Pharmaceutical composition for treating Alzheimer disease

Abstract

The invention belongs to the field of medicine application, and particularly relates to a medicine composition for treating Alzheimer disease. The RIAFP and the AFGP8 have a remarkable improvement effect on cognitive impairment in an AD model, and the proteins can be used as novel biological therapeutic agents and provide a potential treatment strategy for neurodegenerative diseases such as AD and the like.

Description

Technical Field

[0001] The present invention belongs to the field of drug applications, and more particularly, relates to a pharmaceutical composition for treating Alzheimer's disease. Background Art

[0002] Neurodegenerative diseases (NDDs) refer to diseases in which neurons in the nervous system degenerate, including Alzheimer's disease (AD), Parkinson's disease (PD), multiple sclerosis (MS), and amyotrophic lateral sclerosis (ALS), etc. (Bell SM, Burgess T, Lee J, et al. Peripheral Glycolysis in Neuro-degenerative Diseases [J]. Int J Mol Sci, 2020, 21(23):8924). Depending on the location and degenerating neuronal populations, different pathological changes such as oxidative stress, excitotoxicity, mitochondrial dysfunction, and autophagy damage are caused, leading to the progressive degeneration of neurons in the central or peripheral nervous system, and ultimately resulting in the death of neurons in patients (Guerreiro S, Privat AL, Bressac L, et al. CD38 in Neurodegenera-tion and Neuroinfla-mmation [J]. Cells, 2020, 9(2):471).

[0003] Alzheimer's disease (AD), also known as senile dementia, mostly occurs in the elderly and is a common degenerative disorder of the central nervous system. Clinically, it is mainly manifested as progressive memory and cognitive decline, behavioral abnormalities, social impairment, sleep disorders, etc., accompanied by personality changes to varying degrees (SCHELTENS P, DE STROOPER B, KIVIPELTO M, et al. Alzheimer's disease[J]. Lancet, 2021, 397(10284): 1577-1590.). In 2018, the National Institute on Aging and the Alzheimer's Association in the United States promoted the construction of the ATN (Huang S, Wang Y J, Guo J. Biofluid Biomarkers of Alzheimer's Disease: Progress, Problems, and Perspectives[J]. Neurosci Bull, 2022, 38(6): 677-691.) framework for the pathological diagnosis of AD again. However, with the in-depth research and clinical practice, it was found that the ATN framework was difficult to provide a comprehensive explanation for the pathological changes of AD. Therefore, more biomarkers needed to be incorporated into this framework for supplementation, and thus the ATX(N) diagnostic framework (Wang S, Greene R, Song Y, et al. Postoperative delirium and its relationship with biomarkers for dementia: a meta-analysis[J]. Int Psychogeriatr, 2022: 1-14.) was formed to gain a more comprehensive understanding of AD, where X represents a relatively wide range involving other pathogenic mechanisms of AD such as synaptic structure changes, neuroinflammatory responses, and blood-brain barrier disruption. The pathogenesis of AD is complex, and there is currently no radical cure. Existing therapeutic drugs mainly focus on alleviating symptoms, and the curative effects are limited. Therefore, exploring new treatment strategies and targets has become a research hotspot at present.

[0004] The main pathological features of AD include β-amyloid plaque formation, tau protein phosphorylation, neurofibrillary tangles, granulovacuolar degeneration of hippocampal pyramidal cells, mitochondrial energy metabolism disorders, synaptic damage, and neuronal loss, etc. (JIAL, DUY, CHUL, et al. Prevalence, risk factors, and management of dementia and mild cognitive impairment in adults aged 60 years or older in China: a cross-sectional study[J]. Lancet Public Health. 2020, 5(12): e661-e671.). Oxidative stress is considered to be one of the important factors triggering the pathological changes of AD. Oxidative stress refers to the excessive accumulation of highly oxidizing molecules such as reactive oxygen species (ROS) and reactive nitrogen species (RNS) in cells, resulting in cell dysfunction and even cell death. The accumulation of long-term oxidative stress can induce abnormal Aβ protein folding and promote its deposition, thus exacerbating the neurotoxic reaction. In addition, oxidative stress can also activate the neuroinflammatory response through multiple pathways, further accelerating the process of neurodegenerative diseases. Therefore, antioxidant therapy is considered to be one of the important strategies for delaying or preventing AD.

[0005] Among the antifreeze protein family, Antarctic fish antifreeze proteins have received extensive attention due to their structural characteristics and biological activities. Currently, 5 types of antifreeze proteins have been found in polar fish, including antifreeze glycoprotein (AFGP) and 4 types of antifreeze proteins (AFPI, AFP II, AFP III, and AFP IV). AFPs and AFGPs are proteins found in some organisms living in extremely low-temperature environments. Their main function is to protect cells from low-temperature damage by inhibiting the formation of ice crystals. Due to their unique structure and biological activities, these proteins also exhibit significant antioxidant and cytoprotective functions under non-freezing conditions, and can effectively reduce cell damage caused by oxidative stress. Therefore, antifreeze proteins and antifreeze glycoproteins may become therapeutic drugs for preventing and treating various neurodegenerative diseases including AD.

[0006] RiAFP exists in the tissues and hemolymph of insects. This beetle can survive at temperatures below -25°C. Like other antifreeze proteins, RiAFP prevents freezing by adsorbing to the surface of ice crystals and lowering the temperature of ice crystal growth.

[0007] Antifreeze glycoprotein AFGP8 is one of the glycoprotein-rich proteins. It has been found that when ice crystals grow in an AFGP8 solution, AFGP8 molecules will adsorb on the prism face or conical face of the ice crystal, and while reducing the temperature of the ice crystal growth point through the adsorption inhibition mechanism, the melting point temperature of the ice crystal remains unchanged. Summary of the Invention

[0008] The present invention discovers that RIAFP and AFGP8 have a significant improvement effect on cognitive dysfunction in AD models. Based on this, the present invention is completed.

[0009] In a first aspect, the present invention provides a pharmaceutical composition for preventing and / or treating Alzheimer's disease, the pharmaceutical composition comprising one or both of RIAFP and / or AFGP8 protein; the amino acid sequence of AFGP8 is as shown in SEQ ID NO.1; the amino acid sequence of RIAFP is as shown in SEQ ID NO.3.

[0010] Furthermore, the pharmaceutical composition further contains other active ingredients, and the other active ingredients refer to drugs that are different from the RIAFP and AFGP8 proteins and can promote the body to improve and / or treat neurodegenerative diseases.

[0011] Furthermore, the pharmaceutical composition can be made into various dosage forms, including but not limited to one or more of tablets, capsules, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, and / or suppositories.

[0012] Even further, the administration methods of the pharmaceutical composition include oral administration, injection, implantation, external application, spraying, or inhalation.

[0013] In a second aspect, the present invention provides the use of a pharmaceutical composition in the preparation of a preparation for preventing and / or treating Alzheimer's disease, the pharmaceutical composition comprising RIAFP and / or AFGP8; the amino acid sequence of AFGP8 is as shown in SEQ ID NO.1; the amino acid sequence of RIAFP is as shown in SEQ ID NO.3.

[0014] Furthermore, the preparation further comprises pharmaceutically acceptable excipients.

[0015] Furthermore, the dosage forms of the preparation include but not limited to tablets, capsules, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, and / or suppositories.

[0016] Furthermore, the preparation can achieve the prevention and / or treatment of Alzheimer's disease by oral administration or injection.

[0017] Advantageous Effects

[0018] The present invention discovers that RIAFP and AFGP8 have a significant improvement effect on cognitive dysfunction in the AD model by enhancing the antioxidant stress ability of cells and improving the behavioral performance of zebrafish.

[0019] This discovery provides a theoretical basis for the development of new biological agents for the treatment of AD. At the same time, it also provides experimental data for understanding the potential mechanism of antifreeze proteins in neuroprotection, and provides new ideas and methods for the treatment of neurodegenerative diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 RIAFP and AFGP8 enhance the antioxidant stress ability of 293T cells: (A) CCK8 assay for cell viability; (B) detection of relative ROS content.

[0021] Figure 2 RIAFP and AFGP8 improve the locomotor ability of zebrafish larvae after AlCl3 treatment.

[0022] Figure 3 RIAFP and AFGP8 increase the moving distance of zebrafish larvae after AlCl3 treatment: (A) total moving distance statistics; (B) moving distance per minute of zebrafish under light-dark cycle.

[0023] Figure 4 RIAFP and AFGP8 increase the average moving speed of zebrafish larvae after AlCl3 treatment: (A) average speed statistics; (B) average speed statistics per minute under light-dark cycle.

[0024] Figure 5 RIAFP and AFGP8 reduce the Aβ secretion of APP cells.

[0025] Figure 6 RIAFP and AFGP8 proteins increase the mitochondrial mass of APP cells. Note: (A) Mitochondrial TOM20 staining of cells transfected with RIAFP and AFGP8 genes and empty vector plasmid; (B) relative fluorescence intensity statistics after TOM20 staining. DETAILED DESCRIPTION OF THE INVENTION

[0026] The following further describes the specific embodiments of the present invention. It should be noted here that the description of these embodiments is for helping to understand the present invention, but does not limit the present invention. In addition, the technical features involved in the following described embodiments can be combined with each other as long as they do not conflict with each other.

[0027] The experimental methods in the following examples are all conventional methods unless otherwise specified, and the test materials used in the following examples are all commercially available through conventional channels unless otherwise specified.

[0028] Table 1 Related sequences of AFGP8 and RIAFP

[0029]

[0030] Example 1

[0031] 1. Materials and Methods

[0032] 1.1 Detection of the antioxidant capacity of RIAFP and AFGP8 by H2O2 treatment

[0033] To evaluate the effects of RIAFP and AFGP8 on cellular antioxidant stress, expression plasmids of these proteins were introduced into 293T cells by chemical transfection. After transfection, the cells were treated with 0.3 mmol / L hydrogen peroxide (H2O2) to induce oxidative stress. Untransfected 293T cells used as a control were also treated with 0.3 mmol / L hydrogen peroxide (H2O2) to induce oxidative stress. Subsequently, fresh medium was replaced and the cells were further cultured to recover their state. Next, the cell viability was detected by the CCK-8 method, and the level of intracellular reactive oxygen species (ROS) was measured by flow cytometry.

[0034] (1) Detection of cell viability by the CCK-8 method

[0035] After the treated cells were replaced with fresh medium, they were further cultured. Medium was added to each well, and CCK-8 reagent was added. The well plate was shaken and incubated. Subsequently, the OD value was read at a wavelength of 450 nm using a multifunctional microplate reader, and the cell viability was calculated. The OD value of the H2O2 treatment group was ratio-processed with that of the untreated group, and the cell viability was statistically analyzed.

[0036] (2) Detection of ROS level by flow cytometry

[0037] The intracellular ROS level was measured using the CM-H2DCFDA probe. CM-H2DCFDA was diluted with serum-free culture medium at a ratio of 1:1000, and the final concentration was 5 μM. After the cells were collected, they were suspended in the diluted probe solution and incubated. After incubation, the cells were washed with serum-free culture medium to remove the probe that did not enter the cells. Flow cytometry was used to detect the ROS level under the conditions of 495 nm excitation light and 530 nm emission light, and the fluorescence intensity was recorded to evaluate the degree of oxidative stress.

[0038] 1.2 Construction of an Alzheimer's disease (AD) model by treating zebrafish embryos with AlCl3 and detection of behavioral ability

[0039] To establish an Alzheimer's disease (AD) model and evaluate the effects of two proteins on cognitive function and motor ability, zebrafish embryos were treated with AlCl3 to induce AD-like pathological changes. First, the zebrafish embryos were divided into two groups. The first group was treated with an aqueous solution, and the second group was exposed to a certain concentration of aluminum chloride solution to induce neurotoxicity and simulate the pathological characteristics of Alzheimer's disease. Subsequently, by means of microinjection technology, the expression plasmids of RIAFP and AFGP8 were injected into the one-cell stage of some zebrafish embryos in the first and second groups, and the embryos were continuously cultured until the 5th day. Behavioral detection methods were used to evaluate the cognitive function and motor ability of zebrafish, including the following experiments:

[0040] (1) Open-field swimming assay

[0041] This assay was used to evaluate the activity level of zebrafish. By observing the swimming frequency, activity range, and motor ability of zebrafish in water, it was determined whether aluminum chloride treatment caused motor retardation or behavioral abnormalities. Measuring the activity level helped to evaluate the improvement effect of antifreeze proteins and antifreeze glycoproteins on motor ability.

[0042] (2) Phototaxis response assay

[0043] This assay was used to detect the learning and memory ability of zebrafish. By observing the light-seeking behavior of zebrafish, it was evaluated whether there were learning and memory impairments in zebrafish after aluminum chloride treatment. This assay could further reflect the potential effects of antifreeze proteins and antifreeze glycoproteins on cognitive function.

[0044] 1.3 Detection of Aβ42 secretion in APP cells

[0045] The detection of Aβ42 secreted by cells was performed by enzyme-linked immunosorbent assay (ELISA). First, the constructed RIAFP and AFGP8 expression plasmids were transfected into APP cells using a chemical transfection reagent, and the cell supernatant was collected after 48 h. Then, an ELISA kit was used to quantitatively analyze Aβ42 in the supernatant. First, the capture antibody specific for Aβ42 was pre-coated on the microplate. After incubation, the sample was added, and Aβ42 would bind to the capture antibody. Then, the labeled detection antibody was added. After this antibody bound to Aβ42, a color change occurred through the substrate reaction. The absorbance value was measured and compared with the standard curve to quantify the content of Aβ42.

[0046] 1.4 Immunofluorescence detection of mitochondrial mass

[0047] To evaluate the effect of NPAFP protein on mitochondrial mass, SH-SY5Y / WT cells and cells stably expressing Swedish mutant APP (SH-SY5Y / APP695swe) were selected as AD models. The expression plasmids of RIAFP and AFGP8 genes were transfected into WT (SY5Y) and APP (SH-SY5Y / APP695swe) cells using the photochemical transfection reagent lipo3000. After 48 h of transfection, the cells were subjected to immunofluorescence staining. First, the cells were fixed on slides with 4% paraformaldehyde to preserve their structure; then, the cell membranes were treated with 0.1% Triton X-100 to allow antibodies to enter the cells. Next, the primary antibody (anti-TOM20) was added and incubated at room temperature. Subsequently, it was washed with PBS buffer to remove the unbound primary antibody. Then, the secondary antibody (AlexaFluor 594-labeled goat anti-rabbit IgG (H+L)) was added and incubation continued. Finally, it was washed again with PBS to remove the excess secondary antibody, and the slide was sealed with an anti-fluorescence quencher. After completion, the expression of TOM20 in the cells was observed and photographed using a fluorescence microscope.

[0048] 1.5 Data analysis

[0049] All experimental data were analyzed using SPSS statistical software. One-way analysis of variance (ANOVA) was used for inter-group differences, and the significance level was set at p < 0.05. Through statistical analysis, the differences between different experimental groups were evaluated, and the effects of antifreeze proteins and antifreeze glycoproteins in the AD model were verified.

[0050] 2. Experimental results

[0051] 2.1 RIAFP and AFGP8 improve the antioxidant stress ability of 293T cells

[0052] RIAFP and AFGP8 were respectively cloned into expression vectors, and the expression plasmids were introduced into 293T cells by chemical transfection. After treatment with H2O2 for 4 hours, the cell viability was detected by CCK-8 and the intracellular reactive oxygen species (ROS) level was detected by DCFH-DA probe. The results showed that compared with the control group EV, the cell viability of 293T cells expressing RIAFP and AFGP8 was significantly increased ( Figure 1 in A), and the ROS level was significantly decreased ( Figure 1 in B), indicating that these proteins have a significant protective effect on antioxidant stress. This finding suggests that RIAFP and AFGP8 have potential application value and may be used for oxidative stress management in the prevention and treatment of AD.

[0053] 2.2 RIAFP and AFGP8 improve the behavioral ability of zebrafish larvae

[0054] The results showed that in the first group, there was no significant difference in the movement trajectories between zebrafish without injection of the expression plasmids of RIAFP and AFGP8 and zebrafish injected with the expression plasmids of RIAFP and AFGP8, indicating that these proteins had no toxic effects on zebrafish. In the second group, after treatment with AlCl3, the transgenic zebrafish injected with the expression plasmids of RIAFP and AFGP8 showed more active movement trajectories( Figure 2 ), indicating that these proteins could improve the motor and cognitive functions of zebrafish AD models.

[0055] 2.3 RIAFP and AFGP8 increase the moving distance of zebrafish larvae

[0056] Under the same experimental design, the total moving distances of transgenic zebrafish and the control group were detected. The results showed that in the group without AlCl3 treatment (the first group), there was no significant difference between the transgenic group and the empty vector control group( Figure 3 in A). However, in the AlCl3 treatment group (the second group), the total moving distance of the transgenic zebrafish( Figure 3 in A) and the moving distance per minute( Figure 3 in B) were significantly increased, further confirming the potential of these proteins in improving behavioral ability and cognitive function.

[0057] 2.4 RIAFP and AFGP8 increase the moving speed of zebrafish larvae

[0058] The average moving speed of zebrafish larvae was further analyzed. When not treated with AlCl3, there was no significant difference between the transgenic group and the empty vector control group; while in the AlCl3 treatment group, the average moving speed of the transgenic zebrafish( Figure 4 in A) and the moving speed per minute( Figure 4 in B) were significantly higher than those of the control group. These results indicate that the improvement effect of RIAFP and AFGP8 on motor ability further supports their potential application in AD prevention.

[0059] 2.5 RIAFP and AFGP8 proteins reduce the secretion of Aβ42 in APP cells

[0060] Aβ protein is secreted into the interstitial fluid of cells and aggregates to form oligomers, protofibrils, and finally plaques in an environment with enhanced neuronal activity, which is an important cause of Alzheimer's disease.

[0061] The expression plasmids of RIAFP and AFGP8 proteins were transfected into APP695 cells (SHSY5Y / APP695swe) using a chemical transfection reagent. After 48 hours, the content of Aβ42 in the cell culture medium was detected, and it was found that RIAFP and AFGP8 proteins could significantly reduce the secretion of Aβ42 in APP695 cells( Figure 5), The results indicate that RIAFP and AFGP8 proteins have the potential to reduce Aβ accumulation, providing new ideas for the treatment of AD.

[0062] 2.6 RIAFP and AFGP8 proteins improve the mitochondrial quality of APP cells

[0063] Wild-type cells (SH-SY5Y / WT) and cells stably expressing human APP695 with the Swedish familial mutation (SHSY5Y / APP695swe) were used as Alzheimer's disease cell models with endogenous high expression of APP. The expression plasmids of RIAFP and AFGP8 proteins were transfected into these two types of cells using a chemical transfection reagent ( Figure 6 in A-B), and immunofluorescence staining was performed on the mitochondrial quality protein TOM20. The results show that RIAFP and AFGP8 proteins can significantly improve the mitochondrial quality of APP cells. This finding indicates the potential application value of RIAFP and AFGP8 in preventing AD.

[0064] 3. Conclusions and application prospects

[0065] This study shows that RIAFP and AFGP8 improve the antioxidant stress capacity of cells, improve the behavioral performance of zebrafish, reduce the secretion of Aβ42 in APP695 cells, and increase their mitochondrial quality. They have a significant improvement effect on cognitive dysfunction in AD models. These proteins may play a role by regulating the oxidative stress pathway and protecting neurons from oxidative damage. In the future, these proteins can be used as novel biological therapeutics to provide potential treatment strategies for neurodegenerative diseases such as AD.

Claims

1. Use of a pharmaceutical composition in the preparation of a preparation for preventing and / or treating Alzheimer's disease, wherein the pharmaceutical composition comprises RIAFP and / or AFGP8; the amino acid sequence of AFGP8 is as shown in SEQ ID NO.1; the amino acid sequence of RIAFP is as shown in SEQ ID NO.

3.

2. The use according to claim 1, wherein the RIAFP and / or AFGP8 can improve the motor and cognitive functions of Alzheimer's disease patients.

3. The use according to claim 1, wherein the preparation further comprises a pharmaceutically acceptable excipient.

4. The use according to claim 1, wherein the dosage form of the preparation includes tablets, capsules, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents and / or suppositories.

5. The use according to claim 1, wherein the preparation can achieve prevention and / or treatment of Alzheimer's disease by oral administration or injection.

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