WO2023078325A1 - 一种含有依达拉奉右莰醇组合物在治疗认知障碍中的应用 - Google Patents

一种含有依达拉奉右莰醇组合物在治疗认知障碍中的应用 Download PDF

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WO2023078325A1
WO2023078325A1 PCT/CN2022/129456 CN2022129456W WO2023078325A1 WO 2023078325 A1 WO2023078325 A1 WO 2023078325A1 CN 2022129456 W CN2022129456 W CN 2022129456W WO 2023078325 A1 WO2023078325 A1 WO 2023078325A1
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edaravone
dexborneol
composition
mice
test
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French (fr)
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张正平
王磊
陈荣
杨士豹
任晋生
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南京宁丹新药技术有限公司
先声药业有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/415—1,2-Diazoles
    • A61K31/4152—1,2-Diazoles having oxo groups directly attached to the heterocyclic ring, e.g. antipyrine, phenylbutazone, sulfinpyrazone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia

Definitions

  • the invention belongs to the field of pharmacy and relates to the application of the composition of 3-methyl-1-phenyl-2-pyrazolin-5-one or a pharmaceutically acceptable salt thereof and borneol in treating cognitive impairment.
  • Dementia is a syndrome in which acquired cognitive function impairment is the core and leads to significant decline in patients' daily life, learning, work and social interaction skills.
  • the cognitive function impairment of patients involves memory, learning, orientation, comprehension, judgment, calculation, language, visuospatial function, analysis and problem-solving ability, and is often accompanied by mental, behavioral and personality abnormalities at a certain stage of the disease process.
  • Dementia includes degenerative dementia and non-degenerative dementia, the former includes Alzheimer (AD), Lewis dementia (DLB), frontotemporal lobar degeneration (FTLD), etc.; the latter includes vascular dementia (VaD), normal pressure hydrocephalus Dementia caused by water and other diseases such as craniocerebral injury, infection, immunity, tumor, poisoning and metabolic diseases (2018 Chinese diagnostic guidelines for dementia and cognitive impairment, Chinese Journal of Medicine, 2018.98(13), p:965-970 ).
  • AD Alzheimer's disease
  • the most common early symptom of AD is loss of short-term memory (difficulty remembering recent events).
  • symptoms may gradually appear and include language impairment, disorientation (including getting lost easily), emotional lability, loss of motivation, inability to Self-care and many behavioral issues.
  • the condition worsens, the patient is often disconnected from family or society, and gradually loses physical function, eventually leading to death.
  • the course of the disease varies from person to person, the average remaining life after diagnosis is about 3 to 9 years.
  • AD patients worldwide According to the "2021 World Alzheimer's Report" released by the International Alzheimer's Association, as of 2019, there were 55 million Alzheimer's patients worldwide, and with the trend of population aging, it is expected that by 2030 AD patients worldwide will reach 78 million, and it is expected to reach 129 million by 2050. China has entered a stage of rapid population aging, and the number of elderly people continues to rise. As of the end of 2018, my country's elderly population aged 60 and over reached 249 million, accounting for 17.9% of the total population; the elderly population aged 65 and over reached 166 million, accounting for 11.9% of the total population.
  • 3-Methyl-1-phenyl-2-pyrazolin-5-one is a small molecular compound that can scavenge free radicals.
  • Edaravone can concentration-dependently inhibit the aggregation of A ⁇ 1-42 monomers into A ⁇ fibrils and depolymerize A ⁇ fibrils in the concentration range of 0.156-5 ⁇ M; Reduces A ⁇ 1-42 neurotoxicity in SH-SY5Y and primary cortical neurons, improves cell viability and maintains neurite outgrowth.
  • edaravone In SY-SY5Y (SH-SY5Y-APP695) stably expressing APP695 protein, edaravone (0.3-3 ⁇ M) can concentration-dependently reduce the expression of BACE1 and the cleavage of APP to generate A ⁇ , inhibit the activation of GSK-3 ⁇ and reduce the level of tau phosphate (Proc Natl Acad Sci USA, 2015.112(16):p.5225-30). In addition, edaravone can reduce H 2 O 2 and glutamate-induced death of primary hippocampal neurons, protect neuronal synapse growth, reduce excessive oxidation and excitotoxicity (Neurotoxicology, 2021.85: p.68-78 ).
  • Edaravone can improve the cognitive function of various dementia model rats or mice, and reduce brain oxidation indicators, neuroinflammation, A ⁇ plaque progression, tau phosphorylation levels, etc.
  • oral administration of edaravone 15 mg/kg/day for 7 consecutive days can improve the spatial learning and long-term memory impairment induced by scopolamine (International Journal of Pharmacology, 2013.9 (4): p. 271-276).
  • sporadic AD sporadic AD
  • ICV-STZ sporadic AD
  • edaravone 9 mg/kg/day was administered intraperitoneally for 14 consecutive days after ICV-STZ model establishment, and Significantly improved ICV-STZ model rats in the water maze test (Morris water maze test) spatial learning and memory ability, and non-spatial long-term memory ability in the step-down passive avoidance test (step-down passive avoidance test); and can significantly restore Changes in the levels of various oxidative stress indicators in the hippocampus and cortex caused by STZ modeling, including malondialdehyde (MDA), 4-HNE adducts, OH, protein carbonyl (PC), H 2 O 2 , superoxide Dismutase (SOD), reduced glutathione (GSH), glutathione peroxidase (GPx) (NeuroToxicology, 2013.38: p.136-145).
  • MDA malondialdehyde
  • SOD superoxid
  • edaravone (1, 3 and 10 mg/kg/day) for 28 consecutive days can also improve the cognitive function of ICV-STZ rats in the water maze and passive avoidance test, and reduce the STZ-induced brain damage. Oxidative stress, cholinergic abnormalities and inflammation (Eur J Neurosci, 2017.45(7):p.987-997).
  • intraperitoneal administration of edaravone for 15 days significantly improved the cognitive function of mice in the water maze and platform jumping test (Neurotoxicology, 2021.85: p.68 -78).
  • Edaravone 12.6mg/kg (peritoneal injection, 2 times a week) preventive administration (3-9 months old) and therapeutic administration (9-12 months old) can Improve the cognitive function deficits of mice in the water maze test, reduce the deposition of A ⁇ plaques in the brain, neuron and synapse loss and neuroinflammation, tau protein phosphorylation (pSer396, pSer262, pSer199 and pThr231) and oxidation Stress etc.
  • oral administration of 33.2mg/kg/day (administration in drinking water) to mice (month-old 3-12 months) can also reduce the intracerebral A ⁇ plaque deposition and improved cognitive function in mice (Proc Natl Acad Sci USA, 2015.112(16):p.5225-30).
  • oral administration of edaravone (8, 24, and 72 mg/kg/day, administered in drinking water) in APP/PS aged mice (age 14-17 months) significantly improved the cognitive function of aged transgenic mice (Drug Design, Development and Therapy, 2018. Volume 12: p. 2111-2128).
  • FTD frontotemporal dementia
  • Dexborneol ((+)-borneol) is the main component of natural borneol (the 2020 Chinese Pharmacopoeia stipulates that the content of dexborneol in natural borneol should not be less than 96%). It is a bicyclic monoterpene compound that exists in many Chinese herbal medicines in the volatile oil.
  • Dextamphenol can regulate NF- ⁇ B and p38 signaling pathways, inhibit inflammatory neuroinflammation, and reduce various pro-inflammatory factors (TNF- ⁇ , IL-1 ⁇ , etc.) and inflammatory proteins (iNOS and COX-2) in the brain (Eur J Pharmacol, 2014.740: p.522-31; Eur J Pharmacol, 2017.811: p.1-11); in addition, it can stimulate the inhibitory GABAAR receptor, which can regulate nerve excitability damage (Front Pharmacol, 2021.12: p.606682 ).
  • Non-clinical animal experiments show that the composition of 3-methyl-1-phenyl-2-pyrazolin-5-one and dexborneol (mass ratio 4:1 ⁇ 1:1) can synergistically reduce the size of cerebral infarction ( Authorized patent CN101848711B) and improve amyotrophic lateral sclerosis (ALS) (authorized patent CN107613976B).
  • edaravone dexborneol concentrated solution for injection (3-methyl-1-phenyl-2-pyrazolin-5-one and dexborneol mass ratio 4:1 composition ) has been approved by NMPA in 2020 for the treatment of ischemic stroke;
  • Y-2 sublingual tablet (3-methyl-1-phenyl-2-pyrazolin-5-one and dexborneol mass ratio 5 :1 composition) has been conducted in Phase III study in China (China Clinical Registry No. CTR20210233) and Phase I study in the United States (ClinicalTrials.govNCT03495206).
  • the purpose of the present invention is to provide a pharmaceutical composition used in the preparation of drugs for improving or treating cognitive dysfunction, said pharmaceutical composition containing 3-methyl-1-phenyl-2-pyrazolin-5-one (Edaravone) or its pharmaceutically acceptable salt and borneol.
  • said pharmaceutical composition containing 3-methyl-1-phenyl-2-pyrazolin-5-one (Edaravone) or its pharmaceutically acceptable salt and borneol.
  • Another object of the present invention is to provide an application of a pharmaceutical composition in improving or treating cognitive dysfunction, wherein the administration of 3-methyl-1-phenyl-2-pyrazoline- A pharmaceutical composition of 5-ketone or its pharmaceutically acceptable salt and borneol.
  • Another object of the present invention is to provide a pharmaceutical composition for the treatment of cognitive dysfunction, wherein the composition comprises 3-methyl-1-phenyl-2-pyrazoline-5- Ketone or its pharmaceutically acceptable salt and borneol.
  • Another object of the present invention is to provide the application of 3-methyl-1-phenyl-2-pyrazolin-5-one or its pharmaceutically acceptable salt in combination with natural borneol for the treatment of cognitive dysfunction.
  • the cognitive dysfunction is Alzheimer's disease (AD), vascular dementia (VD), mild cognitive impairment (MCI) and other types of dementia; preferably, the The aforementioned cognitive impairments are Alzheimer's disease, vascular dementia, and mild cognitive impairment.
  • the pharmaceutical composition can improve cognitive function in Alzheimer's disease.
  • the pharmaceutical composition can restore the long-term potentiation (LTP) in the hippocampal region of Alzheimer's disease, and restore the function of the hippocampal synapse in the brain.
  • LTP long-term potentiation
  • the pharmaceutical composition can improve the pathology of Alzheimer's disease, including amyloid plaques, astrogliosis, microgliosis, tau protein hyperphosphatemia and the like.
  • the weight ratio of 3-methyl-1-phenyl-2-pyrazolin-5-one or its pharmaceutically acceptable salt:borneol is 10:1 ⁇ 1:8; preferably 7.5:1 to 2.5:1; more preferably 5:1 or 4:1.
  • the borneol of the present invention is preferably natural borneol, which is also called dexborneol.
  • composition of the present invention may further include pharmaceutically acceptable excipients.
  • Excipients are generally pharmacologically inactive substances formulated with the active pharmaceutical ingredient ("API") of the drug therapy.
  • API active pharmaceutical ingredient
  • excipients are often used to bulk up the formulation containing the active active ingredient (thus often referred to as “bulking agents", “fillers” or “diluents”) to allow convenient and accurate Allocation of APIs. It can also serve different therapeutic enhancing purposes, such as facilitating drug absorption or dissolution, or other pharmacodynamic considerations.
  • composition of the present invention can be formulated as a pharmaceutical composition alone or in combination with a pharmaceutically acceptable carrier or diluent, and can be formulated as a preparation in solid, semi-solid, liquid or gaseous form, Such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres and aerosols.
  • administration of the compounds can be accomplished in a variety of ways, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, transdermal, intraductal, intraarticular, and the like.
  • Administration of the active agent can be systemic, or can be localized through the use of regional administration, intramural administration, or the use of implants that retain the active dose at the implantation site.
  • Combination therapies used in the present invention may allow lower doses of each monotherapy than are currently used in standard practice, while achieving significant efficacy, including efficacy over that when either monotherapy is routinely administered. Dosage levels may vary with the particular compound, the severity of symptoms and the susceptibility of the subject to side effects, as will be readily appreciated by those skilled in the art. Some specific compounds are more effective than others. Preferred dosages for a given compound can be readily determined by one of skill in the art by a variety of means. A preferred means is to measure the physiological potency of a given compound. The use of combination therapy may allow lower doses of each monotherapy than is currently used in standard practice, while achieving significant efficacy, including greater efficacy than is achieved when either monotherapy is routinely administered.
  • edaravone dexborneol composition protects neuron damage induced by A ⁇ 1-42 ;
  • Fig. 2 edaravone dexborneol composition improves ICV-STZ spatial learning and memory cognitive function
  • Fig. 6 Effect of edaravone-dexamphenol composition on AD-related pathology in the brain of 5 ⁇ FAD transgenic mice.
  • Example 1 The combination of edaravone and dexborneol protects primary cortical neuron damage induced by A ⁇ 1-42
  • Pregnant SD rats at 18 days of pregnancy were sacrificed by cervical dislocation, and the uterus was dissected to take out the brain of E18 fetal rats.
  • the cerebral cortex tissue of the fetal rats was separated into ice-cold DMEM, and the meninges and blood vessels on the cortical tissue were removed under a dissecting microscope. Cut into pieces (about 1 mm 3 ) in ice-cold DMEM, digest with trypsin at 37°C for 10 minutes, stop digestion with FBS, blow gently with a Pasteur pipette and pass through a 200-mesh sieve. Transfer the filtered cell suspension to a 15mL centrifuge tube, and centrifuge at 1000rpm for 5min.
  • the culture medium of primary neurons matured in vitro was replaced with different concentrations (1.2, 3.7, 11.1, 33.3, 100.0 ⁇ M) of edaravone (E), dexborneol (B) or edaravone-dexborneol
  • E edaravone
  • B dexborneol
  • oligomerized A ⁇ 1-42 final concentration 50 ⁇ M
  • neuron relative activity V (%) (LUM-LUM background )/(LUM normal control group -LUM background group ) ⁇ 100%.
  • the LUM background is the background reading of the Luminescent Cell Viability Assay reagent added to the cell-free complete medium well.
  • E stands for Edaravone
  • B stands for Dexborneol
  • ExBy stands for the combination of Edaravone and Dexborneol
  • x/y data 1-5 is the concentration of each compound, respectively 1.2, 3.7, 11.1, 33.3 , 100.0 ⁇ M.
  • Oligomerized A ⁇ 1-42 leads to decreased cell viability in primary neuronal viability.
  • Pre-incubation administration of edaravone (3.7-100 ⁇ M) or dexborneol (1.2-100 ⁇ M) before A ⁇ 1-42 induction can concentration-dependently inhibit A ⁇ -induced neurotoxicity and improve cell viability.
  • the orthogonal composition of different concentrations of edaravone (1.2, 3.7, 11.1, 33.3, 100.0 ⁇ M) and dexaborneol (1.2, 3.7, 11.1, 33.3, 100.0 ⁇ M) can inhibit A ⁇ from damaging neurons, especially E5B4 ( 3.3:1), E5B3(9:1), E4B5(1:3.3), E4B4(1:1), E4B3(3.3:1), E3B5(1:9), E3B4(1:3.3), E3B3(1 :1), E3B2(3.3:1), E3B1(9:1) and E2B2(1:1) combinations provided significantly better protection than the corresponding individual components (Error! Reference source not found.). Therefore, it is preliminarily judged that the composition of edaravone and dexborneol in the molar ratio range of 9:1-1:9 (mass ratio 10:1-1:8) has a synergistic effect.
  • Example 2 Composition of Edaravone and Dexborneol Improves Cognitive Function of Dementia Rats Induced by Intracerebroventricular Injection of STZ
  • Wistar rats Wistar rats, male, weighing 250-300 g, 110 rats, clean grade, provided by Shanghai Slack Experimental Animal Co., Ltd., license number: SCXK (Shanghai) 2012-0002. Routine feeding before and after the operation, the room temperature was kept at 23-25°C, and free access to food and water.
  • the experimental animals were divided into 5 groups, namely sham operation group, model group, Edaravone group (6mg/kg), Edaravone dexborneol (7.5:1) composition (Edaravone 6mg/kg, right Dexborneol 0.8mg/kg), Edaravone Dexborneol (5:1) composition (Edaravone 6mg/kg, Dexborneol 1.2mg/kg), Edaravone Dexborneol (2.5: 1) Composition (edaravone 6 mg/kg, dexborneol 2.4 mg/kg). Animals in the model and sham operation groups were given corresponding volumes of blank solvent (8% propylene glycol), and all administrations were administered by intraperitoneal injection, and the administration volume was 10 mL/kg.
  • Animals in the model and sham operation groups were given corresponding volumes of blank solvent (8% propylene glycol), and all administrations were administered by intraperitoneal injection, and the administration volume was 10 mL/kg.
  • the animals with successful operation were randomly assigned to the model and drug administration groups in sequence.
  • the animals in each group were intravenously injected with the corresponding drugs 4 hours after the operation, and then administered once a day for 14 consecutive days.
  • the Morris water maze test was started on the 15th day. Administer once a day before the experiment until the end of the experiment as a whole. Animals in each group were tested in the water maze, and the detection indicators were the hidden platform escape latency and the residence time in the quadrant of the original platform after the platform was withdrawn.
  • the rats were anesthetized, they were placed prone on a stereotaxic apparatus, and a dorsal neck midline incision was made to expose the skull.
  • the anterior bregma was used as the coordinate, and the posterior ventricle was 0.8mm, 1.5mm lateral, and 3.6mm deep as the lateral ventricle injection point.
  • the skin was sutured after injection.
  • the dose on the first day is 1.5mg/kg, repeat the STZ injection method on the first day on the third day, then the total amount of STZ injection is 3mg/kg.
  • the animals in the sham-operated group were injected with an equal volume of artificial cerebrospinal fluid into the bilateral lateral ventricle.
  • the Morris water maze pool has a diameter of 160cm, a height of 50cm, and a water depth of 29cm.
  • the platform has a diameter of 12cm and a height of 27cm, and is fixed 2cm below the water surface in the SE quadrant (target quadrant).
  • the camera lens was placed above the center of the pool at a distance of 2m from the bottom of the pool to simultaneously record the movement trajectory of the rats.
  • the water temperature was kept at 22 ⁇ 1°C.
  • the experiment lasted for 5 days, including the hidden platform test and the space exploration test. The first 4 days are hidden platform experiments, and the last day is space exploration experiments.
  • the experiment lasted for 4 days.
  • the position of the platform was fixed in the target quadrant (SE quadrant), and the water was entered from the water entry points of the four quadrants respectively.
  • the animals were gently put into the water facing the pool wall mark, and the time when the animals found the platform was recorded. (escape latency, escape latency), and then allow the animal to stay on the platform for 10s. If the platform cannot be found within 90s, the incubation period is recorded as 90s, and the animal guide is placed on the platform to stay for 10s.
  • the rats were put back into the cage and kept warm. Train once a day at each of the 4 water entry points, and use the average of the 4 incubation periods as the results of the day for statistical analysis. The order of water entry points for each animal was consistent every day, but different animals in the same cage entered the water in different order to eliminate the communication of animal information within the group.
  • the platform was removed 24 hours after the concealed platform test. Then enter the water from the water entry point on the opposite side of the platform, record the swimming path of the rat within 120s, and perform statistical analysis on the residence time of the rat in the target quadrant. Observe the spatial orientation ability of the tested animals and the change rule in the process of space exploration.
  • the retention time of the target quadrant of ICV-STZ model rats was significantly shortened.
  • the rats in the intraperitoneal injection of edaravone (6mg/kg) or edaravone dexborneol composition (7.5:1, 5:1 and 2.5:1) retained in the target quadrant
  • the time was significantly prolonged, and the edaravone dexborneol composition (5:1 and 2.5:1) group was significantly better than the edaravone group (error! Reference source not found. B). Therefore, the edaravone dexborneol composition (7.5:1, 5:1 and 2.5:1) can significantly improve the cognitive function deficit of ICV-STZ rats, and its efficacy is significantly better than that of edaravone.
  • Example 3 Effects of long-term administration of edaravone-dexborneol composition on AD cognitive function and AD pathology in 5 ⁇ FAD transgenic mice
  • Wild-type WT mice and 5 ⁇ FAD transgenic mice were provided by Sima Jian’s research group at China Pharmaceutical University.
  • Edaravone that is, Edaravone injection (must store , Simcere Pharmaceuticals), the active ingredient 3-methyl-1-phenyl-2-pyrazolin-5-one.
  • Edaravone dexborneol composition i.e. Edaravone dexborneol concentrated solution for injection (cementexin , Simcere Pharmaceuticals)
  • the mass ratio of 3-methyl-1-phenyl-2-pyrazolin-5-one to dexborneol is 4:1.
  • the solvent control was 8% propylene glycol.
  • the experiment was divided into 4 groups.
  • the mice in each group were administered from the age of 8 weeks. After 16 weeks of continuous daily administration, the water maze and Y maze behavioral experiments were carried out. After the behavioral detection was completed, the animals were killed to detect the electrophysiological and AD pathology in the brain, including detection of A ⁇ plaques, biomarkers such as GFAP and Iba-1, and p-Tau, etc.
  • WT solvent control group: 10 wild-type mice were given i.p. solvent control at a dose of 10 mL/kg, once a day.
  • 5 ⁇ FAD solvent group: 10 5 ⁇ FAD mice were given i.p. the solvent control at a dose of 10 mL/kg, once a day.
  • Edaravone group 10 5 ⁇ FAD mice were given Edaravone ip (must store )6mg/kg, the dose is 10.0mL/kg, once a day.
  • mice Before the start of the experiment, the mice need to be placed in the test room for about 30 minutes to adapt to the environment.
  • Experiment preparation pour clean water into the constant temperature pool (120cm inner diameter, 50cm height) of the water maze, control the water temperature at about 18-22°C, and select the four vertices of the pool as “S”, “N”, “W”, “ E", the platform is placed in the "SW” direction and 1-2cm below the water surface, adding titanium dioxide to the water to make the water opaque milky white.
  • Parameter setting the test time is set to 1min, and the time for mice to stay on the platform is set to 2s.
  • mice The first 5 days were the training phase of the water maze. The mice were placed in the water pool from four different positions every day, and their trajectory, time, and distance and time in each quadrant were recorded before they boarded the platform. For mice that have not found the platform within 1 minute, guide them to the platform after 1 minute and stay there for 10-15 seconds. The specific orientation of the mice is shown in Table 1.
  • Test Test on the first day after training. During the test, the platform was removed, the test time was 60s, the mice were placed in the NE position, and the number of times the mice crossed the platform and the time spent in the quadrant of the platform during the test was recorded.
  • mice Before the start of the experiment, the mice need to be placed in the test room for about 30 minutes to adapt to the environment.
  • Training block one arm of the Y maze with a baffle, place the mouse at the end of one of the remaining two arms, let it freely explore for 15 minutes and record it.
  • Test After training for 1 hour, remove the baffle, wipe the channel of the Y maze with alcohol to remove the smell of the last mouse, put the mouse to be tested in the same position for 5 minutes and record the time, and record the mouse for the test Entries into newly opened arms as a percentage of the total number of entries into the three arms.
  • Electrophysiological recordings were performed after the behavioral testing of the mice. All mice were euthanized after anesthesia, and brain tissue was collected rapidly and stored in artificial cerebrospinal fluid (ACSF) (75 mM sucrose, 87 mM NaCl, 2.5 mM KCl) at 4 °C in oxygenated (95% O 2 /5% CO 2 ), 1.25 mM NaH 2 PO 4 , 21.4 mM NaHCO 3 , 0.5 mM CaCl 2 , 7 mM MgCl 2 and 20 mM glucose). Hippocampal slices were cut by vibrating knife in oxygenated ACSF at 4 °C and oxygenated ACSF at 32 °C. Next, slices were incubated at room temperature for 1 hr before recording. Hippocampal LTP was induced by two electrical theta burst stimulation (TBS). Data acquisition and analysis were performed using Clampex and Clampfit 8.2 (Molecular Devices).
  • mice were perfused with the heart and the brain was taken out, fixed in 4% paraformaldehyde for 24 hours, dehydrated with 30% sucrose, embedded in quick-frozen embedding medium, and sliced (thickness: 30 ⁇ M).
  • 5 ⁇ FAD mice were given 6mg/kg edaravone or 7.5mg/kg edaravone dexborneol composition by intraperitoneal injection every day from the age of 8 weeks (containing 6mg/kg edaravone and 1.5mg/kg dexaborneol) 1 time, continuous administration for 16 weeks, can significantly shorten the escape latency of 5xFAD mice in the water maze concealed platform test (error! No reference source was found. A), and prolong the withdrawal period in the withdrawal test. Target quadrant dwell time and number of platform crossings (ERROR! Reference source not found. B, C).
  • the administration of the composition had a shorter escape latency time on the 5th day of the hidden platform test, a significantly longer residence time in the target quadrant in the withdrawal platform test, and a higher number of times of crossing the platform. Multiple (Error! Reference source not found. A, B and C). This shows that both Edaravone and the Edaravone composition can improve the learning, memory and cognitive functions of 5 ⁇ FAD mice in the water maze test, and the improvement effect of the composition is significantly better than that of Edaravone.
  • the edaravone dexborneol composition has a significantly higher efficacy in improving the new arm entry rate of 5 ⁇ FAD mice (error! Reference source not found.). This shows that both Edaravone and the Edaravone composition can improve the spatial reference memory cognitive function of 5 ⁇ FAD mice in the Y maze test, and the improvement effect of the composition is significantly better than that of Edaravone.
  • LTP Long-term potentiation
  • the 24-week-old 5 ⁇ FAD mice had already developed obvious AD pathology in the brain, including ⁇ -amyloid plaques, astrogliosis, microgliosis, and hyperphosphorylation of tau protein in neuronal cells in the hippocampus.
  • 5 ⁇ FAD mice were administered intraperitoneally with 6 mg/kg edaravone or 7.5 mg/kg edaravone dexborneol composition (containing 6 mg/kg edaravone) every day from the age of 8 weeks.
  • Bong and 1.5mg/kg dexborneol) 1 time, continuous administration for 16 weeks, can significantly reduce AD pathology ( ⁇ -amyloid plaque, astrocyte hyperplasia, microgliosis) and hippocampal AD pathology in the brain tau phosphorylation level.
  • AD pathology ⁇ -amyloid plaque, astrocyte hyperplasia, microgliosis
  • hippocampal AD pathology in the brain tau phosphorylation level.
  • the edaravone dexborneol composition improved AD pathology (beta amyloid plaques, astrogliosis, microgliosis) and reduced tau phosphorylation levels
  • Error! No reference source found. There is a significant advantage in the efficacy of (Error! No reference source found.).

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Abstract

一种组合物在改善或治疗认知障碍中的应用，该组合物含有3-甲基-1-苯基-2-吡唑啉-5-酮或其药学上可接受的盐和冰片或右莰醇，该认知障碍包括阿尔茨海默病（AD）、血管性痴呆症（VD）、轻度认知障碍（MCI）和其他类型痴呆。

Description

一种含有依达拉奉右莰醇组合物在治疗认知障碍中的应用
本申请要求于2021年11月08日提交中国专利局、申请号为202111311631.1、发明名称为“一种含有依达拉奉右莰醇组合物在治疗认知障碍中的应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明属于制药领域，涉及3-甲基-1-苯基-2-吡唑啉-5-酮或其药学上可接受的盐和冰片的组合物在治疗认知障碍中的应用。
背景技术
痴呆(dementia)是一种以获得性认知功能损伤为核心，并导致患者日常生活能力、学习能力、工作能力和社会交往能力明显减退的综合征。患者的认知功能损害涉及记忆、学习、定向、理解、判断、计算、语言、视空间功能、分析及解决问题的能力，在病程某一阶段常伴有精神、行为和人格异常。痴呆包括变性病和非变性病痴呆，前者包括阿尔茨海默(AD)、路易斯痴呆(DLB)、额颞叶变性(FTLD)等；后者包括血管性痴呆(VaD)、正常压力性脑积水以及其他疾病如颅脑损伤、感染、免疫、肿瘤、中毒和代谢性疾病引起的的痴呆(2018年中国痴呆与认知障碍诊断指南，中华医学杂志，2018.98(13),p:965-970)。
AD是一种发病进程缓慢、随着时间不断恶化的神经退行性疾病，是目前唯一无法治愈、预防甚至延缓病情发展的疾病。2020年WHO公布的全球前十位死亡原因中AD位列第七。AD最常见的早期症状为丧失短期记忆(难以记住最近发生的事)，当疾病逐渐进展，症状可能逐渐出现，包括语言障碍、定向障碍(包括容易迷路)、情绪不稳、丧失动机、无法自理和许多行为问题。当情况恶化时，患者往往会因此和家庭或社会脱节，并逐渐丧失身体机能，最终导致死亡。虽然疾程因人而异，但诊断后的平均余命约为3～9年。
根据国际阿尔茨海默症协会发布的《2021年世界阿尔茨海默症报告》，截至2019年，全球共有5500万阿尔茨海默症患者，而随着人口老龄化的趋势，预计到2030年全球AD患者将会达到7800万人，预计到2050会达到1.29亿人。中国已经进入人口老龄化快速发展阶段，老年人口数量持续攀升。截至2018年底，我国60岁及以上老年人口达2.49亿，占总人口的17.9％；65岁及以上老年人口达1.66亿，占总人口的11.9％。2017年全国精神疾病流行病学调查显示，我国65岁及以上人群老年期痴呆患病率为5.56％(The Lancet Psychiatry,2019.6(3):p.211-224)。近年荟萃分析显示，中国60岁及以上人群的痴呆症总体患病率为5.3％。60岁以上痴呆症患者约为1000-1100万人，其中60％为AD(The Lancet  Neurology,2020.19(1):p.81-92.)。
美国FDA批准五款“老药”可缓解AD患者某些症状，包括四个胆碱酯酶抑制剂他克林(因肝毒性已退市)、多奈哌齐、加兰他敏、卡巴拉丁和一个NMDAR拮抗剂美金刚(Molecules,2020.25(24))。此外，2019年11月中国NMPA有条件批准的甘露特钠胶囊(九期一
Figure PCTCN2022129456-appb-000001
，GV-971，低分子酸性寡糖)，靶向脑—肠轴改善轻中度AD患者认知功能(First Approval.Drugs,2020.80(4):p.441-444)。2021年6月7日美国FDA有条件批准的aducanumab(
Figure PCTCN2022129456-appb-000002
anti-Aβ单抗)靶向可溶性和不可溶性的Aβ淀粉样蛋白的IgG1单克隆抗体药物，用于治疗轻度认知障碍或轻度AD患者(First Approval.Drugs,2021.81(12):p.1437-1443.)。但是，目前所有获批药物尚无法治愈AD，临床急需更有效的、更能延缓疾病进程治疗AD的药物。
3-甲基-1-苯基-2-吡唑啉-5-酮(依达拉奉，edaravone)是一种能够清除自由基的小分子化合物。在体外水平上(in vitro)，依达拉奉在0.156～5μM浓度范围能浓度依赖性地抑制Aβ 1-42单体聚集成Aβ原纤维(fibrils)和解聚Aβ原纤维；依达拉奉能减少Aβ 1-42对SH-SY5Y和原代皮层神经元的神经毒性，提高细胞活力和维持神经突的生长。在稳定表达APP695蛋白的SY-SY5Y(SH-SY5Y-APP695)中，依达拉奉(0.3～3μM)能浓度依赖性地减少BACE1表达和APP切割生成Aβ，抑制GSK-3β激活降低tau磷酸水平(Proc Natl Acad Sci U S A,2015.112(16):p.5225-30)。此外，依达拉奉能够减少H 2O 2和谷氨酸诱导的原代海马神经元死亡，保护神经元突触生长，减少过度氧化和兴奋性神经毒性(Neurotoxicology,2021.85:p.68-78)。在体内水平上(in vivo)，依达拉奉能够改善多种痴呆模型大鼠或小鼠的认知功能，减少脑内氧化指标、神经炎症、Aβ斑块行程、tau磷酸化水平等。在东莨菪碱诱导的大鼠痴呆模型中，连续7天口服给予依达拉奉15mg/kg/day能够改善东莨菪碱诱导的空间学习和长时程记忆障碍(International Journal of Pharmacology,2013.9(4):p.271-276)。在大鼠海马齿状回注射寡聚化的Aβ 1-40模型中，Aβ脑内注射同时同位点给予依达拉奉(5mg/kg)能显著改善大鼠在水迷宫中空间学习和记忆能力，减少海马中4-羟基-2-壬烯醛(4-HNE)加合物水平，恢复Aβ注射升高的AChE和ChAT酶活(Neurological Sciences,2015.36(11):p.2067-2072)。在侧脑室注射STZ(ICV-STZ)诱导学习记忆障碍的散发性AD(SAD)大鼠模型中，ICV-STZ造模后连续14天腹腔注射给药依达拉奉9mg/kg/day，能显著提高ICV-STZ模型大鼠在水迷宫试验(Morris water maze test)空间学习和记忆能力，以及在跳台被动回避试验(step-down passive avoidance test)中非空间长时间记忆能力；并 且能显著恢复STZ造模引起的海马和皮层中多种氧化应激指标水平改变，包括丙二醛(MDA)、4-HNE加合物、·OH、蛋白羰基(PC)、H 2O 2、超氧物歧化酶(SOD)、还原性谷胱甘肽(GSH)、谷胱甘肽过氧化物酶(GPx)(NeuroToxicology,2013.38:p.136-145)。此外，连续28天口服给药依达拉奉(1、3和10mg/kg/day)同样能改善ICV-STZ大鼠在水迷宫和被动回避试验中的认知功能，降低STZ引起的脑内氧化应激、胆碱能异常和炎症(Eur J Neurosci,2017.45(7):p.987-997)。在AlCl 3/D-半乳糖化学诱导小鼠认知障碍模型中，连续15天腹腔给予依达拉奉显著改善小鼠在水迷宫和跳台试验中的认知功能(Neurotoxicology,2021.85:p.68-78)。此外，在L-甲硫氨酸诱导大鼠血管性痴呆模型中，连续9周腹腔给予依达拉奉6mg/kg/day能够显著提高模型大鼠在八臂水迷宫试验中空间学习和记忆能力(Naunyn Schmiedebergs Arch Pharmacol,2020.393(7):p.1221-1228)。在APP/PS1转基因小鼠模型中，依达拉奉12.6mg/kg(腹腔注射，每周2次)预防给药(3～9月龄)和治疗给药(9～12月龄)均能改善小鼠在水迷宫试验中的认知功能的缺陷，减少脑内Aβ斑块的沉积、神经元和神经突触丢失和神经炎症、tau蛋白磷酸化(pSer396，pSer262，pSer199和pThr231)和氧化应激等。同样根据依达拉奉在小鼠口服生物利用度(38％)折算后，小鼠(月龄3-12月)口服给药33.2mg/kg/day(饮用水给药)同样可减少脑内Aβ斑块沉积和改善小鼠认知功能(Proc Natl Acad Sci U S A,2015.112(16):p.5225-30)。此外，在APP/PS老年小鼠(月龄14～17月)口服给予依达拉奉(8、24和72 mg/kg/day，饮用水给药)显著改善老年转基因小鼠的认知功能(Drug Design,Development and Therapy,2018.Volume 12:p.2111-2128)。在tau P301L转基因小鼠额颞叶痴呆(FTD)模型中，连续3个月口服给予依达拉奉24mg/kg/day(饮用水给药)9～10月龄和21月龄转基因小鼠，能显著改善动物在水迷宫试验和新物体识别试验中的认知功能，并改善动物运动缺陷，降低脑组织4-HNE和3-NT加合物产生，tau磷酸化水平和神经炎症(DOI:https://doi.org/10.21203/rs.3.rs-306628/v1)。
依达拉奉的化学结构式如下：
Figure PCTCN2022129456-appb-000003
依达拉奉
(分子式C 10H 10N 2O；分子量174.20)
右莰醇((+)-borneol)是天然冰片的主要成分(2020版中国药典规定天然冰片中右旋莰醇含量不低于96％)，是一种双环单萜类化合物，存在于许多中草药的挥发油中。右莰醇能调控NF-κB和p38信号通路，抑制炎症神经炎症，减少脑内多种促炎症因子(TNF-α、IL-1β等)和炎性蛋白(iNOS和COX-2)(Eur J Pharmacol,2014.740:p.522-31；Eur J Pharmacol,2017.811:p.1-11)；此外，还能激动抑制性的GABAAR受体，可以调节神经兴奋性损伤(Front Pharmacol,2021.12:p.606682)。
右旋莰醇的化学结构式如下：
Figure PCTCN2022129456-appb-000004
右旋莰醇
(分子式C 10H 18O；分子量154.25)
非临床动物实验表明，3-甲基-1-苯基-2-吡唑啉-5-酮和右莰醇组合物(质量比4:1～1:1)能协同性减少脑梗死面积(授权专利CN101848711B)和改善肌萎缩侧索硬化症(ALS)(授权专利CN107613976B)。在治疗脑卒中新药研发方面，依达拉奉右莰醇注射用浓溶液(3-甲基-1-苯基-2-吡唑啉-5-酮和右莰醇质量比4:1组合物)已于2020年由NMPA获批用于治疗缺血脑卒中；Y-2舌下片(3-甲基-1-苯基-2-吡唑啉-5-酮和右莰醇质量比5:1组合物)已在中国开展III期研究(中国临床登记号CTR20210233)和在美国开展I期研究(ClinicalTrials.govNCT03495206)。
依据现有技术，无法预期3-甲基-1-苯基-2-吡唑啉-5-酮和右莰醇的组合物是否对痴呆症及其相关的认知功能障碍具有治疗作用，也无法判断现有3-甲基-1-苯基-2-吡唑啉-5-酮和右莰醇的组合物是否能产生协同增效作用。
发明内容
本发明目的是提供一种药物组合物在制备改善或治疗认知功能障碍药物中的应用，所述的药物组合物含有3-甲基-1-苯基-2-吡唑啉-5-酮(依达拉奉)或其药学上可接受的盐和冰片。
本发明的另一目的在于提供一种药物组合物在改善或治疗认知功能障碍中的应用，其中向有此需要的患者施用含有3-甲基-1-苯基-2-吡唑啉-5-酮或其在药学上可接受的盐和冰片的药物组合物。
本发明的又一目的在于提供一种药物组合物，所述组合物用于治疗认知功能障碍，其中所述组合物包含3-甲基-1-苯基-2-吡唑啉-5-酮或其在药学上可接受的盐和冰片。
本发明的再一目的在于提供3-甲基-1-苯基-2-吡唑啉-5-酮或其在药学上可接受的盐和天然冰片联合治疗认知功能障碍的应用。
更进一步地，所述药物组合物中3-甲基-1-苯基-2-吡唑啉-5-酮或其在药学上可接受的盐和冰片的配合使用能协同性增加治疗认知功能障碍的药效。
在本发明的一些实施方案中，所述认知功能障碍为阿尔茨海默病(AD)、血管性痴呆症(VD)、轻度认知障碍(MCI)和其它类型痴呆；优选地，所述认知功能障碍为阿尔茨海默病、血管性痴呆症和轻度认知障碍。
在本发明的一个实施方案中所述药物组合物能改善阿尔茨海默病认知功能。
在本发明另一个实施方案中所述药物组合物能恢复阿尔茨海默病海马区长时程增强(LTP)，恢复大脑海马突触的功能。
在本发明再一个实施方案中所述药物组合物能改善阿尔茨海默病病理，包括淀粉样斑块、星形胶质细胞增生、小胶质细胞增生和tau蛋白超磷酸等。
本发明所述组合物中，3-甲基-1-苯基-2-吡唑啉-5-酮或其药学上可接受的盐：冰片的重量比10：1～1：8；优选为7.5：1～2.5：1；进一步优选为5：1或4：1。
本发明所述的冰片优选为天然冰片，所述天然冰片又称右莰醇。
本发明所述的药物组合物，可以进一步包括药学上可接受的赋形剂。
赋形剂一般是与药物治疗的活性药物成分(“API”)一起配制的药理学非活性物质。当生产剂型时，常使用赋形剂来对含有效活性成分的制剂进行增量(因而经常被称为“增量剂”、“填充剂”或“稀释剂”)，以允许便利又准确地分配原料药。其还可以服务于不同的治疗增强目的，诸如促进药物吸收或溶解，或其他药效动力学考虑。
更具体来说，本发明的组合物可以通过单独或与药学上可接受的载剂或稀释剂组合而 配制成药物组合物，并且可以配制成呈固体、半固体、液体或气体形式的制剂，诸如片剂、胶囊剂、粉剂、颗粒剂、软膏、溶液、栓剂、注射液、吸入剂、凝胶剂、微球体和气雾剂。因而，化合物的施用可以用多种方式实现，包括经口、颊、直肠、肠胃外、腹膜内、皮内、透皮、管内、关节内等施用。活性剂在施用后可以是系统性的，或可以通过使用区域性施用、壁内施用或使用能将活性剂量保留在植入部位的植入物加以定位。
本发明中使用的组合疗法可以允许各单一疗法的剂量低于标准操作中目前所使用的，同时实现显著效力，包括超过常规给予任一种单一疗法时的效力。本领域技术人员应容易了解，剂量水平可以随特定化合物、症状严重程度和受试者对副作用的敏感性而变化。一些特定化合物比其他的更有效。指定化合物的优选剂量可由本领域技术人员通过多种手段容易地确定。优选手段是测量指定化合物的生理学效力。使用组合疗法可以允许各单一疗法的剂量低于标准操作中目前所使用的，同时实现显著效力，包括比常规给予任一种单一疗法时所实现的更大的效力。
附图说明
图1依达拉奉右莰醇组合物保护Aβ 1-42诱导神经元损伤；
图2依达拉奉右莰醇组合物改善ICV-STZ空间学习记忆认知功能；
图3依达拉奉右莰醇组合物对AD转基因小鼠在水迷宫试验中认知功能的影响；
图4依达拉奉右莰醇组合物对AD转基因小鼠在Y迷宫试验中认知功能的影响；
图5依达拉奉右莰醇组合物对AD转基因小鼠脑片电生理的影响；
图6依达拉奉右莰醇组合物对5×FAD转基因小鼠脑内AD相关的病理影响。
具体实施方式
下述实施例举例说明本发明，不应被认为是对本发明的限制。实施例中提及的依达拉奉是3-甲基-1-苯基-2-吡唑啉-5-酮。
实施例1：依达拉奉和右莰醇组合物保护Aβ 1-42诱导的原代皮层神经元损伤
1材料和方法
1.1动物
SD孕大鼠，上海斯莱克实验动物有限责任公司(生产许可证号：SCXK(沪)2017-0005) 1.2试剂和耗材
Figure PCTCN2022129456-appb-000005
1.3原代皮层神经元的制备
怀孕18天的SD孕大鼠颈椎脱臼处死，解剖大鼠子宫取出E18胎鼠的大脑，分离胎鼠大脑皮层组织至冰冷DMEM中，解剖镜下去除皮层组织上脑膜和血管，将皮层组织转至冰冷DMEM中剪碎(约1mm 3)，胰酶37℃消化10min，FBS终止消化，巴氏吸管轻轻吹散后过200目筛。将过滤后的细胞悬液转至15mL离心管，1000rpm离心5min，吸弃上清后用37℃预热的完全培养基(Neurobasal+B27+GlutaMax+1％P/S)轻轻吹散离心管底的细胞团，血球计数板计数，用完全培养基稀释至5×10 5细胞/mL，接种于包被PDL的96孔板内培养(100μL/孔)，隔天用完全培养基半量换液，体外培养至第11天神经元分化成熟，用于氧糖剥夺试验。
1.4Aβ 1-42诱导原代皮层神经元兴奋性损伤试验
体外成熟的原代神经元培养基更换成含有不同浓度(1.2、3.7、11.1、33.3、100.0μM)的依达拉奉(E)、右旋莰醇(B)或依达拉奉右莰醇组合物(EB)的完成培养基，37℃孵育30min后，加入寡聚化的Aβ 1-42(终浓度50μM)继续培养24h，检测神经元活力。采用Luminescent Cell Viability Assay试剂盒检测原代神经元细胞活力。按照说明书加入100μL/孔试剂，震荡10min，在SpectraMax i3X(Molecule Device)多功能读板仪上读取化学发光值(LUM)，并计算神经元相对活力。计算公式：神经元相对活力V(％)＝(LUM-LUM 本底)/(LUM 正常对照组-LUM 本底组)×100％。LUM 本底为Luminescent Cell Viability Assay试剂加入到无细胞的完全培养基孔本底读数。
表1依达拉奉和右莰醇组合物正交设计
DMSO E1 E2 E3 E4 E5
B1 E1B1 E2B1 E3B1 E4B1 E5B1
B2 E1B2 E2B2 E3B2 E4B2 E5B2
B3 E1B3 E2B3 E3B3 E4B3 E5B3
B4 E1B4 E2B4 E3B4 E4B4 E5B4
B5 E1B5 E2B5 E3B5 E4B5 E5B5
注：E代表依达拉奉，B代表右莰醇，ExBy代表依达拉奉和右莰醇组合物，x/y数据1-5为浓度化合物各自浓度，分别为1.2、3.7、11.1、33.3、100.0μM。各组药物5个重复孔(n＝5)。所有化合物均先溶于DMSO，细胞培养液DMSO终浓度为0.2％。
1.5数据统计
采用Prism软件(GraphPad)进行One-way ANOVA单因素方差分析后，用Uncorrected Fisher's LSD分析两组之间的差异。P<0.05表示为有显著差异。#p<0.05,####p<0.0001,Ex或By与DMSO组相比；**p<0.01,***p<0.001,****p<0.0001,ExBy同时与Ex和By组相比有显著性差异。
2实验结果
寡聚化Aβ 1-42导致原代神经元活力细胞活力下降。Aβ 1-42诱导前预孵育给药依达拉奉(3.7～100μM)或右莰醇(1.2～100μM)能浓度依赖性地抑制Aβ诱导的神经毒性，提高细 胞活力。不同浓度的依达拉奉(1.2、3.7、11.1、33.3、100.0μM)和右莰醇(1.2、3.7、11.1、33.3、100.0μM)正交组合物能抑制Aβ损伤神经元，尤其是E5B4(3.3：1)、E5B3(9：1)、E4B5(1：3.3)、E4B4(1：1)、E4B3(3.3：1)、E3B5(1：9)、E3B4(1：3.3)、E3B3(1：1)、E3B2(3.3：1)、E3B1(9：1)和E2B2(1：1)组合物的保护作用显著优于相应的单独组分的保护作用(错误！未找到引用源。)。由此，初步判断依达拉奉和右莰醇在摩尔比9：1～1：9(质量比10：1～1：8)比例范围的组合物有协同增效作用。
实施例2：依达拉奉和右莰醇组合物改善侧脑室注射STZ诱导痴呆大鼠的认知功能
1实验材料
1.1实验动物
Wistar大鼠，雄性，体重250-300g，110只，清洁级，由上海斯莱克实验动物有限责任公司提供，许可证号：SCXK(沪)2012-0002。手术前、后常规饲养，室温保持23-25℃，自由进食、进水。
1.2试验仪器
Morris水迷宫(Topscan lite 2.0分析软件，Clever Systems)；大鼠脑立体定位仪(型号51600，Stoeling)；分析天平(型号AL104，Melttler Toledo)。
1.3受试药物
依达拉奉注射液(必存
Figure PCTCN2022129456-appb-000006
)，活性成分3-甲基-1-苯基-2-吡唑啉-5-酮。依达拉奉右莰醇组合物(重量比为7.5：1，5：1和2.5：1)为现配现用，溶剂对照为8％丙二醇。
2实验设计
2.1实验分组及给药
实验动物分为5组，即假手术组、模型组、依达拉奉组(6mg/kg)、依达拉奉右莰醇(7.5：1)组合物(依达拉奉6mg/kg，右莰醇0.8mg/kg)、依达拉奉右莰醇(5：1)组合物(依达拉奉6mg/kg，右莰醇1.2mg/kg)、依达拉奉右莰醇(2.5：1)组合物(依达拉奉6mg/kg，右莰醇2.4mg/kg)。模型及假手术组动物给于相应体积空白溶剂(8％丙二醇)，所有给药均为腹腔注射给药，给药体积为10mL/kg。
2.2实验程序
实验中除假手术组外，手术成功的动物按顺序随机分至模型及给药各组。按上述剂量设置原则，各组动物于术后4小时静脉注射给于相应药物，而后每日给药一次，连续给药14天，于第15日开始进行Morris水迷宫实验，水迷宫检测期间每日实验前给药一次，直至试验整体结束。各组动物均进行水迷宫测试，检测指标为隐藏平台逃避潜伏期、撤台后原站台所在象限停留时间。
3实验方法
3.1大鼠双侧侧脑室注射链脲霉素(STZ)致痴呆模型制备
大鼠用麻醉后，俯卧固定于立体定位仪,行背侧颈正中切口，暴露颅骨，以前囟点为坐标，前囟点后0.8mm，旁开1.5mm，深3.6mm为侧脑室注射点。用微量进样器于双侧脑室内注射STZ(以人工脑脊液配置)各10μL，1min内注射完成，留针3min。注射完毕后缝合皮肤。首日剂量为1.5mg/kg，第三日重复首日STZ注射方法，则STZ注射总量为3mg/kg。假手术组动物双侧侧脑室注射等体积的人工脑脊液。
3.2Morris水迷宫实验
Morris水迷宫水池直径160cm，高50cm，水深29cm。平台直径12cm，高27cm，固定于SE象限(目标象限)水面下2cm处。摄像镜头安放于水池中心上方距离水池底部2m处，用以同步记录大鼠运动轨迹。水温保持22±1℃。实验共计5天，包括隐藏平台试验和空间探索试验。前4天为隐藏平台试验，最后1天为空间探索实验。
(1)隐蔽平台试验(hidden platform trial)
本实验为期4天，试验时平台位置固定在目标象限(SE象限)，分别从四个象限的入水点入水，训练时将动物面朝池壁标示轻轻放入水中，记录动物找到平台的时间(逃避潜伏期，escape latency)，然后让动物在平台上停留10s。如果90s内找不到平台，潜伏期记为90s，并将动物引导置于平台上停留10s。训练结束后，将大鼠放回饲养笼中并注意保暖。每天在4个入水点各训练1次，以4次潜伏期的均值作为这一天的成绩进行统计分析。每天每只动物入水点顺序保持一致，但是同一笼内不同动物入水顺序不同，以排除组内动物信息交流。
(2)空间探索试验(probe trial)
隐蔽平台试验结束24h后撤除平台。然后从平台的对面入水点入水，记录大鼠在120s内的游泳路径，对大鼠目标象限停留时间进行统计分析。观察受试动物的空间定位能力及在空间探索过程中的变化规律。
3.3数据统计
数据表示为Mean±SEM。各组之间统计学分析使用Prism软件(GraphPad，USA)进行分析：两组以上比较采用One-way或Two-way ANOVA检测后，再选用Fisher’s LSD test进行分析。P<0.05表示有显著性差异。####p<0.0001,与假手术组相比；*p<0.05,**p<0.01,***p<0.001,****p<0.0001,与模型组相比；&p<0.05,&&p<0.01,指示两组相比。
4实验结果
在水迷宫隐蔽平台试验中，与假手术组相比，侧脑室注射STZ(ICV-STZ)模型逃避潜伏期显著延长，说明ICV-STZ大鼠发生认知功能障碍。腹腔注射依达拉奉(6mg/kg)或依达拉奉右莰醇组合物(7.5：1、5：1和2.5：1)能够显著缩短ICV-STZ大鼠逃避潜伏期。与依达拉奉给药组相比，依达拉奉右莰醇(7.5：1、5：1和2.5：1)组合物对ICV-STZ模型大鼠的学习功能提升显著优于依达拉奉(错误！未找到引用源。A)。在水迷宫空间探索试验中，ICV-STZ模型大鼠目标象限的保留时间显著缩短。与模型组相比，腹腔注射依达拉奉(6mg/kg)或依达拉奉右莰醇组合物(7.5：1、5：1和2.5：1)给药组的大鼠在目标象限保留时间显著延长，并且依达拉奉右莰醇组合物(5：1和2.5：1)组显著优于依达拉奉组(错误！未找到引用源。B)。因此，依达拉奉右莰醇组合物(7.5：1、5：1和2.5：1)能显著改善ICV-STZ大鼠认知功能缺陷，并且其药效明显优于依达拉奉。
实施例3：长期给药依达拉奉右莰醇组合物对5×FAD转基因小鼠AD认知功能和AD病理的影响
1材料与方法
1.1试验动物
野生型WT小鼠和5×FAD转基因小鼠均由中国药科大学司马健课题组提供。
1.2受试药物与试剂
依达拉奉，即依达拉奉注射液(必存
Figure PCTCN2022129456-appb-000007
，先声药业)，活性成分3-甲基-1-苯基-2-吡唑啉-5-酮。依达拉奉右莰醇组合物，即依达拉奉右莰醇注射用浓溶液(先必新
Figure PCTCN2022129456-appb-000008
，先声药业)，3-甲基-1-苯基-2-吡唑啉-5-酮和右莰醇质量比为4：1。溶剂对照为8％丙二醇。
1.3实验方法
本试验分为4组，各组小鼠从8周龄开始给药，连续每天给药16周后，进行水迷宫和Y迷宫行为学实验，行为学检测完成后处死动物检测脑片电生理和脑内AD病理，包括检测Aβ斑块，GFAP和Iba-1等生物标志物以及p-Tau等。
1.3.1动物分组和给药
WT:溶剂对照组：野生型小鼠10只，i.p.给予溶剂对照，剂量为10mL/kg，每天给药1次。
5×FAD:溶剂组：5×FAD小鼠10只，i.p.给予溶剂对照，剂量为10mL/kg，每天给药1次。
5×FAD:依达拉奉组：5×FAD小鼠10只，i.p.给予依达拉奉(必存
Figure PCTCN2022129456-appb-000009
)6mg/kg，剂量为10.0mL/kg，每天给药1次。
5×FAD:组合物组：5×FAD小鼠10只，i.p.给予依达拉奉右莰醇(先必新
Figure PCTCN2022129456-appb-000010
)7.5mg/kg(依达拉奉：右莰醇＝4：1，含6mg/kg依达拉奉和1.5mg/kg右莰醇)，剂量为10.0mL/kg，每天给药1次。
1.3.2水迷宫实验：
实验开始前，小鼠需在测试房间放置30min左右适应环境。
实验准备：向水迷宫的恒温水池(内径120cm，高50cm)注入清水，水温控制在18-22℃左右，选取水池的四个顶点分别记作“S”，“N”，“W”，“E”，平台放置在“SW”方向且低于水面1-2cm，向水中加入二氧化钛使水成为不透明的乳白色。
参数设置：测试时间设为1min，小鼠在平台停留时间设为2s。
训练：前5天为水迷宫的训练阶段，每天分别从四个不同的方位将小鼠放置在水池里，记录其登上平台前的轨迹，时间，以及在各个象限的路程和时间。对于1min之内仍未找到平台的小鼠，在1min结束后，将其引导至平台上，停留10-15s。小鼠投放的具体方位如表1。
表1水迷宫训练试验在不同天数中小鼠投放方位汇总
Figure PCTCN2022129456-appb-000011
测试：训练结束后的第一天进行测试。测试时，撤去平台，测试时间为60s，在NE方位放置小鼠，记录在测试过程中小鼠穿越平台的次数以及在平台所在象限的时间。
1.3.3Y迷宫实验：
实验开始前，小鼠需在测试房间放置30min左右适应环境。
训练：将Y迷宫的一条臂用挡板挡住，将小鼠放置在剩余两条臂的中的一条臂的末端，让其自由探索15min并录像。
测试：训练1h后，将挡板撤掉，用酒精擦拭Y迷宫的通道以去除上一只小鼠的气味，将待测试的小鼠放在相同的位置计时5min并录像，测试要记录小鼠进入新开放的臂的次数占进入三条臂总次数的百分比。
1.3.4脑片电生理检测
小鼠行为学检测结束后进行电生理记录。麻醉后对所有小鼠实施安乐死，迅速收集脑组织并储存在4℃含氧(95％O 2/5％CO 2)人工脑脊液(ACSF)(75mM蔗糖、87mM NaCl、2.5mM KCl)中、1.25mM NaH 2PO 4、21.4mM NaHCO 3、0.5mM CaCl 2、7mM MgCl 2和20mM葡萄糖)。海马切片通过振动刀在4℃含氧ACSF中切割，并在32℃含氧ACSF。接下来，切片在记录前在室温下孵育1小时。海马LTP是由两个电θ脉冲刺激(TBS)诱导的。使用Clampex和Clampfit 8.2(Molecular Devices)进行数据采集和分析。
1.3.5 AD病理检测(免疫组化试验)
动物行为学检测结束后，将小鼠进行心脏灌流后取脑，4％多聚甲醛中固定24h，并使 用30％的蔗糖脱水，包埋剂速冻包埋，并进行脑切片(厚度30μM)。选取有海马区域的脑切片，PBS清洗5min×3次，使用0.2％Triton透化，PBS清洗7min×3次，使用Blocking buffer(2.5％BSA,0.3％Triton)封闭1h，将脑片放置在一抗中4℃过夜。第二天取出一抗中的脑片，PBS清洗5min×3次，将脑片放入二抗中，室温避光孵育1h，取出脑片，PBS清洗5min×3次，滴加含DAPI的抗荧光淬灭剂，封片。
1.4统计分析
数据表示为Mean±SEM。各组之间统计学分析使用Prism软件(GraphPad，USA)进行分析：两组以上比较采用One-way或Two-way ANOVA检测后，再选用Fisher’s LSD test进行分析。P<0.05表示有显著性差异。##p<0.01，###p<0.001,####p<0.0001，与WT组相比；*p<0.05,**p<0.01,***p<0.0001，****p<0.0001，与模型组相比；&p<0.05,&&p<0.01,&&&&p<0.0001，指示的两组相比。
2.实验结果
2.1依达拉奉右莰醇组合物改善AD转基因小鼠的认知功能缺陷
在Morris水迷宫试验中，相比于同龄野生型小鼠(WT)，24周龄的5×FAD小鼠出现认知功能缺陷，具体表现为：在隐台训练试验中，5×FAD小鼠从2～5天逃避期显著长于WT小鼠；在撤台检测试验中，5×FAD小鼠在目标象限停留时间和平台穿越次数上均明显少于WT小鼠。与溶剂对照相比，5×FAD小鼠从8周龄开始每天腹腔注射给予6mg/kg依达拉奉或7.5mg/kg依达拉奉右莰醇组合物(含6mg/kg依达拉奉和1.5mg/kg右莰醇)1次，连续给药16周，能显著缩短5xFAD小鼠在水迷宫隐台试验中的逃避潜伏期(错误！未找到引用源。A)，延长撤台试验中目标象限停留时间和穿越平台的次数(错误！未找到引用源。B，C)。与依达拉奉给药组相比，组合物给药在隐台试验第5天逃避潜伏期时间更短，在撤台试验中目标象限停留时间显著性的更长，在穿越平台次数上也更多(错误！未找到引用源。A，B和C)。这说明，依达拉奉和依达拉奉组合物均能改善5×FAD小鼠在水迷宫试验中的学习记忆认知功能，并且组合物的改善作用显著优于依达拉奉。
在Y-迷宫中，相比于同龄野生型小鼠(WT)，24周龄的5×FAD小鼠出现认知功能缺陷，具体表现为：5×FAD小鼠新臂进臂率显著下降，即空间参考记忆功能下降。与溶剂对 照相比，5×FAD小鼠从8周龄开始每天腹腔注射给予6mg/kg依达拉奉或7.5mg/kg依达拉奉右莰醇组合物(含6mg/kg依达拉奉和1.5mg/kg右莰醇)1次，连续给药16周，能显著增加5×FAD小鼠新臂进臂率。与依达拉奉相比，依达拉奉右莰醇组合物提高5×FAD小鼠新臂进臂率药效显著更高(错误！未找到引用源。)。这说明，依达拉奉和依达拉奉组合物均能改善5×FAD小鼠在Y迷宫试验中的空间参考记忆认知功能，并且组合物的改善作用显著优于依达拉奉。
2.2依达拉奉右莰醇组合物恢复AD小鼠海马区突触传递功能
长时程增强(LTP)反映了突触功能的可塑性，是学习和记忆的主要机制之一。水迷宫和Y-迷宫检测结束后，电生理检测发现5×FAD小鼠海马的区LTP显著受损。与5×FAD小鼠组相比，组合物给药能显著恢复了小鼠海马LTP，但依达拉奉给药对5×FAD小鼠海马LTP没有明显的恢复作用，并且组合物给药LTP恢复也显著性地优于依达拉奉组(错误！未找到引用源。A，B和C)。这表明24周龄的5×FAD转基因小鼠海马区突触可塑性已受损，而依达拉奉右莰醇组合物可以促进海马神经可塑性，并使海马LTP恢复到较好水平。
2.3依达拉奉右莰醇组合物改善AD小鼠脑内AD病理
24周龄的5×FAD小鼠脑内已经产生明显的AD病理，包括β淀粉蛋白斑块、星形胶质细胞增生、小胶质细胞增生，和海马区神经细胞内tau蛋白过度磷酸化。与溶媒对照组相比，5×FAD小鼠从8周龄开始每天腹腔注射给予6mg/kg依达拉奉或7.5mg/kg依达拉奉右莰醇组合物(含6mg/kg依达拉奉和1.5mg/kg右莰醇)1次，连续给药16周，能显著降低脑内的AD病理(β淀粉蛋白斑块、星形胶质细胞增生、小胶质细胞增生)以及海马区tau磷酸化水平。与依达拉奉给药组相比，依达拉奉右莰醇组合物在改善AD病理(β淀粉蛋白斑块、星形胶质细胞增生、小胶质细胞增生)和降低tau磷酸化水平上的药效上有显著性的优势(错误！未找到引用源。)。

Claims (10)

  1. 一种组合物在改善或治疗认知障碍中的应用，所述组合物含有3-甲基-1-苯基-2-吡唑啉-5-酮或其在药学上可接受的盐和冰片或右莰醇。
  2. 根据权利要求1所述的应用，其特征在于，所述组合物含有3-甲基-1-苯基-2-吡唑啉-5-酮或其在药学上可接受的盐和右莰醇。
  3. 根据权利要求1所述的应用，其特征在于，所述3-甲基-1-苯基-2-吡唑啉-5-酮或其在药学上可接受的盐和冰片或右莰醇的重量比为10：1～1：8。
  4. 根据权利要求1所述的应用，其特征在于，所述3-甲基-1-苯基-2-吡唑啉-5-酮或其在药学上可接受的盐和冰片或右莰醇的重量比为7.5：1～2.5：1。
  5. 根据权利要求1所述的应用，其特征在于，所述3-甲基-1-苯基-2-吡唑啉-5-酮或其在药学上可接受的盐和冰片或右莰醇的重量比为5：1。
  6. 根据权利要求1所述的应用，其特征在于，所述3-甲基-1-苯基-2-吡唑啉-5-酮或其在药学上可接受的盐和冰片或右莰醇的重量比为4：1。
  7. 根据权利要求1～6所述的应用，其特征在于，所述组合物进一步包括药学上可接受的赋形剂。
  8. 根据权利要求1～6所述的应用，其特征在于，所述认知障碍是指阿尔茨海默病、血管性痴呆症、轻度认知障碍和其它类型痴呆。
  9. 根据权利要求1～6所述的应用，其特征在于，所述认知障碍是指阿尔茨海默病、血管性痴呆症和轻度认知障碍。
  10. 根据权利要求1～6所述的应用，其特征在于，所述认知障碍是指阿尔茨海默病。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116421647A (zh) * 2023-06-14 2023-07-14 广州白云山和记黄埔中药有限公司 复方丹参制剂的新应用

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101848711A (zh) 2008-03-04 2010-09-29 江苏先声药物研究有限公司 一种药物组合物及其在制备治疗脑血管病药物中的应用
CN102579432A (zh) * 2011-01-12 2012-07-18 江苏先声药物研究有限公司 3-甲基-1-苯基-2-吡唑啉-5-酮与2-莰醇组合物的新应用
CN107613976A (zh) 2015-06-10 2018-01-19 江苏先声药业有限公司 一种组合物在制备治疗肌萎缩性侧索硬化症药物中的应用
CN107773545A (zh) * 2016-08-29 2018-03-09 烟台益诺依生物医药科技有限公司 依达拉奉与(+)‑2‑莰醇的舌下用药物组合物
US20190083463A1 (en) * 2016-03-16 2019-03-21 Suzhou Auzone Biological Technology Co., Ltd. Edaravone dosage form

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101848711A (zh) 2008-03-04 2010-09-29 江苏先声药物研究有限公司 一种药物组合物及其在制备治疗脑血管病药物中的应用
CN102579432A (zh) * 2011-01-12 2012-07-18 江苏先声药物研究有限公司 3-甲基-1-苯基-2-吡唑啉-5-酮与2-莰醇组合物的新应用
CN107613976A (zh) 2015-06-10 2018-01-19 江苏先声药业有限公司 一种组合物在制备治疗肌萎缩性侧索硬化症药物中的应用
US20190083463A1 (en) * 2016-03-16 2019-03-21 Suzhou Auzone Biological Technology Co., Ltd. Edaravone dosage form
CN107773545A (zh) * 2016-08-29 2018-03-09 烟台益诺依生物医药科技有限公司 依达拉奉与(+)‑2‑莰醇的舌下用药物组合物

Non-Patent Citations (19)

* Cited by examiner, † Cited by third party
Title
"Chinese Pharmacopoeia", 2020
"First Approval", DRUGS, vol. 80, no. 4, 2020, pages 441 - 444
"First Approval", DRUGS, vol. 81, no. 12, 2021, pages 1437 - 1443
"Guidelines for the diagnosis and treatment of dementia and cognitive impairment in China", NATIONAL MEDICAL JOURNAL OF CHINA, vol. 98, no. 13, 2018, pages 965 - 970
CHEN ZHONGJIAN, ZHANG YUAN, ZHENG YEJIAO, YANG HAIYAN, GU LIQIANG: "Research progress of borneol in promoting drug penetration through blood-brain barrier", CHINESE TRADITIONAL PATENT MEDICINE, GUOJIA YIYAO GUANLIJU, ZHONGCHENGYAO QINGBAO ZHONGXINZHAN, CN, vol. 41, no. 9, 1 September 2019 (2019-09-01), CN , pages 2170 - 2173, XP055885376, ISSN: 1001-1528, DOI: 10.3969/j.issn.1001-1528.2019.09.031 *
DRUG DESIGN, DEVELOPMENT AND THERAPY, vol. 12, 2018, pages 2111 - 2128
EUR J NEUROSCI, vol. 45, no. 7, 2017, pages 987 - 997
EUR J PHARMACOL, vol. 740, 2014, pages 522 - 31
EUR J PHARMACOL, vol. 811, 2017, pages 1 - 11
FRONT PHARMACOL, vol. 12, 2021, pages 606682
HUR JINYOUNG, PAK SOK CHEON, KOO BYUNG-SOO, JEON SONGHEE: "Borneol alleviates oxidative stress via upregulation of Nrf2 and Bcl-2 in SH-SY5Y cells", PHARMACEUTICAL BIOLOGY, SWETS AND ZEITLINGER, LISSE,, NL, vol. 51, no. 1, 1 January 2013 (2013-01-01), NL , pages 30 - 35, XP055975430, ISSN: 1388-0209, DOI: 10.3109/13880209.2012.700718 *
MOLECULES, vol. 25, no. 24, 2020
NAUNYN SCHMIEDEBERGS, ARCH PHARMACOL, vol. 393, no. 7, 2020, pages 1221 - 1228
NEUROLOGICAL SCIENCES, vol. 36, no. 11, 2015, pages 2067 - 2072
NEUROTOXICOLOGY, vol. 38, 2013, pages 136 - 145
NEUROTOXICOLOGY, vol. 85, 2021, pages 68 - 78
PROC NATL ACAD SCI U S A, vol. 112, no. 16, 2015, pages 5225 - 30
THE LANCET NEUROLOGY, vol. 19, no. 1, 2020, pages 81 - 92
THE LANCET PSYCHIATRY, vol. 6, no. 3, 2019, pages 211 - 224

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116421647A (zh) * 2023-06-14 2023-07-14 广州白云山和记黄埔中药有限公司 复方丹参制剂的新应用
CN116421647B (zh) * 2023-06-14 2023-08-29 广州白云山和记黄埔中药有限公司 复方丹参制剂的新应用

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