Application of 4-isopropylphenyl acetyl piperazine in preparation of anti-Alzheimer disease drugs

Abstract

The invention relates to an application of 4-isopropyl phenyl acetyl piperazine in preparation of an anti-Alzheimer disease drug, and a pharmaceutical composition of the 4-isopropyl phenyl acetyl piperazine comprises 4-isopropyl phenyl acetyl piperazine (P-355) or pharmaceutically acceptable salt, ester, solvate, hydrate or prodrug thereof, and at least one pharmaceutically acceptable excipient or auxiliary material, and is used for preparing an anti-Alzheimer disease (AD) pharmaceutical preparation. The P-355 preparation and the compound dosage form thereof not only fill up the research blank in related fields, but also bring new hope for AD treatment due to the characteristics of rapidness, high efficiency and safety, and have extremely high social value and economic benefit.

Description

Technical Field

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

[0002] Alzheimer's disease (AD), as a progressive neurodegenerative disease, poses a severe health challenge to the world, especially the aging society. The disease is characterized by brain atrophy, brain cell death and characteristic pathological changes in the brain - senile plaques and neurofibrillary tangles. The clinical manifestations are diverse, including cognitive decline, memory loss, mood disorders, mental abnormalities and movement disorders, and the course of the disease is insidious, long and irreversible. With the deepening of the aging of the world and China, the incidence of AD continues to rise. China has become one of the countries with the largest number of AD patients in the world, accounting for about a quarter of the world's patients, which has brought a heavy burden to families and society.

[0003] The etiology of AD is complex, involving dysfunction of multiple systems such as the nervous system, immune system, and blood circulation. Although studies have revealed multiple factors closely related to the occurrence and development of AD, such as dysfunction of the central nervous system cholinergic system, entanglement and precipitation of β-amyloid protein (Aβ), hyperphosphorylation of Tau protein, persistent immune response and inflammation, and disorders of the central metabolic system, the development of drugs targeting these pathological mechanisms has been slow and the success rate is extremely low. Currently, clinically available anti-AD drugs are mainly limited to cholinesterase inhibitors and NMDAR blockers. These drugs have certain efficacy in patients with mild and moderate AD, but they have limitations such as peripheral adverse reactions and limited efficacy. Although the Aβ monoclonal antibody, which has attracted much attention in recent years, has shown positive effects in clinical trials, its side effects and expensive treatment costs limit its widespread application.

[0004] In this context, it is imperative to explore new anti-AD drugs that are safe, effective, and economical. Pantothenate kinase (PANK), as a key regulatory enzyme in the biosynthesis of coenzyme A (CoA), plays a vital role in intracellular metabolism. In particular, the PANK2 gene is highly expressed in human neurons, and its mutation can lead to pantothenate kinase-associated neurodegeneration (PKAN), a neurological disease with similar clinical symptoms to AD. This discovery provides a new perspective for the development of drugs for AD.

[0005] P-355, an oral PANK regulator that can penetrate the blood-brain barrier, has been shown to increase PANK2 and CoA levels in the liver and brain of mice, and is used to improve movement disorders caused by insufficient pantothenate kinase activity in neurons.

[0006]

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

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

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

[0010] Technical problem to be solved: In view of the defects of the existing Alzheimer's disease, such as unclear key mechanisms and targets, and poor therapeutic effects, the present invention provides a drug for treating Alzheimer's disease, namely: 4-isopropylphenylacetylpiperazine can play a therapeutic and anti-Alzheimer's disease role by regulating PANK. The present invention provides the use of 4-isopropylphenylacetylpiperazine (P-355) or its preparation composition 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 comprises 4-isopropylphenylacetylpiperazine (P-355) or a pharmaceutically acceptable salt, ester, solvate, hydrate or prodrug thereof, and at least one pharmaceutically acceptable excipient or auxiliary material, for preparing a pharmaceutical preparation for treating Alzheimer's disease (AD).

[0013] The above-mentioned preparation is 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, pills, oral patches, suppositories, micropills, microcapsules, injections, freeze-dried preparations, liposomes, microspheres or other sustained-release or controlled-release preparations.

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

[0015] An oral solution comprises 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 ratio of cyclodextrin to P-355 enables P-355 to have good solubility in the oral solution.

[0017] A method for preparing a pharmaceutical preparation, comprising 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 auxiliary material to prepare the preparation according to claim 3 or 4.

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

[0019] A pharmaceutical composition comprises 4-isopropylphenylacetylpiperazine (P-355) or a pharmaceutically acceptable salt, ester, solvate, hydrate or prodrug thereof, and at least one existing clinical anti-AD drug, for preparing a compound anti-AD drug preparation.

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

[0021] Beneficial effects: The present invention successfully developed the P-355 preparation. This innovative achievement not only fills the gap in the field of P-355 anti-Alzheimer's disease (AD) preparations, but also marks a major breakthrough in the research and development of AD therapeutic drugs. Through rigorous animal model experiments, P-355 showed a very significant reduction effect on AD symptoms only one week after administration. And no obvious toxic side effects were observed during the entire experiment, which fully demonstrated its safety and effectiveness. In addition, the present invention further verified the improvement effect of P-355 through in vitro neural cell experiments, and the experimental results were highly consistent with in vivo animal experiments, which provided a strong basis for our in-depth understanding of the anti-AD mechanism of P-355. These experimental results collectively show that P-355, as a safe, fast and efficient anti-AD drug, has extremely high clinical application potential. It is worth mentioning that the present invention also explored the combined use effect of P-355 and existing clinical anti-AD drugs (such as donepezil). Through scientific matching, it was found that at half the dose, 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 plan. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1Results of water maze behavioral tests on mice. A: latency on the fifth day of water maze training (positioning navigation experiment); B: number of platform crossings on the sixth day of water maze training (spatial exploration experiment); C: movement trajectory on the fifth day of water maze training (positioning navigation with platform); D: movement trajectory on the sixth day of water maze training (spatial exploration without platform). One-way ANOVA analysis was used to analyze the results. p <0.05,** p <0.01,*** p <0.001.

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

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

[0025] Figure 4 The protective effect of P-355 on galactose-induced neuronal cell injury model. A: The protective effect of P-355 on PC12 cells; B: The protective effect of P-355 on HT22 cells. One-way ANOVA analysis was used to analyze the difference between the two groups. p <0.05,** p <0.01,*** p <0.001.

[0026] Figure 5 Results of the determination of AD biomarker levels in plasma. A: NFL level in plasma; B: GFAP level in plasma. One-way ANOVA analysis was used to analyze the differences between the two groups. p <0.01,*** p <0.001. DETAILED DESCRIPTION

[0027] The following embodiments can enable those skilled in the art to more fully understand the present invention, but the present invention is not limited to the scope of the embodiments.

[0028] Example 1 Preparation of P-355 Oral Solution P-355 is poorly soluble in water. A pharmaceutical method was used to add solubilizing means such as cosolvents, and a prescription study was conducted according to the dosage in Table 1. The EP tubes were divided into approximately 1.5 ml tubes, and were placed at room temperature and refrigerated at 4°C to observe the state of the solution. It was found that prescriptions 1, 2, and 3 were turbid or had a small amount of undissolved precipitate, and no significant changes were observed after being placed at room temperature or refrigerated at 4°C. Prescription No. 4 was clear and transparent after preparation, and slightly turbid after being placed at room temperature for 3 days or refrigerated at 4°C for three days. Prescription No. 5 was stable, clear, and transparent after preparation, being placed at room temperature for 3 days, and being refrigerated at 4°C for three days (Table 1).

[0029] Table 1 Study on the solubilization formula of P-355 liquid preparation

[0030] Based on the research results of prescriptions 4 and 5 in Example 1, P-355 oral solution was prepared using sulfobutyl-β-cyclodextrin sodium salt as a cosolvent and purified water as a dispersing solvent, and finally a solution system with a cosolvent content of 10% or 30% was formed, and specifications ①-② were 10 and 30 mg / 10 mL, respectively. The prescription is as follows: P-355 10 / 30 mg Sulfonbutyl-β-cyclodextrin sodium 1.0 / 3.0 g Purified water Add to 10 mL Preparation process: Weigh 1.0 g or 3.0 g of sodium sulfobutyl-β-cyclodextrin and dissolve it in 5 mL of pure water. Then weigh 10 mg or 30 mg of P-355 and dissolve it in the above solution, and add pure water to 10 mL. Mix by ultrasonic vibration to finally form a 10% or 30% cyclodextrin system, with each 10 mL of solution containing 10 mg or 30 mg of P-355.

[0031] Example 2 P-355 against Aβ 1-42 Study on drug efficacy in induced AD model mice Animal species selection: C57 mice (male, 6-7 weeks old, purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd., weighing 18-22 g) were used and adaptively raised for one week under a standard feeding environment (free diet and drinking water, alternating day and night, 12 hours each).

[0032] Aβ 1-42 Modeling agent preparation: Aβ 1-42 purchased from Shanghai Biyuntian Biotechnology Co., Ltd., and 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) was purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd. 1-42The protein solid powder was dissolved in HFIP to make a 1 mg / mL solution and vortexed until completely dissolved. The solution was moved to a fume hood and evaporated to form a transparent and colorless peptide film. 1-42 The solid was dissolved in sterile saline to make a 4 mg / mL solution and incubated in a 37°C incubator for 96 h to promote its oligomerization.

[0033] Aβ 1-42 Establishment of induced AD mouse model: One of the important pathological characteristics of AD is the massive deposition of Aβ in senile plaques of brain tissue. Aβ peptide is a proteolytic fragment containing 39−43 amino acids, which is cleaved by β- and γ-secretase of transmembrane amyloid precursor protein (APP). Soluble dimers (Aβ oligomers) are the smallest synaptic toxic substances, which can weaken synaptic structure and function. Excessive accumulation of Aβ and imbalance of clearance dysfunction destroy the neural homeostasis in the brain, and then a large number of Aβ aggregate to form amyloid plaques, accompanied by progressive synaptic dysfunction, neuronal degeneration and neuritic damage, which eventually cause cognitive impairment in patients. Except for the blank group, all mice underwent intracerebral ventricle injection (icv.) surgery. After anesthesia, the hair on the head of the mouse was cut with sterile surgical instruments, the scalp was disinfected with iodine, the scalp was gently cut, and the periosteum was removed with 3% hydrogen peroxide solution. According to the mouse brain stereotaxic atlas (coordinates: AP -2.0 mm; ML +2.0 mm; DV -2.0 mm), a micro-skull drill was used to drill the bone at the location. A microinjection needle was used to slowly inject 2.5 μL of Aβ into the mouse hippocampus. 1-42 (10 μg) of normal saline solution (injection speed: 0.5 μL / min). After the injection, the needle was retained for 5 min to prevent the liquid from overflowing, and the needle was withdrawn for 2 min. The sham operation group performed the same operation except that the injected liquid was replaced with 2.5 μL normal saline. The scalp was then sutured with absorbable surgical sutures and disinfected with iodine. The postoperative status of the mice was observed, and fixed-point disinfection was performed daily to prevent mutual biting between groups and promote scalp regrowth. Two weeks later, the modeled mice were randomly divided into groups ( Figure 1 : A).

[0034] Dosage of donepezil: Taking 20 g body weight as an example, AD model mice were gavaged with 0.2 mL of donepezil solution (0.5 mg / mL, 5 mg / kg) once a day for one week.

[0035] P-355 administration method: Taking 20 g body weight as an example, AD model mice were gavaged with 0.2 mL P-355 solution (3 mg / mL, 30 mg / kg), and other groups of mice were given an equal volume of solvent, once a day for one week.

[0036] Administration of the compound preparation: Taking 20 g body weight as an example, 0.2 mL of the compound preparation solution (each mL of solution contains 1.5 mg P-355 and 0.25 mg donepezil) was administered orally to AD model mice, and the other groups of mice were given an equal volume of solvent, once a day for one week.

[0037] Morris water maze experiment: In order to explore the effect of P-355 on the motor behavior of mice, the Morris water maze was used to evaluate the learning and memory ability of experimental animals. One week after administration, water maze positioning and navigation training was performed. The circular pool is 45 cm high and 60 cm in radius, and it is divided into 4 quadrants. The platform (radius 5 cm) is placed in the fourth quadrant, the water level is slightly higher than the platform by 1 cm, and the water temperature is maintained at 24°C. Pay attention to avoid light and keep quiet during the experiment. During the training period, the mouse is placed in the pool with its back to the platform and facing the pool wall from any of the four starting points on the pool wall. The swimming time is set to 60 s. If the mouse can find the platform and stay within 60 s, the collection is automatically terminated, and the mouse's swimming trajectory and the time to find the platform (escape latency) are recorded. If the mouse still does not find the platform after more than 60 s, the experimenter will guide it to the platform and let it stay on the platform and remember it. The mice were placed in the water from four different starting points for 4 training sessions, and the average value of the latency of the mice in 4 training sessions was used as the learning performance of the mice on that day. 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 a spatial exploration test. The number of times the mice crossed the platform and the swimming path within 60 s were recorded.

[0038] The experimental results show that ( Figure 1 ), after five days of training, compared with the blank mice, the platform latency and platform crossing times of the sham-operated mice were comparable, and the swimming tracks were clear, indicating that the injection into the hippocampus itself did not have an adverse effect on the mice, and the differences in results caused by the operation factors were excluded. Compared with the blank and sham-operated mice, the model mice took longer to find the platform, the platform crossing times decreased significantly, and the swimming tracks were chaotic, indicating that the injection of Aβ into the hippocampus 1-42 It can cause cognitive impairment and decreased learning and memory ability in C57 mice. Compared with the model group mice, the escape latency of mice treated with P-355 was significantly shortened and the degree of confusion in swimming trajectories was significantly reduced. Similarly, the positive drug donepezil can also reduce the escape latency of mice. Compared with the two groups of mice treated with the compound preparation, the escape latency of mice was shorter ( Figure 1:A), the difference was more significant, and the results were closer to the blank group and sham operation group. The sixth day space exploration experiment showed that compared with the model group mice, the blank control, sham operation, donepezil, P-355 alone or combined administration mice showed an increase in the number of platform area crossings, the number of times entering the target quadrant, and the increase in the movement time and distance in the target quadrant. These results show that P-355 can improve Aβ 1-42 The induced cognitive impairment in mice and improved the learning and spatial memory abilities of experimental animals, and the effect was comparable to that of the positive drug donepezil. The compound preparation could further enhance the efficacy of the drug, and the effect was comparable to the blank level.

[0039] Y-maze test: The Y-maze test is used to evaluate the spatial memory and cognitive ability of experimental mice. Alternation behavior is to use the experimental animals' nature to explore new environments. During the search process, the experimental animals need to remember the direction they explored last time to avoid repeated entry. Therefore, the Y-maze test can effectively measure the spatial memory ability of the experiment. The experimental mouse is placed at the end of any arm and allowed to explore freely in the Y-maze for 5 minutes. The total number of times the mouse enters each arm is recorded. All four feet of the mouse enter the arm once. Alternation refers to entering three different arms of the maze in sequence (123, 132, 312, etc.). The maximum number of turns refers to the total number of arms - 2. The alternation percentage = total number of turns / maximum number of turns × 100%.

[0040] The experimental results show that ( Figure 2 :A)Compared with the blank group and sham operation group mice, Aβ 1-42 The mice in the modeling group showed a decrease in the alternation percentage, indicating that the model mice had cognitive impairment and memory loss. The administration of P-355 reversed this phenomenon, and the alternation percentage increased significantly, alleviating their spatial memory cognitive impairment and enhancing their desire to explore, showing a significant statistical difference compared with the model group. Similarly, mice administered with donepezil also showed a higher alternation percentage. Compared with the two groups of mice treated with the drug alone, the mice treated with the compound preparation had a higher alternation percentage, a more significant difference, and the results were closer to the blank group. The results of this behavioral experiment showed that P-355 can improve cognitive impairment and memory impairment in AD-like mice, and the effect is comparable to that of the positive drug donepezil. The compound preparation has more advantages than single medication, and cognitive memory ability is almost restored to the same level as the blank group.

[0041] New object recognition experiment: The cognitive memory ability of animals is evaluated by behavioral methods of the length of time the experimental animals explore familiar objects and new unfamiliar objects. First, place the mouse in an open field and let it move freely for 10 minutes. Then put two identical objects in the open field, and put the mouse in the open field with its back facing the objects. The exploration time of the mouse on each object is recorded, and the number, time and distance of the mouse exploring each object are measured within 5 minutes. One hour later, a new object recognition test is conducted, and one of the objects is replaced with a different object and placed in the open field. Similarly, put the mouse in the open field with its back facing the object for 5 minutes, and record the number, time and distance of its exploration of each object. If the mouse has poor cognitive ability, there is no difference in the exploration of new and old objects; if the mouse has normal cognitive ability, the exploration of new objects is longer than that of old objects. Recognition index (RI) = new object / (new object+old object)*100%.

[0042] The experimental results show that ( Figure 2 :B)Compared with the blank group and sham operation group mice, Aβ 1-42 The modeling group mice showed a decrease in cognitive index, indicating that the model mice had cognitive impairment and low ability to explore new things. P-355 administration can significantly improve the cognitive index of mice, alleviate their cognitive impairment and enhance their desire to explore, with significant statistical differences compared with the model group. Mice treated with donepezil also showed a higher cognitive index and preference for new objects. Compared with the two groups of mice treated with the compound preparation, the mice treated with the compound preparation had a higher cognitive index of new objects and a more significant difference, and the results were comparable to those of the blank group mice. The results of this behavioral experiment showed that P-355 can improve the cognitive memory ability of AD-like mice, and the effect is comparable to that of positive drugs. Not only that, compared with mice treated with the compound preparation alone, mice treated with the compound preparation showed superior behavior and learning, memory, and cognitive abilities, approaching those of blank mice. This shows that the compound preparation not only exerts the anti-AD advantages of P-355 and donepezil, but also improves the therapeutic effect through drug synergy when both P-355 and donepezil are halved, providing a more efficient anti-AD treatment plan.

[0043] In addition, it is worth mentioning that in the preliminary dose exploration experiment, mice showed obvious salivation, tremors, and muscle spasms after being given 10 mg / kg of donepezil, while the P-355 group mice at a dose of 30 mg / kg showed normal related behaviors. This shows that donepezil has a narrow therapeutic window and is prone to cause peripheral adverse reactions, which are similar to the side effects of clinical patients, while P-355 shows fewer side effects, indicating good safety. Furthermore, by reducing the dose and combining P-355 with donepezil, not only good efficacy was achieved, but also it was helpful to reduce the adverse side effects of donepezil when it was administered at high doses.

[0044] Example 3 Study on the effect of P-355 on liver and kidney function

[0045] The same animals, dosage and administration method as in Example 2 were used. Whole blood was collected from animals in each group after 7 consecutive days of administration to prepare serum for the following tests.

[0046] Selection of blood biochemical indicators: Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) will overflow from damaged liver cells, so they are sensitive indicators for detecting liver cell damage. Blood urea nitrogen (BUN) and creatinine (CRE) are both commonly used clinical evaluation indicators of renal function, and their content reflects the glomerular filtration function to a certain extent. ALT, AST, BUN, and CRE kits were purchased from Nanjing Jiancheng Bioengineering Institute.

[0047] ALT kit: ALT acts on a substrate composed of alanine and α-ketoglutarate at 37°C and pH = 7.4 to generate pyruvate and glutamate. After 30 minutes of reaction, add 2,4-dinitrophenylhydrazine (DNPH) hydrochloric acid solution to terminate the reaction. At the same time, DNPH adds to the carbonyl group in the keto acid to generate pyruvate phenylhydrazone. Phenylhydrazone is reddish brown under alkaline conditions. The absorbance is tested at a wavelength of 505 nm, and the enzyme activity is calculated by inserting it into the standard curve.

[0048] AST Kit: AST can transfer the amino and keto groups of a-ketoglutarate and aspartic acid to generate glutamic acid and oxaloacetic acid. Oxaloacetic acid can decarboxylate to pyruvic acid during the reaction. Pyruvic acid reacts with DNPH to generate phenylhydrazone, which appears reddish brown in alkaline solution. Measure the absorbance at a wavelength of 510 nm, insert it into the standard curve and calculate the enzyme activity.

[0049] BUN kit: Urea is hydrolyzed by urease to produce ammonia ions and carbon dioxide. The ammonia ions react with the phenol color developer in an alkaline medium to produce a blue substance, the amount of which is proportional to the urea content. The absorbance is tested at a wavelength of 640 nm and the BUN concentration is calculated.

[0050] CRE kit: CRE generates creatine under the catalysis of creatinine amidohydrolase, creatine is hydrolyzed into sarcosine and urea under the catalysis of creatine aminohydrolase, and sarcosine is then catalyzed by sarcosine oxidase to generate glycine, formaldehyde and hydrogen peroxide. Hydrogen peroxide, 2,4-(6-triiodo-3-hydroxybenzoic acid) and 4-aminoantipyrine react under the catalysis of peroxidase to generate quinoneimine, a purple-red compound. The absorbance is measured at a wavelength of 546 nm and the CRE concentration is calculated.

[0051] Blood biochemical index test results: The experimental results show that ( Figure 3 ), the four blood biochemical indices of mice in the donepezil group and the P-355 group were not significantly different from those in the blank group. However, except for AST, the ALT, BUN and CRE levels in the donepezil group were slightly higher than those in the blank group; while compared with the donepezil group, the ALT, BUN and CRE levels in the P-355 group were slightly lower, closer to those of the blank mice. This shows that P-355 has good safety, and indicators such as ALT, BUN, and CRE indicate that its effects on liver and kidney function are smaller than those of donepezil, and it has more advantages.

[0052] Example 4 Study on the protective effect of P-355 on D-galactose-induced AD-like cell model

[0053] Modeling agent selection: D-galactose is a reducing sugar that exists naturally in the human body and is also found in 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 a large number of superoxide anions and oxygen free radicals, thereby inducing oxidative stress and inflammatory responses, damaging the structure and function of cells, leading to cell apoptosis, and ultimately producing aging-like changes and mild cognitive impairment.

[0054] In vitro cell and neuron protection experiment: D-(+)-galactose was used to construct a neuron injury model. D-(+)-galactose can cause oxidative stress and lead to neuron damage. Two neuron cell lines, PC12 (rat adrenal medulla pheochromoma cells) and HT22 (mouse hippocampal neuron cells), were selected as the research objects. Cells in the logarithmic growth phase were plated on 96-well plates. When seeding the plate, 100 µL of liquid was added to each well, and there were about 8,000 cells per well. The 96-well plate was placed in a constant temperature incubator at 5% CO2 and 37°C for 24 h. The original culture medium in the 96-well plate was discarded, and it was careful not to touch the bottom to avoid damaging the cells. In addition to the blank wells, different concentrations of the test compound solution and galactose solution were added to the wells, and the final concentrations of the compound were 0.2 and 0.5 µM; the final concentration of D-(+)-galactose was 40 mg / mL. No compound was added to the modeling group; no modeling agent and compound were added to the blank group, and the same volume of DMEM was used instead. Place the 96-well plate in a constant temperature incubator at 5% CO2 and 37°C for 24 h. Add 10 µL of CCK-8 solution to each well (do not generate bubbles) and incubate the plate at 37°C for 1 hour. Measure the absorbance at 450 nm with an ELISA reader and calculate the cell viability = [(As-Ab) / (Ac-Ab)] × 100%. Where As: absorbance of the experimental well (containing cells, culture medium, CCK-8 solution and drug solution); Ac: absorbance of the control well (containing cells, culture medium, CCK-8 solution, but no drug); Ab: absorbance of the blank well (containing culture medium, CCK-8 solution, but no cells or drug).

[0055] The experimental results showed that 40 mg / mL galactose could cause damage to PC12 and HT22 cells, and the survival rates were reduced to about 60% and 45% of the blank control group, respectively. The survival rates of PC12 and HT22 cells co-incubated with P-355 were significantly improved compared with the model group ( Figure 4 ), and the protective effect showed dose dependence. Among them, PC12 cells can recover to about 80% under the action of 0.5 μM P-355, and HT22 cells can recover to about 55% under the action of 0.5 μM P-355, indicating that P-355 can show good neuronal cell protection at a low dose and improve the survival rate of cells under oxidative stress damage.

[0056] Example 5 Study on the Effect of P-355 on Plasma Biomarkers in AD Model Mice

[0057] The same animals, dosage and administration method as in Example 2 were used. Whole blood was collected from animals in each group after 7 consecutive days of administration to prepare plasma for the following tests.

[0058] Biomarker selection: Neurofilament light (NFL) and glial fibrillary acidic protein (GFAP) are considered plasma biomarkers for AD. Increased levels of NfL or GFAP in plasma are highly correlated with cognitive decline and increased risk of AD. NFL and GFAP ELISA kits were purchased from Jiangsu Enzyme Immunity Industry Co., Ltd.

[0059] ELISA experiment: Add the sample and standard to the microwells pre-coated with the target capture antibody, add the biotinylated antibody, add the detection antibody labeled with horseradish peroxidase (HRP), incubate and wash thoroughly. Add the substrate tetramethylbenzidine (3,3',5,5'-Tetramethylbenzidine, TMB) for color development. TMB is converted to blue by peroxidase catalysis and finally converted to yellow by acid. Use an enzyme reader to measure the OD value at a wavelength of 450 nm. The color depth is positively correlated with the concentration of the substance to be detected in the sample. Substitute it into the standard curve and calculate the content of the target substance.

[0060] The experimental results showed that compared with the blank group mice, Aβ 1-42 The levels of NFL and GFAP in the plasma of mice in the model group were increased ( Figure 5 ), the increase of these two plasma markers indicates that Aβ 1-42 Modeling caused a decline in the cognitive ability of mice. Compared with the model group, administration of P-355 significantly reduced the levels of NFL and GFAP in plasma, with statistically significant differences, indicating that P-355 can improve the pathological state of AD-like mice.

[0061] In summary, P-355 is a specific PANK agonist that can significantly improve Aβ 1-42 The induced AD-like model mice had cognitive impairment, spatial exploration and memory learning ability, and reduced the expression levels of AD plasma biomarkers NFL and GFAP, without obvious toxic side effects on animals. This indicates that P-355 is a new anti-AD candidate drug that is fast, efficient, safe, and has potential clinical application value.

Claims

Application of 1.4-isopropylphenylacetylpiperazine in the preparation of anti-Alzheimer's disease drugs.

2. A pharmaceutical composition, characterized in that The invention comprises 4-isopropylphenylacetylpiperazine or a pharmaceutically acceptable salt, ester, solvate, hydrate or prodrug thereof, and at least one pharmaceutically acceptable excipient or auxiliary material, and is used for preparing a pharmaceutical preparation for resisting Alzheimer's disease.

3. The pharmaceutical composition according to claim 2, characterized in that The preparation is 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 films, suppositories, micropills, microcapsules, injections, freeze-dried preparations, liposomes or microspheres.

4. An oral solution, characterized in that The invention comprises 4-isopropylphenylacetylpiperazine or a pharmaceutically acceptable salt, ester, solvate, hydrate or prodrug thereof, and sulfobutyl-β-cyclodextrin as a solubilizer.

5. The oral solution according to claim 4, characterized in that The ratio of sulfobutyl-β-cyclodextrin to 4-isopropylphenylacetylpiperazine enables 4-isopropylphenylacetylpiperazine to have good solubility in the oral solution.

6. A method for preparing a pharmaceutical preparation, characterized in that: The method comprises the steps of mixing 4-isopropylphenylacetylpiperazine or a pharmaceutically acceptable salt, ester, solvate, hydrate or prodrug thereof with at least one pharmaceutically acceptable excipient or auxiliary material to prepare the preparation according to claim 3.

7. The preparation method according to claim 6, characterized in that: When preparing the oral solution, a step of adding sulfobutyl-β-cyclodextrin as a solubilizer is also included.

8. A pharmaceutical composition, characterized in that The invention comprises 4-isopropylphenylacetylpiperazine or a pharmaceutically acceptable salt, ester, solvate, hydrate or prodrug thereof, and at least one existing clinical anti-Alzheimer's disease drug, and is used for preparing a compound anti-Alzheimer's disease drug preparation.

9. The pharmaceutical composition according to claim 8, characterized in that The compound anti-Alzheimer's disease pharmaceutical preparation is selected from tablets, capsules, granules, dispersible tablets, effervescent tablets, oral liquids, solutions or injections.

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