Use of bardoxolone and combinations thereof with donepezil in the manufacture of a medicament for the treatment of alzheimer's disease

Abstract

The application discloses application of a drug composition of bacosol and donepezil in preparation of a drug for treating Alzheimer's disease. The application first discovers that bacosol has double-target point inhibitory activity on BACE1 and AChE. Through cell and animal model verification, the bacosol single drug and the composition can exert an anti-AD effect through a multi-target point mechanism, and the composition with a molar ratio of 1:1 to 1:10 has a significant synergistic effect, wherein the synergistic effect is the most significant when the molar ratio is 1:1. The application provides a new treatment method for Alzheimer's disease.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and more particularly to a novel use of a known compound, bardosolone, specifically the use of bardosolone or its pharmaceutically acceptable salts and derivatives in the preparation of medicaments for the prevention and / or treatment of Alzheimer's disease. Furthermore, this invention also relates to a pharmaceutical composition comprising bardosolone and donepezil, and its use in the preparation of medicaments for the prevention and / or treatment of Alzheimer's disease. Background Technology

[0002] Alzheimer's disease (AD) is a neurodegenerative disease of the central nervous system, clinically characterized by progressive memory impairment, cognitive decline, and behavioral abnormalities, severely impacting patients' quality of life. With the increasing aging of the global population, the incidence of AD continues to rise, becoming a major public health issue.

[0003] Currently, the pathogenesis of Alzheimer's disease (AD) mainly involves multiple pathological processes, including β-amyloid (Aβ) deposition, abnormal phosphorylation of Tau protein, neuroinflammation, oxidative stress, and cholinergic dysfunction. These mechanisms are interconnected and work synergistically to accelerate disease progression.

[0004] Clinically, drugs used to treat Alzheimer's disease (AD) mainly fall into three categories: cholinesterase inhibitors (such as donepezil), glutamate receptor antagonists (such as memantine), and anti-amyloid monoclonal antibodies (such as lencanezumab). However, most existing drugs act on a single target, making it difficult to comprehensively address the multiple pathological mechanisms of AD; long-term use of some drugs is also accompanied by adverse reactions such as dizziness, hallucinations, and hepatotoxicity, resulting in unsatisfactory overall treatment outcomes.

[0005] In recent years, multi-target drug strategies have become an important direction in the development of drugs for complex diseases because they can act on multiple pathological stages of the disease simultaneously. However, to date, no multi-target AD treatment drug that can simultaneously activate the Nrf2 antioxidant pathway, inhibit BACE1 and AChE activity, and also has anti-inflammatory and anti-Aβ deposition effects has been approved for marketing.

[0006] Bardoxolone, as an Nrf2 pathway activator, has been patented for its applications in myocarditis, pneumonia, and tumors (see patent documents CN202310049322.4, CN202410625437.8, and CN202410672876.4), but no technical solutions for its use in the treatment of Alzheimer's disease have been reported. Donepezil, as a representative drug of cholinesterase inhibitors, has had numerous related patents published, with its protection scope focusing on pharmaceutical salt form, formulation process, and application in dementia-like diseases. Its core mechanism of action is to improve the function of the cholinergic system. Existing patents related to donepezil combination compositions cover synergistic schemes centered on memantine (see patent documents CN202510521585.X and CN202311339425.0), acetylcholine-related combinations (see patent document TW113149961), and combination strategies that reduce the inflammatory factor TNF-α (see patent document CN202410796338.6). However, the aforementioned donepezil monotherapy and combination patents all have mechanistic limitations. They cannot effectively intervene in the core pathological changes of AD, such as Aβ deposition and abnormal phosphorylation of Tau protein, nor can they simultaneously regulate key pathological links such as antioxidation and anti-inflammation.

[0007] Therefore, developing a monotherapy of bardoxolone that can cover multiple pathological aspects of Alzheimer's disease (AD) and has a synergistic effect, as well as a drug combination with donepezil, has significant clinical value and industrialization prospects. Summary of the Invention

[0008] The technical problem to be solved by the present invention is to provide a drug or drug composition that can effectively intervene in multiple pathological links of Alzheimer's disease (AD) to overcome the technical defects of existing AD treatment drugs that have a single target and are difficult to block the disease process.

[0009] To achieve the above objectives, the present invention provides bardosulolon monotherapy, the pharmaceutical composition thereof, and its use in the prevention and / or treatment of Alzheimer's disease (AD), the specific technical solutions of which are as follows:

[0010] In a first aspect, the present invention provides the use of bardosorlon or pharmaceutically acceptable salts or derivatives thereof in the preparation of medicaments for the prevention and / or treatment of Alzheimer's disease.

[0011] The structure of Bardoxolone (CAS No.: 218600-44-3) is shown in formula (I):

[0012]

[0013] (I)

[0014] The drug also contains pharmaceutically acceptable carriers or excipients.

[0015] The method of administration of the drug is selected from one or more of oral, injection, implantation, spray or inhalation.

[0016] The dosage form of the drug is injection, oral liquid, powder, tablet, granule, capsule, syrup, sustained-release preparation, enteric solvent, aerosol or suspension.

[0017] In a second aspect, the present invention provides a pharmaceutical composition for the prevention and / or treatment of Alzheimer's disease, the pharmaceutical composition comprising a therapeutically effective amount of bardosulolon or a pharmaceutically acceptable salt or derivative thereof, and a therapeutically effective amount of donepezil or a pharmaceutically acceptable salt thereof.

[0018] The structure of Bardoxolone (CAS No.: 218600-44-3) is shown in formula (I):

[0019]

[0020] (I)

[0021] The structure of donepezil (CAS No.: 120014-06-4) is shown in formula (II):

[0022]

[0023] (II)

[0024] The molar ratio of badosolone to donepezil is 1:0.01 to 1:100; preferably, the molar ratio of badosolone to donepezil is 1:0.3 to 1:30; more preferably, the molar ratio of badosolone to donepezil is 1:1 to 1:10; and most preferably, the molar ratio is 1:1.

[0025] The pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient. The pharmaceutically acceptable carrier or excipient is selected from one or more of fillers, binders, disintegrants, lubricants, solubilizers, preservatives, antioxidants, pH adjusters, emulsifiers, and stabilizers.

[0026] The dosage form of the pharmaceutical composition is an injection, oral liquid, powder, tablet, granule, capsule, syrup, sustained-release preparation, enteric solvent, aerosol, or suspension.

[0027] The administration method of the pharmaceutical composition is selected from one or more of oral, injection, implantation, spray, or inhalation.

[0028] In a third aspect, the present invention also provides the use of the above-described pharmaceutical composition in the preparation of a medicament for the prevention and / or treatment of Alzheimer's disease.

[0029] The pharmaceutical composition achieves its therapeutic effect through the following multi-target synergistic mechanism: activating the Nrf2-Keap1-ARE antioxidant pathway, inhibiting the activity and expression of β-secretase 1 (BACE1), inhibiting the activity of acetylcholinesterase (AChE), reducing Aβ protein deposition, inhibiting abnormal phosphorylation of Tau protein, reducing neuroinflammation, and alleviating oxidative stress damage.

[0030] The administration method of the pharmaceutical composition is selected from one or more of oral, injection, implantation, spray, or inhalation.

[0031] The dosage form of the pharmaceutical composition is an injection, oral liquid, powder, tablet, granule, capsule, syrup, sustained-release preparation, enteric solvent, aerosol, or suspension.

[0032] A fourth aspect of the present invention provides a medicine box for the prevention and / or treatment of Alzheimer's disease, characterized in that it comprises:

[0033] (i) A first preparation containing the above-mentioned bardosorlon or its pharmaceutically acceptable salts or derivatives;

[0034] (ii) A second preparation containing donepezil or a pharmaceutically acceptable salt thereof;

[0035] The first and second formulations are used for simultaneous, separate or sequential administration.

[0036] In a fifth aspect, the present invention also provides a combined administration method for the prevention and / or treatment of Alzheimer's disease, the method comprising administering a therapeutically effective amount of bardosulolon and a therapeutically effective amount of donepezil simultaneously, separately, or sequentially.

[0037] Preferably, the bardosolizuron and donepezil are administered in a molar ratio of 1:1.

[0038] Preferably, the dosage of bardosorlon is 1-10 mg / kg·d, and the dosage of donepezil is 1-10 mg / kg·d;

[0039] In a preferred embodiment of the present invention, the dosage of bardosolizuron is 2 mg / kg·d, and the dosage of donepezil is 2 mg / kg·d.

[0040] Preferably, the administration method is oral.

[0041] Preferably, the dosing cycle is once daily for 4, 8, or 12 weeks.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] I. First public disclosure of a new application for Bardosolone in AD treatment

[0044] In the prior art, bardosulon is only recognized as an Nrf2 pathway activator, and its patent protection scope is concentrated in the fields of chronic kidney disease, myocarditis, pneumonia, and tumors. This invention is the first to apply bardosulon to the prevention and treatment of Alzheimer's disease, expanding the indications for this compound and providing a novel treatment approach in this field.

[0045] II. First discovery of the dual-target enzyme inhibitory activity of bardosorone

[0046] In vitro enzymatic experiments have verified that this invention is the first to discover that bardosulomon has dual-target inhibitory activity against β-secretase 1 (BACE1) and acetylcholinesterase (AChE), with a half-maximal inhibitory concentration (IC50) of [missing information]. 50 The concentrations were 44.28 μM and 48.65 μM, respectively. This discovery breaks through the limitations of existing technology in understanding bardosulolon as merely an Nrf2 activator, providing a novel mechanism to support its anti-AD effect and also providing a target basis for subsequent drug combination therapy.

[0047] III. Significant Synergistic Effects

[0048] Using the SH-SY5Y APPswe / Tau double transgenic cell model (including basal state and rotenone-induced stress state) and FAD 4T In screening AD mouse models, this invention determined that the preferred molar ratio of badosole to donepezil is 1:1 to 1:10. At this ratio:

[0049] At the cellular level (1 μM each): the combination of bardoxazoline and donepezil in equal proportions significantly enhanced cell viability compared to either drug alone (P<0.001).

[0050] Animal level (administered bardossolon and donepezil hydrochloride 2 mg / kg·d, corresponding to a molar ratio of approximately 1:1.18): The synergistic therapeutic effect of the drug combination was significantly better than that of the proportional high-dose group (4 mg / kg·d each) and better than that of the positive control group (2 mg / kg·d donepezil hydrochloride + 6 mg / kg·d memantine hydrochloride) (P<0.001).

[0051] IV. Multi-target coverage, comprehensive mechanism

[0052] The pharmaceutical composition of the present invention can simultaneously intervene in multiple pathological processes of AD, such as Aβ deposition, Tau phosphorylation, neuroinflammation, oxidative stress, and cholinergic dysfunction, overcoming the limitation of the limited range of action of existing single-target drugs.

[0053] V. Strong universality in pathological stages

[0054] Cell model validation showed that the 1:1 equimolar ratio of badosole and donepezil exhibited a stable synergistic effect under both basal conditions (without inducer) and rotenone-induced stress. This result indicates that the pharmaceutical composition described in this invention is consistently effective regardless of whether the Alzheimer's disease is in the early stage with mild oxidative stress or the middle to late stage with severe oxidative stress, demonstrating good general applicability across different pathological stages.

[0055] VI. Superiority of Low-Dose

[0056] In FAD 4T In an AD mouse model, this invention found that the low-dose group (2 mg / kg·d each) of the drug composition showed significantly better synergistic therapeutic effects than the high-dose proportional combination group (4 mg / kg·d each). This "non-monotonicity of the dose-response curve" indicates that the composition of this invention possesses unique pharmacodynamic characteristics, achieving optimal therapeutic effects at low doses and avoiding the potential toxicity risks associated with high doses.

[0057] VII. Differentiated Protection of Brain Regions

[0058] This invention detected FAD separately. 4T The levels of GSH, SOD, and inflammatory factors in the cortex and hippocampus of AD mice were measured. Results showed that the drug composition produced a protective effect in both brain regions, with a more significant improvement in the hippocampus. Since the hippocampus is a core pathological brain region for cognitive impairment in AD, this differential protective effect further confirms the precise targeting of the drug composition described in this invention in improving cognitive function.

[0059] 8. Good safety

[0060] After a 12-week continuous dosing experiment, neither bardosulone monotherapy (2 mg / kg·d) nor the drug combination (2 mg / kg·d each) had a significant effect on the weight gain of AD mice (P>0.05), and no obvious organ toxicity or behavioral abnormalities were observed, indicating good safety. Attached Figure Description

[0061] Figure 1 : In vitro inhibitory activity curves of bardosupine against BACE1 and AChE. Among them, Figure 1 A represents the half-maximal inhibitory concentration (IC50) of bardosorlon against BACE1. 50 ) curve, IC 50 =44.28 μM; Figure 1 B is the half-maximal inhibitory concentration (IC50) of bardosorlon against AChE. 50 ) curve, IC 50=48.65 μM.

[0062] Figure 2 : Effects of badoxazoline and donepezil monotherapy on cell viability at basal levels in the SH-SY5Y APPswe / Tau double-transgenic cell model. Figure 2 A represents the effect of bardosorlon concentration gradients (0.03–100 μM) on cell viability; Figure 2 B represents the effect of donepezil concentration gradients (0.03–100 μM) on cell viability.

[0063] Figure 3 : Effects of badoxazoline and donepezil monotherapy on cell viability under rotenone-induced stress in the SH-SY5Y APPswe / Tau double transgenic cell model. Figure 3 A represents the effect of bardosorlon concentration gradients (0.03–100 μM) on cell viability; Figure 3 B represents the effect of donepezil concentration gradients (0.03–100 μM) on cell viability.

[0064] Figure 4 : The effect of different concentrations of badosole and donepezil on cell viability under basal conditions in the SH-SY5Y APPswe / Tau double transgenic cell model. Figure 4 A represents the effect of a fixed badoxazoline concentration of 0.3 μM combined with donepezil gradient concentrations (0.01–30 μM) on cell viability; Figure 4 B represents the effect of a fixed badoxazoline concentration of 1 μM combined with donepezil gradient concentrations (0.01–30 μM) on cell viability; Figure 4 C represents the effect of a fixed bardosulomon concentration of 10 μM combined with donepezil gradient concentrations (0.01–100 μM) on cell viability.

[0065] Figure 5 : The effect of different concentrations of bardosorlon and donepezil on cell viability under rotenone-induced stress in the SH-SY5Y APPswe / Tau double transgenic cell model. Figure 5 To illustrate the effect of fixed 1 μM bardosulorin combined with donepezil gradient concentrations (0.1–100 μM) on cell viability, the synergistic Q values ​​for each combination are labeled below the figure.

[0066] Figure 6 The graph shows the effects of different drug administration groups on GSH content and SOD activity induced by rotenone in the SH-SY5Y APPswe / Tau double transgenic cell model. Figure 6 A shows the comparison of GSH content among the groups; Figure 6 B represents a comparison of SOD activity among the groups.

[0067] Figure 7 : Effect of each drug administration group on ARE activity induced by rotenone in the SH-SY5Y APPswe / Tau double transgenic cell model.

[0068] Figure 8 : A diagram showing the regulation of Nrf2 pathway-related protein expression under rotenone stress in different drug-treated groups in the SH-SY5Y APPswe / Tau double transgenic cell model. Among them, Figure 8 A is a Western blotting plot; Figure 8 B is a statistical graph showing the relative expression levels of nuclear Nrf2 protein; Figure 8 C represents the statistical graph of the relative expression level of Nrf2 protein; Figure 8 D is a statistical graph showing the relative expression levels of HO-1 protein; Figure 8 E is a statistical graph showing the relative expression levels of NQO1 protein.

[0069] Figure 9 : A graph showing the regulation of Aβ and BACE1 expression and p-Tau / Tau ratio under rotenone stress in the SH-SY5Y APPswe / Tau double transgenic cell model. Among them, Figure 9 A is a Western blotting plot; Figure 9 B is a statistical graph showing the relative expression levels of Aβ protein; Figure 9 C is a statistical chart of the p-Tau / Tau ratio; Figure 9 D is a statistical graph showing the relative expression levels of BACE1 protein.

[0070] Figure 10 The effect of each drug-treated group on LPS-induced mRNA expression of inflammatory factors in BV2 cells is shown in the figure. Figure 10 A represents the relative expression level of TNF-α mRNA; Figure 10 B represents the relative expression level of IL-6 mRNA; Figure 10 C represents the relative expression level of IL-1β mRNA.

[0071] Figure 11 : Trend of body weight change in AD mice during drug administration in each group.

[0072] Figure 12 : Effects of each group on the novel object recognition ability of AD mice. Among them, Figure 12 A is a statistical chart of total exploration time; Figure 12 B is a statistical chart of the preference index; Figure 12 C is a statistical chart of the identification index; Figure 12 D shows the movement trajectories of mice in each group during the new object recognition test.

[0073] Figure 13A comparative graph showing the effects of each group on the spontaneous alternation rate in the Y-maze of AD mice. (The graph includes figures for each group.) Figure 13 A is a statistical graph showing the spontaneous alternation rate of mice in each group; Figure 13 B shows the movement trajectories of mice in the Y-maze spontaneous alternation test for each group.

[0074] Figure 14 : Effects of each group on the learning and memory abilities of AD mice in the Morris water maze. (The graph shows the effects of each group on the learning and memory abilities of AD mice in the Morris water maze.) Figure 14 A represents the average swimming speed curve; Figure 14 B represents the change curve of the escape latency period during the acquired training period; Figure 14 C represents the percentage of time spent in the target quadrant during the space exploration phase; Figure 14 D represents the number of platform crossings during the space exploration phase; Figure 14 E represents the movement trajectory of mice during the spatial exploration period in each group.

[0075] Figure 15 The effects of different drug groups on GSH content and SOD activity in the cortical and hippocampal regions of AD mice are shown in the figure. Figure 15 A represents the cortical GSH content; Figure 15 B represents the GSH content in the hippocampus; Figure 15 C represents cortical SOD activity; Figure 15 D represents hippocampal SOD activity.

[0076] Figure 16 : Effects of each group on the expression levels of inflammatory factors in the cortex and hippocampus of AD mice. (The diagram shows the effects of each group on these effects.) Figure 16 A represents the cortical TNF-α expression level; Figure 16 B represents the hippocampal TNF-α expression level; Figure 16 C represents the level of IL-6 expression in the cortex; Figure 16 D represents the hippocampal IL-6 expression level; Figure 16 E represents the cortical IL-1β expression level; Figure 16 F represents the hippocampal IL-1β expression level. Detailed Implementation

[0077] Terminology Definition

[0078] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0079] In this invention, "bardosolone" refers to 2-cyano-3,12-dioxoolean-1,9(11)-dien-28-oic acid, CAS No.: 218600-44-3, with the molecular formula C2. 31 H41 NO4 has a molecular weight of approximately 491.66 g / mol.

[0080] The structure of the Badosoron is shown in equation (I):

[0081]

[0082] Formula (I)

[0083] In this invention, the term "pharmaceutically acceptable salt of bardoxalon" refers to a salt formed by bardoxalon with a pharmaceutically acceptable acid or base. The acid is selected from one or more of the following: hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, formic acid, acetic acid, propionic acid, butyric acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, tartaric acid, citric acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, glutamic acid, and aspartic acid. The base is selected from one or more of the following: sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate, sodium bicarbonate, ammonia, triethylamine, diethylamine, ethanolamine, diethanolamine, triethanolamine, and meglumine.

[0084] In this invention, "badosolone derivatives" refers to compounds in which badosolone retains its pharmacological activity after chemical modification, including but not limited to: ester derivatives, amide derivatives, ether derivatives, hydroxyl derivatives, halogenated derivatives, alkyl-substituted derivatives, and prodrugs. The prodrug refers to a compound that releases badosolone after metabolism or chemical transformation in vivo.

[0085] In this invention, "donepezil" refers to 1-benzyl-4-[(5,6-dimethoxyindanone-2-yl)methyl]piperidine (2-[(1-benzylpiperidin-4-yl)methyl]-5,6-dimethoxy-2,3-dihydroinden-1-one), CAS No.: 120014-06-4, molecular formula C 24 H 29 NO3 has a molecular weight of approximately 379.49 g / mol.

[0086] The structure of donepezil is shown in formula (II):

[0087]

[0088] Equation (II)

[0089] In this invention, the "pharmaceutically acceptable salt of donepezil" refers to a salt formed by donepezil and a pharmaceutically acceptable acid. The acid is selected from one or more of hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, formic acid, acetic acid, propionic acid, butyric acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, tartaric acid, citric acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid. Preferably, the pharmaceutically acceptable salt of donepezil is donepezil hydrochloride (CAS No.: 120011-70-3).

[0090] In this invention, "derivatives of donepezil" refers to compounds that retain the pharmacological activity of donepezil after chemical modification, including but not limited to: ester derivatives, amide derivatives, ether derivatives, hydroxyl derivatives, halogenated derivatives, alkyl-substituted derivatives, and prodrugs. The prodrug refers to a compound that releases donepezil after metabolism or chemical transformation in vivo.

[0091] In this invention, "pharmaceuticalally acceptable excipients" refers to components in a pharmaceutical preparation other than the active ingredient, including but not limited to: fillers, binders, disintegrants, lubricants, solubilizers, preservatives, antioxidants, pH adjusters, emulsifiers, stabilizers, colorants, and flavoring agents. The fillers are selected from one or more of starch, lactose, microcrystalline cellulose, mannitol, sorbitol, and dicalcium phosphate; the binders are selected from one or more of starch paste, hydroxypropyl cellulose, gelatin, povidone, and polyethylene glycol; the disintegrants are selected from one or more of sodium carboxymethyl starch, crospovidone, crospovidone carboxymethyl cellulose, and low-substituted hydroxypropyl cellulose; and the lubricants are selected from one or more of magnesium stearate, stearic acid, talc, micronized silica gel, and polyethylene glycol. In this invention, "therapeuticly effective amount" refers to the amount of badosolone monotherapy or a combination of badosolone and donepezil that produces the expected therapeutic effect when administered to a subject. The therapeutic effects include, but are not limited to: alleviating Alzheimer's disease symptoms, slowing disease progression, improving cognitive function, reducing Aβ deposition, inhibiting Tau phosphorylation, reducing neuroinflammation, and alleviating oxidative stress damage. The effective therapeutic dose can be adjusted according to factors such as the subject's age, weight, sex, disease severity, and route of administration. In this invention, "prevention" refers to administering badosolone monotherapy or a combination of badosolone and donepezil before or in the early stages of Alzheimer's disease to reduce the risk of disease onset, delay disease onset, or reduce the severity of disease after onset. In this invention, "treatment" refers to administering badosolone monotherapy or a combination of badosolone and donepezil to subjects diagnosed with Alzheimer's disease to achieve one or more of the following objectives: alleviating clinical symptoms, improving cognitive function, slowing disease progression, preventing disease deterioration, prolonging survival, and improving quality of life. In this invention, "pharmaceutically acceptable carrier" refers to an excipient component formulated with the active ingredient into a pharmaceutical preparation that is compatible with the active ingredient and harmless to the subject. The carriers include, but are not limited to: water, ethanol, glycerol, propylene glycol, polyethylene glycol, vegetable oil, mineral oil, liposomes, cyclodextrin and their derivatives. In this invention, the "Nrf2-Keap1-ARE antioxidant pathway" refers to the signal transduction pathway in which nuclear factor E2-associated factor 2 (Nrf2) binds to Kelch-like ECH-associated protein 1 (Keap1), dissociates under oxidative stress, translocates to the cell nucleus, binds to antioxidant response elements (AREs), and initiates the expression of downstream antioxidant proteins (including HO-1, NQO1, etc.). In this invention, the "β-secretase 1 (BACE1)" is an aspartic protease capable of cleaving the β site of amyloid precursor protein (APP) to generate the N-terminus of β-amyloid protein (Aβ), and is a key rate-limiting enzyme in Aβ production.In this invention, "acetylcholinesterase (AChE)" is a serine hydrolase that hydrolyzes acetylcholine into choline and acetic acid, and is a key enzyme regulating the levels of cholinergic neurotransmitters. In this invention, "Aβ protein deposition" refers to the process by which β-amyloid protein aggregates in brain tissue to form oligomers, fibrils, and amyloid plaques, which is one of the core pathological features of Alzheimer's disease. In this invention, "abnormal phosphorylation of Tau protein" refers to the process by which Tau protein is overphosphorylated at multiple sites, leading to a decreased ability to bind to microtubules and its own aggregation to form neurofibrillary tangles, which is one of the core pathological features of Alzheimer's disease. In this invention, "neuroinflammation" refers to the inflammatory response mediated by the activation of microglia and astrocytes in the central nervous system, characterized by the overexpression of pro-inflammatory factors (including TNF-α, IL-6, and IL-1β), which is one of the important pathological mechanisms of Alzheimer's disease. In this invention, "oxidative stress" refers to a state in which the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in the body is imbalanced with the antioxidant defense system, leading to cell damage. It is one of the important pathological mechanisms of Alzheimer's disease.

[0092] "Combination therapy" refers to the administration of two or more drugs in the same formulation (i.e., a drug combination) or in separate formulations, administered simultaneously, separately (at different times), or sequentially (one drug is administered at a certain time interval before another drug is given). (FAD) 4T In an AD mouse model, the concurrent dosing regimen (badosolone 2 mg / kg·d + donepezil hydrochloride 2 mg / kg·d, orally, for 12 consecutive weeks) produced a significant synergistic effect, significantly superior to any single-drug treatment (P<0.001). The preferred molar ratio of badosolone to donepezil hydrochloride is 1:1 to 1:10, corresponding to a preferred dosage of badosolone 2 mg / kg·d and donepezil hydrochloride 2 mg / kg·d. The preferred dosing cycle is once daily for 12 consecutive weeks.

[0093] In this invention, the combined dosing regimen can be appropriately adjusted according to clinical needs, including but not limited to the following modifications:

[0094] Depending on the administration time, simultaneous administration, separate administration, or sequential administration can be used. When administration is sequential, the interval between the two drugs is 15 minutes to 12 hours.

[0095] Depending on the dosing cycle, it can be administered once daily, twice daily, or multiple times daily; it can be administered continuously or intermittently (e.g., administer for 5 days and then stop for 2 days); it can be used for short-term treatment (4-12 weeks), medium-term treatment (3-6 months), or long-term maintenance (more than 6 months).

[0096] Dosage adjustments can be made using a fixed-dose regimen (e.g., 2 mg / kg / day for each dose), or a dose-escalation regimen (e.g., badosoxone escalated from 0.5 mg / kg / day to 2 mg / kg / day, donepezil hydrochloride escalated from 1 mg / kg / day to 2 mg / kg / day), or individualized adjustments based on the patient's age, weight, liver and kidney function, and tolerance.

[0097] Depending on the route of administration, administration can be done via the same route (e.g., both are oral) or via different routes (e.g., oral administration of bardosoxone combined with donepezil hydrochloride injection).

[0098] Based on dosage form combinations, the same dosage form or different dosage forms can be used (such as ordinary tablets combined with sustained-release tablets, oral liquids combined with tablets, injections combined with oral preparations, etc.).

[0099] Depending on the stage of the disease, appropriate treatment plans can be developed for early AD (1-2 mg / kg·d each), mid-stage AD (badosoxone 2 mg / kg·d combined with donepezil hydrochloride 2-3 mg / kg·d) or late-stage AD (2-3 mg / kg·d each, preferably oral solution or injection).

[0100] Depending on individual patient differences, the dosage or dosing interval may be adjusted based on age (under 65 years, 65-75 years, over 75 years), renal function (mild to severe renal insufficiency), liver function (Child-Pugh A / B / C grade), and concomitant medications.

[0101] Based on efficacy and tolerability, the dosing regimen can be dynamically adjusted according to cognitive function scores (MMSE, MoCA, ADAS-Cog, etc.), activities of daily living (ADL) scores, and adverse reactions.

[0102] Long-term dosing can be achieved using a 4-week short-term regimen, a 12-week medium-term regimen, a 24-week long-term regimen, or a 52-week or longer long-term maintenance regimen.

[0103] Special dosing regimens may include a holiday discontinuation regimen (5 days of dosing followed by 2 days of discontinuation), a morning / nighttime differentiated regimen (badosulone in the morning and donepezil at night), a pre-meal / mid-meal / post-meal regimen, or an intermittent intensive regimen (1-2 days of intensive treatment per week).

[0104] "Medicine box": refers to a product that packages two or more drugs separately in the same package, including a separate first and second preparation, and optional instructions for use in combination.

[0105] The technical solution of the present invention will be described in more detail below with reference to specific embodiments and accompanying drawings. In implementing the present invention, except for the experimental steps, conditions, and methods described in detail below, all other techniques and common knowledge in the art are employed, and no additional special limitations are imposed.

[0106] It should be noted that the embodiments described herein are only some typical examples of the present invention, and not all implementation methods. The following description of exemplary embodiments is for illustrative purposes only and does not constitute any limitation on the present invention or its application scenarios.

[0107] In the demonstration and discussion of the various embodiments, the specific numerical values ​​and parameters involved are for illustrative purposes only and are not intended to be limiting. Therefore, other embodiments based on the concept of this invention can have their relevant numerical values ​​adjusted according to actual needs.

[0108] In addition, unless otherwise specified, the experimental materials used in the examples are all commercially available biochemical reagents, and the experimental operations also follow the standard procedures in this field.

[0109] I. Experimental Materials

[0110] 1. Main reagents

[0111]

[0112] 2. Antibodies

[0113]

[0114] 3. Primer sequences

[0115] The primer sequences used for qPCR detection were designed using conventional methods in the field and synthesized by Suzhou Genewiz Biotechnology Co., Ltd. The sequence information is as follows:

[0116]

[0117] Note: Specific primer sequences can be designed according to conventional methods in this field, or customized by a primer synthesis company.

[0118] 4. Cell lines

[0119]

[0120] Note: The stable cell line for the ARE reporter gene can be a commercially available ARE-Luciferase stable cell line (such as the CignalARE Reporter Kit), or it can be constructed by yourself according to conventional methods in this field.

[0121] 5. Laboratory animals

[0122]

[0123] Animal Ethics Statement: All animal experiments have been approved by our laboratory animal ethics committee, and the experimental procedures comply with relevant national regulations on laboratory animal welfare and ethics.

[0124] 6. Major Instruments and Equipment

[0125]

[0126] Example 1: Preparation of Bardoxapro monotherapy and pharmaceutical combinations

[0127] 1. Preparation of samples for cell experiments

[0128] Accurately weigh 49.17 mg of bardoxolone (purchased from MedChemExpress (MCE), molecular weight approximately 491.66 g / mol, CAS: 218600-44-3, catalog number: HY-14909), dissolve in an appropriate amount of dimethyl sulfoxide (DMSO), and bring the volume to 1 mL to prepare a 100 mM bardoxolone stock solution. Vortex mix well and store at -20°C protected from light. For experiments, use cell culture medium to serially dilute to the required concentrations (0.03 μM, 0.3 μM, 1 μM, 3 μM, 10 μM, 30 μM, etc.).

[0129] Accurately weigh 37.95 mg of donepezil (purchased from MedChemExpress (MCE), molecular weight approximately 379.49 g / mol, CAS: 120014-06-4, catalog number: HY-14566), dissolve in an appropriate amount of DMSO, and bring the volume to 1 mL to prepare a 100 mM donepezil stock solution. Vortex mix well and store at -20°C protected from light. For experiments, use cell culture medium to serially dilute to the required concentrations (0.03 μM, 0.3 μM, 1 μM, 3 μM, 10 μM, 30 μM, etc.).

[0130] Preparation of working solutions: Take 1 μL of 100 mM bardoxazoline stock solution and 1 μL of 100 mM donepezil stock solution, add them to 998 μL of culture medium, and mix thoroughly to obtain working solutions with a final concentration of 100 μM for both bardoxazoline and donepezil. Further dilute to the required concentration for experiments.

[0131] 2. Preparation of samples for animal experiments

[0132] Weigh out 400 mg of bardosulon and 400 mg of donepezil hydrochloride (purchased from MedChemExpress (MCE), molecular weight 415.95, CAS: 120011-70-3, catalog number: HY-B0034), and dissolve each in 3.333 mL of DMSO to prepare a stock solution of 120 mg / mL. Weigh out 150 mg of memantine hydrochloride (purchased from MedChemExpress (MCE), molecular weight 215.76, CAS: 41100-52-1, catalog number: HY-B0365A), and dissolve each in 0.8333 mL of DMSO to prepare a stock solution of 180 mg / mL. The badosolone and donepezil hydrochloride stock solutions were diluted 100-fold to obtain an intermediate solution of 1.2 mg / mL; the memantine hydrochloride stock solution was diluted 100-fold to obtain an intermediate solution of 1.8 mg / mL. The badosolone and donepezil hydrochloride intermediate solutions were further diluted to single-drug solutions with final concentrations of 0.3 mg / mL (corresponding to a dose of 2 mg / kg) and 0.6 mg / mL (corresponding to a dose of 4 mg / kg). The memantine hydrochloride intermediate solution was further diluted to a single-drug solution with a final concentration of 0.9 mg / mL (corresponding to a dose of 6 mg / kg). Mixed solutions were also prepared, namely, donepezil hydrochloride 0.3 mg / mL + badosolone 0.3 mg / mL (2+2 mg / kg), donepezil hydrochloride 0.6 mg / mL + badosolone 0.6 mg / mL (4+4 mg / kg), and donepezil hydrochloride 0.3 mg / mL + memantine hydrochloride 0.9 mg / mL (2+6 mg / kg). The normal control group and the model group were given pure water containing 1% DMSO. All groups were administered the drug once daily via gavage at a volume of 200 μL per animal.

[0133] Example 2: Detection of the in vitro inhibitory activity of bardosulomon against BACE1 and AChE

[0134] 1. Detection of AChE activity and inhibitory effect

[0135] The Elman colorimetric assay was used to detect AChE activity and the inhibitory effect of bardoxazoline on it. An in vitro enzymatic reaction system was established using acetylthiocholine iodide (ATChI) (Sigma-Aldrich, catalog number: A5751) as the specific substrate and 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) (Sigma-Aldrich, catalog number: D8130) as the chromogenic agent. Under the catalysis of AChE (Sigma-Aldrich, catalog number: C3389), the substrate ATChI hydrolyzes to generate thiocholine. Thiocholine reacts rapidly with DTNB to generate the yellow product 5-mercapto-2-nitrophenylacetic acid, which has a characteristic maximum absorption peak at 412 nm. The change in absorbance (OD value) at 412 nm was dynamically monitored using a microplate reader. The rate of increase in absorbance quantitatively reflected the AChE enzyme activity, and the inhibition rate and half-maximal inhibitory concentration (IC50) of bardoxazoline on AChE were calculated. 50 ).

[0136] 2. Detection of BACE1 activity and inhibitory effect

[0137] Fluorescence resonance energy transfer (FRET) technology was used to detect BACE1 activity and the inhibitory effect of bardosulomon on it. A specific peptide (Beyotime Biotechnology, catalog number: P0363S) linked to the fluorescent donor 7-methoxycoumarin-4-acetic acid (MCA) and the fluorescent acceptor 2,4-dinitrophenyl (Dnp) was selected as the BACE1 substrate. When not cleaved by BACE1, MCA and Dnp were close together, and the fluorescence emitted by MCA was specifically quenched by Dnp. After cleavage by BACE1, MCA and Dnp spatially separated, the fluorescence quenching effect was relieved, and the fluorescence intensity of MCA significantly increased. The change in MCA fluorescence intensity was measured using a fluorescence detector, and the inhibition rate and IC50 of bardosulomon against BACE1 were calculated. 50 .

[0138] 3. Experimental Results

[0139] Experimental results are as follows Figure 1 As shown. Bardozoron's IC against BACE1 50 44.28 μM ( Figure 1 A), IC of AChE 50 48.65 μM ( Figure 1 (B) indicates that bardosulomon has a clear dual-target inhibitory activity against BACE1 and AChE. This discovery breaks through the existing technical understanding that bardosulomon is only an Nrf2 activator and provides a new mechanism to support its anti-AD effect.

[0140] Example 3: Cellular-level validation of badoxapro monotherapy and pharmaceutical combinations

[0141] 1. Experimental Model and Grouping

[0142] Using the SH-SY5Y APPswe / Tau double transgenic cell model as the research object, experiments were conducted under basal conditions and under rotenone-induced stress.

[0143] The SH-SY5Y APPswe / Tau double-transgenic cell line (co-expressing Swedish mutant APP and Tau proteins) was constructed and preserved in our laboratory according to the literature method (Velagapudi et al., 2018). Construction method: Human neuroblastoma cells (SH-SY5Y) (purchased from Shanghai Fuheng Biotechnology Co., Ltd., catalog number: FH0156) were used as host cells. The cells were transfected with plasmids containing Swedish mutant amyloid precursor protein (APPswe) and Tau protein (P301L mutant). Stable cell lines expressing these proteins were obtained after selection with puromycin (Beyotime Biotechnology, catalog number ST551).

[0144] Cell viability assay grouping:

[0145] Monotherapy group: Bardosulon (0.03–100 μM) or donepezil (0.03–100 μM)

[0146] Composition group: fixed bardosulomon concentrations (0.3 μM, 1 μM, 10 μM), combined with donepezil gradient concentrations (0.01~100 μM)

[0147] Mechanism detection groups (all drug concentrations were 1 μM):

[0148] Group 1: Double-transformed cell control group (no drug administration, no rotenone induction)

[0149] Group 2: Rotenone (Rot) induced model group

[0150] Group 3: Donepezil monotherapy group

[0151] Group 4: Bardosulone monotherapy group

[0152] Group 5: Combination therapy (badossolon 1 μM + donepezil 1 μM)

[0153] Each group had 3 replicates, and the experiment was repeated three times. Data are expressed as mean ± standard deviation and analyzed using statistical methods (P < 0.05 was considered statistically significant).

[0154] 2. Cell viability assay

[0155] Cell viability was assessed using the CCK-8 assay (CCK-8 kit, Beyotime Biotechnology, catalog number: C0037). The synergistic effect of badosolone combined with donepezil in a rotenone-induced cellular stress model was evaluated using the King's Law formula (Q = E(A + B) / (EA + EB − EA·EB)). In the formula, EA represents the net protective effect of badosolone monotherapy, EB represents the net protective effect of different concentrations of donepezil monotherapy, and EA+B represents the net protective effect of the combination therapy. Judgment criteria: Q > 1.15: significant synergistic effect; 0.85 ≤ Q ≤ 1.15: additive effect; Q < 0.85: antagonistic effect. Results are as follows:

[0156] (1) Under basal conditions, bardosorone increases cell viability in a concentration-dependent manner within the range of 0.03–1 μM. Figure 2 A); donepezil significantly improves cell viability only at a concentration of 1 μM, and cell viability decreases at concentrations of 3–100 μM. Figure 2 B).

[0157] (2) Under rotenone-induced stress, the concentration-dependent enhancement of cell viability by bardosoprolol extended to the range of 0.03–10 μM, and the increase in viability was greater than that under basal conditions. Figure 3 A).

[0158] (3) Under basal conditions, bardoxazoline concentrations of 0.3 μM, 1 μM, and 10 μM were fixed and combined with donepezil in the concentration range of 0.3–30 μM. The combination of bardoxazoline and donepezil at equal proportions of 1 μM each showed the best effect in improving cell viability, which was superior to the use of either drug alone. Figure 4 A- Figure 4 C).

[0159] (4) Under rotenone-induced stress, when bardoxazoline was fixed at 1 μM, donepezil in combination with it in the range of 0.1~100 μM could further enhance cell viability. Verification using the King's Q-value method showed that the Q values ​​of each combination group were greater than 1.15, demonstrating that the combination of the two drugs had a significant synergistic protective effect. Figure 5 ).

[0160] (5) The 1 μM equal proportion combination can effectively exert a synergistic effect in both the basic and stress models, suggesting that it has universality in the pathological stage. Compared with the dosage combination of 3~100μM donepezil, the cell viability of donepezil monotherapy gradually decreased at concentrations of 3~100μM, with the most significant decrease in cell viability at 100μM (P<0.0001) (Figure 2B). Considering the rationality of the in vitro pharmacological concentration and the universality of the dual models, the 1 μM equal proportion combination was finally selected as the core ratio for subsequent studies.

[0161] 3. Detection of Nrf2 pathway and antioxidant proteins

[0162] (1) SOD activity was detected using the WST-1 method. The SOD detection kit was purchased from Nanjing Jiancheng Bioengineering Institute (catalog number: A001-3). Following the kit instructions, cell lysis buffer was mixed with WST-1 working solution and enzyme working solution, incubated at 37℃ for 30 minutes, and the absorbance was measured at 450 nm to calculate SOD activity. GSH content was detected using the DTNB colorimetric method. The GSH detection kit was purchased from Nanjing Jiancheng Bioengineering Institute (catalog number: A006-2-1). Following the kit instructions, cell lysis buffer was reacted with DTNB colorimetric reagent, and the absorbance was measured at 412 nm. GSH content was calculated based on the standard curve. The results showed that, compared with the model group, the combined treatment group significantly increased cell GSH and SOD activities (P<0.001), and the antioxidant effect was superior to both the badoxolotone monotherapy group and the donepezil monotherapy group. Figure 6 ).

[0163] (2) The activation of the ARE gene was detected using the luciferase reporter gene assay. The stable ARE reporter gene cell line was constructed in our laboratory (host cells were SH-SY5Y APPswe / Tau). The luciferase reporter gene assay kit was purchased from Promega (product name: Dual-Luciferase® Reporter Assay System, catalog number: E1910). Cells were seeded in 96-well plates and cultured overnight. Afterward, cells were treated with badoxalon (1 μM), donepezil (1 μM), or a combination of both (1 μM each) for 6 hours. Luciferase activity was detected after lysis. The results showed that both the badoxalon monotherapy group and the combination therapy group significantly activated ARE gene expression (P<0.0001). Figure 7 Furthermore, compared with the bardoxol monotherapy group, the combination therapy group showed a significantly enhanced ARE gene activation effect.

[0164] (3) Western blotting was used to detect the expression of nuclear Nrf2 (Abcam, catalog number: ab62352), HO-1 (Abcam, catalog number: ab68477), and NQO1 (Abcam, catalog number: ab80588) proteins. β-actin (Proteintech, catalog number: 66009-1-Ig) and Lamin B1 (Abcam, catalog number: ab229025) were used as internal references for nuclear protein normalization. The results showed that the combination therapy synergistically activated the Nrf2 pathway, significantly upregulating the expression level of Nrf2 protein in the cell nucleus (P<0.05), and simultaneously significantly upregulating the expression of downstream antioxidant target proteins HO-1 (P<0.0001) and NQO1 (P<0.001). This upregulation effect was significantly better than that of the badoxolotone monotherapy group and the donepezil monotherapy group (P<0.0001). Figure 8 ).

[0165] 4. Detection of Aβ, BACE1, Tau, and p-Tau proteins

[0166] Western blotting was used to detect the expression of Aβ (BioLegend, catalog number: SIG-39320), BACE1 (Abcam, catalog number: ab183612), and the p-Tau (Proteintech, catalog number: 80333-1-RR) / Tau (Proteintech, catalog number: 10274-1-AP) ratio, with β-actin (Proteintech, catalog number: 66009-1-Ig) used as an internal control protein for normalization. Results showed that the Aβ and BACE1 protein levels in the combination therapy group were significantly downregulated compared to the model group (P<0.001), and the p-Tau / Tau ratio was decreased, both significantly better than those in the badoxolotl monotherapy group and donepezil monotherapy group. Figure 9 A~ Figure 9 D).

[0167] 5. Inflammatory factor detection

[0168] The mRNA expression of TNF-α, IL-6, and IL-1β in LPS-induced BV2 cells (purchased from Shanghai Fuheng Biotechnology Co., Ltd., catalog number: FH0355) was detected by qPCR. The results showed that the combination therapy group had a significantly better inhibitory effect on inflammatory factors than the badoxolone monotherapy group and the donepezil monotherapy group. Compared with the model group, the expression levels of TNF-α, IL-6, and IL-1β in the combination therapy group were reduced by 60.47% ± 8.40%, 66.71% ± 5.52%, and 44.37% ± 1.50%, respectively (P < 0.01).

[0169] Compared with the badoxolotl monotherapy group (reductions of 49.01% ± 8.51%, 54.78% ± 9.37%, and 37.46% ± 5.36%) and the donepezil monotherapy group (reductions of 13.13% ± 5.32%, 17.77% ± 7.94%, and 17.48% ± 10.14%), the combination therapy group showed a more significant reduction in phospholipin levels. Figure 10 ).

[0170] Example 4: Animal-level validation of bardosulon monotherapy and pharmaceutical combinations

[0171] 1. Animal models and grouping

[0172] Adopting FAD 4T AD mouse model (strain name: B6 / JGpt-Tg(Thy-APP / Thy-PSEN1)5 / Gpt, purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.), 80 SPF-grade AD model mice aged 6-8 weeks, weighing 20-22 g, were randomly divided into the following groups (n=10 per group):

[0173] Model group

[0174] Low-dose badoxolomide monotherapy (2 mg / kg·d)

[0175] High-dose badosulone monotherapy group (4 mg / kg·d)

[0176] Donepezil hydrochloride monotherapy low-dose group (2 mg / kg·d)

[0177] Donepezil hydrochloride monotherapy high-dose group (4 mg / kg·d)

[0178] Low-dose combination (badossolon 2 mg / kg·d + donepezil hydrochloride 2 mg / kg·d)

[0179] High-dose combination (badossolon 4 mg / kg·d + donepezil hydrochloride 4 mg / kg·d)

[0180] Positive control group (donepezil hydrochloride 2 mg / kg·d + memantine hydrochloride 6 mg / kg·d)

[0181] Ten age- and weight-matched wild-type (WT) mice were selected as a normal control group.

[0182] 2. Administration method

[0183] The normal control group and the model group were given gavage with an equal volume of pure water containing 1% DMSO. The single drug groups and the combination groups were given the corresponding doses by gavage once a day for 12 consecutive weeks.

[0184] 3. Detection Indicators and Methods

[0185] (1) Weight monitoring

[0186] Mice were weighed at fixed times each week during the drug administration period, and the trend of weight change was recorded. Results showed that the weight of mice in all groups increased with the number of weeks over 12 weeks, with no statistically significant difference in the rate of weight gain among the groups (P>0.05). Figure 11 This indicates that the pharmaceutical composition has no significant adverse effects on mouse growth.

[0187] (2) New object recognition experiment

[0188] The experimental setup consisted of a 40 cm × 40 cm × 40 cm soundproof box. During the training period, two identical objects were placed in fixed positions within the box each day, allowing mice to explore freely for 10 minutes. On the fourth day of the testing period, one of the objects was replaced with a new object, and the time taken by the mice to explore both the old and new objects within 5 minutes was recorded. Evaluation metrics: Recognition Index (DI) = (Exploration time of new object - Exploration time of familiar object) / Total exploration time; Preference Index (RI) = Exploration time of new object / Total exploration time.

[0189] The results showed no significant difference in total exploration time among the groups of mice. Figure 12 Based on this, the results showed that AD model mice had significant impairment in novel object recognition memory; all drug-treated groups improved their novel object recognition ability; the preference index (RI) of mice in the low-dose combination group was >55%, and the novel-old object exploration difference index was close to that of the WT group, which was significantly better than all single-drug groups, the positive control group, and the high-dose combination group (P<0.0001). Figure 12 B, 12C), representative motion trajectories are shown in Figure 12 D.

[0190] (3) Y-maze experiment

[0191] The Y-maze consists of three equidistant arms (arm length 25 cm, width 8 cm, height 15 cm), labeled A, B, and C. A mouse is placed at the end of one arm and allowed free movement for 8 minutes, and the order in which it enters the arm is recorded. Spontaneous alternation rate (%) = [Number of consecutive entries into three different arms / (Total number of arm entries - 2)] × 100%.

[0192] The results showed that AD model mice had significant working memory impairment (decreased spontaneous alternation rate, P<0.001); all drug-treated groups increased the spontaneous alternation rate of AD mice; the low-dose combination group had the highest spontaneous alternation rate, which was higher than that of the WT group and significantly better than other groups (P<0.0001). Figure 13 A), the movement trajectories of mice in each group during the spontaneous alternation test period are shown in Figure 1. Figure 13 B.

[0193] (4) Morris water maze experiment

[0194] The experimental setup consisted of a circular water tank with a diameter of 120 cm, and the water temperature was kept constant at 22-24℃. The experiment was divided into three stages:

[0195] Platform visibility period (1 day): The platform is 1 cm above the water surface to help the mice become familiar with the pool environment.

[0196] Acquired training period (4 days): The platform was hidden underwater, and the escape latency of mice within 60 seconds was recorded.

[0197] Probe test period (1 day): Remove the platform and record the number of times the mouse crosses the original platform position within 60 seconds and the percentage of time spent in the target quadrant.

[0198] The results showed no significant difference in swimming speed among the groups of mice. Figure 14 A, P>0.05), excluding the interference of motor ability on cognitive outcomes. AD model mice showed significant spatial learning and memory impairment; all drug administration groups improved the spatial learning and memory abilities of AD mice; the low-dose group of the composition showed a synergistic effect, with the shortest escape latency during the acquired training phase ( Figure 14 B), the target quadrant had the highest percentage of time spent in the space exploration testing phase and the most platform crossings. Figure 14 C and 14D were significantly better than the single-drug groups (P<0.001). The spatial exploration trajectory diagrams of mice in each group are shown in 14E.

[0199] (5) Detection of GSH and SOD in brain tissue

[0200] Brain tissue homogenates and hippocampal homogenates from AD mice were collected. SOD activity was detected by WST-1 method, and GSH content was detected by DTNB colorimetric method.

[0201] The results showed that significant oxidative stress imbalance was present in the cortex and hippocampus of AD model mice (decreased GSH content and SOD activity, P<0.001); each single-drug group increased GSH content and enhanced SOD activity to varying degrees; the low-dose combination group showed a clear synergistic effect, and its effect on improving oxidative stress was significantly better than that of each single-drug group (P<0.0001). Figure 15 ).

[0202] (6) Detection of inflammatory factors in brain tissue

[0203] The expression levels of TNF-α, IL-6, and IL-1β in the cortical and hippocampal tissues of AD mice were detected by ELISA.

[0204] The results showed that the levels of three pro-inflammatory factors in the cortex and hippocampus of the AD model group were significantly higher than those in the WT group (P<0.001); all single-drug groups significantly reduced the levels of pro-inflammatory factors; the low-dose combination group showed a more pronounced effect in reducing pro-inflammatory factors, significantly better than the single-drug groups (P<0.001). Figure 16 ).

[0205] This embodiment demonstrates in animal studies that: (1) both bardosulone monotherapy and the drug combination can significantly improve cognitive function in AD mice; (2) the synergistic therapeutic effect of the low-dose group (2 mg / kg·d each) of the drug combination is significantly better than that of the high-dose group (4 mg / kg·d each) and the positive control group (donepezil hydrochloride + memantine hydrochloride); (3) the drug combination can significantly improve oxidative stress and neuroinflammatory status in the brain tissue of AD mice, and has a more significant protective effect on the hippocampus; (4) the drug combination has good safety within the effective dose range.

[0206] In summary, the following conclusions can be drawn:

[0207] I. Bardosorone possesses anti-AD activity, and its mechanism involves multi-target regulation.

[0208] Example 2 is the first demonstration that badocoxone has dual-target inhibitory activity against BACE1 and AChE, IC50. 50 The concentrations were 44.28 μM and 48.65 μM, respectively. Example 3 further verified that bardosuloone can activate the Nrf2-Keap1-ARE antioxidant pathway at the cellular level (…). Figure 7 , Figure 8 ), inhibit Aβ ( Figure 9 B), BACE1 expression ( Figure 9 D) Inhibition of Tau phosphorylation ( Figure 9 C) and reducing the expression of inflammatory factors ( Figure 10 It exerts its anti-AD effect through multiple mechanisms, including [list of mechanisms]. These results indicate that the anti-AD effect of bardosorone is not achieved through a single mechanism, but rather through the synergistic regulation of multiple targets and pathways.

[0209] II. The combination of bardosulozoline and donepezil exhibits significant synergistic effects.

[0210] The cell viability test results of Example 3 showed that, under the basal state ( Figure 4 B) and rotenone-induced stress ( Figure 5 Under A), the equal-ratio combination of 1 μM each of bardoxazoline and donepezil significantly enhanced cell viability compared to either drug alone (P<0.001). Animal experiments in Example 4 further validated this synergistic effect: the low-dose group of the composition (2 mg / kg·d each) showed improved cell viability in new object recognition (…). Figure 12 Y Maze ( Figure 13 Morris Water Maze Figure 14 The performance in behavioral tests such as [list of tests] was significantly better than that of each single-drug group and the positive control group (P<0.001). The above results consistently confirm the synergistic effect of the composition at both the cellular and animal levels.

[0211] III. Optimal Molar Ratio

[0212] This invention discovers through systematic screening at the cellular level ( Figure 5 A): When badosole is fixed at 1 μM, donepezil exhibits a significant synergistic effect in the range of 1 μM to 10 μM (molar ratio 1:1 to 1:10), with the optimal effect observed at a 1:1 ratio. Furthermore, animal experiments have verified that badosole (molecular weight 491.68) 2 mg / kg·d and donepezil hydrochloride (molecular weight 415.95) 2 mg / kg·d (molar ratio 0.85:1, i.e., 1:1.18) also produce a significant synergistic effect, with this ratio falling within the range of 1:1 to 1:10. Therefore, the preferred molar ratio of badosole to donepezil described in this invention is 1:1 to 1:10, with 1:1 being the most preferred.

[0213] Example 4 further verified that, at the animal level, the synergistic therapeutic effect of each 2 mg / kg·d dose group (low-dose group of composition) was significantly better than that of the proportional high-dose group (4 mg / kg·d each), suggesting that the composition does not follow the conventional "dose-effect positive correlation" relationship, but rather exhibits a non-linear characteristic of "low-dose superiority", which is non-obvious.

[0214] IV. The composition has universal applicability across pathological stages.

[0215] In Example 3, the 1 μM equal-proportion combination effectively exerted a synergistic effect in both the basal state (without inducer) and the rotenone-induced stress state. Figure 4 B. Figure 5 A). This result indicates that the pharmaceutical composition described in this invention can be stably effective regardless of whether the AD disease is in the early stage with mild oxidative stress or in the middle or late stage with severe oxidative stress, demonstrating good general applicability across different pathological stages.

[0216] V. The composition has good safety.

[0217] The weight monitoring results of Example 4 showed that during the 12-week continuous administration period, there was no statistically significant difference in weight gain between the treatment groups and the normal control group (P>0.05). Figure 11 This indicates that the pharmaceutical composition of the present invention has no significant adverse effects on mouse growth within the effective dose range, and no obvious organ toxicity or behavioral abnormalities were observed.

[0218] The embodiments described in this specification are only intended to help those skilled in the art better understand and implement the present invention. For those skilled in the art, appropriate adjustments, substitutions, or extensions can be made to these embodiments based on the content disclosed in this invention without requiring creative effort, and the basic principles of the invention can be applied to other similar scenarios. Therefore, the disclosed embodiments should not be considered as limiting the scope of protection of this invention. Any equivalent modifications, improvements, or alternatives made based on this invention without departing from its essential spirit should fall within the scope of protection of this invention.

Claims

1. The use of bardosulon or its pharmaceutically acceptable salts or derivatives in the preparation of medicaments for the prevention and / or treatment of Alzheimer's disease, wherein the structure of bardosulon is shown in formula (I): （I）。 2. The application according to claim 1, characterized in that, The drug also contains pharmaceutically acceptable carriers or excipients.

3. The application according to claim 1, characterized in that, The method of administration of the drug is selected from one or more of oral, injection, implantation, spray or inhalation.

4. The application according to claim 1 or 2, characterized in that, The dosage form of the drug is injection, oral liquid, powder, tablet, granule, capsule, syrup, sustained-release preparation, enteric solvent, aerosol or suspension.

5. A pharmaceutical composition for the prevention and / or treatment of Alzheimer's disease, characterized in that, The product comprises bardosulon as claimed in claim 1, or a pharmaceutically acceptable salt or derivative thereof, and donepezil or a pharmaceutically acceptable salt thereof, wherein the structure of donepezil is shown in formula (II): (II).

6. The pharmaceutical composition according to claim 5, characterized in that, The molar ratio of bardosorlon to donepezil is from 1:0.01 to 1:

100.

7. The pharmaceutical composition according to claim 5, characterized in that, The molar ratio of the badosole to the donepezil is 1:0.3 to 1:

30.

8. The pharmaceutical composition according to claim 5, characterized in that, The molar ratio of bardosorone to donepezil is 1:1 to 1:

10.

9. The pharmaceutical composition according to claim 5, characterized in that, The molar ratio of bardosorone to donepezil is 1:

1.

10. The pharmaceutical composition according to claim 5, characterized in that, The pharmaceutical composition also includes a pharmaceutically acceptable carrier or excipient.

11. The pharmaceutical composition according to claim 10, characterized in that, The pharmaceutically acceptable carrier or excipient is selected from one or more of the following: fillers, binders, disintegrants, lubricants, solubilizers, preservatives, antioxidants, pH adjusters, emulsifiers, and stabilizers.

12. The pharmaceutical composition according to claim 5, characterized in that, The dosage form of the pharmaceutical composition is an injection, oral liquid, powder, tablet, granule, capsule, syrup, sustained-release preparation, enteric solvent, aerosol, or suspension.

13. The pharmaceutical composition according to claim 5, characterized in that, The administration method of the pharmaceutical composition is selected from one or more of oral, injection, implantation, spray, or inhalation.

14. Use of the pharmaceutical composition of any one of claims 5 to 13 in the preparation of a medicament for the prevention and / or treatment of Alzheimer's disease.

15. The application according to claim 14, characterized in that, The pharmaceutical composition achieves its therapeutic effect through the following multi-target synergistic mechanism: activating the Nrf2-Keap1-ARE antioxidant pathway, inhibiting the activity and expression of β-secretase 1 (BACE1), inhibiting the activity of acetylcholinesterase (AChE), reducing Aβ protein deposition, inhibiting abnormal phosphorylation of Tau protein, reducing neuroinflammation, and alleviating oxidative stress damage.

16. A medicine box for the prevention and / or treatment of Alzheimer's disease, characterized in that, Include: (i) A first preparation containing bardosorlon or a pharmaceutically acceptable salt or derivative thereof; (ii) A second preparation containing donepezil or a pharmaceutically acceptable salt thereof; The first and second formulations are used for simultaneous, separate or sequential administration.

Images