A pharmaceutical composition for treating alzheimer's disease

Abstract

The present application belongs to the field of pharmaceutical application, and particularly relates to a pharmaceutical composition for treating Alzheimer's disease. The present application finds that RIAFP and AFGP8 have a significant improvement effect on cognitive dysfunction in AD models, and the proteins can be used as novel biological therapeutic agents to provide potential treatment strategies for AD and other neurodegenerative diseases.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical applications, and more specifically, relates to a pharmaceutical composition for treating Alzheimer's disease. Background Technology

[0002] Neurodegenerative disease (NDD) refers 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). Based on the location and degenerated neuronal groups, different pathological changes such as oxidative stress, excitotoxicity, mitochondrial dysfunction and autophagy damage are caused, leading to progressive degeneration of neurons in the central or peripheral nervous system, ultimately resulting in neuronal death in patients (Guerreiro S, Privat AL, Bressac L, et al. CD38 in Neurodegeneration and Neuroinflammation[J]. Cells,2020,9(2):471).

[0003] Alzheimer's disease (AD), also known as senile dementia, is a common degenerative disease of the central nervous system that mainly affects the elderly. Its main clinical manifestations include progressive decline in memory and cognitive function, behavioral abnormalities, social impairment, and sleep disorders, accompanied by varying degrees of personality changes (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 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. However, with the deepening of research and clinical practice, it was found that the ATN framework could not provide a comprehensive explanation of the pathological changes in AD. Therefore, the framework needed to be supplemented with more biomarkers, thus forming the ATX(N) diagnostic framework (Wang S, Greene R, Song Y, et al. Postoperative delirium and its relationship with biomarkers for dementia: ameta-analysis[J]. Int Psychogeriatr, 2022: 1-14.) to achieve a more comprehensive understanding of AD. The X represents a broader scope involving other pathogenesis mechanisms of AD, such as synaptic structural changes, neuroinflammatory responses, and blood-brain barrier disruption. The pathogenesis of Alzheimer's disease (AD) is complex, and there is currently no cure. Existing treatments are mostly focused on relieving symptoms, and their efficacy is limited. Therefore, exploring new treatment strategies and targets has become a current research hotspot.

[0004] The main pathological features of Alzheimer's disease (AD) include β-amyloid plaque formation, tau protein phosphorylation, neurofibrillary tangles, vacuolar degeneration of hippocampal pyramidal cells, mitochondrial energy metabolism disorders, synaptic damage, and neuronal loss (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 one of the important factors inducing pathological changes in AD. Oxidative stress refers to the excessive accumulation of highly oxidizing molecules such as reactive oxygen species (ROS) and nitrogenous substances (RNS) in cells, leading to cell dysfunction and even cell death. Long-term accumulation of oxidative stress can induce abnormal folding of Aβ protein and promote its deposition, thereby exacerbating neurotoxicity. In addition, oxidative stress can also activate neuroinflammatory responses through multiple pathways, further accelerating the progression of neurodegenerative diseases. Therefore, antioxidant therapy is considered one of the important strategies for delaying or preventing AD.

[0005] Among the antifreeze protein family, Antarctic fish antifreeze proteins have attracted widespread attention due to their structural characteristics and biological activities. Currently, five types of antifreeze proteins have been identified in polar fish, including antifreeze glycoproteins (AFGPs) and four antifreeze proteins (AFPI, AFP II, AFP III, and AFP IV). AFPs and AFGPs are proteins found in organisms living in extremely low-temperature environments. Their main function is to protect cells from cryogenic damage by inhibiting water crystal formation. Due to their unique structure and biological activities, these proteins also exhibit significant antioxidant and cytoprotective functions under non-freezing conditions, effectively reducing cell damage caused by oxidative stress. Therefore, antifreeze proteins and antifreeze glycoproteins may become therapeutic agents for the prevention and treatment of various neurodegenerative diseases, including Alzheimer's disease (AD).

[0006] RiAFP is found in the tissues and hemolymph of insects, including beetles that can survive temperatures below -25°C. Like other antifreeze proteins, RiAFP prevents freezing by adsorbing onto the surface of ice crystals and lowering the temperature at which ice crystals grow.

[0007] Antifreeze glycoprotein AFGP8 is one such glycosyl-rich protein. Studies have found that when ice crystals grow in AFGP8 solution, AFGP8 molecules adsorb onto the prism faces or cone faces of the ice crystals, lowering the temperature at the ice crystal growth point through an adsorption inhibition mechanism without changing the melting point temperature of the ice crystals. Summary of the Invention

[0008] This invention discovered that RIAFP and AFGP8 have a significant effect on improving cognitive dysfunction in AD models. Based on this, this invention was completed.

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

[0010] Furthermore, the pharmaceutical composition also contains other active ingredients, which are drugs that are different from the RIAFP and AFGP8 proteins and can promote the improvement and / or treatment of neurodegenerative diseases.

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

[0012] Furthermore, the drug composition can be administered orally, by injection, implantation, external application, spraying, or inhalation.

[0013] Secondly, the present invention provides the use of a pharmaceutical composition in the preparation of an agent for the prevention and / or treatment of Alzheimer's disease, the pharmaceutical composition comprising RIAFP and / or AFGP8; the amino acid sequence of the AFGP8 is shown in SEQ ID NO.1; the amino acid sequence of the RIAFP is shown in SEQ ID NO.3.

[0014] Furthermore, the formulation also contains pharmaceutically acceptable excipients.

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

[0016] Furthermore, the formulation can be administered orally or by injection to prevent and / or treat Alzheimer's disease.

[0017] Beneficial effects

[0018] This invention found that RIAFP and AFGP8 significantly improve cognitive dysfunction in AD models by enhancing cellular antioxidant capacity and improving zebrafish behavioral performance.

[0019] This discovery provides a theoretical basis for developing new biologics to treat Alzheimer's disease (AD). It also provides experimental data for understanding the potential mechanisms of antifreeze proteins in neuroprotection, offering new ideas and methods for the treatment of neurodegenerative diseases. Attached Figure Description

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

[0021] Figure 2 RIAFP and AFGP8 improved the mobility of zebrafish juveniles after AlCl3 treatment.

[0022] Figure 3 RIAFP and AFGP8 increased the movement distance of zebrafish juveniles after AlCl3 treatment: (A) Total movement distance statistics; (B) Movement distance of zebrafish per minute under light and dark cycles.

[0023] Figure 4 RIAFP and AFGP8 increased the average movement speed of zebrafish juveniles after AlCl3 treatment: (A) average speed statistics; (B) average speed statistics per minute under light and dark cycles.

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

[0025] Figure 6 RIAFP and AFGP8 proteins improve mitochondrial quality in APP cells. Note: (A) TOM20 staining of mitochondria transfected with RIAFP and AFGP8 genes and empty vector plasmids; (B) Statistical analysis of relative fluorescence intensity after TOM20 staining. Detailed Implementation

[0026] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the embodiments described below can be combined with each other as long as they do not conflict with each other.

[0027] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0028] Table 1. Correlation sequences of AFGP8 and RIAFP

[0029]

[0030] Example 1

[0031] 1. Materials and Methods

[0032] 1.1 H2O2 treatment to detect the antioxidant capacity of RIAFP and AFGP8

[0033] To evaluate the effects of RIAFP and AFGP8 on cellular antioxidant stress, expression plasmids of these proteins were introduced into 293T cells via chemical transfection. After transfection, cells were treated with 0.3 mmol / L hydrogen peroxide (H2O2) to induce oxidative stress. Untransfected 293T cells, serving as a control, were also treated with 0.3 mmol / L hydrogen peroxide (H2O2) to induce oxidative stress. Subsequently, the cells were replaced with fresh culture medium and cultured further to restore cell viability. Cell viability was then assessed using the CCK-8 assay, and intracellular reactive oxygen species (ROS) levels were measured by flow cytometry.

[0034] (1) CCK-8 assay for cell viability

[0035] After changing the culture medium, the treated cells were cultured again. Culture medium and CCK-8 reagent were added to each well. The plates were shaken well and incubated. Subsequently, OD values ​​were read at 450 nm using a multi-functional plate reader, and cell viability was calculated. The OD values ​​of the H2O2-treated group were compared with those of the untreated group to determine cell viability.

[0036] (2) Flow cytometry detection of ROS levels

[0037] Intracellular ROS levels were determined using the CM-H2DCFDA probe. CM-H2DCFDA was diluted 1:1000 with serum-free culture medium to a final concentration of 5 μM. After cell collection, cells were suspended in the diluted probe solution and incubated. After incubation, cells were washed with serum-free culture medium to remove any probe that had not entered the cells. ROS levels were detected using flow cytometry under 495 nm excitation and 530 nm emission conditions, and fluorescence intensity was recorded to assess the degree of oxidative stress.

[0038] 1.2 AlCl3 treatment of zebrafish embryos to construct an Alzheimer's disease (AD) model and test behavioral abilities

[0039] To establish an Alzheimer's disease (AD) model and assess the effects of two proteins on cognitive function and motor ability, zebrafish embryos were treated with AlCl3 to induce AD-like pathological changes. First, zebrafish embryos were divided into two groups: the first group was treated with an aqueous solution, and the second group was exposed to a specific concentration of aluminum chloride solution to induce neurotoxicity and mimic the pathological features of Alzheimer's disease. Subsequently, expression plasmids of RIAFP and AFGP8 were injected into the 1-cell stage of some zebrafish embryos from both groups using microinjection, and cultured until day 5. Behavioral assays were used to assess the cognitive function and motor ability of the zebrafish, including the following experiments:

[0040] (1) Experiment in an open swimming pool

[0041] This experiment was used to assess the activity level of zebrafish. By observing the frequency, range of motion, and mobility of zebrafish in the water, it was determined whether aluminum chloride treatment caused sluggishness or abnormal behavior. Measuring activity levels helps assess the effects of antifreeze proteins and antifreeze glycoproteins on improving mobility.

[0042] (2) Phototaxis experiment

[0043] This experiment was used to assess the learning and memory abilities of zebrafish. By observing the zebrafish's attraction to light sources, the study evaluated whether aluminum chloride treatment caused learning and memory impairments. This experiment can further reflect the potential impact of antifreeze proteins and antifreeze glycoproteins on cognitive function.

[0044] 1.3 Detection of Aβ42 secretion in APP cells

[0045] The detection of cell-secreted Aβ42 was performed using 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 hours. Then, the Aβ42 in the supernatant was quantitatively analyzed using an ELISA kit. A specific anti-Aβ42 capture antibody was pre-coated onto a microplate, incubated, and then the sample was added; Aβ42 bound to the capture antibody. Next, a labeled detection antibody was added. Upon binding to Aβ42, a color change occurred through a substrate reaction. The absorbance value was measured and compared with a standard curve to quantify the Aβ42 content.

[0046] 1.4 Immunofluorescence detection of mitochondrial quality

[0047] To assess the impact of NPAFP protein on mitochondrial quality, SH-SY5Y / WT cells and cells stably expressing the Swedish mutant APP (SH-SY5Y / APP695swe) were selected as AD models. Expression plasmids for RIAFP and AFGP8 genes were transfected into WT (SY5Y) and APP (SH-SY5Y / APP695swe) cells using the photochemical transfection reagent Lipo3000. 48 h after transfection, the cells were subjected to immunofluorescence staining. First, cells were fixed on slides with 4% paraformaldehyde to maintain their structure; then, the cell membrane was treated with 0.1% Triton X-100 to allow antibody entry into the cells. Next, primary antibody (anti-TOM20) was added, and the cells were incubated at room temperature. Following this, the cells were washed with PBS buffer to remove unbound primary antibody. Subsequently, secondary antibody (Alexa Fluor 594-labeled goat anti-rabbit IgG (H+L)) was added, and incubation continued. Finally, the cells were washed again with PBS to remove excess secondary antibody, and the slides were mounted with an anti-fluorescence attenuator. 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 ANOVA was used to assess differences between groups, with a significance level set at p < 0.05. Statistical analysis was conducted to evaluate the differences between different experimental groups and to verify the effectiveness of antifreeze proteins and antifreeze glycoproteins in the AD model.

[0050] 2. Experimental Results

[0051] 2.1 RIAFP and AFGP8 enhance the antioxidant stress resistance of 293T cells

[0052] RIAFP and AFGP8 were cloned into expression vectors, and the expression plasmids were introduced into 293T cells via chemical transfection. After treatment with H2O2 for 4 hours, cell viability was assessed using CCK-8 assay and intracellular reactive oxygen species (ROS) levels were detected using the DCFH-DA probe. The results showed that, compared with the control EV group, the viability of 293T cells expressing RIAFP and AFGP8 was significantly increased. Figure 1 In A), ROS levels were significantly reduced ( Figure 1 The presence of RIAFP and AFGP8 (as shown in Figure B) indicates that these proteins have a significant protective effect against oxidative stress. This finding suggests that RIAFP and AFGP8 have potential applications, potentially for managing oxidative stress in the prevention and treatment of Alzheimer's disease (AD).

[0053] 2.2 RIAFP and AFGP8 enhance the behavioral abilities of zebrafish juveniles

[0054] The results showed that in the first group, there was no significant difference in trajectories between zebrafish without RIAFP and AFGP8 expression plasmids and those with RIAFP and AFGP8 expression plasmids, indicating that these proteins have no toxic effect on zebrafish. In the second group, transgenic zebrafish injected with RIAFP and AFGP8 expression plasmids after AlCl3 treatment exhibited more active trajectories. Figure 2 This indicates that these proteins can improve motor and cognitive functions in zebrafish, an AD model.

[0055] 2.3 RIAFP and AFGP8 increase the movement distance of zebrafish juveniles

[0056] Under the same experimental design, the total migration distance of transgenic zebrafish and the control group was measured. The results showed that in the group without AlCl3 treatment (Group 1), there was no significant difference between the transgenic group and the empty vector control group. Figure 3 (A). However, in the AlCl3 treatment group (Group 2), the total distance traveled by the transgenic zebrafish ( Figure 3 (A) and the distance moved per minute ( Figure 3 The significant increase in B1 protein further confirms the potential of these proteins in improving behavioral abilities and cognitive function.

[0057] 2.4 RIAFP and AFGP8 increase the movement speed of juvenile zebrafish.

[0058] Further analysis was conducted on the average movement speed of zebrafish juveniles. Without AlCl3 treatment, there was no significant difference between the transgenic group and the empty vector control group; however, in the AlCl3-treated group, the average movement speed of the transgenic zebrafish was significantly higher. Figure 4 (A) and speed of movement per minute ( Figure 4 The levels of RIAFP and AFGP8 were significantly higher in the middle group than in the control group. These results indicate that the improving effects of RIAFP and AFGP8 on motor function further support their potential application in AD prevention.

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

[0060] Aβ protein is secreted and released into the intercellular fluid in an environment of enhanced neuronal activity, where it aggregates to form oligomers and fibrils, eventually forming plaques, which is an important cause of Alzheimer's disease.

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

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

[0063] Wild-type cells (SH-SY5Y / WT) and cells stably expressing the Swedish familial mutation of human APP695 (SHSY5Y / APP695swe) served as Alzheimer's cell models with high endogenous expression of APP. RIAFP and AFGP8 protein expression plasmids were transfected into these two cell types using chemical transfection reagents. Figure 6 Immunofluorescence staining of the mitochondrial mass protein TOM20 (in AB) showed that RIAFP and AFGP8 proteins significantly improved mitochondrial mass in APP cells. This finding suggests the potential application value of RIAFP and AFGP8 in the prevention of Alzheimer's disease (AD).

[0064] 3. Conclusions and Application Prospects

[0065] This study demonstrates that RIAFP and AFGP8 significantly improve cognitive function in an AD model by enhancing cellular antioxidant capacity, improving zebrafish behavioral performance, reducing Aβ42 secretion in APP695 cells, and improving mitochondrial quality. These proteins may function by regulating oxidative stress pathways and protecting neurons from oxidative damage. In the future, these proteins could serve as novel biotherapeutic agents, providing potential therapeutic strategies for neurodegenerative diseases such as AD.

Claims

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

3.

2. The use of claim 1, wherein the medicament comprises a pharmaceutically acceptable excipient.

3. The use of claim 1, wherein the medicament is in a dosage form selected from the group consisting of a tablet, a capsule, an aerosol, a pill, a powder, a solution, a suspension, an emulsion, a granule, and / or a suppository.

Images