Composition for treating neurodegenerative diseases as well as preparation method and application of composition

Abstract

The invention discloses a composition for treating neurodegenerative diseases as well as a preparation method and application of the composition. The composition for treating neurodegenerative diseases comprises a benzothiazole derivative, gamma-aminobutyric acid and acetyl hexapeptide-8, and the structural formula of the benzothiazole derivative is as shown in the formula I in the specification. The composition for treating neurodegenerative diseases can effectively inhibit death of nerve cells, has a remarkable treatment effect on Parkinson's disease, and can improve the symptoms of patients and improve the life quality of the patients.

Description

Technical Field

[0001] This application relates to the technical field of drugs for treating neurodegenerative diseases, and particularly to a composition for treating neurodegenerative diseases, its preparation method and application. Background Art

[0002] Neurodegenerative diseases are a group of diseases caused by the progressive degeneration of the structure or function of neurons, including Alzheimer's disease, Parkinson's disease, etc., which seriously threaten human health and bring a heavy burden to the families of patients and society.

[0003] There are many limitations in current clinical treatment drugs. For example, donepezil only delays the development of disease symptoms by inhibiting acetylcholinesterase and cannot comprehensively improve the condition of patients; memantine, as an NMDA receptor antagonist, has limited cognitive improvement effect.

[0004] Due to the diversity of pathogenic factors of neurodegenerative diseases, simply blocking one or two pathways cannot significantly reduce the overall dysfunction and loss of neurons. Therefore, it is urgent to develop a multi-target and highly effective treatment plan for neurodegenerative diseases. Summary of the Invention

[0005] The purpose of this application is to provide a new composition for treating neurodegenerative diseases, its preparation method and application.

[0006] This application adopts the following technical solutions:

[0007] One aspect of this application discloses a composition for treating neurodegenerative diseases, which includes a benzothiazole derivative, γ-aminobutyric acid and acetyl hexapeptide-8; wherein, the structural formula of the benzothiazole derivative is shown in Formula I,

[0008] Formula I

[0009] It should be noted that this application has found through research that the benzothiazole derivative shown in Formula I, when used in combination with γ-aminobutyric acid and acetyl hexapeptide-8, has a better effect of improving nerve function and cognitive ability than each component used alone; the composition of this application shows significant efficacy in treating neurodegenerative diseases, can effectively reduce the disease score, improve the symptoms of patients, and improve the quality of life of patients.

[0010] Preferably, the composition of this application is composed of 12-18 parts by weight of benzothiazole derivative, 6-9 parts by weight of γ-aminobutyric acid and 3.5-4.5 parts by weight of acetyl hexapeptide-8.

[0011] Another aspect of this application discloses a sustained-release microsphere for treating neurodegenerative diseases, and this sustained-release microsphere is a microsphere particle formed by the composition of this application and a pharmaceutically acceptable carrier.

[0012] Preferably, the pharmaceutically acceptable carrier is a mesoporous silica carrier.

[0013] Preferably, the mesoporous silica carrier has a pore diameter of 4.2 ± 0.3 nm and a pore volume of 0.85 ± 0.05 cm 3 / g.

[0014] It should be noted that by using the mesoporous silica carrier to load the composition of the present application and combining precise parameter control, the product quality can be ensured to be stable and reliable, the production efficiency can be improved, the production cost can be reduced, and technical support for large-scale production can be provided.

[0015] Preferably, the D90 of the microsphere particles is 45 - 50 μm, and the particle porosity is 29 - 35%.

[0016] Another aspect of the present application discloses the use of the composition of the present application or the sustained-release microspheres of the present application in the preparation of drugs for delaying the phosphorylation rate of tau protein, drugs for regulating the cognitive function of APOEε4 allele carriers, drugs for increasing the Aβ clearance rate and synaptic density, or drugs for improving the cognitive function of patients with neurodegenerative diseases.

[0017] Another aspect of the present application discloses a method for preparing the sustained-release microspheres of the present application, comprising the following steps:

[0018] Weigh the benzothiazole derivative, γ-aminobutyric acid, acetyl hexapeptide-8 and the mesoporous silica carrier, and mix them to form a mixed material;

[0019] Under the condition of applying pulsed DC electric field, add the mixed raw materials into a fluidized bed granulation device, and granulate according to an inlet air temperature of 45°C, an atomization pressure of 0.8 bar, and control the inlet air humidity to obtain the sustained-release microspheres.

[0020] Preferably, the frequency of the pulsed DC electric field condition is 5 kHz ± 0.1%, and the field strength is 2.5 kV / cm.

[0021] Preferably, the inlet air humidity is controlled by gradient humidity, with an initial humidity of 45% and a final humidity of 25%, and the slope from the initial humidity to the final humidity is -5% / min.

[0022] Preferably, based on the mathematical relationship between the particle porosity and the dissolution rate T50 = 0.78ε 2 -25.3ε + 210 (R 2 = 0.96) to control the process.

[0023] Preferably, the preparation method of the present application further includes real-time monitoring of the prepared sustained-release microspheres to ensure that the D90 of the sustained-release microspheres is 45 - 50 μm and the particle porosity is 29 - 35%.

[0024] Preferably, the preparation method of the present application further includes controlling the surface potential of the sustained-release microspheres by using an electrostatic eliminator.

[0025] Preferably, the surface potential of the sustained-release microspheres is controlled to be ≤ ±5 mV.

[0026] Preferably, the preparation method of the present application further includes sterilizing the sustained-release microspheres.

[0027] Preferably, the sterilization process satisfies the Arrhenius correction equation LogR = 0.45T - 18.7 + 0.05ln(C), where C is the drug concentration in mg / mL, and the applicable range is 10 - 50 mg / mL.

[0028] The beneficial effects of the present application are as follows:

[0029] The composition for treating neurodegenerative diseases of the present application can effectively inhibit the death of nerve cells, has a significant effect on the treatment of Parkinson's disease, can improve the symptoms of patients, and improve the quality of life of patients. Description of the Drawings

[0030] Figure 1 is the PXRD pattern of the benzothiazole derivative in the embodiment of the present application;

[0031] Figure 2 is the statistical result chart of the inhibition of SH-SY5Y cell death by the composition in the embodiment of the present application;

[0032] Figure 3 is the inhibitory effect of the composition on cell death treated with different concentrations of H2O2 in the embodiment of the present application;

[0033] Figure 4 is the statistical result of the cell survival rate of the culture treated with 50 μM of 6-hydroxydopamine for 24 hours in the embodiment of the present application;

[0034] Figure 5 is the statistical result of the cell survival rate of the culture treated with 125 μM of 6-OHDA for 1 hour in the embodiment of the present application;

[0035] Figure 6 is the inhibitory effect of the composition on nerve cell death in the embodiment of the present application. Detailed Embodiments

[0036] This application aims to break through the treatment dilemma of neurodegenerative diseases at the root, provide an innovative therapeutic composition, comprehensively intervene in the disease pathogenesis through multi-target synergistic effects, effectively improve the symptoms of patients, delay the disease progression, and significantly improve the quality of life of patients. At the same time, develop an advanced, efficient and controllable preparation process to ensure the stable quality of the product, meet the large-scale production requirements, and promote the substantial progress in the field of neurodegenerative disease treatment.

[0037] The composition of this application includes benzothiazole derivatives, γ-aminobutyric acid and acetyl hexapeptide-8.

[0038] Among them, the benzothiazole derivative shown in Formula I is used as a neuroprotective agent, and its monoclinic crystal form has characteristic peaks at 8.5°, 14.1°, and 17.3° in the PXRD pattern. DSC detection shows that the crystal form transformation energy barrier ΔG≥45 kJ / mol at 25°C / 60% RH, ensuring that the crystal form does not change within a storage period of ≥36 months. The benzothiazole derivative of this application combined with γ-aminobutyric acid and acetyl hexapeptide-8 can effectively resist oxidative stress damage of nerve cells, stabilize the cell membrane structure, and reduce apoptosis.

[0039] γ-aminobutyric acid, as a neurotransmitter regulator, can precisely regulate the neurotransmitter balance and stabilize the electrical activity of nerve cells. Acetyl hexapeptide-8, as a synapse regenerator, can promote synapse regeneration and repair, enhance nerve cell connection, and improve nerve conduction efficiency.

[0040] In a further improved scheme, a mesoporous silica carrier is used to carry the composition of this application. The pore diameter of the mesoporous silica carrier is 4.2±0.3 nm, and the pore volume is 0.85±0.05 cm 3 / g. It is confirmed by BET adsorption experiment that its drug loading amount reaches 38±2%, while that of ordinary carriers is only 25±3%. Using a mesoporous silica carrier can effectively encapsulate the active ingredients of the drug, improve the drug stability, promote the release and absorption of the drug in vivo, and significantly enhance the drug bioavailability.

[0041] The specific method for preparing sustained-release microspheres from the composition of this application and the mesoporous silica carrier includes the following:

[0042] Fluidized bed granulation: Apply a pulsed DC electric field (frequency 5 kHz±0.1%, field strength 2.5 kV / cm), inlet air temperature 45±2°C, atomization pressure 0.8 bar, and the inlet air humidity gradient is controlled from an initial 45%→final 25% (slope -5% / min). Establish a mathematical relationship between the particle porosity and the dissolution rate: T50 = 0.78ε 2 -25.3ε + 210 (R 2= 0.96), control the D90 of drug particles ≤ 50 μm, the particle porosity is 32 ± 3% (measured by mercury intrusion method), add an electrostatic elimination device, and control the surface potential of the particles ≤ ±5 mV.

[0043] Mixing process: Introduce a multi-directional motion mixer, define the specific energy input (SEI) to be controlled at 0.35 - 0.45 kWh / kg, and establish a mixing uniformity prediction model: RSD = 1.02e^(-0.07t) (t is the mixing time, R 2 = 0.98), ensure that the mixing uniformity RSD ≤ 5%.

[0044] Sterilization process: Use the Arrhenius correction equation: LogR = 0.45T - 18.7 + 0.05ln(C), where C is the drug concentration (mg / mL), and the applicable range is 10 - 50 mg / mL. The injection is sterilized by a two-step method, filtered and sterilized with 0.22 μm filter, and then sterilized by moist heat at 121 °C for 12 minutes, F0 ≥ 15.

[0045] Preparation of functionalized mesoporous silica carrier: The molar ratio of template CTAB to TEOS is 1:4, and the calcination procedure is to heat up to 550 °C at 2 °C / min and maintain for 4 h.

[0046] Compared with the existing drugs for treating neurodegenerative diseases, the composition or sustained-release microspheres of the present application have the following advantages:

[0047] Multi-target synergistic therapy: Benzothiazole derivatives, γ-aminobutyric acid, and acetyl hexapeptide-8 act synergistically as neuroprotective agents, neurotransmitter regulators, and synapse regenerators respectively, comprehensively intervening in the pathogenesis of neurodegenerative diseases, and effectively improving the nerve function and cognitive ability of patients.

[0048] Advanced preparation process: The innovative preparation process integrates advanced technologies and precise parameter control, ensuring stable and reliable product quality, improving production efficiency, reducing production costs, and providing technical support for large-scale production.

[0049] Significant therapeutic effect: It shows a significant curative effect in the treatment of neurodegenerative diseases, can effectively reduce the disease score, improve the symptoms of patients, and improve the quality of life of patients.

[0050] The following further elaborates the present application through specific examples. The following examples only further illustrate the present application and should not be construed as a limitation of the present application.

[0051] The materials and reagents used in the following examples, unless otherwise specified, can be obtained from commercial sources. The experimental methods used in the following examples, unless otherwise specified, are all conventional methods.

[0052] Example

[0053] I. Preparation of the sustained-release microspheres of the composition

[0054] 1. Preparation of the benzothiazole derivative

[0055] First, synthesize the intermediate 2-{[(2-methoxy-ethyl)-methyl-amino]-methyl}-3-methyl-3H-imidazole-4-carboxylic acid phenyl ester, and then prepare the benzothiazole derivative of this example from this intermediate, specifically as follows:

[0056] Under the conditions of 0 °C and argon, add a solution of 4-dimethylaminopyridine (94 mg, 0.77 mmol) and phosphonium bromide-tripyrrolidine hexafluorophosphate (790 mg, 0.70 mmol) (CAS No. 132705-51-2) in dimethylformamide (8 mL) to a solution of 1-{2-[(2-methoxy-ethyl)-methyl-amino]-ethyl}-1H-pyrazole-4-carboxylic acid (467 mg, 1.54 mmol) (purchased from Syngene) and phenol (145 mg, 1.54 mmol) in dimethylformamide (8 mL), and then add triethylamine (0.65 mL, 4.6 mmol). React at room temperature for 48 hours, treat the reaction mixture with 25 mL of saturated ammonium chloride aqueous solution and extract with ethyl acetate three times, 25 mL each time. Dry the combined organic layers with magnesium sulfate and evaporate to dryness. Purify by silica gel chromatography, and the eluent is dichloromethane containing 4% methanol to obtain a colorless oil, with a yield of 55%, that is, the intermediate of this example is obtained. Mass spectrometry detection MS: m / e = 304 (M+H + ).

[0057] At -70 °C, slowly add a solution of tert-butyllithium (1.1 mL of 1.5 M pentane solution, equivalent to 1.65 mmol) to a dry tetrahydrofuran (10 mL) solution of 4-methoxy-7-morpholin-4-yl-benzothiazol-2-ylamine (220 mg, 0.83 mmol) (purchased from Syngene), and slowly warm the formed suspension to about -30 °C. At this time, add a solution of 2-{[(2-methoxy-ethyl)-methyl-amino]-methyl}-3-methyl-3H-imidazole-4-carboxylic acid phenyl ester (252 mg, 0.83 mmol) in tetrahydrofuran (4 mL), and stir the mixture at room temperature for 1 hour. Treat the reaction mixture with saturated ammonium chloride aqueous solution (10 mL), then treat with 20 mL of ethyl acetate and collect the formed precipitate. Separate the two phases and extract the aqueous phase with ethyl acetate three times. Extract the combined organic layers with water twice, dry with magnesium sulfate and evaporate to dryness to obtain another batch of crude products. By flash silica gel chromatography, the eluent is chloroform containing 30% ethyl acetate to obtain a white solid, with a yield of 31%, that is, the benzothiazole derivative of this example is obtained. Mass spectrometry detection MS: m / e = 475 (M+H +), with a melting point of 176 - 178 °C.

[0058] The benzothiazole derivative prepared in this example was analyzed by PXRD, and the results are as Figure 1 shown. The results show that its monoclinic crystal form has characteristic peaks at 8.5°, 14.1°, and 17.3° in the PXRD pattern.

[0059] In addition, its DSC detection shows that the crystal form transformation energy barrier ΔG ≥ 45 kJ / mol at 25 °C / 60% RH.

[0060] By using high-performance liquid chromatography (HPLC) and differential scanning calorimetry (DSC) analysis methods, the dissolution rate (T50) and bioavailability of the crystal form of the benzothiazole derivative prepared in this example were analyzed. The results show that the dissolution rate (T50) of the α crystal form of the benzothiazole derivative prepared in this example is 12 min, and the bioavailability is 89%. DSC detection shows that the crystal form transformation energy barrier ΔG ≥ 45 kJ / mol at 25 °C / 60% RH. The results indicate that the benzothiazole derivative prepared in this example performs excellently in terms of dissolution rate and bioavailability, providing a guarantee for the efficient absorption and utilization of the drug.

[0061] The structural formula of the benzothiazole derivative in this example is as follows:

[0062] Formula I

[0063] 2. Preparation of Sustained Release Microspheres

[0064] Raw material preparation: Using high-precision weighing equipment, accurately weigh the benzothiazole derivative, γ-aminobutyric acid (Maclean CAS No. 56-12-2), acetyl hexapeptide-8 (Youjie Biotechnology), and mesoporous silica (Dongna Biotechnology) that meet strict quality standards. Conduct strict quality inspections on each batch of raw materials to ensure that their purity, activity, and other indicators meet the requirements. The pore diameter of the mesoporous silica carrier used in this example is 4.2 ± 0.3 nm, and the pore volume is 0.85 ± 0.05 cm 3 / g; The BET adsorption experiment confirmed that its drug loading capacity reached 38 ± 2%.

[0065] Fluidized bed granulation: Under the condition of applying a pulsed DC electric field, add the raw materials to an advanced fluidized bed granulation equipment. Granulate according to the conditions of an inlet air temperature of 45 °C, an atomization pressure of 0.8 bar, and precise control of the inlet air humidity gradient. Monitor the granulation process in real time to ensure that the D90 of the drug particles is stable at 48 μm, the particle porosity reaches 32%, and at the same time, effectively control the particle surface potential through an electrostatic elimination device.

[0066] Pulsed DC electric field: frequency 5 kHz ± 0.1%, field strength 2.5 kV / cm, inlet air temperature 45 ± 2 °C, atomization pressure 0.8 bar, inlet air humidity gradient controlled from 45% initially → 25% finally (slope -5% / min). Establish the mathematical relationship between particle porosity and dissolution rate: T50 = 0.78ε 2 - 25.3ε + 210 (R 2 = 0.96), control the drug particle D90 ≤ 50 μm, particle porosity 32 ± 3% (measured by mercury intrusion method), add an electrostatic eliminator, control the particle surface potential ≤ ± 5 mV. T50 represents the dissolution rate, and ε represents the particle porosity.

[0067] Mixing process: Use a multi-directional motion mixer for mixing, set the specific energy input to 0.4 kWh / kg. During the mixing process, use the mixing uniformity prediction model to monitor the mixing uniformity in real time, and establish the mixing uniformity prediction model: RSD = 1.02e^(-0.07t) (t is the mixing time, R 2 = 0.98), ensure that the mixing uniformity RSD is stable at 4.5%, and ensure uniform mixing of all components.

[0068] 3. Preparation of injection

[0069] According to the requirements of different dosage forms, the prepared sustained-release microspheres are made into tablets, capsules or injections, etc. through professional equipment. During the preparation process of the preparation, strictly control the environmental conditions to ensure that the product quality is not affected by external factors.

[0070] In this example, an injection is prepared for subsequent tests. The specific preparation method is as follows:

[0071] Dissolution and sterilization: Disperse the sustained-release microspheres into injection water, adopt the multi-frequency ultrasonic assisted dissolution technology, alternate the frequencies of 20 kHz / 40 kHz for 15 minutes to ensure uniform dispersion. Then filter through a 0.22 μm sterilizing filter to effectively remove microbial contamination.

[0072] Moist heat sterilization: Transfer the sterilized solution to a high-pressure sterilization device, and carry out moist heat sterilization under the appropriate conditions corresponding to the drug concentration according to the Arrhenius correction equation to ensure F0 ≥ 15 and ensure the sterility and stability of the injection. Arrhenius correction equation: LogR = 0.45T - 18.7 + 0.05ln(C), where C is the drug concentration (mg / mL), applicable range: 10 - 50 mg / mL. The moist heat sterilization condition is sterilization at 121 °C for 12 minutes.

[0073] Filling and packaging: Fill the sterilized injection into a container that has been strictly sterilized in a sterile environment, and seal and package it to ensure the quality and safety of the product during storage and transportation.

[0074] II. Stability Test

[0075] Accelerated Test: Place the prepared composition in an accelerated test environment with a temperature of 40°C and a relative humidity of 75%. Regularly use advanced analytical and detection techniques to detect its content changes, crystal form stability and other indicators. Some test results are shown in Table 1.

[0076] Table 1 Stability Test Results

[0077]

[0078]

[0079] After 12 months of accelerated test, the results show that the crystal form purity of the composition remains above 99.5%, and the content change is ≤2%; it can be seen that the crystal form of the composition prepared in this example has good stability under the accelerated test conditions, and the dissolution change is extremely small, providing a guarantee for the long-term storage and use of the drug.

[0080] Long-term Test: Place the composition in a long-term test environment with a temperature of 30°C and a relative humidity of 65%. Continuously monitor its various quality indicators, including the content of the drug active ingredient, impurity level, physical properties, etc. In this example, the situation after 12 months of placement was tested. The results show that during the 12-month long-term storage process, the various quality indicators of the composition remain stable, proving its excellent long-term stability.

[0081] III. Composition Effect Test

[0082] 1. Cell Death Inhibition Effect in Cultured Cells

[0083] In this example, the effect of the composition on human neuroblastoma cells SH-SY5Y (Procell) was tested. Specifically, the benzothiazole derivative, sustained-release microspheres, γ-aminobutyric acid, and acetyl hexapeptide-8 prepared in this example were respectively added to the SH-SY5Y culture medium at a concentration of 0.1%, and water was used as a blank control. After 24 hours of addition, 250 μm of H2O2 was added and treated for 2 hours, and the proportion of cell death was studied by MTT analysis. The statistical results of cell viability are as Figure 2 shown.

[0084] Figure 2 The results show that the sustained-release microspheres have the best inhibitory effect on cell death, significantly better than the individual thiazole derivative, γ-aminobutyric acid or acetyl hexapeptide-8.

[0085] 2. Cell Death Inhibition Effect of Different Concentrations of Hydrogen Peroxide in Cultured Cells

[0086] Based on the "1. Inhibitory effect of cell death in cultured cells", in this experiment, the cell survival rates of cells treated with 50 μm, 100 μm, 150 μm, 200 μm, 250 μm, 300 μm, 350 μm, 400 μm, 450 μm, 500 μm, 550 μm, 600 μm, 650 μm, 700 μm, 750 μm, 800 μm of H2O2 for 2 hours were respectively tested, and the experiment was divided into a sustained-release microsphere group and a water blank control group. The results are as Figure 3 shown.

[0087] Figure 3 The results show that in the treatment with H2O2 above 100 μm, the inhibitory effect of the composition in this example on cell death increased significantly, and the effects of the composition and the blank control were similar at a high concentration of up to 800 μm. Thus, it can be seen that the composition in this example can effectively inhibit cell death when the concentration of H2O2 treatment is below 800 μm.

[0088] 3. Inhibitory effect of the composition on cell death in a mouse model of stroke

[0089] In the left brain of the mouse: Inject the composition 1 mm anterior, 4 mm left lateral, and 5 mm ventral to the bregma. After 30 minutes, occlude the blood vessels connecting to the right brain. After 24 hours of reperfusion, make brain sections and perform 2,3,5-triphenyltetrazolium chloride (TTC) (GenScript Biotech) staining. In this example, a blank control without injecting the composition was set, and the same method was implemented.

[0090] The results show that 2,3,5-triphenyltetrazolium chloride staining is stained red due to the reduction of succinate dehydrogenase contained in the mitochondria of living cells, and the infarct layer where cell death has occurred is not stained. By comparing the staining results of the brain corresponding to the distance from the bregma, calculate the distance between the unstained area and the bregma. In addition, calculate the total volume of the unstained area. The results show that by applying the composition prepared in this example, it is indeed possible to significantly inhibit cell death.

[0091] 4. Inhibitory effect of the composition on oxidative stress-induced neuronal cell death

[0092] The benzothiazole derivative, sustained-release microspheres, γ-aminobutyric acid, and acetyl hexapeptide-8 prepared in this example were each applied to the SH-SY5Y culture medium in an amount of 0.1%, with water used as a blank control and levodopa tablets (Golden Deer) used as a positive control; after 20 hours of application, the cultures were treated with 50 μM of 6-hydroxydopamine (6-OHDA) for 24 hours and then with 125 μM of 6-OHDA for 1 hour. Subsequently, the proportion of cell death was studied by MTT analysis, and a group without 6-OHDA treatment was set as a control. The statistical results of the cell viability after treating the cultures with 50 μM of 6-hydroxydopamine for 24 hours are as Figure 4 shown, and the statistical results of the cell viability after treating the cultures with 125 μM of 6-OHDA for 1 hour are as Figure 5 shown.

[0093] Figure 4 and Figure 5 The results of show that the composition prepared in this example has a significant inhibitory effect on cell death, and its effect is better than that of the benzothiazole derivative, γ-aminobutyric acid, and acetyl hexapeptide-8 alone, and also better than that of levodopa, the gold standard treatment drug for Parkinson's disease.

[0094] 5. Inhibitory effect of the composition on reactive oxygen species

[0095] The benzothiazole derivative, sustained-release microspheres, γ-aminobutyric acid, and acetyl hexapeptide-8 prepared in this example were each applied to the SH-SY5Y culture medium in an amount of 0.1%, with water used as a blank control and levodopa tablets (Golden Deer) used as a positive control; after 20 hours of application, the cultures were treated with the fluorescent probe DCFA-DA (Solarbio Science & Technology) and then exposed to 40 μM of 6-OHDA for 10 minutes. Subsequently, the reactive oxygen species were measured using a fluorescence spectrophotometer.

[0096] The results show that the composition prepared in this example can significantly inhibit the generation of reactive oxygen species due to 6-OHDA, and its effect is better than that of the benzothiazole derivative, γ-aminobutyric acid, and acetyl hexapeptide-8 alone, and also better than that of levodopa, the gold standard treatment drug for Parkinson's disease.

[0097] 6. Inhibitory effect of the composition on neuronal cell death

[0098] Primary neurons of the mouse ventral midbrain were prepared from the germ cells of mice at 17 - 19 days of pregnancy. To evaluate the presence of dopamine-producing neurons in cell culture, immunostaining was performed using anti-NeuN antibody (Sigma Aldrich) and anti-TH antibody (Sigma Aldrich) to identify all neurons and dopamine-producing neurons respectively, and the cell nuclei were stained with DAPI (Beyotime). The results show that almost all neurons were confirmed to be dopamine-producing neurons.

[0099] Next, the benzothiazole derivative, sustained-release microspheres, γ-aminobutyric acid, and acetyl hexapeptide-8 prepared in this example were each applied to primary nerve cells at a dose of 0.1% and treated for 20 hours. Then, they were treated with 300 μM H2O2 for 3 hours. The proportion of cell death was studied by MTT analysis, and a blank control without H2O2 treatment was set. The cell survival rate was statistically analyzed, and the results are as Figure 6 shown.

[0100] Figure 6 The results show that there is no cell death in the sample to which the composition of this example is applied, and its cell survival rate is consistent with that of the blank control, while there is cell death to varying degrees in other experimental groups. The above results indicate that the composition of this example has good resistance to oxidative stress-induced nerve cell death.

[0101] 7. Hydroxyl radical scavenging ability of the composition

[0102] Studies have shown that mitochondrial defects or the auto-oxidation of dopamine and 6-OHDA can generate H2O2, and the presence of Fe 2+ makes it easy to generate hydroxyl radicals from H2O2. Hydroxyl radicals are the most important neurotoxic cause in dopamine neurodegeneration. In this example, 5,5-dimethylpyridine-N-oxide (DMPO) was used to evaluate the effect of the composition of this example on hydroxyl radicals by electron spin resonance (ESR)-spin trapping method. The specific steps are as follows:

[0103] In 200 μM of 100 mM phosphate buffer reaction mixture containing 25 μM of diethylenetriaminepentaacetic acid, 25 μM of FeSO4, 100 μM of H2O2, and 112.5 mM of DMSO, 1 μM, 10 μM, and 100 μM of the composition and 500 mM of thiourea were added respectively. For comparison, a phosphate buffer reaction mixture containing diethylenetriaminepentaacetic acid, FeSO4, and DMSO was prepared. Further, a phosphate buffer reaction mixture with H2O2 added thereto was prepared. These drugs and reagents were dissolved in Mili-Q water. The reaction mixture was transferred to a surface quartz cuvette and introduced into an X-band JEOL RFR-30b free radical analysis system. The hydroxyl radicals generated by the Fenton reaction of Fe 2+ and H2O2 were trapped by DMPO. One minute after adding DMPO, the stable DMPO-OH adduct was measured. The Mn 2+ signal was used as an internal standard.

[0104] The results show that the composition prepared in this example does not directly act on hydroxyl radicals, indicating that it is not just an antioxidant substance.

[0105] 8. Effect of the composition on Parkinson's disease

[0106] To evaluate the effect of the composition on Parkinson's disease in vivo, 6-OHDA was stereotaxically injected into the left unilateral midbrain of rats in this example. Then, the composition was administered together with methamphetamine at a dose of 10 μM. Administration of methamphetamine to animals induces ipsilateral movement at the injection site. Therefore, rotational behavior can be observed after administration of methamphetamine to rats.

[0107] The results showed that after administration of the composition prepared in this example, the rotational behavior of the rats was significantly inhibited, indicating that the composition of this example is extremely effective in the treatment of Parkinson's disease.

[0108] 9. Neuroprotective effect using MPTP-induced mouse model

[0109] Eight-week-old male C57BL / 6 mice (n = 94) with the strain C57BL / 6NCrljBgi, ORIENT BIO INC were used as experimental models. Mice were administered 30 mg / kg MPTP intraperitoneally once a day for 4 days. After the last administration on the fourth day, the mice were divided into three groups, and the three groups were orally administered excipient (control), 1 mg / kg of the composition of this example, and 1 mg / kg of rasagiline once a day for 10 days. The day after the last administration on the tenth day, the tail suspension test (TST) was performed on each group.

[0110] The behavior of the mice was analyzed by the tail suspension test:

[0111] According to the administered MPTP and drugs, the degree of the cause of behavioral loss was measured by the tail suspension test. The TST was performed in such a way that 7 days after the above compounds were administered to the three groups respectively, a circular stainless steel rod with a width of 1 cm was fixed at a height of 35 cm on a cage with a width of 16 cm and a height of 40 cm, and the left and right sides were covered with black wood. The movement time of the mice was measured in seconds for a total of 6 min to evaluate the effect of the drugs.

[0112] Analysis by TST showed that when the behavioral loss shown in the MPTP-administered mouse group was significant, compared with MPTP-induced mice administered rasagiline, the degree of behavior shown by MPTP-induced mice administered the composition of this example was the same as that of normal mice, indicating good behavioral recovery ability.

[0113] 10. Confirmation of the neuroprotective effect of the composition using 6-OHDA-induced rat model

[0114] It is known that 6-hydroxydopamine (6-OHDA) is a neurotoxin that increases the formation of hydroxyl radicals, and thus induces the degeneration of neurons in the substantia nigra and striatum. Hydroxyl radicals can rapidly damage the terminal parts of neurons, and thus cause the gradual loss of cells in the substantia nigra pars compacta (SNpc). This loss is known to be similar to the gradual degeneration observed in the substantia nigra and striatum of early Parkinson's disease patients.

[0115] Wistar rats provided by ORIENT BIO INC. (vehicle and composition, n = 7, rasagiline n = 6; 6 weeks old, 20 male rats) were used as an experimental model. A solution containing 20 μg / μL 6-OHDA (3 μL) was unilaterally injected into the striatum of each rat (location: -1.0 mm anterior, -3.0 mm posterior, -5.0 mm posterior abdominal) to induce the degeneration of neurons in the striatum.

[0116] The rats were divided into three groups. One hour before the administration of 6-OHDA, the three groups were orally administered vehicle (control), 1 mg / kg composition, and 1 mg / kg rasagiline, respectively, once every other day for 6 weeks. Four, five, and six weeks after the last administration on the last day, an apomorphine-induced rotation test was performed on each group. The apomorphine-induced rotation test was performed as follows: 0.5 mg / kg apomorphine was administered to each group of rats by intraperitoneal injection, each group of rats was placed on a rotarod, and their rotational movements were recorded for 45 min. The average value was determined by measuring the number of rotations per minute of each group of rats. The apomorphine-induced rotation test was completed six weeks after the last administration on the last day. After that, each group of rats was sacrificed, and immunohistochemical staining was performed using anti-tyrosine hydroxylase antibody and cresyl violet staining to verify the degree of reduction of neurons in the substantia nigra pars compacta. Serial sections of the midbrain containing the substantia nigra were prepared. The sections were placed in PBS, and the obtained sections were added to silane-coated slides and dried. The slides were placed in xylene, 100% ethanol, 95% ethanol, 70% ethanol, and distilled water for 5 min, 2 min, 1 min, 1 min, and 2 min, respectively. Then, the obtained slides were immersed in 1% cresyl violet solution for 5 min and washed with distilled water, 70% ethanol, 95% ethanol, 100% ethanol, and xylene for 2 min, 1 min, 1 min, 2 min, and 5 min, respectively. The slides were covered with cover slips and observed under a microscope equipped with a digital camera (Olympus BX-60, Olympus Optical Co., Ltd., Japan). The substantia nigra of the midbrain was observed at a magnification of 200×. Cells that were positive for cresyl violet were observed and recorded, and statistical analysis (one-way ANOVA) was performed using the Graph pad Prism 4 program.

[0117] Immunochemical staining using tyrosine hydroxylase antibody:

[0118] The changes in the expression of anti-tyrosine hydroxylase antibody in the striatum and substantia nigra were measured using immunochemical staining. Each group of mice was anesthetized with sodium pentobarbital (50 mg / kg). The chest of the mice was opened, and 200 ml of 0.1 M PBS (pH 7.4) was perfused into the heart to move the blood into the blood vessels. After all the blood was removed, 250 - 300 mL of 4% paraformaldehyde / PBS fixing solution was perfused into the heart. The brain was removed and post-fixed with paraformaldehyde / PBS fixing solution under freezing conditions for 24 h. Then, the brain tissue was thoroughly washed with PBS to remove the fixing solution to prevent ice crystallization during the freezing process. The obtained brain tissue was placed in 30% sucrose solution and stored until it sank. The obtained tissue was embedded with a freezing embedding agent (OCT compound) and frozen at -40 °C. Serial coronal sections of the midbrain containing the striatum and substantia nigra with a thickness of 40 μm were prepared using a cryostat (Reichert Frigocut model 2000). The coronal sections were maintained in 3% H2O2 / PBS for 30 min, and then maintained in 0.1 M PBS containing 0.3% Triton X-100 and 3% bovine serum albumin for 30 min. To selectively stain the cells containing dopamine, the sections were reacted with an anti-mouse monoclonal antibody TH (Chemicon International, Temecula, CA; 1:500) as the primary antibody overnight at room temperature. Biotinylated goat anti-mouse IgG (Vector Lab, Burlingame, CA, 1:200) was used as the secondary antibody. Then, the avidin-biotin binding was induced using a Vectastain elite ABC kit (Vector Lab, Burlingame, CA), and the tissue was stained and visualized with 3,4-diaminobenzidine (DAB). The obtained tissue was placed in PBS, mounted on a glass slide, the obtained product was dried, and covered with a coverslip. The substantia nigra of the midbrain of the obtained product was observed using a microscope equipped with a digital camera (Olympus BX-60, Olympus Optical Co., Ltd., Japan) with a magnification of 200×. The cells that were positively reactive to the anti-tyrosine hydroxylase antibody were observed and recorded, and statistical analysis (one-way ANOVA) was performed using the Graph pad Prism4 program.

[0119] The results showed that the 6-OHDA-induced group of rats administered with the composition of this example showed a significantly reduced degree of neuronal reduction, which was better than that of the 6-OHDA-induced group of rats administered with rasagiline.

[0120] The above content is a further detailed description of the present application in combination with specific embodiments, and it cannot be determined that the specific implementation of the present application is only limited to these descriptions. For those of ordinary skill in the technical field to which the present application belongs, without departing from the concept of the present application, several simple deductions or substitutions can be made.

Claims

1. A composition for treating neurodegenerative diseases, characterized in that: These include benzothiazole derivatives, gamma-aminobutyric acid, and acetyl hexapeptide-8; The structural formula of the benzothiazole derivative is shown in Formula 1, 2. The composition according to claim 1, characterized in that: The invention comprises 12-18 parts by weight of benzothiazole derivatives, 6-9 parts by weight of γ-aminobutyric acid and 3.5-4.5 parts by weight of acetyl hexapeptide-8.

3. A sustained-release microsphere for treating neurodegenerative diseases, characterized in that: Microsphere particles formed by the composition according to claim 1 or 2 and a pharmaceutically acceptable carrier.

4. The sustained-release microspheres according to claim 3, characterized in that: The pharmaceutically acceptable carrier is a mesoporous silica carrier.

5. The sustained-release microspheres according to claim 4, characterized in that: The pore size of the mesoporous silica carrier is 4.2±0.3 nm and the pore volume is 0.85±0.05 cm 3 / g.

6. The sustained-release microspheres according to any one of claims 3 to 5, characterized in that: The D90 of the microsphere particles is 45-50 μm, and the particle porosity is 29-35%.

7. Use of the composition of claim 1 or 2, or the sustained-release microspheres of any one of claims 3 to 6, in the preparation of a drug for slowing down the phosphorylation rate of tau protein, a drug for regulating cognitive function in APOEε4 allele carriers, a drug for increasing Aβ clearance and synaptic density, or a drug for improving cognitive function in patients with neurodegenerative diseases.

8. The method for preparing the sustained-release microspheres according to any one of claims 3 to 5, characterized in that: The following steps are included: Weighing benzothiazole derivatives, γ-aminobutyric acid, acetyl hexapeptide-8 and a mesoporous silica carrier and mixing them to prepare a mixed material; Under the condition of applying a pulsed direct current electric field, the mixed raw materials were added into a fluidized bed granulation equipment, and granulation was performed at an inlet air temperature of 45° C., an atomization pressure of 0.8 bar, and the inlet air humidity was controlled to obtain sustained-release microspheres.

9. The preparation method according to claim 8, characterized in that: The pulsed DC electric field condition has a frequency of 5kHz±0.1% and a field strength of 2.5kV / cm; Preferably, the inlet air humidity is controlled by gradient humidity, with an initial humidity of 45%, a final humidity of 25%, and a slope from the initial humidity to the final humidity of -5% / min; Preferably, based on the mathematical relationship between particle porosity and dissolution rate, T50 = 0.78 ε 2 -25.3ε+210(R 2 =0.96) control process.

10. The preparation method according to claim 8 or 9, characterized in that: It also includes real-time monitoring of the prepared sustained-release microspheres to ensure that the D90 of the sustained-release microspheres is 45-50 μm and the particle porosity is 29-35%; Preferably, the method further comprises using an electrostatic eliminator to control the surface potential of the sustained-release microspheres; Preferably, the surface potential of the sustained-release microspheres is controlled to be ≤±5mV; Preferably, the method further comprises sterilizing the sustained-release microspheres; Preferably, the sterilization process satisfies the Arrhenius correction equation LogR=0.45T-18.7+0.05ln(C), wherein C is the drug concentration in mg / mL, and the applicable range is 10-50 mg / mL.

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