Medicament for treating Alzheimer disease and application thereof

Abstract

The invention provides a therapeutic agent for Alzheimer's disease and application thereof, and belongs to the technical field of neurobiology. Each 100 [mu] L of the medicament comprises the following active ingredients: 0.25 mg to 0.35 mg of epigallocatechin gallate, 1.8 mg to 2.0 mg of citicoline and 0.2 mg to 0.35 mg of pterostilbene, and the balance of water. The medicament has positive influence on patients with Alzheimer's disease by regulating and controlling Abeta42 deposition, improving exploration ability and relieving anxiety mood. Therefore, the medicament has a wide clinical prospect in application, and a novel and synergistic treatment strategy can be provided for treating the Alzheimer disease.

Description

Technical Field

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

[0002] Alzheimer's Disease (AD) is a common neurodegenerative disease, mainly manifested as progressive memory loss, cognitive impairment, behavioral changes, and decline in daily living ability. With the aggravation of global aging, the prevalence of Alzheimer's disease has been increasing year by year, and it has become an important public health problem worldwide. Currently, the main drugs used clinically include cholinesterase inhibitors and NMDA receptor antagonists, which can improve the symptoms of patients to a certain extent, but due to limited efficacy and large side effects, the long-term use effect is not good. In addition, the treatment methods targeting pathological features such as amyloid protein deposition and abnormal tau protein are still in the clinical research stage and have not achieved significant breakthroughs.

[0003] In recent years, more and more researchers have begun to pay attention to the potential of natural plant components and some neuroprotective molecules in the treatment of Alzheimer's disease. These natural compounds have been widely studied for the treatment of neurodegenerative diseases due to their antioxidant, anti-inflammatory, and neuroprotective effects. Research shows that these components can improve the symptoms of Alzheimer's disease through multiple pathways, especially their potential for clearing amyloid plaques, neuroprotection, and anti-inflammatory effects, which has received extensive attention.

[0004] However, the therapeutic effect of a single component is still limited, and long-term application may produce drug resistance or side effects. Therefore, developing a compound agent that combines multiple components to improve the efficacy and safety of drugs has become an important direction in the current research on the treatment of Alzheimer's disease. Summary of the Invention

[0005] The present invention provides a therapeutic agent for Alzheimer's disease and its application, aiming to overcome the problem of limited efficacy of single drugs in the prior art. The present 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 the symptoms of AD patients.

[0006] Specifically, the core content of the present invention includes: First of all, the present invention provides a therapeutic agent for Alzheimer's disease. Every 100 μL of the agent consists of the following active ingredients: It is composed of 0.25 mg - 0.35 mg of epigallocatechin gallate, 1.8 mg - 2.0 mg of citicoline, 0.2 mg - 0.35 mg of pterostilbene and a pharmaceutically acceptable solvent.

[0007] Preferably, the solvent is composed of 5% ethanol and 95% normal saline.

[0008] Preferably, in a preferred embodiment, every 100 μL of the medicament consists of the following active ingredients: It is composed of 0.25 mg of epigallocatechin gallate, 2.0 mg of citicoline, 0.25 mg of pterostilbene and a pharmaceutically acceptable solvent.

[0009] This specific combination of active ingredients has been experimentally verified to show excellent effects in reducing the concentration of Aβ42 in the brain tissue of Alzheimer's disease model mice and improving behavioral indicators (such as exploratory ability and anxiety).

[0010] Preferably, the Alzheimer's disease is of the type with abnormal deposition of Aβ42.

[0011] Secondly, the present invention not only provides the specific composition of the above-mentioned therapeutic medicament, but also further clarifies its application value in multiple therapeutic fields.

[0012] In the first aspect, the present invention provides the application of the therapeutic medicament in the preparation of a drug for treating Alzheimer's disease.

[0013] Preferably, the drug is used to improve the exploratory ability and anxiety of Alzheimer's disease patients.

[0014] Preferably, the drug is used to improve the abnormal deposition of Aβ42 in the brain of Alzheimer's disease patients.

[0015] In the second aspect, the present invention provides the application of the therapeutic medicament in the preparation of a therapeutic drug for improving the exploratory ability and anxiety of Alzheimer's disease patients.

[0016] Alzheimer's disease patients are often accompanied by 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.

[0017] In the third aspect, the present invention provides the application of the therapeutic medicament in the preparation of a therapeutic drug for improving the abnormal deposition of Aβ42 in the brain of Alzheimer's disease patients.

[0018] Aβ42 is a key toxic protein in the pathological process of Alzheimer's disease. Its abnormal deposition can trigger the formation of amyloid plaques, leading to neuronal damage and cognitive decline. This medicament effectively alleviates this core pathological feature by significantly reducing the concentration of Aβ42.

[0019] The beneficial effects of the present invention are as follows: The present invention combines three active ingredients, EGCG, Citicoline, and Pterostilbene, for the first time, giving full play to their multi-pathway and multi-target mechanism of action. EGCG mainly regulates the accumulation of Aβ42, Citicoline enhances the synthesis of acetylcholine, and Pterostilbene has antioxidant and anti-inflammatory effects. The present invention discovers for the first time that the synergistic effect of combining the three is significantly higher than the therapeutic effects of single or dual components, and significantly higher than the expected combined effect, thus being able to more effectively alleviate the symptoms of AD.

[0020] The experimental results show that the medicament 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 neurodegenerative diseases. The combined medicament can significantly reduce the deposition of Aβ42, thereby alleviating the intracerebral pathological changes in AD mice.

[0021] In addition, the medicament of the present invention also shows excellent effects in improving the exploratory ability and anxiety of AD model mice. In the open field experiment, the exploratory ability and the movement time in the middle area of the treated AD mice are significantly improved, showing better behavioral performance than the model group, proving that the medicament can effectively improve the anxiety and cognitive function of AD patients. Description of the Drawings

[0022] Figure 1 It is a result graph of the average movement distance in the middle area of mice in different treatment groups; Figure 2 It is a result graph of the average movement time in the middle area of mice in different treatment groups; Figure 3 It is a result graph of the concentration of Aβ42 in the brain tissue of mice in different treatment groups. Detailed Embodiments

[0023] Example 1: Alzheimer's disease treatment medicament a of the present invention Every 100 μL of the medicament consists of the following components: Epigallocatechin gallate (EGCG, CAS No.: 989-51-5): 0.25 mg; Citicoline (CAS No.: 987-78-0): 2.0 mg; Pterostilbene (CAS No.: 537-42-8): 0.25 mg; Solvent: 5% ethanol (v / v) and 95% normal saline (0.9% NaCl).

[0024] Preparation steps: First, mix 5% ethanol (v / v) and 95% normal saline (0.9% NaCl) thoroughly to ensure the uniformity of the solvent; Add epigallocatechin gallate (EGCG), citicoline, and pterostilbene to the prepared solvent and stir well on a stirrer until dissolved; Filter and sterilize using a 0.22 μm filter membrane to obtain the therapeutic agent a.

[0025] Example 2: Alzheimer's disease therapeutic agent b of the present invention Each 100 μL of the agent consists of the following components: Epigallocatechin gallate (EGCG): 0.3 mg; Citicoline: 2.0 mg; Pterostilbene: 0.2 mg; Solvent: 5% ethanol (v / v) and 95% normal saline (0.9% NaCl).

[0026] The preparation method is the same as that in Example 1.

[0027] Example 3: Alzheimer's disease therapeutic agent c of the present invention Each 100 μL of the agent consists of the following components: Epigallocatechin gallate (EGCG): 0.35 mg; Citicoline: 1.8 mg; Pterostilbene: 0.35 mg; Solvent: 5% ethanol (v / v) and 95% normal saline (0.9% NaCl).

[0028] The preparation method is the same as that in Example 1.

[0029] Example 4: Use the open field test to detect the effects of the Alzheimer's disease therapeutic agent of the present invention on the exploratory ability and anxiety of AD model mice (1) Six wild-type mice (6 months old) were used as the normal control group, and 42 APP / PS1 mice (6 months old) were randomly divided into 7 groups, namely the model group, the EGCG group (the drug only contains EGCG), the Citicoline group (the drug only contains Citicoline), the Pterostilbene group (the drug only contains Pterostilbene), the EGCG + Citicoline group (the drug contains EGCG and Citicoline), the EGCG + Pterostilbene group (the drug contains EGCG and Pterostilbene), and the combined therapeutic agent group (therapeutic agent a) of the present invention.

[0030] (2) After one week of adaptive feeding, at 9 o'clock every day, 100 μL of the corresponding drug was intragastrically administered to each mouse according to the above grouping, and the normal control group was intragastrically administered with the corresponding normal saline. The administration time was 8 weeks.

[0031] (3) The open field experiment was used to detect the exploratory ability and anxiety of the mice. The specifications of the open field box were 50 cm × 50 cm × 50 cm, and the bottom surface was evenly divided into 25 10 cm × 10 cm square grids; (4) The treated mice were placed in the center of the bottom of the open field box, observed for 5 min, and the walking distance and time of the mice in the middle area of the open field were recorded; (5) After each experiment was completed, the bottom surface of the open field was wiped with 75% ethanol to remove the odor and prevent interference with the next mouse. Finally, the average movement distance and average movement time in the middle area of all treatment groups of mice were statistically analyzed.

[0032] Table 1 Average movement distance in the middle area of mice in different treatment groups

[0033] From the results in Table 1, it can be seen that the average movement distance of the mice in the AD model group in the middle area was significantly reduced, indicating that the exploratory ability of the AD model mice decreased. And all treatment groups after drug treatment had a certain level of improvement compared with the model group. This result shows that using EGCG, Citicoline, Pterostilbene alone or in combination can treat AD mice to a certain extent.

[0034] Through the expected effect formula of the combination of 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 detection result, indicating that there was no synergistic effect in the treatment of the exploratory ability of AD mice when EGCG and Citicoline were combined.

[0035] The expected therapeutic effect of the EGCG+Pterostilbene group was 32.32%, which was also similar to the actual test results, indicating that the combination of EGCG and Pterostilbene also had no synergistic effect on the exploration ability of AD mice.

[0036] 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 therapeutic effect of the combined treatment agent group was 42.49%, and the actual test results were significantly higher than the expected therapeutic effect, indicating that when EGCG, Citicoline, and Pterostilbene were used in combination to treat AD mice, the three drugs had a significant synergistic effect and could effectively treat the problem of decreased exploration ability of AD mice.

[0037] Table 2 Average movement time of mice in the middle area in different treatment groups

[0038] From the results in Table 2, it can be seen that compared with the control group, the movement time of mice in the model group in the middle area was significantly reduced, indicating an increase in the anxiety level of AD mice. Similar to the results of the movement distance, all treatment groups after drug treatment had a certain level of improvement compared with the model group, further proving that using EGCG, Citicoline, Pterostilbene alone or in combination could treat AD mice to a certain extent.

[0039] Further analysis found that the expected therapeutic effect of the EGCG+Citicoline group was 36.58%, and the expected therapeutic effect of the EGCG+Pterostilbene group was 39.29%. By comparison, the difference could be seen, and the actual test effect was basically the same as the expected effect, indicating that when EGCG was combined with Citicoline or Pterostilbene, it could not produce a synergistic effect on the treatment of AD.

[0040] The expected therapeutic effect of the combination of the three drugs was 50.41%, and the actual drug effect was significantly higher than the expected therapeutic effect, indicating that when the three drugs were used in combination, a significant synergistic effect could be produced, thereby more effectively reducing the anxiety level of AD mice. Moreover, compared with the therapeutic effect on the exploration ability, the therapeutic effect of this drug on relieving the anxiety level of mice was more significant.

[0041] Example 5: To further verify the effect of the therapeutic drug provided by the present invention, the content of Aβ42 in the brain tissue of the treated mice was detected in this example.

[0042] (1)On the premise of conforming to the ethical norms of animal experiments, the mice that had completed the open field experiment were decapitated to remove the brain. One cerebral hemisphere was taken and added to Solution A in the Aβ42 ELISA kit of Invitrogen at 8 times the volume, and then thoroughly ground using a homogenizer. (2)After vortex mixing, it was incubated at room temperature for 3.5 h, then Solution B of the reaction solution was added, and centrifuged at 4 °C and 160,000 g for 20 min. (3)Carefully aspirate the supernatant into another EP tube and place it on ice for later use. (4)Dilute the Aβ42 standard product in the kit into 8 different concentrations. (5)Add the samples and standard products of different concentrations to the enzyme-linked immunosorbent assay (ELISA) plate, 50 μl per well. Both the standard products and samples were set with 3 replicates. (6)Add 50 μl of Aβ42 detection antibody to each well. After sealing with sealing tape, incubate at room temperature on a shaker for 3 h. (7)After removing the liquid in the plate, wash it 3 times with Wash Buffer. Add 100 μl of anti-rabbit IgG HRP working solution to each well. After pasting the sealing tape, incubate at room temperature on a shaker for 30 min. (8)After removing the liquid in the plate, wash it 3 times with Wash Buffer. Add 100 μl of chromogenic solution to each well and incubate at room temperature in the dark for 30 min. (9)Add 100 μl of termination solution to each well, and use an enzyme-linked immunosorbent assay reader to detect the OD value at a wavelength of 450 nm to obtain the Aβ42 concentration of the sample.

[0043] Table 3 Aβ42 concentration in the brain tissues of mice in different treatment groups

[0044] According to the results in Table 3, the expected combined effect of the EGCG + Citicoline group was 42.03%, which was similar to the actual drug treatment effect. The expected combined effect of the EGCG + Pterostilbene group was 42.95%, which was similar to the actual drug treatment effect. The expected combined effect of the combined treatment agent group was 55.27%, and the actual drug treatment effect was significantly higher than the expected combined effect. This fully demonstrates that the combined treatment agent provided by the present invention exhibits excellent synergistic effects in reducing the Aβ42 concentration. This significant synergistic effect not only breaks through the limitations of single-drug or two-drug combination treatments but also provides a more efficient strategy for the treatment of AD, especially showing outstanding advantages in dealing with 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 and 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% 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 drug 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 deposition of Aβ42 in the brain of patients with Alzheimer's disease.

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