the acidic environment of the lysosome, it is cleaved by active hydrolases to release red fluorescent fragments. Autophagy inducers (such as Torin1) can accelerate the degradation process of DQ-Red BSA and promote the release of red fluorescence, indicating that the autophagic flux is enhanced. In contrast, autophagy inhibitors (such as Bafilomycin A1) inhibit autophagy by disrupting lysosomal acidification, resulting in incomplete degradation of DQ-Red BSA and blocked release of red fluorescence. Experimental observations show that the red fluorescence intensity was significantly enhanced in cells treated with smilax glabra, 2-hydroxycinnamic acid, clevidipine butyrate and 3',4',7-trimethoxyquercetin ( Figure 1 This indicates that the above compounds can effectively enhance autophagic flux, thereby promoting the degradation of substrates in autolysosomes.

[0030] Example 2: Phytolaccarin, 2-hydroxycinnamic acid, clevidipine butyrate, and 3',4',7-trimethoxyquercetin can alleviate pathological changes in Alzheimer's disease-related proteins Tau is a microtubule-associated protein that is crucial for maintaining neuronal stability and function. Mutations in the gene encoding tau lead to hyperphosphorylation. Hyperphosphorylated tau loses its ability to bind to microtubules, leading to the formation of neurofibrillary tangles (NFTs). Furthermore, mutations in the amyloid precursor protein (APP) gene can trigger aberrant cleavage, leading to the overproduction and aggregation of β-amyloid (Aβ) peptides into amyloid plaques. These genetic mutations collectively contribute to synaptic dysfunction, neuronal death, and the progressive cognitive decline of Alzheimer's disease.

[0031] In PC12 cells overexpressing mutant Tau and APP, phytosan, 2-hydroxycinnamic acid, clevidipine butyrate, and 3',4',7-trimethoxyquercetin significantly reduced the hyperphosphorylation level of Tau protein at key phosphorylation sites (Thr181, Ser202, Thr205, Thr231). Figure 2 At the same time, these compounds also significantly reduced the protein levels of APP cleavage products CTF-β and CTF-α ( Figure 2 Further testing showed that after treatment with the four compounds, LC3-II levels were slightly increased, while SQSTM1 / p62 protein levels were significantly decreased ( Figure 2 Among them, phytolanthin and 2-hydroxycinnamic acid were particularly effective.

[0032] Example 3: Phytolaccarin and 2-hydroxycinnamic acid can improve cognitive function and prolong lifespan in Alzheimer's disease model nematodes without developmental toxicity Caenorhabditis elegans ( Caenorhabditis elegans ) has a simple structure, a clear genetic background and a short life cycle, and is a commonly used model for evaluating candidate drugs for Alzheimer's disease. In the chemotaxis experiment, hunger serves as an aversive stimulus, and isopentanol (IA) serves as a "no food" cue. When wild-type nematodes are pre-exposed to IA and encounter IA again, they show obvious learned avoidance; while transgenic nematodes hTau[P301L] (overexpressing human Tau protein carrying the P301L mutation) show significant learning and memory defects: even if they are pre-exposed to IA, they cannot form effective avoidance behavior, simulating the cognitive impairment of neurodegenerative diseases. After administration of safflower oil and 2-hydroxycinnamic acid, the chemotaxis index of hTau[P301L] nematodes was significantly improved, indicating that learning and memory abilities were restored ( Figure 3 A in ).

[0033] Compared with wild-type nematodes, hTau[P301L] nematodes had a significantly shortened median lifespan, reflecting the systemic damage caused by pathological Tau. This shortened lifespan was significantly reversed after intervention with phytosolic acid and 2-hydroxycinnamic acid. Figure 3BD in the worm), suggesting that it has neuroprotective potential and can antagonize the negative effects associated with Tau protein disease. In addition, testing of key developmental nodes such as egg hatching, L4 larval transition and adult emergence showed that neither compound produced observable toxicity ( Figure 3 EG in).

[0034] Example 4: Phytolaccarin and 2-hydroxycinnamic acid significantly alleviate cognitive impairment in 3×Tg-AD mice 3×Tg-AD mice have become a classic model for studying Alzheimer's disease (AD) because they simultaneously overexpress mutant APP, Tau, and presenilin-1. This example systematically evaluated the protective effects of safflower oil and 2-hydroxycinnamic acid on the neurological function of this model through Morris water maze, Y maze, and novel object recognition experiments. The results of the Morris water maze showed that during 5 consecutive days of training, the latency of wild-type mice to find the hidden platform decreased day by day, while 3×Tg-AD mice showed no significant improvement, suggesting that their spatial learning ability was impaired. After intervention with safflower oil and 2-hydroxycinnamic acid, the exploratory behavior of 3×Tg-AD mice after the platform was removed was significantly enhanced: the number of times they crossed the original platform area, the time they stayed in the target quadrant, and the distance they traveled were all significantly increased ( Figure 4 AF in ), indicating that the two compounds can effectively restore spatial memory in Alzheimer's disease models.

[0035] To further verify its effect on short-term memory, this example conducted a Y-maze and novel object recognition test. Compared with wild-type mice, 3×Tg-AD mice performed poorly in both experiments, with obvious cognitive deficits. After drug intervention, the spontaneous alternation rate of 3×Tg-AD mice in the Y-maze was significantly increased ( Figure 4 GH in the brain), suggesting an improvement in working memory; the new object recognition experiment also showed that the two compounds could significantly enhance the mice's preference for exploring new objects ( Figure 4 In conclusion, phytosan and 2-hydroxycinnamic acid can improve cognitive dysfunction in 3×Tg-AD mice at multiple levels.

[0036] Example 5: Phytolaccarin and 2-hydroxycinnamic acid can clear abnormal protein aggregates and activate autophagy in the brain tissue of Alzheimer's disease model mice After the behavioral experiment, tissue from the hippocampal CA1 region of 3×Tg-AD mice was frozen and sectioned. Immunofluorescence results showed that after intervention with phytosan and 2-hydroxycinnamic acid, the amount of phosphorylated Tau protein and Aβ deposition in this region was significantly reduced ( Figure 5 AB in ). Immunoblotting further confirmed that the protein levels of total Tau and its major phosphorylated isoforms, as well as APP cleavage products CTF-β and CTF-α, were significantly decreased ( Figure 5Critically, drug treatment increased the LC3-II / LC3-I ratio and decreased the SQSTM1 / p62 level ( Figure 5 E), indicating that the autophagy pathway was effectively activated.

[0037] Example 6: Phytolaccarin and 2-hydroxycinnamic acid can penetrate the blood-brain barrier of mice.

[0038] The blood-brain barrier is a highly selective "moat" that not only shields peripheral harmful substances but also blocks most potential central nervous system drugs. Therefore, whether it can successfully cross this barrier has become a key indicator for evaluating the drugability of neuroprotective agents. In this example, C57BL / 6 mice were used as a model to systematically investigate the brain penetration ability of phytosine and 2-hydroxycinnamic acid. After intraperitoneal administration of the two compounds, brain tissue was collected for mass spectrometry quantification 1 hour and 3 hours after full perfusion. The results showed that the target drug ( Figure 5 This finding suggests that phytosolic acid and 2-hydroxycinnamic acid can cross the blood-brain barrier, activate autophagy, clear abnormal protein aggregates, and ultimately reshape cognitive functions such as learning and memory, exerting significant neuroprotective effects.

[0039] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. Use of a target compound in preparing a product for treating or preventing Alzheimer's disease, characterized in that: The target compound includes at least one of phytolanthin, 2-hydroxycinnamic acid, clevidipine butyrate and 3',4',7-trimethoxyquercetin.

2. The use according to claim 1, characterized in that The target compound includes at least one of phytolanthin and 2-hydroxycinnamic acid.

3. The use according to claim 1, characterized in that The treatment of Alzheimer's disease includes one or more of the following: (1) delaying the pathological progression of Alzheimer's disease; (2) improving the symptoms caused by Alzheimer's disease.

4. The use according to claim 3, characterized in that The improvement of symptoms caused by Alzheimer's disease includes improvement of any one or more of cognitive dysfunction, decreased independent exploratory behavior, decreased learning ability and memory loss.

5. The use according to any one of claims 1 to 4, characterized in that The product includes a medicine or a food.

6. The use according to any one of claims 1 to 4, characterized in that The product is used to reduce the accumulation of β-amyloid protein and / or hyperphosphorylated tau protein in the brain.

7. The use according to any one of claims 1 to 4, characterized in that The product exerts neuroprotective effects by enhancing autophagy.

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