A therapeutic agent for Alzheimer's disease and its application

Abstract

The present invention provides a therapeutic agent for Alzheimer's disease and its application, belonging to the field of neurobiology technology. Each 100 μL of the agent contains the following active ingredients: 0.25mg-0.35mg of epigallocatechin gallate, 1.8mg-2.0mg of citicoline, and 0.2mg-0.35mg of pterostilbene. The agent has a positive impact on Alzheimer's patients by regulating Aβ42 deposition, improving exploration ability, and alleviating anxiety. Therefore, the application of this agent has broad clinical prospects and can provide a new, synergistic therapeutic strategy for the treatment of Alzheimer's disease.

Description

Technical Field

[0001] The present invention belongs to the technical field of neurobiology, and in particular relates to a therapeutic agent for Alzheimer's disease and an application thereof. Background Art

[0002] Alzheimer's disease (AD) is a common neurodegenerative disorder characterized by progressive memory loss, cognitive impairment, behavioral changes, and a decline in the ability to function in daily life. With the increasing global aging population, the prevalence of AD has increased annually, becoming a major global public health issue. Currently, the main medications used clinically include cholinesterase inhibitors and NMDA receptor antagonists, which can improve patients' symptoms to a certain extent. However, due to their limited efficacy and significant side effects, long-term use is ineffective. Furthermore, treatments targeting pathological features such as amyloid deposition and tau protein abnormalities are still under clinical research, with no significant breakthroughs yet.

[0003] In recent years, researchers have increasingly focused on the potential of natural plant compounds and neuroprotective molecules in the treatment of Alzheimer's disease. These natural compounds, due to their antioxidant, anti-inflammatory, and neuroprotective properties, have been extensively studied for their potential in treating neurodegenerative diseases. Studies have shown that these compounds can improve Alzheimer's symptoms through multiple pathways, particularly their potential to clear amyloid plaques, provide neuroprotection, and exert anti-inflammatory effects, which have garnered widespread attention.

[0004] However, the therapeutic effect of a single ingredient is still limited, and long-term use may lead to drug resistance or side effects. Therefore, the development of compound drugs that combine multiple ingredients to improve the efficacy and safety of drugs has become an important direction in current Alzheimer's disease treatment research. Summary of the Invention

[0005] This invention provides a therapeutic agent for Alzheimer's disease and its use, aiming to overcome the limited efficacy of single drugs in existing technologies. This invention combines three active ingredients—epigallocatechin gallate (EGCG), citicoline, and pterostilbene—in a specific ratio to form a synergistic therapeutic agent, thereby more effectively improving symptoms in AD patients.

[0006] Specifically, the core contents of the present invention include:

[0007] First, the present invention provides a therapeutic agent for Alzheimer's disease, wherein each 100 μL of the agent comprises the following active ingredients:

[0008] The invention comprises 0.25mg-0.35mg of epigallocatechin gallate, 1.8mg-2.0mg of citicoline, 0.2mg-0.35mg of pterostilbene and a pharmaceutically acceptable solvent.

[0009] Preferably, the solvent consists of 5% ethanol and 95% saline.

[0010] Preferably, in a preferred embodiment, each 100 μL of the medicament comprises the following active ingredients:

[0011] The invention is composed of 0.25 mg of epigallocatechin gallate, 2.0 mg of citicoline and 0.25 mg of pterostilbene and a pharmaceutically acceptable solvent.

[0012] This specific combination of active ingredients has been experimentally verified to be able to show excellent effects in reducing Aβ42 concentration in the brain tissue of Alzheimer's disease model mice and improving behavioral indicators such as exploratory ability and anxiety.

[0013] Preferably, the Alzheimer's disease is a type of Alzheimer's disease characterized by abnormal deposition of Aβ42.

[0014] Secondly, the present invention not only provides the specific composition of the above-mentioned therapeutic agent, but also further clarifies its application value in multiple therapeutic fields.

[0015] In a first aspect, the present invention provides use of the therapeutic agent in the preparation of a drug for treating Alzheimer's disease.

[0016] Preferably, the drug is used to improve the exploration ability and anxiety of patients with Alzheimer's disease.

[0017] Preferably, the drug is used to improve abnormal deposition of Aβ42 in the brain of patients with Alzheimer's disease.

[0018] In a second aspect, the present invention provides the use of the therapeutic agent in the preparation of a therapeutic drug for improving the exploratory ability and anxiety of patients with Alzheimer's disease.

[0019] Alzheimer's patients often experience behavioral changes, such as decreased exploratory ability and increased anxiety. The present invention significantly improves these behavioral manifestations through the synergistic effect of three drugs.

[0020] In a third aspect, the present invention provides a use of the therapeutic agent in the preparation of a therapeutic drug for improving abnormal Aβ42 deposition in the brain of Alzheimer's patients.

[0021] Aβ42 is a key toxic protein in the pathology of Alzheimer's disease. Its abnormal deposition triggers the formation of amyloid plaques, leading to neuronal damage and cognitive decline. This agent effectively alleviates this core pathological feature by significantly reducing Aβ42 concentration.

[0022] The beneficial effects of the present invention are:

[0023] This invention combines the three active ingredients of EGCG, Citicoline, and Pterostilbene for the first time, leveraging their multi-pathway, multi-target mechanisms of action. EGCG primarily regulates the accumulation of Aβ42, Citicoline enhances acetylcholine synthesis, and Pterostilbene has antioxidant and anti-inflammatory properties. This invention demonstrates for the first time that the synergistic effect of these three ingredients combined is significantly greater than the therapeutic effects of either or both of the ingredients, and significantly greater than the expected combined effect, resulting in more effective relief of AD symptoms.

[0024] Experimental results show that the agent of the present invention can significantly reduce the concentration of Aβ42 in the brain tissue of AD model mice. Aβ42 is one of the core pathological features of Alzheimer's disease, and its abnormal deposition is one of the main causes of neurodegeneration. The combined agent can significantly reduce Aβ42 deposition, thereby alleviating pathological changes in the brains of AD mice.

[0025] Furthermore, the drug also demonstrated remarkable efficacy in improving the exploratory ability and anxiety of AD model mice. In an open field test, treated AD mice demonstrated significantly improved exploratory ability and mid-range locomotion time, demonstrating better behavioral performance than the model group, demonstrating the drug's effectiveness in improving anxiety and cognitive function in AD patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The results of the average middle zone movement distance of mice in different treatment groups are shown in the figure;

[0027] Figure 2 The results of the average middle zone movement time of mice in different treatment groups are shown in the figure;

[0028] Figure 3 The graph shows the results of Aβ42 concentration in brain tissue of mice in different treatment groups. DETAILED DESCRIPTION

[0029] Example 1: Alzheimer's disease therapeutic agent a of the present invention

[0030] Each 100 μL of the solution consists of the following ingredients:

[0031] Epigallocatechin gallate (EGCG, CAS No.: 989-51-5): 0.25 mg; Citicoline (Citicoline, CAS No.: 987-78-0): 2.0 mg; Pterostilbene (CAS No.: 537-42-8): 0.25 mg;

[0032] Solvents: 5% ethanol (v / v) and 95% saline (0.9% NaCl).

[0033] Preparation steps:

[0034] First, 5% ethanol (v / v) was thoroughly mixed with 95% saline (0.9% NaCl) to ensure the homogeneity of the solvent;

[0035] Add epigallocatechin gallate (EGCG), citicoline and pterostilbene to the prepared solvent and stir thoroughly on a stirrer to dissolve;

[0036] The solution was sterilized by filtration using a 0.22 μm filter membrane to obtain therapeutic agent a.

[0037] Example 2: Alzheimer's disease therapeutic agent b of the present invention

[0038] Each 100 μL of the solution consists of the following ingredients:

[0039] Epigallocatechin gallate (EGCG): 0.3 mg; Citicoline: 2.0 mg; Pterostilbene: 0.2 mg;

[0040] Solvents: 5% ethanol (v / v) and 95% saline (0.9% NaCl).

[0041] The preparation method is the same as that of Example 1.

[0042] Example 3: Alzheimer's disease therapeutic agent c of the present invention

[0043] Each 100 μL of the solution consists of the following ingredients:

[0044] Epigallocatechin gallate (EGCG): 0.35 mg; Citicoline: 1.8 mg; Pterostilbene: 0.35 mg;

[0045] Solvents: 5% ethanol (v / v) and 95% saline (0.9% NaCl).

[0046] The preparation method is the same as that of Example 1.

[0047] Example 4: Using the open field test to detect the effects of the Alzheimer's disease therapeutic agent of the present invention on the exploration ability and anxiety of AD model mice

[0048] (1) Six wild-type mice (6 months old) were used as a normal control group, and 42 APP / PS1 mice (6 months old) were randomly divided into 7 groups, namely, a model group, an EGCG group (the drug only contained EGCG), a citicoline group (the drug only contained citicoline), a pterostilbene group (the drug only contained pterostilbene), an EGCG+cterostilbene group (the drug contained EGCG and citicoline), an EGCG+pterostilbene group (the drug contained EGCG and pterostilbene), and a combination treatment agent group of the present invention (treatment agent a).

[0049] (2) After one week of adaptive feeding, each mouse was gavaged with 100 μL of the corresponding drug at 9:00 a.m. daily according to the above groups, and the normal control group was gavaged with the corresponding saline solution. The drug administration period was 8 weeks.

[0050] (3) The open field test was used to detect the exploratory ability and anxiety of mice. The open field box was 50 cm × 50 cm × 50 cm in size, and the bottom was evenly divided into 25 square grids of 10 cm × 10 cm.

[0051] (4) Place the treated mice in the center of the bottom of the open field box and observe for 5 minutes. Record the walking distance and time of the mice in the middle area of ​​the open field.

[0052] (5) After each experiment, the bottom of the open field was wiped with 75% ethanol to remove odors and prevent interference with the next mouse. Finally, the average middle area movement distance and average middle area movement time of all treatment groups of mice were calculated.

[0053] Table 1 Average middle zone movement distance of mice in different treatment groups

[0054]

[0055] As shown in Table 1, the average mid-range distance traveled by mice in the AD model group was significantly reduced, indicating a decrease in their exploratory ability. However, all treatment groups showed some improvement compared to the model group after drug treatment. This suggests that EGCG, Citicoline, Pterostilbene, or their combination can all be used to treat AD mice to some extent.

[0056] Using the expected effect formula of the combination of the two drugs: Eexp=EA+EB−EA⋅EB, it was calculated that the expected drug treatment effect of the EGCG+Citicoline group was 30.78%, which was close to the actual test results, indicating that the combination of EGCG and Citicoline did not have a synergistic effect on the treatment of the exploratory ability of AD mice.

[0057] The expected drug treatment effect of the EGCG + Pterostilbene group was 32.32%, which was also close to the actual test results, indicating that the combination of EGCG and Pterostilbene did not have a synergistic effect on the treatment of the exploratory ability of AD mice.

[0058] Through the expected effect formula of the combination of three drugs: Eexp=E1+E2+E3−(E1E2+E1E3+E2E3)+E1E2E3, it was calculated that the expected drug treatment effect of the combination treatment group was 42.49%. The actual test results were significantly higher than the expected drug treatment effect, indicating that when EGCG, Citicoline and Pterostilbene are used together to treat AD mice, the three drugs have a significant synergistic effect and can effectively treat the problem of decreased exploration ability of AD mice.

[0059] Table 2 Average middle zone movement time of mice in different treatment groups

[0060]

[0061] As shown in Table 2, compared to the control group, the model group mice significantly decreased their middle zone movement time, indicating increased anxiety in AD mice. Similar to the results for movement distance, all treatment groups showed some improvement compared to the model group after drug treatment, further demonstrating that EGCG, Citicoline, Pterostilbene, or their combination can treat AD mice to a certain extent.

[0062] Further analysis found that the expected drug treatment effect of the EGCG + Citicoline group was 36.58%, and the expected drug treatment effect of the EGCG + Pterostilbene group was 39.29%. The difference can be seen in the comparison. The actual test effect is basically the same as the expected effect, indicating that when EGCG is used in combination with Citicoline or Pterostilbene, it cannot produce a synergistic effect on the treatment of AD.

[0063] The expected therapeutic effect of the three drugs combined was 50.41%, and the actual therapeutic effect was significantly higher than the expected therapeutic effect, indicating that the combination of the three drugs can produce a significant synergistic effect, thereby more effectively reducing anxiety in AD mice. Moreover, compared with the therapeutic effect on exploratory ability, the drug's therapeutic effect on alleviating mice's anxiety was more significant.

[0064] Example 5: To further verify the effect of the therapeutic drug provided by the present invention, this example detected the content of Aβ42 in the brain tissue of mice after treatment.

[0065] (1) Under the premise of complying with the ethical standards of animal experiments, the mice that have completed the open field experiment were decapitated and the brains were removed. One cerebral hemisphere was taken and 8 times the volume of the A solution in the Invitrogen Aβ42 ELISA kit was added and the brain was thoroughly ground using a homogenizer;

[0066] (2) Vortex to mix, incubate at room temperature for 3.5 h, add reaction solution B, and centrifuge at 4°C and 160,000 g for 20 min;

[0067] (3) Carefully pipette the supernatant into another EP tube and place it on ice for later use;

[0068] (4) Dilute the Aβ42 standard in the kit into 8 different concentrations;

[0069] (5) Add samples and standards of different concentrations to the ELISA plate, add 50 μl to each well, and set up 3 replicate wells for both standards and samples;

[0070] (6) Add 50 μl of Aβ42 detection antibody to each well, seal with adhesive tape, and incubate on a shaker at room temperature for 3 h;

[0071] (7) After removing the liquid from the plate, wash it three times with Wash Buffer, add 100 μl of anti-rabbit IgG HRP working solution to each well, apply sealing tape, and incubate on a shaker at room temperature for 30 min;

[0072] (8) After removing the liquid from the plate, wash it three times with Wash Buffer, add 100 μl of color development solution to each well, and incubate at room temperature in the dark for 30 min;

[0073] (9) Add 100 μl of stop solution to each well and use a microplate reader to detect the OD value at a wavelength of 450 nm to obtain the Aβ42 concentration of the sample.

[0074] Table 3 Aβ42 concentration in brain tissue of mice in different treatment groups

[0075]

[0076] According to the results in Table 3, the expected combined effect of the EGCG + Citicoline group was 42.03%, which is similar to the actual drug treatment effect. The expected combined effect of the EGCG + Pterostilbene group was 42.95%, which is similar to the actual drug treatment effect. The expected combined effect of the combination treatment group was 55.27%, and the actual drug treatment effect was significantly higher than the expected combined effect. This fully demonstrates that the combination treatment agent provided by the present invention exhibits an excellent synergistic effect in reducing Aβ42 concentration. This significant synergistic effect not only breaks through the limitations of single drug or two-drug combination therapy, but also provides a more efficient strategy for AD treatment, especially when addressing Aβ42, a key pathological marker.

Claims

1. A therapeutic agent for Alzheimer's disease, characterized in that: Each 100 μL of the drug contains the following active ingredients: The invention comprises 0.25mg-0.35mg of epigallocatechin gallate, 1.8mg-2.0mg of citicoline, 0.2mg-0.35mg of pterostilbene and a pharmaceutically acceptable solvent.

2. The therapeutic agent according to claim 1, characterized in that The solvent consists of 5% ethanol and 95% physiological saline.

3. The therapeutic agent according to claim 1, characterized in that Each 100 μL of the drug contains the following active ingredients: The invention is composed of 0.25 mg of epigallocatechin gallate, 2.0 mg of citicoline and 0.25 mg of pterostilbene and a pharmaceutically acceptable solvent.

4. The therapeutic agent according to claim 3, characterized in that The Alzheimer's disease is a type of Alzheimer's disease characterized by abnormal deposition of Aβ42.

5. Use of the therapeutic agent according to any one of claims 1 to 4 in the preparation of a medicament for treating Alzheimer's disease.

6. The use according to claim 5, characterized in that The drug is used to improve the exploration ability and anxiety of patients with Alzheimer's disease.

7. The use according to claim 6, characterized in that The drug is used to improve the abnormal deposition of Aβ42 in the brain of patients with Alzheimer's disease.

8. Use of the therapeutic agent according to any one of claims 1 to 4 in the preparation of a therapeutic drug for improving the exploratory ability and anxiety of patients with Alzheimer's disease.

9. Use of the therapeutic agent according to any one of claims 1 to 4 in the preparation of a therapeutic drug for improving abnormal Aβ42 deposition in the brain of patients with Alzheimer's disease.

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