4-isopropylphenylacetyl piperazine for the preparation of a medicament for the treatment of alzheimer's disease

Abstract

4-Isopropylphenylacetyl piperazine in the preparation of anti-Alzheimer's disease drugs, its pharmaceutical composition contains 4-isopropylphenylacetyl piperazine (P-355) or its pharmaceutically acceptable salt, ester, solvate, hydrate or prodrug, and at least one pharmaceutically acceptable excipient or adjuvant, for preparing anti-Alzheimer's disease (AD) drug preparation. The P-355 preparation and its compound dosage form of the application not only fill the research gap in the related field, but also bring new hope for the treatment of AD with its rapid, efficient and safe characteristics, and have very high social value and economic benefits.

Description

Technical Field

[0001] This invention belongs to the field of medicine, specifically relating to the application of 4-isopropylphenylacetylpiperazine (P-355) in the preparation of drugs for treating Alzheimer's disease. Background Technology

[0002] Alzheimer's disease (AD), a progressive neurodegenerative disease, poses a serious health challenge globally, especially to aging societies. It is characterized by brain atrophy, brain cell death, and distinctive brain pathological changes—senile plaques and neurofibrillary tangles. Clinical manifestations are diverse, including cognitive decline, memory loss, mood disorders, mental abnormalities, and motor impairments. The disease course is insidious, protracted, and irreversible. With the deepening of global and Chinese aging populations, the incidence of AD continues to rise. China has become one of the countries with the largest number of AD patients globally, accounting for approximately one-quarter of the world's cases, placing a heavy burden on families and society.

[0003] Alzheimer's disease (AD) has a complex etiology, involving dysfunction of multiple systems, including the nervous, immune, and circulatory systems. Although research has revealed several factors closely related to the development and progression of AD, such as dysfunction of the central nervous system's cholinergic system, tangles and deposition of β-amyloid (Aβ), hyperphosphorylation of Tau protein, persistent immune responses and inflammation, and disorders of the central metabolic system, drug development targeting these pathological mechanisms has progressed slowly with extremely low success rates. Currently, clinically available anti-AD drugs are mainly limited to cholinesterase inhibitors and NMDAR blockers. These drugs have some efficacy in patients with mild to moderate AD, but have limitations such as peripheral adverse reactions and limited efficacy. While Aβ monoclonal antibodies, which have attracted much attention in recent years, have shown positive effects in clinical trials, their side effects and high treatment costs limit their widespread application.

[0004] Against this backdrop, exploring safe, effective, and affordable novel anti-AD drugs has become an urgent priority. Pantothenic acid kinase (PANK), a key regulatory enzyme in coenzyme A (CoA) biosynthesis, plays a crucial role in intracellular metabolism. In particular, the PANK2 gene, highly expressed in human neurons, leads to pantothenic acid kinase-associated neurodegeneration (PKAN), a neurological disorder with similar clinical symptoms to AD. This discovery provides a new perspective for AD drug development.

[0005] P-355, an orally administered PANK modulator that can penetrate the blood-brain barrier, has been shown to increase PANK2 and CoA levels in the liver and brain of mice, thereby improving motor disorders caused by insufficient pantothenic acid kinase activity in neurons.

[0006]

[0007] Given the similarity of AD and PKAN in clinical symptoms, and the discovery of decreased acetyl-CoA levels and abnormal histone acetylation levels in AD patients, P-355, as a PANK regulator, is expected to improve AD-like symptoms such as cognitive impairment by regulating CoA synthesis and metabolism.

[0008] Furthermore, the emerging role of ferroptosis in AD research further supports the potential of P-355 as an anti-AD drug. Ferroptosis affects Aβ deposition, Tau protein phosphorylation, and neuronal loss through multiple pathways and is closely related to the progression of AD. As a PANK modulator, P-355 may provide a new strategy for AD treatment by modulating ferroptosis-related pathways.

[0009] In conclusion, P-355, as an effective PANK modulator, has the potential to improve AD-like symptoms such as cognitive impairment and is expected to become a novel anti-AD small molecule drug, bringing new treatment hope to AD patients. Summary of the Invention

[0010] Technical Problem Solved: Addressing the shortcomings of existing Alzheimer's disease treatments, such as unclear key mechanisms and targets, and poor efficacy of existing drugs, this invention provides a drug for treating Alzheimer's disease: 4-isopropylphenylacetylpiperazine exerts its anti-Alzheimer's effect by regulating PANK. This invention provides the application of 4-isopropylphenylacetylpiperazine (P-355) or its formulations in anti-AD.

[0011] Technical solution: Application of 4-isopropylphenylacetylpiperazine (P-355) in the preparation of anti-Alzheimer's disease (AD) drugs.

[0012] A pharmaceutical composition comprising 4-isopropylphenylacetylpiperazine (P-355) or a pharmaceutically acceptable salt, ester, solvate, hydrate or prodrug thereof, and at least one pharmaceutically acceptable excipient or excipient, for use in the preparation of a pharmaceutical formulation for Alzheimer's disease (AD).

[0013] The above-mentioned preparations are selected from tablets, enteric-coated tablets, sustained-release tablets, capsules, enteric-coated capsules, powders, dispersible tablets, effervescent tablets, chewable tablets, oral liquids, solutions, syrups, granules, pellets, oral patches, suppositories, microcapsules, microcapsules, injections, lyophilized preparations, liposomes, microspheres, or other sustained-release or controlled-release preparations.

[0014] The above-mentioned preparations preferably include oral solutions, injections, granules, tablets, capsules, dispersants, and effervescent tablets.

[0015] An oral solution comprising 4-isopropylphenylacetylpiperazine (P-355) or a pharmaceutically acceptable salt, ester, solvate, hydrate or prodrug thereof, and sulfobutyl-β-cyclodextrin as a solubilizer.

[0016] The above-mentioned ratio of cyclodextrin to P-355 results in good solubility of P-355 in oral solutions.

[0017] A method for preparing a pharmaceutical formulation includes the step of mixing 4-isopropylphenylacetylpiperazine (P-355) or a pharmaceutically acceptable salt, ester, solvate, hydrate or prodrug thereof with at least one pharmaceutically acceptable excipient or excipient to prepare the formulation of claim 3 or 4.

[0018] When preparing oral solutions, the step of adding cyclodextrin as a solubilizer is also included.

[0019] A pharmaceutical composition comprising 4-isopropylphenylacetylpiperazine (P-355) or a pharmaceutically acceptable salt, ester, solvate, hydrate or prodrug thereof, and at least one existing clinically used anti-AD drug, for the preparation of a combination anti-AD drug formulation.

[0020] The above-mentioned compound anti-AD drug preparations are selected from tablets, capsules, granules, dispersible tablets, effervescent tablets, oral liquids, solutions, injections, or other dosage forms suitable for compound administration.

[0021] Beneficial Effects: This invention successfully developed a P-355 formulation. This innovative achievement not only fills the gap in the field of P-355 anti-Alzheimer's disease (AD) formulations but also marks a significant breakthrough in the development of AD treatment drugs. Rigorous animal model experiments verified that P-355 showed a highly significant reduction in AD symptoms just one week after administration. Furthermore, no obvious toxic side effects were observed throughout the experiment, fully demonstrating its safety and efficacy. In addition, this invention further verified the ameliorative effect of P-355 through in vitro nerve cell experiments. The experimental results were highly consistent with in vivo animal experiments, providing strong evidence for a deeper understanding of the anti-AD mechanism of P-355. These experimental results collectively indicate that P-355, as a safe, rapid, and highly effective anti-AD drug, has extremely high clinical application potential. More importantly, this invention also explored the combined effects of P-355 with existing clinical anti-AD drugs (such as donepezil). Through scientific formulation, it was found that when the dosage was halved, the combined administration of P-355 and donepezil produced a good synergistic therapeutic effect, providing AD patients with a more comprehensive, effective and safe combination treatment option. Attached Figure Description

[0022] Figure 1Results of behavioral studies in mouse water maze. A: Latency on day 5 of water maze training (orientation and navigation experiment); B: Number of platform crossings on day 6 of water maze training (spatial exploration experiment); C: Movement trajectory on day 5 of water maze training (orientation and navigation with platform); D: Movement trajectory on day 6 of water maze training (spatial exploration without platform). One-way ANOVA analysis was used.* p <0.05,** p <0.01, *** p <0.001.

[0023] Figure 2 Results of mouse Y-maze and open field behavioral tests. A: Y-maze spontaneous alternation recognition rate; B: Novel object recognition index. One-way ANOVA analysis was used.* p <0.05,** p <0.01, *** p <0.001.

[0024] Figure 3 Effects of P-355 on animal blood biochemical parameters. A: ALT level; B: AST level; C: BUN level; D: CRE level. One-way ANOVA analysis was used, ns: no significance, no significant difference.

[0025] Figure 4 Protective effect of P-355 on a galactose-induced neuronal cell injury model. A: Protective effect of P-355 on PC12 cells; B: Protective effect of P-355 on HT22 cells. One-way ANOVA analysis was used.* p <0.05,** p <0.01, *** p <0.001.

[0026] Figure 5 Results of plasma AD biomarker assays. A: Plasma NFL level; B: Plasma GFAP level. One-way ANOVA analysis was used. p <0.01, *** p <0.001. Detailed Implementation

[0027] The following embodiments are intended to enable those skilled in the art to more fully understand the present invention, but are not intended to limit the invention to the scope of the embodiments described.

[0028] Example 1: Preparation of P-355 Oral Solution

[0029] P-355 is poorly soluble in water. Solubilization methods, such as adding co-solvents, were employed using pharmaceutical methods, and formulation studies were conducted according to the dosages in Table 1. EP tubes were dispensed, approximately 1.5 ml per tube, and the solutions were observed at room temperature and refrigerated at 4°C. Formulations 1, 2, and 3 showed turbidity or a small amount of undissolved precipitate, but no significant changes were observed after room temperature or refrigeration at 4°C. Formulation 4 was clear and transparent after preparation, but slight turbidity appeared after 3 days at room temperature or 3 days refrigerated at 4°C. Formulation 5 remained stable and clear after 3 days at room temperature and 3 days refrigerated at 4°C (Table 1).

[0030] Table 1. Study on the solubilization formulation of P-355 liquid dosage form

[0031]

[0032] Based on the research results of formulations 4 and 5 in Example 1, P-355 oral solution was prepared using sodium sulfonyl-β-cyclodextrin as a cosolvent and purified water as the dispersing solvent, ultimately forming a solution system with a cosolvent content of 10% or 30%, with specifications ①-② of 10 and 30 mg / 10 mL, respectively. The formulation is as follows:

[0033] P-355 10 / 30 mg

[0034] Sodium sulfonyl-β-cyclodextrin 1.0 / 3.0 g

[0035] Appropriate amount of purified water

[0036] Add to 10 mL

[0037] Preparation process: Weigh 1.0 g or 3.0 g of sodium sulfobutyl-β-cyclodextrin and dissolve it in 5 mL of purified water. Then weigh 10 mg or 30 mg of P-355 and dissolve it in the above solution, and add purified water to 10 mL. Mix thoroughly by ultrasonication to form a 10% or 30% cyclodextrin system, with each 10 mL solution containing 10 mg or 30 mg of P-355.

[0038] Example 2 P-355 on Aβ 1-42 Pharmacological studies on AD-induced mouse models

[0039] Animal species selection: C57 mice (male, 6-7 weeks old, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., weighing 18-22 g) were used and acclimatized for one week in a standard feeding environment (free access to food and water, 12 hours of day and night).

[0040] Aβ 1-42 Preparation of molding agent: Aβ 1-421,1,1,3,3,3-Hexafluoroisopropanol (HFIP) was purchased from Shanghai Beyotime Biotechnology Co., Ltd., and from Shanghai Bid Pharmaceutical Technology Co., Ltd. Aβ... 1-42 Protein solid powder was dissolved in HFIP to prepare a 1 mg / mL solution, and vortexed until completely dissolved. The solution was then transferred to a fume hood and evaporated to dryness, forming a transparent, colorless peptide film. Subsequently, the thin film of Aβ was... 1-42 The solid was dissolved in sterile physiological saline to prepare a 4 mg / mL solution and incubated in a 37°C incubator for 96 h to promote its oligomerization.

[0041] Aβ 1-42 Establishment of an Alzheimer's Disease (AD) mouse model: One of the key pathological features of AD is the massive deposition of Aβ in senile plaques in brain tissue. The Aβ peptide is a proteolytic fragment containing 39–43 amino acids, obtained by the cleavage of transmembrane amyloid precursor protein (APP) by β- and γ-secretases. Soluble dimers (Aβ oligomers) are minimally synaptic toxic substances, capable of weakening synaptic structure and function. The imbalance between excessive accumulation and inability to clear Aβ disrupts neuronal homeostasis in the brain, leading to the massive accumulation of amyloid plaques, accompanied by progressive synaptic dysfunction, neuronal degeneration, and neuroinflammatory damage, ultimately causing cognitive impairment in patients. Except for the control group, all mice underwent intracerebral ventricle injection (ICV). After anesthesia, the hair on the mouse's head was clipped using sterilized surgical instruments, the scalp was disinfected with iodine, the scalp was gently cut open, and the periosteum was removed with 3% hydrogen peroxide solution. Based on the stereotactic mapping of the mouse brain (coordinates: AP -2.0 mm; ML +2.0 mm; DV -2.0 mm), a mini-skull drill was used to drill bone at the designated location. 2.5 μL of Aβ was then slowly injected into the mouse hippocampus using a micro-injection needle. 1-42 (10 μg) of physiological saline solution (injection rate: 0.5 μL / min). After injection, leave the needle in place for 5 min to prevent backflow, then withdraw the needle for 2 min. The sham-operated group underwent the same procedure as the sham-operated group, except that the injected fluid was replaced with 2.5 μL of physiological saline. The scalp was then sutured with absorbable surgical sutures and disinfected with povidone-iodine. The postoperative condition of the mice was observed, and disinfection was carried out daily at fixed points to prevent biting among the group and promote scalp regrowth. Two weeks later, the model mice were randomly divided into groups (…). Figure 1 :A).

[0042] Donepezil administration: For example, AD model mice weighing 20 g were given 0.2 mL of donepezil solution (0.5 mg / mL, 5 mg / kg) by gavage once daily for one week.

[0043] P-355 administration method: Taking a body weight of 20 g as an example, AD model mice were given 0.2 mL of P-355 solution (3 mg / mL, 30 mg / kg) by gavage, and other groups of mice were given the same volume of solvent, once a day for one week.

[0044] Administration of the compound preparation: Taking a body weight of 20 g as an example, AD model mice were given 0.2 mL of compound preparation solution by gavage (each mL of solution contains 1.5 mg P-355 and 0.25 mg donepezil), and other groups of mice were given the same volume of solvent once a day for one week.

[0045] Morris water maze experiment: To investigate the effects of P-355 on the motor behavior of mice, the Morris water maze was used to evaluate the learning and memory abilities of experimental animals. One week after administration, water maze navigation training was conducted. A circular pool was 45 cm high and 60 cm in radius, divided into four quadrants. A platform (5 cm in radius) was placed in the fourth quadrant, with the water level slightly higher than the platform by 1 cm, and the water temperature maintained at 24℃. During the experiment, the mice were kept out of direct sunlight and kept quiet. During the training period, mice were placed in the pool from any of the four starting points on the pool wall, facing away from the platform and towards the pool wall. The swimming time was set to 60 seconds. If the mouse found the platform and stayed within 60 seconds, the data collection was automatically terminated, and the mouse's swimming trajectory and the time to find the platform (escape latency) were recorded. If the mouse failed to find the platform after 60 seconds, it was guided to the platform by the experimenter and allowed to stay and memorize the information. Four training sessions were conducted, in which mice were placed in the water from four different starting points each day. The average latency of the four training sessions was used as the mouse's learning performance for that day, and the training lasted for five days. After the training period, the original platform was removed, the guidance was stopped, and the mice were subjected to spatial exploration tests. The number of times the mice crossed the platform and their swimming path were recorded within 60 seconds.

[0046] Experimental results show that ( Figure 1 After five days of training, compared with the control mice, the sham-operated group mice showed comparable platform latency and platform crossing frequency, and clear swimming trajectories, indicating that the injection into the hippocampus itself did not have an adverse effect on the mice, ruling out differences in results caused by operational factors. Compared with the control and sham-operated groups, the model mice required more time to find the platform, had a significantly reduced number of platform crossings, and exhibited chaotic swimming trajectories, indicating that the injection of Aβ into the hippocampus... 1-42It can cause cognitive impairment and decreased learning and memory abilities in C57 mice. Compared with the model group mice, mice treated with P-355 showed a significantly shorter escape latency and a significantly reduced degree of swimming trajectory disorder. Similarly, donepezil, a positive control drug, also reduced the escape latency in mice. Compared with the two groups of mice treated with the single drug, mice treated with the combination preparation had a shorter escape latency. Figure 1 (A) The differences were more significant, and the results were closer to those of the blank control group and the sham-operated group. The space exploration experiment on day six showed that, compared with the model group mice, mice treated with blank control, sham-operated mice, donepezil, and P-355 alone or in combination all exhibited increased platform crossings, increased entry into the target quadrant, and increased movement time and distance in the target quadrant. These results indicate that P-355 can improve Aβ. 1-42 The induced cognitive impairment in mice improved the learning and spatial memory abilities of experimental animals, with effects comparable to that of donepezil, a positive control drug. The combination formulation further enhanced efficacy, achieving results comparable to the control group.

[0047] Y-maze test: The Y-maze test is used to evaluate the spatial memory and cognitive abilities of experimental mice. Alternating behavior utilizes the natural instinct of experimental animals to explore new environments. During the search process, experimental animals need to remember the directions they have explored before to avoid re-entering the same area. Therefore, the Y-maze test can effectively measure spatial memory ability. The experimental mouse is placed at the end of any arm and allowed to freely explore the Y-maze for 5 minutes. The total number of times the mouse enters each arm is recorded. Entering all four paws is considered one arm entry. One alternation refers to entering three different arms of the maze in sequence (123, 132, 312, etc. are all acceptable). The maximum number of alternations is the total number of arm entries minus 2. The percentage of alternations = total number of alternations / maximum number of alternations × 100%.

[0048] Experimental results show that ( Figure 2 A), compared with the blank control group and sham-operated group mice, Aβ 1-42 The model group mice exhibited a decreased alternation percentage, indicating cognitive impairment and memory decline. P-355 administration reversed this phenomenon, significantly increasing the alternation percentage, alleviating spatial memory and cognitive impairment, and enhancing their exploratory drive, showing a statistically significant difference compared to the model group. Similarly, donepezil-treated mice also showed a higher alternation percentage. Compared to mice treated with either drug alone, mice treated with the combination formulation had a higher and more significant alternation percentage, and the results were closer to those of the control group. These behavioral results indicate that P-355 can improve cognitive impairment and memory loss in AD-like mice, with effects comparable to the positive control drug donepezil. The combination formulation showed greater advantages than single-drug administration, with cognitive and memory abilities almost recovering to levels comparable to the control group.

[0049] The new object recognition experiment evaluates an animal's cognitive memory ability by measuring the time it takes to explore familiar and unfamiliar objects. Mice are first placed in an open field and allowed free movement for 10 minutes. Then, two identical objects are placed in the field, with the mice placed back-to-back. The exploration time for each object is recorded, including the number of times, time, and distance explored within 5 minutes. One hour later, a new object recognition test is performed, replacing one of the objects with a different one. The mice are again placed back-to-back for 5 minutes, and the number of times, time, and distance explored are recorded. If the mouse has poor cognitive ability, there will be no difference in exploration time between the old and new objects; if the mouse has normal cognitive ability, the exploration time for the new object will be longer than that for the old object. The recognition index (RI) is calculated as: (new object / (new object + old object)) * 100%.

[0050] Experimental results show that ( Figure 2 (B) Compared with the blank control group and the sham-operated group, Aβ 1-42 Mice in the model group exhibited a decline in cognitive index, indicating cognitive impairment and low ability to explore new objects. P-355 administration significantly improved the cognitive index, alleviating cognitive impairment and enhancing exploratory desire, showing a statistically significant difference compared to the model group. Mice treated with donepezil also showed higher cognitive indices and a preference for new objects. Compared to mice treated with either drug alone, mice treated with the combination formulation showed a higher cognitive index for new objects, with a more significant difference, comparable to the control group. These behavioral results indicate that P-355 can improve cognitive and memory abilities in AD-like mice, with effects comparable to the positive control drug. Furthermore, compared to mice treated with either drug alone, mice treated with the combination formulation exhibited superior behavior, learning, memory, and cognitive abilities, approaching those of the control group. This suggests that the combination formulation not only leverages the individual anti-AD advantages of P-355 and donepezil, but also enhances the therapeutic effect through synergistic effects when both P-355 and donepezil are at half the dose, providing a more efficient anti-AD treatment regimen.

[0051] Furthermore, it is worth mentioning that in the preliminary dose-exploration experiments, mice administered 10 mg / kg donepezil exhibited significant salivation, tremors, and muscle spasms, while mice in the P-355 group at a dose of 30 mg / kg showed normal related behaviors. This indicates that donepezil has a narrow therapeutic window and is prone to causing peripheral adverse reactions, similar to the side effects experienced by patients using the drug in clinical settings, while P-355 showed fewer side effects, suggesting good safety. Further, by reducing the dose and combining P-355 with donepezil, not only was good efficacy achieved, but the adverse side effects of donepezil at high doses were also reduced.

[0052] Example 3: Study on the effects of P-355 on liver and kidney function

[0053] Using the same animals, dosage, and administration method as in Example 2, whole blood was collected from each group of animals after 7 consecutive days of administration, serum was prepared, and the following tests were performed.

[0054] Selection of blood biochemical indicators: Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) leak from damaged hepatocytes, making them sensitive indicators for detecting hepatocyte damage. Blood urea nitrogen (BUN) and creatinine (CRE) are commonly used clinical indicators for evaluating renal function, and their levels reflect glomerular filtration function to some extent. ALT, AST, BUN, and CRE reagent kits were purchased from Nanjing Jiancheng Bioengineering Institute.

[0055] ALT kit: ALT reacts with a substrate composed of alanine and α-ketoglutarate at 37℃ and pH 7.4 to generate pyruvate and glutamate. After 30 min of reaction, 2,4-dinitrophenylhydrazine (DNPH) hydrochloric acid solution is added, which stops the reaction. Simultaneously, DNPH adds to the carbonyl group of the keto acid to generate pyruvate phenylhydrazone. Phenyrohydrazone is reddish-brown under alkaline conditions. The absorbance is measured at 505 nm, and the results are used to calculate enzyme activity using a standard curve.

[0056] AST kit: AST converts the amino and ketone groups of α-ketoglutarate and aspartic acid to glutamate and oxaloacetic acid. Oxaloacetic acid undergoes spontaneous decarboxylation to pyruvate during the reaction. Pyruvate reacts with DNPH to form phenylhydrazone, which appears reddish-brown in alkaline solution. Absorbance was measured at 510 nm, and the results were used to calculate enzyme activity using a standard curve.

[0057] BUN kit: Urea hydrolyzes under the action of urease to produce ammonia ions and carbon dioxide. The ammonia ions react with a phenol chromogenic agent in an alkaline medium to form a blue substance; the amount produced is directly proportional to the urea content. The absorbance is measured at 640 nm, and the BUN concentration is calculated.

[0058] CRE kit: CRE is converted to creatine by creatine amide hydrolase, creatine is hydrolyzed to sarcosine and urea by creatine aminohydrolase, and sarcosine is then catalyzed by sarcosine oxidase to glycine, formaldehyde, and hydrogen peroxide. Hydrogen peroxide, 2,4-(6-triiodo-3-hydroxybenzoic acid), and 4-aminoantipyrine react with peroxidase to form the purple-red compound quinone imine. The absorbance is measured at 546 nm, and the CRE concentration is calculated.

[0059] Blood biochemistry test results: The experimental results show ( Figure 3 In the donepezil and P-355 groups, there were no significant differences in four blood biochemical indicators compared to the control group. However, except for AST, the levels of ALT, BUN, and CRE in the donepezil group were slightly higher than those in the control group; while the levels of ALT, BUN, and CRE in the P-355 group were slightly lower than those in the donepezil group, more closely resembling those in the control group. This indicates that P-355 has good safety, and the ALT, BUN, and CRE levels suggest that it has less impact on liver and kidney function than donepezil, making it more advantageous.

[0060] Example 4: Study on the protective effect of P-355 on a D-galactose-induced AD-like cell model

[0061] Modeling agent selection: D-galactose is a naturally occurring reducing sugar found in the human body and various foods. Under normal circumstances, it is completely metabolized by D-galactokinase (GALK) and galactose-1-phosphate uridyltransferase (GALT). However, at high concentrations, it is converted into aldehydes (sugars) and hydrogen peroxide by galactose oxidase, leading to the production of large amounts of superoxide anions and oxygen free radicals. This induces oxidative stress and inflammatory responses, damages cell structure and function, causes apoptosis, and ultimately results in age-like changes and mild cognitive impairment.

[0062] In vitro cell and nerve cell protection experiments: A nerve cell injury model was constructed using D-(+)-galactose, which induces oxidative stress, leading to nerve cell damage. Two nerve cell lines, PC12 (rat adrenal medullary pheochromocytoma cells) and HT22 (mouse hippocampal neurons), were selected as the research subjects. Cells in the logarithmic growth phase were seeded into 96-well plates. Approximately 8000 cells were added to each well during seeding. The 96-well plates were incubated at 37°C with 5% CO2 for 24 h. The original culture medium in the 96-well plates was discarded, taking care not to touch the bottom to avoid damaging the cells. Except for the blank wells, different concentrations of the test compound solution and galactose solution were added to the wells, with the final concentrations of the compounds being 0.2 and 0.5 µM; the final concentration of D-(+)-galactose was 40 mg / mL. No compound was added to the modeling group; the blank group received no modeling agent or compound, but was replaced with the same volume of DMEM. Incubate the 96-well plate in a 5% CO2 incubator at 37°C for 24 h. Add 10 µL of CCK-8 solution to each well (avoiding air bubbles), and incubate the plate at 37°C for 1 hour. Measure the absorbance at 450 nm using a microplate reader and calculate the cell viability = [(As-Ab) / (Ac-Ab)] × 100%. Where, As: absorbance of experimental wells (containing cells, culture medium, CCK-8 solution, and drug solution); Ac: absorbance of control wells (containing cells, culture medium, and CCK-8 solution, but no drug); Ab: absorbance of blank wells (containing culture medium and CCK-8 solution, but no cells or drug).

[0063] Experimental results showed that 40 mg / mL of galactose caused damage to PC12 and HT22 cells, reducing their survival rates to approximately 60% and 45% of the control group, respectively. However, the survival rates of PC12 and HT22 cells co-incubated with P-355 were significantly higher than those in the model group. Figure 4 Furthermore, the protective effect showed a dose-dependent relationship. Specifically, PC12 cells recovered to approximately 80% recovery rate after treatment with 0.5 μMP-355, and HT22 cells recovered to approximately 55% recovery rate, indicating that P-355 can exhibit good neuroprotective effects at low doses and improve cell survival under oxidative stress.

[0064] Example 5: Study on the effect of P-355 on plasma biomarkers in AD model mice

[0065] Using the same animals, dosage, and administration method as in Example 2, whole blood was collected from each group of animals after 7 consecutive days of administration, plasma was prepared, and the following tests were performed.

[0066] Biomarker selection: Neurofilament light chain (NfL) and glial fibrillary acidic protein (GFAP) are considered plasma biomarkers for Alzheimer's disease (AD). Elevated plasma NfL or GFAP levels are highly correlated with cognitive decline and increased risk of AD. NfL and GFAP ELISA kits were purchased from Jiangsu Enzyme Immunoassay Co., Ltd.

[0067] ELISA Assay: Samples and standards were sequentially added to microwells pre-coated with target capture antibodies and incubated. Biotinylated antibodies were then added for antibody incubation. After incubation and thorough washing, horseradish peroxidase (HRP)-labeled detection antibodies were added, followed by incubation and thorough washing. Tetramethylbenzidine (TMB) was added for color development. TMB was converted to blue by peroxidase catalysis, and finally to yellow under acidic conditions. The OD value was measured at 450 nm using a microplate reader. The color intensity was positively correlated with the concentration of the analyte in the sample. The results were then used to calculate the target analyte concentration using a standard curve.

[0068] Experimental results showed that, compared with the control group mice, Aβ 1-42 The levels of NFL and GFAP in the plasma of mice in the model group were increased. Figure 5 Elevated levels of these two plasma markers indicate Aβ 1-42 The model induced a decline in cognitive abilities in mice. Compared with the model group, P-355 administration significantly reduced plasma levels of NFL and GFAP, with statistically significant differences, indicating that P-355 can improve the pathological state of AD-like mice.

[0069] In summary, P-355 is a specific PANK agonist that significantly improves Aβ at a dose of 30 mg / kg. 1-42 The induced AD-like model mice exhibited cognitive impairment, spatial exploration, and memory learning abilities, and the expression levels of AD plasma biomarkers NFL and GFAP were reduced without significant toxic side effects. This indicates that P-355 is a novel, rapid, efficient, and safe anti-AD candidate drug with potential clinical application value.

Claims

1. Application in the preparation of drugs for treating Alzheimer's disease.

2. The application according to claim 1, characterized in that... The drug is an oral solution, which contains... Or a pharmaceutically acceptable salt thereof, and sulfobutyl-β-cyclodextrin as a solubilizer.

3. The application according to claim 2, characterized in that, The sulfobutyl-β-cyclodextrin and The proportion makes It has good solubility in oral solutions.

Images