Drug combination for treating alzheimer's disease and pharmaceutical composition thereof

Abstract

A drug combination for treating Alzheimer's disease and a pharmaceutical composition thereof. The pharmaceutical composition comprises: (a) a prophylactically or therapeutically effective amount of HDAC6 inhibitor; and (b) a prophylactically or therapeutically effective amount of GSK-3β inhibitor. The components of the drug combination are used in combination, so that the therapeutic effect of each single drug on Alzheimer's disease can be synergistically enhanced. Moreover, significant weight loss or abnormal behavior does not appear in mice after drug administration, showing that the drug combination has good efficacy and safety.

Description

Combination drug and pharmaceutical composition for treating Alzheimer's disease Technical Field

[0001] The present invention belongs to the field of medicine, and specifically relates to a combination drug and a pharmaceutical composition thereof for treating Alzheimer's disease. Background Art

[0002] The pathogenesis of Alzheimer's disease (AD) is complex, involving multiple pathological processes, including the generation and accumulation of amyloid-β (Aβ), abnormal phosphorylation and aggregation of tau, oxidative stress, and inflammatory responses. Single-target drug interventions often have limited efficacy and fail to completely control the disease. Histone deacetylase 6 (HDAC6) is involved in regulating Aβ and tau proteolysis, neurotransmitter release and reuptake, microtubule network stability, inflammation, and antioxidant activity. Inhibition of HDAC6 activity can enhance Aβ degradation and tau proteolysis, and exert neuroprotective effects. Glycogen synthase kinase 3β (GSK-3β) is involved in the phosphorylation of amyloid-β precursor protein (APP) and tau, as well as the production and release of inflammatory mediators. Inhibition of GSK-3β can reduce Aβ deposition, tau phosphorylation, and inflammatory damage.

[0003] Although the use of GSK-3β inhibitors or HDAC6 inhibitors alone can alleviate AD pathology to a certain extent, the anti-AD effect needs to be improved.

[0004] Summary of the Invention

[0005] The following is a summary of the subject matter described in detail herein. This summary is not intended to limit the scope of the claims.

[0006] The present invention aims to address at least one of the technical problems existing in the aforementioned prior art. To this end, the present invention provides a combination drug for the treatment of Alzheimer's disease, a pharmaceutical composition thereof, and a formulation comprising the pharmaceutical composition. The present invention combines an HDAC6 inhibitor with a GSK-3β inhibitor, synergistically enhancing the therapeutic efficacy of each agent alone for Alzheimer's disease. Mice treated with the drugs showed no significant weight loss or behavioral abnormalities, demonstrating the effectiveness and safety of the combination drug.

[0007] Specifically, the present invention provides the following embodiments:

[0008] In one aspect, the present invention provides a pharmaceutical composition comprising:

[0009] (a) a preventively or therapeutically effective amount of an HDAC6 inhibitor; and

[0010] (b) a prophylactically or therapeutically effective amount of a GSK-3β inhibitor.

[0011] In some embodiments of the present invention, the HDAC6 inhibitor includes a compound represented by the following formula, or a pharmaceutically acceptable salt, isotope label, hydrate, solvate, diastereomer or enantiomeric form thereof:

[0012] In some preferred embodiments of the present invention, the HDAC6 inhibitor includes a compound represented by the following formula, or a pharmaceutically acceptable salt, isotope label, hydrate, solvate, diastereomer or enantiomer thereof:

[0013] Compound EH-1 is a specific HDAC6 inhibitor that has shown potential to alleviate AD symptoms in experimental models. EH-1 has neuroprotective activity against oxidative stress, apoptosis, and abnormal tau phosphorylation, thereby alleviating AD symptoms. However, this HDAC6 inhibitor requires high doses to demonstrate any efficacy. The present invention combines it with a GSK-3β inhibitor, and the combination, at a specific ratio, produces a synergistic anti-AD effect without side effects.

[0014] In some embodiments of the present invention, the GSK-3β inhibitor comprises LiCl or a compound represented by the following formula, or a pharmaceutically acceptable salt, isotope label, hydrate, or solvate thereof:

[0015] In some preferred embodiments of the present invention, the GSK-3β inhibitor comprises LiCl.

[0016] LiCl is a specific GSK-3β inhibitor that has been clinically used for many years and is widely used in bipolar disorder. LiCl exhibits neuroprotective activities, including anti-inflammatory and antioxidant properties, and promotes neurogenesis and synaptic plasticity. It can also reduce Aβ production and abnormal tau protein phosphorylation. The present invention combines it with an HDAC6 inhibitor, and the two, at a certain ratio, can produce a synergistic anti-AD effect without side effects.

[0017] In some embodiments of the present invention, the ratio of the effective amount of the HDAC6 inhibitor to the effective amount of the GSK-3β inhibitor is about 1:(1-20000), for example: about 1:(1-20000), about 1:(1-10000), about 1:(1-5000), about 1:(1-4000), about 1:(5-20000), about 1:(5-10000), about 1:(5-5000), about 1:(5-4000), about 1:(1-25), about 1:(1-20), about 1:(1-15), about 1:(1-10), about 1:(1-5), about 1:(100-20000), about 1:(1000-20000), about 1:(2000-20000), about 1:(2000-10000), about 1:(4000-20000), about 1:(4000-10000), about 1:(1000-10000), about 1:(2000-8000), about 1:(3000-5000), about 1:(8000-12000), about 1:5, about 1:4000, about 1:10000.

[0018] The present invention limits the ratio of the effective amount of the HDAC6 inhibitor to the effective amount of the GSK-3β inhibitor to about 1:(1-20000). Exceeding this range may result in excessive drug efficacy, causing side effects or adverse reactions, which may have adverse effects on the body, such as cardiovascular system problems, nervous system problems, etc.

[0019] In some preferred embodiments of the present invention, the ratio of the effective amount of the HDAC6 inhibitor to the effective amount of the GSK-3β inhibitor is about 1:(5-10000), about 1:(1-25), or about 1:(2000-10000).

[0020] In some more preferred embodiments of the present invention, the ratio of the effective amount of the HDAC6 inhibitor to the effective amount of the GSK-3β inhibitor is about 1:5, about 1:4000, or about 1:10000.

[0021] In some embodiments of the present invention, the pharmaceutical composition is used for preventing and / or treating Alzheimer's disease in a subject in need thereof.

[0022] The Alzheimer's disease has one or more pathological changes selected from the following: cognitive decline, senile plaques formed by deposition of β-amyloid protein, and neurofibrillary tangles formed by abnormal protein byssus.

[0023] In another aspect, the present invention provides a pharmaceutical preparation, which comprises the pharmaceutical composition of the present invention and pharmaceutically acceptable excipients.

[0024] In some embodiments of the present invention, the components of the pharmaceutical preparation can work systemically and / or locally. For this purpose, they can be administered by suitable routes, such as by injection (for example, intravenous, intraarterial, subcutaneous, intraperitoneal, intramuscular administration, including instillation) or transdermal administration; or by oral, oral, nasal, transmucosal, topical administration, in the form of ophthalmic preparations or by inhalation.

[0025] In some embodiments of the present invention, the pharmaceutical preparation may be administered in a suitable dosage form corresponding to the above-mentioned administration routes, including but not limited to tablets, capsules, granules, syrups, lozenges, cachets, elixirs, pills, powders, suppositories, ointments, creams, emulsions, suspensions, solutions, aerosols, injections, paints, and lotions. Preferably, the pharmaceutical preparation may be in the form of tablets, capsules, granules, syrups, lozenges, cachets, elixirs, pills, powders, suspensions, solutions, aerosols, or injections.

[0026] In another aspect, the present invention provides use of the aforementioned pharmaceutical composition or pharmaceutical preparation in the preparation of a medicament for preventing and / or treating Alzheimer's disease.

[0027] In some embodiments of the present invention, the Alzheimer's disease has one or more pathological changes selected from the following: cognitive decline, senile plaques formed by deposition of β-amyloid protein, and neurofibrillary tangles formed by abnormal protein byssus.

[0028] The drugs for treating Alzheimer's disease described in the present invention include drugs for directly treating Alzheimer's disease or drugs for adjuvant treatment of Alzheimer's disease. Specifically, the drugs for adjuvant treatment of Alzheimer's disease can be used to assist radiotherapy or other anti-Alzheimer's disease drugs, targeted preparations, immunotherapy, biological therapy, etc. in treating Alzheimer's disease.

[0029] In another aspect, the present invention provides a method for preventing and / or treating Alzheimer's disease, comprising administering to a subject in need thereof:

[0030] (a) a preventively or therapeutically effective amount of an HDAC6 inhibitor; and

[0031] (b) a prophylactically or therapeutically effective amount of a GSK-3β inhibitor;

[0032] Optionally, the HDAC6 inhibitor is as defined above,

[0033] The GSK-3β inhibitor is as defined above.

[0034] In some embodiments of the present invention, the amount of the HDAC6 inhibitor administered is about 0.001 mg / d to 5 g / d, for example, about 0.001 mg / d to 1000 mg / d, about 0.001 mg / d to 800 mg / d, about 0.001 mg / d to 600 mg / d, 0.01 mg / d to 5000 mg / d, about 0.01 mg / d to 1000 mg / d, about 0.01 mg / d to 600 mg / d, about 0.1 mg / d to 1000 mg / d, about 0.1 mg / d to 600 mg / d, about 1 mg / d to 1000 mg / d, about 1 mg / d to 600 mg / d, about 10 mg / d to 1000 mg / d, about 10 mg / d to 600 mg / d, about 100 mg / d ~1000mg / d, about 200mg / d~1000mg / d, about 300mg / d~1000mg / d, about 400mg / d / d~1000mg / d, about 200mg / d / d~1000mg / d, about 200mg / d / d~800mg / d, about 400mg / d / d~1000mg / d, about 400mg / d / d~800mg / d, about 500mg / d~700mg / d, about 0.001mg / d, about 0.01mg / d, about 0.1mg / d, about 1mg / d, about 10mg / d, about 100mg / d, about 200mg / d, about 300mg / d, about 400mg / d, about 500mg / d, about 500mg / d, about 600mg / d, about 700mg / d.

[0035] In some embodiments of the present invention, the HDAC6 inhibitor is administered in an amount of about 1 μg / kg to 100 mg / kg per unit dose, for example, about 1 μg / kg to 100 mg / kg, about 1 μg / kg to 10 mg / kg, about 10 μg / kg to 100 mg / kg, about 10 μg / kg to 10 mg / kg, about 0.1 mg / kg to 100 mg / kg, about 0.1 mg / kg to 10 mg / kg, about 1 mg / kg to 100 mg / kg, About 1 mg / kg to 10 mg / kg, about 1 mg / kg to 50 mg / kg, about 1 mg / kg to 40 mg / kg, about 1 mg / kg to 20 mg / kg, about 5 mg / kg to 20 mg / kg, about 8 mg / kg to 12 mg / kg, about 1 μg / kg, about 10 μg / kg, about 0.1 mg / kg, about 1 mg / kg, about 10 mg / kg per unit dose, and administered in one or more (e.g., 1, 2, 3, 4, 5) unit doses per day.

[0036] In some embodiments of the present invention, the amount of the GSK-3β inhibitor administered is about 0.001 mg / d to 25 g / d, for example, about 0.001 mg / d to 5000 mg / d, about 0.001 mg / d to 3000 mg / d, about 0.01 mg / d to 25 g / d, about 0.01 mg / d to 5000 mg / d, about 0.01 mg / d to 3000 mg / d, about 0.1 mg / d to 5000 mg / d, about 0.1 mg / d to 3000 mg / d, about 1 mg / d to 5000 mg / d, about 1 mg / d to 3000 mg / d, about 10 mg / d to 5000 mg / d, about 10 mg / d to 3000 mg / d. mg / d, about 100mg / d~5000mg / d, about 1000mg / d~5000mg / d, about 2000mg / d~5000mg / d, about 3000mg / d~5000mg / d, about 2000mg / d~5000mg / d, about 2000mg / d~5000mg / d, about 2000mg / d~4000mg / d, about 2500mg / d~3500mg / d, about 0.001mg / d, about 0.01mg / d, about 0.1mg / d, about 1mg / d, about 10mg / d, about 1000mg / d, about 1500mg / d, about 2000mg / d, about 2500mg / d, about 3000mg / d.

[0037] In some embodiments of the present invention, the GSK-3β inhibitor is administered in an amount of about 1 μg / kg to 500 mg / kg per unit dose, for example, about 1 μg / kg to 500 mg / kg, about 1 μg / kg to 50 mg / kg, about 10 μg / kg to 500 mg / kg, about 10 μg / kg to 500 mg / kg, about 0.1 mg / kg to 500 mg / kg, about 0.1 mg / kg to 50 mg / kg, about 1 mg / kg to 500 mg / kg, about 1 mg / kg to 50 mg / kg, about 5 mg / kg to 500 mg / kg, About 5 mg / kg to 50 mg / kg, about 10 mg / kg to 500 mg / kg, about 10 mg / kg to 50 mg / kg, about 10 mg / kg to 100 mg / kg, about 10 mg / kg to 50 mg / kg, about 20 mg / kg to 80 mg / kg, about 40 mg / kg to 60 mg / kg, about 1 μg / kg, about 10 μg / kg, about 0.1 mg / kg, about 1 mg / kg, about 10 mg / kg, about 50 mg / kg, per unit dose, and administered in one or more (e.g., 1, 2, 3, 4, 5) unit doses per day.

[0038] In some embodiments of the invention, the HDAC6 inhibitor and the GSK-3β inhibitor are administered continuously for at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, at least 20 days, at least 21 days, at least 22 days, at least 23 days, at least 24 days, at least 25 days, at least 30 days, at least 35 days, at least 40 days, at least 45 days, or at least 50 days.

[0039] In some embodiments of the present invention, the HDAC6 inhibitor and the GSK-3β inhibitor are administered together, simultaneously, sequentially or alternately.

[0040] The terms "including," "comprising," "having," "containing," or "comprising" and other variations thereof herein are inclusive or open-ended and do not exclude other unlisted elements or method steps.

[0041] Unless otherwise stated, the terms "a," "an," "the," and similar designations used herein in the context of describing the invention (especially in the context of the claims) should be interpreted as including both the singular and the plural. Unless otherwise stated, the recitation of numerical ranges herein is merely a shorthand method of referring to each individual value within the range individually, and each individual value is incorporated into the specification as if it were individually recited herein. Unless otherwise stated, the use of any and all examples or exemplary language (such as "for example") provided herein is intended to better illustrate the disclosure of the invention and not to limit the scope of the disclosure. No language in this specification should be construed to indicate that any non-claimed element is essential to the practice of the invention as disclosed.

[0042] As used herein, the terms "prevent," "preventing," and "prevention" refer to methods of preventing the onset of a disease or condition and / or its attendant symptoms or preventing a subject from acquiring the disease. As used herein, "prevent," "preventing," and "prevention" also include delaying the onset of a disease and / or its attendant symptoms and reducing the risk of a subject developing a disease. The terms "prevent," "preventing," and "prevention" may include "prophylactic treatment," which refers to reducing the probability of a disease or symptom developing again or recurring in a subject who does not have, but is at risk for or susceptible to developing again or recurring a previously controlled disease or symptom.

[0043] As used herein, the terms "treat," "treating," "treatment," and the like refer to the elimination, reduction, or amelioration of a disease or condition and / or symptoms associated therewith. Although not excluded, treatment of a disease or condition does not require the complete elimination of the disease, condition, or symptoms associated therewith. The term "treat" and synonyms contemplate the administration of a therapeutically effective amount of a compound disclosed herein to a subject in need of such treatment. Treatment can be symptomatic, for example, to suppress symptoms. It can be effective in the short term, can be carried out over the medium term, or can be a long-term treatment, for example, in the context of maintenance therapy.

[0044] As used herein, the term "combination" refers to a mode of administration that includes administering at least one dose of Compound A and at least one dose of Compound B over a period of time, wherein both agents exhibit a pharmacological effect. The period of time may be within a dosing cycle, such as within 24 hours. Compound A and Compound B may be administered simultaneously or sequentially. This period includes treatments in which Compound A and Compound B are administered via the same route of administration or different routes of administration. The combined modes of administration described herein are selected from simultaneous administration, independently formulated and co-administered, or independently formulated and administered sequentially.

[0045] As used herein, the term "synergistic" refers to an effect of two therapeutic agents, e.g., slowing the progression of Alzheimer's disease, that is greater than the simple sum of the effects of each agent alone. For example, synergistic effects can be calculated using various methods and equations known in the art, such as those listed in the examples herein.

[0046] The terms "individual", "patient" or "subject" in the present invention refer to humans (e.g., patients) and animals (e.g., mice, rats, dogs, cats, rabbits, chickens, monkeys, etc.). When the subject is a human patient (usually calculated based on a body weight of 60 kg), unless otherwise specified, the dosage described in the present invention can be obtained by converting the experimental animal conversion factor (e.g., human dose = mouse dose / 12.3) (Kin Tam. "Estimating the "First in human" dose-a revisit with special evidence on the oncology drug, ADMET & DMPK 1 (4) (2013) 63-75). A person of ordinary skill in the art can reasonably adjust the dosage based on common sense, according to factors such as the weight of the subject, the type and severity of the disease, and these adjusted technical solutions are all within the scope of the technical solutions required by the present invention.

[0047] As used herein, the term "effective amount" or "prophylactically and / or therapeutically effective amount" refers to the amount (e.g., dosage) of a drug or compound to be administered that is sufficient to achieve treatment to alleviate one or more symptoms of a disease or condition. The result can be a reduction and / or alleviation of the cause of the condition or disease or any other desired change in a biological system. For example, an "effective amount" for therapeutic use is an amount of a compound or drug (e.g., a combination product as claimed herein) that significantly alleviates the clinical symptoms of a disease or condition without causing excessive toxic side effects.

[0048] The term "pharmaceutically acceptable salt" in the present invention includes acid addition salts and base addition salts of the compound.

[0049] Suitable acid addition salts are formed from acids that form non-toxic salts. Examples include acetate, adipate, aspartate, benzoate, benzenesulfonate, bicarbonate / carbonate, bisulfate / sulfate, borate, camphorsulfonate, citrate, cyclamate, edisylate, ethanesulfonate, formate, fumarate, glucoheptonate, gluconate, glucuronate, hexafluorophosphate, benzoate, hydrochloride / chloride, hydrobromide / bromide, hydroiodide / iodide, isethionate, lactate, malate, maleate, malonate, methanesulfonate, methylsulfate, naphthoate, 2-naphthalenesulfonate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate / hydrogenphosphate / dihydrogenphosphate, pyroglutamate, aldonic acid, stearate, succinate, tannate, tartrate, toluenesulfonate, trifluoroacetate, and octinoxate.

[0050] Suitable base addition salts are formed from bases which form non-toxic salts. Examples include aluminum, arginine, benzathine benzylpenicillin, calcium, choline, diethylamine, diethanolamine, glycine, lysine, magnesium, meglumine, ethanolamine, potassium, sodium, tromethamine, and zinc salts.

[0051] For a review of suitable salts, see "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" by Stahl and Wermuth (Wiley-VCH, 2002). Methods for preparing pharmaceutically acceptable salts of the compounds of the invention are known to those skilled in the art.

[0052] The term "solvate" in the present invention is a substance formed by a combination, physical combination and / or solvation of the compounds of the present invention and solvent molecules such as disolvates, monosolvates or hemisolvates, wherein the ratio of solvent molecules to the ratio of the compounds of the present invention is about 2:1, about 1:1 or about 1:2. This physical bonding involves ionization and covalent bonding (including hydrogen bonding) to varying degrees. In some cases (for example, when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid), the solvate can be separated. Therefore, solvents include both solution phases and isolatable solvents. The compounds of the present invention can exist in the form of a solvent with a pharmaceutically acceptable solvent (for example, water, methanol and ethanol), and the application is intended to include solvated and non-solvated forms of the compounds of the present invention.

[0053] One type of solvate is a hydrate. "Hydrate" refers to a specific subset of solvates in which the solvent molecule is water. Solvates generally act as pharmacological equivalents. The preparation of solvates is known in the art. A representative, non-limiting method for preparing a solvate involves dissolving a compound of the invention in a desired solvent (an organic solvent, water, or a mixture thereof) at a temperature of greater than 20°C to about 25°C, then cooling the solution at a rate sufficient to form crystals, and isolating the crystals by a known method (e.g., filtration). Analytical techniques such as infrared spectroscopy can be used to confirm the presence of solvent in the crystals of the solvate.

[0054] The term "pharmaceutically acceptable excipient" as used herein refers to a diluent, adjuvant or carrier for use with a therapeutic agent, which is suitable for contact with the tissues of humans and / or other animals within the scope of sound medical judgment and does not cause excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio.

[0055] Pharmaceutically acceptable excipients that can be used in the pharmaceutical preparations of the present invention include, but are not limited to, sterile liquids, such as water and oils, including oils derived from petroleum, animal, vegetable, or synthetic sources, for example, peanut oil, soybean oil, mineral oil, sesame oil, and the like. When the pharmaceutical preparation is administered intravenously, water is an exemplary carrier. Physiological saline and aqueous solutions of glucose and glycerol can also be used as liquid carriers, particularly for injection. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, maltose, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene glycol, water, ethanol, and the like. As needed, the pharmaceutical preparations may also contain a small amount of a wetting agent, emulsifier, or pH buffer. Oral formulations may include standard carriers, for example, pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutically acceptable excipients are described in Remington's Pharmaceutical Sciences (1990).

[0056] The term "about" in the present invention refers to ±10% of the modified value of the term, preferably ±5%, and more preferably ±2%. People with ordinary skills in this technology can clearly determine the scope of "about" based on the modified value.

[0057] According to a preferred embodiment of the present invention, there are at least the following beneficial effects:

[0058] The combined use of the components of the drug combination of the present invention can synergistically enhance the therapeutic effect of each single drug on Alzheimer's disease, synergistically reduce the abnormal phosphorylation of Tau protein, and improve cognitive impairment in mice. No obvious weight loss or behavioral abnormalities occurred in the mice after administration, indicating that the combination drug of the present invention has good efficacy and safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] The present application is further described below with reference to the accompanying drawings and embodiments, wherein:

[0060] FIG1 shows the effect of EH-1 / LiCl combination on cell survival in Glu or STZ-induced AD cell models;

[0061] FIG2 shows the combination index and median effect of the EH-1 / LiCl combination in Glu or STZ-induced AD cell models;

[0062] FIG3 shows the effect of the EH-1 / LiCl combination on cell survival in a Glu-induced AD cell model (supplementation concentration ratio of 1:10000);

[0063] FIG4 shows the phosphorylation of Tau protein by EH-1 / LiCl combination in Glu or STZ-induced AD cell models;

[0064] FIG5 shows the water maze behavioral evaluation of AD mice using the EH-1 / LiCl combination in the STZ-induced AD mouse model;

[0065] FIG6 shows the open field and Y-maze behavioral evaluation of AD mice using the EH-1 / LiCl combination in the STZ-induced AD mouse model.

[0066] FIG. 7 shows the phosphorylation of Tau protein in AD mice induced by intracerebroventricular injection of STZ by the EH-1 / LiCl combination. DETAILED DESCRIPTION

[0067] The following will clearly and completely describe the concept and technical effects of this application in conjunction with the embodiments to fully understand the purpose, features and effects of this application. Obviously, the embodiments described are only part of the embodiments of this application, not all of them. Based on the embodiments of this application, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of this application.

[0068] The experimental methods in the following examples where specific conditions are not specified are generally carried out under conventional conditions or conditions recommended by the manufacturers.

[0069] Unless otherwise specified, all commonly used chemical reagents used in the examples are commercially available products.

[0070] Statistical analysis method in the embodiment:

[0071] Statistical analyses were performed using GraphPad Prism software (version 9.5.0, San Diego, CA, USA). Results are expressed as mean ± standard error (SEM). Repeated measures one-way analysis of variance (ANOVA) with t-test was used to evaluate data from cell-based and animal behavioral experiments, such as cell viability, escape latency, and number of platform crossings. Unless otherwise indicated, a P value ≤ 0.05 indicated significant differences.

[0072] Example 1

[0073] In this example, the MTT method was used to evaluate the effects of EH-1 and / or LiCl on the cell survival rate of AD cell models established by mouse hippocampal neuronal cells (HT22) induced by glutamate (Glu) or streptozotocin (STZ).

[0074] Mouse hippocampal neuronal cells (HT22) were cultured in a DMEM medium containing 1% double antibiotics (penicillin / streptomycin) and 10% fetal bovine serum at 37° C. in a 5% CO 2 cell culture incubator.

[0075] After trypsinization, the cells were prepared with complete culture medium to contain 8 × 10 cells per ml. 4 A cell suspension of 1 μg / mL was seeded into a 96-well culture plate, 100 μL per well. The cells were cultured in a 37°C, 5% CO2 incubator for 24 hours, after which the cell suspension was discarded. A blank control group was treated with complete culture medium containing 0.2% DMSO. Treatment groups were treated with 1, 1.5, 2, and 2.5 μM EH-1, 4, 6, 8, and 10 mM LiCl, and combinations of 1 μM / 4 mM, 1.5 μM / 6 mM, 2 μM / 8 mM, and 2.5 μM / 10 mM EH-1 / LiCl. Three replicate wells were set up for each concentration. After incubation with Glu or STZ for 24 hours, 10 μL of MTT solution (5 mg / mL, in PBS) was added to each well. After a further 4 hours of incubation, the supernatant was aspirated and discarded using a pipette. 150 μL of DMSO was then added to each well. The cells were shaken on a shaker in the dark until the crystals were completely dissolved. Finally, the absorbance of each well was measured using a UV spectrophotometer at a wavelength of 570 nm relative to 630 nm. The survival rate of each cell group was calculated. The results are shown in Table 1 and Figure 1.

[0076] As shown in FIG1 , the combination of compound EH-1 and LiCl can synergistically inhibit Glu- or STZ-induced HT22 cell death (**, P<0.01), and exhibits a significant dose-effect relationship.

[0077] Table 1 Cell viability of each drug group (%)

[0078] The Chou-Talalay method and CalcuSyn 2.1 software were used to calculate the drug combination index (CI) and drug dose reduction index (DIR) to evaluate the efficacy of EH combined with LiCl. A CI > 1 indicates antagonism; a CI = 1 indicates additive effects; a CI < 1 indicates synergism; and a CI < 0.3 indicates strong synergy. The DRI represents the amount by which the dose of each drug can be reduced while maintaining the therapeutic effect. Higher DRI values ​​indicate greater drug synergy. The results are shown in Table 2 and Figure 2.

[0079] As shown in Figure 2, in Glu- or STZ-induced AD cell models, after combined administration at the above doses, the CI was less than 1. Furthermore, the median effect of the combination group was greater than that of the single-dose group. These results indicate that the combination of compound EH-1 and LiCl can produce a synergistic neuroprotective effect.

[0080] Table 2 Cell viability effect, drug combination index and drug dose reduction index of each drug group

[0081] Because low-dose LiCl has poor cytoprotective activity, increasing the LiCl dose is desirable to achieve a more synergistic therapeutic effect. Figure 3 illustrates a cell-protective combination with an EH-1 to LiCl concentration ratio of 1:10,000. While maintaining the same EH-1 concentration, the LiCl concentration was increased. The results demonstrate that increasing the combined LiCl concentration also enhances cell survival, broadening the acceptable range of combined EH-1 and LiCl concentrations.

[0082] The two main histopathological lesions in the brains of AD patients are senile plaques (SP) formed by beta-amyloid protein and neuronal tangles (NFTs) formed by abnormal phosphorylation of Tau protein. Tau protein, the main component of NFT, is normally a highly soluble protein that binds to and stabilizes microtubules. In AD and other tauopathies, Tau undergoes structural changes that lead to its aggregation. Tau aggregation is central to disease progression in tauopathies. Cognitive ability in AD patients is negatively correlated with the degree of Tau phosphorylation and its aggregation load. Inhibiting the hyperphosphorylation of Tau is a potential approach to modulate AD disease.

[0083] Western blotting was used to analyze the phosphorylation level of Tau protein in Glu / STZ-induced AD cell models.

[0084] In the Glu-induced AD cell model, Glu did not significantly induce upregulation of Tau phosphorylation. However, administration of LiCl alone or in combination with the compound EH-1 and LiCl significantly downregulated Tau phosphorylation at Ser396 and Thr231. These results suggest that the GSK-3β inhibitor LiCl plays a primary role in inhibiting Tau phosphorylation. In the STZ-induced AD cell model, STZ significantly upregulated Tau phosphorylation, with phosphorylation at Thr231 being more pronounced than at Ser396. Compared with the model group, the EH-1 group reduced Tau phosphorylation, particularly at Thr231. The LiCl group significantly inhibited Tau phosphorylation at both Ser396 and Thr231. The combined use of the compound EH-1 and LiCl significantly inhibited Tau phosphorylation compared with either group alone. These results indicate that the GSK-3β inhibitor LiCl plays a primary role in inhibiting abnormal Tau protein phosphorylation, while the HDAC6 inhibitor EH-1 enhances the inhibitory effect. Their combined use synergistically inhibits abnormal Tau protein phosphorylation. See Figure 4 for the results.

[0085] Example 2

[0086] In this example, an AD mouse model was established by bilateral intracerebroventricular injection of STZ to evaluate the anti-AD effects of EH-1 and / or LiCl in vivo.

[0087] Male C57BL / 6 mice (approximately 6 weeks old, weighing approximately 25 g) were provided by Zhuhai Bestone Biotechnology Co., Ltd. After one week of constant temperature and natural light in the animal room of Jiangmen Big Health International Innovation Institute, an AD mouse model was established. Following administration, behavioral tests were performed to evaluate the in vivo anti-AD effects of EH-1 and / or LiCl.

[0088] 5 μL of STZ (3 mg / kg) prepared in artificial cerebrospinal fluid was injected into the left / right ventricular hippocampus of mice using a microsyringe on days 1 and 3, respectively. The mice were then randomly divided into 5 groups, 11 in each group, and orally gavaged starting from the first day after modeling. The sham-operated group was given an equal amount of solvent (0.5% CMC-Na solution). The EH-1-treated group, the LiCl-treated group, and the EH-1 / LiCl combination-treated group were orally gavaged at doses of 10 mg / kg, 50 mg / kg, and 10 mg / kg + 50 mg / kg, respectively, once a day, and continued to be administered until the introduction of behavioral experiments. The body weight (g) of the animals was recorded every day during the experiment. After modeling and administration for 14 days, the in vivo anti-AD effects of EH-1 and LiCl were evaluated through behavioral tests such as open field, Y maze, and water maze. The results are shown in Figures 5 and 6.

[0089] The experimental results shown in Figures 5 and 6 demonstrate that continuous administration of EH-1 (10 mg / kg / d) and LiCl (50 mg / kg / d) starting from the first day after model establishment can prevent cognitive impairment in mice to a certain extent, and the EH-1 / LiCl combination significantly inhibits cognitive impairment in mice (**, P < 0.01). In the water maze navigation and positioning behavioral assessment experiment from days 14 to 19 after administration, the escape latency of mice in the EH-1 / LiCl combination group was significantly reduced compared with sham-operated mice (**, P < 0.01). In the water maze spatial exploration behavioral assessment experiment on day 20 after administration, the EH-1 / LiCl combination group spent significantly more time in the target quadrant and crossed the platform compared with sham-operated mice (**, P < 0.01). All animals in the treatment groups did not show significant weight loss or behavioral abnormalities.

[0090] In this example, an AD mouse model was established by bilateral intracerebroventricular injection of STZ to evaluate the in vivo inhibitory effects of EH-1 and / or LiCl on Tau protein phosphorylation.

[0091] In animal experiments, an AD mouse model was established by injecting streptozotocin (3 mg / kg) into the bilateral lateral ventricles of C57BL / 6 mice. After injection of STZ into the lateral ventricles, abnormal increases in the phosphorylation levels of Tau protein at Ser396 and Thr231 sites were induced. After four weeks of treatment with compound EH-1 (10 mg / kg) or LiCl (50 mg / kg), the phosphorylation levels of Tau protein at Ser396 and Thr231 sites in the hippocampus of AD mice decreased. After EH-1 combined with LiCl treatment, the phosphorylation levels of Tau protein at Ser396 and Thr231 sites in the hippocampus of mice decreased significantly, which was significantly different from the single treatment group. EH-1 combined with LiCl can synergistically inhibit abnormal phosphorylation of Tau protein. The results are shown in Figure 7.

[0092] The embodiments of the present application have been described in detail above with reference to the accompanying drawings. However, the present application is not limited to the above embodiments. Various modifications can be made within the scope of knowledge possessed by ordinary technicians in the relevant technical field without departing from the purpose of the present application. In addition, the embodiments of the present application and the features of the embodiments can be combined with each other unless there is a conflict.

Claims

1. A pharmaceutical composition, characterized in that, Comprising: (a) a therapeutically or prophylactically effective amount of an HDAC6 inhibitor; and (b) a therapeutically or prophylactically effective amount of a GSK-3β inhibitor.

2. The pharmaceutical composition according to claim 1, wherein The HDAC6 inhibitor includes a compound represented by the following formula, or a pharmaceutically acceptable salt, isotope-labeled compound, hydrate, solvate, diastereoisomer or enantiomer thereof:

3. The pharmaceutical composition according to claim 2, wherein, The HDAC6 inhibitor includes a compound represented by the following formula, or a pharmaceutically acceptable salt, isotope-labeled compound, hydrate, solvate, diastereoisomer or enantiomer thereof:

4. The pharmaceutical composition according to claim 1, characterized in that, The GSK-3β inhibitor includes LiCl or a compound represented by the following formula, or a pharmaceutically acceptable salt, isotope-labeled substance, hydrate, or solvate thereof:

5. The pharmaceutical composition according to claim 1, wherein The GSK-3β inhibitor includes LiCl.

6. The pharmaceutical composition according to claim 1, wherein The ratio of the effective amount of the HDAC6 inhibitor to the effective amount of the GSK-3β inhibitor is about 1:(1-20000), preferably about 1:(5-10000), about 1:(1-25), about 1:(2000-20000).

7. The pharmaceutical composition according to any one of claims 1 to 6, characterized in that, The pharmaceutical composition is used for preventing and / or treating Alzheimer's disease in a subject in need thereof.

8. A pharmaceutical preparation, characterized in that, The pharmaceutical preparation comprises the pharmaceutical composition according to any one of 1 to 7, and a pharmaceutically acceptable excipient.

9. The pharmaceutical preparation according to claim 8, characterized in that, The dosage form of the pharmaceutical preparation includes tablets, capsules, granules, syrups, lozenges, cachets, elixirs, pills, powders, suspensions, solutions, aerosols, injections.

10. Use of the pharmaceutical composition according to any one of claims 1 to 7, or the pharmaceutical preparation according to claim 8 or 9, in the preparation of a medicament for preventing and / or treating Alzheimer's disease.

11. The use according to claim 10, characterized in that, The Alzheimer's disease has one or more pathological changes selected from the following: cognitive function decline, senile plaques formed by β-amyloid deposition, and neurofibrillary tangles formed by abnormal protein filaments.

12. A method for preventing and / or treating Alzheimer's disease, comprising administering to a subject in need thereof: (a) a therapeutically or prophylactically effective amount of an HDAC6 inhibitor; and (b) a therapeutically or prophylactically effective amount of a GSK-3β inhibitor; Optionally, the HDAC6 inhibitor is as defined in claim 2 or 3, The GSK-3β inhibitor is as defined in claim 4 or 5.

13. The method according to claim 12, wherein The dosage of the HDAC6 inhibitor is about 0.01 mg / d to 5 g / d.

14. The method according to claim 12, wherein The dosage of the GSK-3β inhibitor is about 0.01 mg / d to 25 g / d.

15. The method according to claim 12, wherein The HDAC6 inhibitor and the GSK-3β inhibitor are administered simultaneously, sequentially or alternately.

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