Pharmaceutical composition for treating TDP-43 proteinopathy, and preparation method therefor and use thereof

Abstract

The present invention belongs to the technical field of pharmaceuticals. Disclosed are a pharmaceutical composition for treating a TDP-43 proteinopathy, and a preparation method therefor and the use thereof. The pharmaceutical composition comprises at least one of each of compounds of class A and compounds of class B. The compounds of class A comprise at least one of sodium aescinate, a sodium aescinate derivative, hederagenin or a hederagenin derivative; and the compounds of class B comprise at least one of dehydrated icaritin, a dehydrated icaritin derivative, diosmin or a diosmin derivative. The pharmaceutical composition has a good therapeutic effect on the TDP-43 proteinopathy.

Description

Pharmaceutical composition for treating TDP-43 proteinopathy and preparation method and application thereof TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and particularly relates to a pharmaceutical composition for treating TDP-43 proteinopathy and a preparation method and application thereof. BACKGROUND

[0002] TDP-43 proteinopathy is a collective term for a group of various neurological disease classes, and the common pathological feature of these diseases is that pathological protein inclusions formed by transactive response DNA binding protein 43 (TDP-43) can be detected in the affected area (reference: Chen L. The important functional role of TDP-43 plays in amyotrophic lateral sclerosis-frontotemporal dementia. Neural Regen Res. 2021; 16(4): 682-683).

[0003] TDP-43 is a highly conserved nuclear protein that plays a regulatory role in mRNA transcription, splicing and stability, and RNA metabolism, and its activity regulation is reflected in its controlled amount shuttle between the nucleus and the cytoplasm (reference: Helmold, BR, Pauss, KE and Ozdinler, PH. TDP-43 protein interactome informs about perturbed canonical pathways and may help develop personalized medicine approaches for patients with TDP-43 pathology. Drug Discov Today. 2023; 11: 103769; de Boer EMJ, Orie VK, Williams T, Baker MR, De Oliveira HM, Polvikoski T, et al.. TDP-43 proteinopathies: a new wave of neurodegenerative diseases. J. Neurol. Neurosurg. Psychiatry. 2020; 92: 86-95).

[0004] In the brain of normal people, TDP-43 mainly exists in the nucleus, and there is almost no abnormal deposition, while in the pathological state, TDP-43 usually appears in the form of insoluble inclusion bodies or is massively located in the cytoplasm, and some appear abnormal degradation, so as to lose function, thus producing cytotoxicity, causing damage to the nervous system (references: Dugger BN and Dickson DW. Pathology of neurodegenerative diseases. Cold Spring Harb. Perspect. Biol. 2017; 9: a028035; De Oliveira HM, Polvikoski T, et al.. TDP-43 proteinopathies: a new wave of neurodegenerative diseases. J. Neurol. Neurosurg. Psychiatry. 2020; 92: 86-95).

[0005] Abnormal aggregation and mislocalization of TDP-43 are found in 97% of ALS patients, nearly 50% of frontotemporal lobar degeneration (FTLD / FTD) patients, and more than 30% of Alzheimer’s disease (AD) patients (Ref: Chen L. The important functional role of TDP-43 plays in amyotrophic lateral sclerosis-frontotemporal dementia. Neural Regen Res. 2021; 16(4): 682-683; Dugger BN and Dickson DW. Pathology of neurodegenerative diseases. Cold Spring Harb. Perspect. Biol. 2017; 9: a028035;). In addition, it is also found in some patients with other neurodegenerative diseases, including chronic traumatic encephalopathy (CTE), Lewy body disease (LBD), Huntington’s disease (HD), argyrophilic grain disease (AGD), and hippocampal sclerosis, etc. (Uchino, et al., 2015; de Boer, et al., 2020).

[0006] Over the past decade, the number of reported cases of TDP-43 proteinopathy has increased significantly with the advancement of detection technology (Ref: de Boer EMJ, Orie VK, Williams T, Baker MR, De Oliveira HM, Polvikoski T, et al. TDP-43 proteinopathies: a new wave of neurodegenerative diseases. J. Neurol. Neurosurg. Psychiatry. 2020; 92: 86-95), however, there have been great challenges in drug development for TDP-43 proteinopathy. Despite the huge investment, no effective drug has been obtained so far. The following takes ALS, the most typical representative of TDP-43 proteinopathy, to illustrate its pathogenic characteristics and drug development.

[0007] ALS can cause a combination of upper and lower motor neuron disease, affecting the muscles of the trunk, limbs, and head and face that it innervates. Patients gradually lose control of their muscles, leading to muscle atrophy, and usually die from respiratory failure 3 to 5 years after onset, with a prevalence of about 6 cases per 100,000 people, with slightly more men than women. (Reference: Lin, Cheng GC, Wu LY, Lai WY, Ling TY, Kuo YC, Huang YH. Potential of cellular therapy for ALS: current strategies and future prospects. Front Cell Dev Biol. 2022; 10: 851613). About 10% of ALS patients are familial, caused by genetic mutations such as TDP-43, SOD1, FUS, etc.; and more than 90% are sporadic, i.e., no pathogenic genes have been identified, but their pathological features are highly related to the structural and functional abnormalities of TDP-43 protein (Reference: Eck RJ, Kraemer BC and Liachko NF. Regulation of TDP-43 phosphorylation in aging and disease. GeroScience. 2021; 43: 1605-1614). At least 20 mutations in the TDP-43 gene are pathogenic, and among these TDP-43 mutation genes, the mutation of A315T located in the C-terminal has the most extensive functional impact, involving cytoplasmic localization, fragmentation, phosphorylation, and ubiquitination, and insolubility, so its symptoms are relatively severe compared to other mutations (Reference: Ke YD, van Hummel A, Stevens CH, Gladbach A, Ippati S, Bi M, Lee WS, Kruger S, van der Hoven J, Volkerling A, et al. Short-term suppression of A315T mutant human TDP-43 expression improves functional deficits in a novel inducible transgenic mouse model of FTLD-TDP and ALS. Acta Neuropathol. 2015; 130(5): 661-78).

[0008] Abnormal accumulation and dysfunction of TDP-43 protein are found in the nervous system of most ALS patients, so ALS is a typical representative of TDP-43 proteinopathy. Although the research and development of drugs for treating ALS has continued for many years, the approved drugs have little effect, and some drugs have serious side effects, indicating that there is still a lack of effective drugs for TDP-43 proteinopathy caused by abnormal accumulation and dysfunction of TDP-43 protein.

[0009] Riluzole is the first drug approved by the US FDA and the European Union for the treatment of ALS. Clinical studies have shown that riluzole treatment can reduce the loss of motor neurons, improve the survival status of patients, and slow the progression of the disease, but cannot stop the development of ALS, and the survival period of patients is only increased by about 3 months (reference: Bensimon G, Lacomblez L, Delumeau JC, Bejuit R, Truffinet P, Meininger V. A study of riluzole in the treatment of advanced stage or elderly patients with amyotrophic lateral sclerosis. J Neurol. 2002; 249: 609-615; Doble A. The pharmacology and mechanism of action of riluzole. Neurology. 1996; 47:S233-41). And riluzole can cause dizziness, fatigue, gastrointestinal symptoms, and liver function changes and other side effects (reference: Tzeplaeff L, Wilfling S, Requardt MV, Herdick M. Current state and future directions in the therapy of ALS. Cells. 2023; 12: 1523).

[0010] Edaravone (MCI-186, Edaravone) is another compound approved for the treatment of amyotrophic lateral sclerosis in several countries in Asia, the United States, Canada, and Switzerland since riluzole. (Reference: Hoxhaj P, Hastings N, Kachhadia MP, et al. Exploring Advancements in the Treatment of Amyotrophic Lateral Sclerosis: A Comprehensive Review of Current Modalities and Future Prospects. Cureus. 2023; 15(9): e45489). A number of experimental studies have shown that edaravone has a certain effect on inhibiting motor decline and motor neuron degeneration and slowing disease progression, but the efficacy is limited and not satisfactory. In addition, the drug has strong side effects, and common adverse events include contusion, confusion, dysphagia, constipation, eczema, headache, bruising, gait disturbance, and allergic reactions, etc. (Reference: Cruz MP. Edaravone (Radicava): a novel neuroprotective agent for the treatment of amyotrophic lateral sclerosis. P T. 2018; 43: 25-8; Witzel S, Maier A, Steinbach R, et al. Safety and effectiveness of long-term intravenous administration of edaravone for treatment of patients with amyotrophic lateral sclerosis. JAMA Neurol. 2022; 79: 121-30; Cho H and Shukla S. Role of edaravone as a treatment option for patients with amyotrophic lateral sclerosis. Pharmaceuticals (Basel). 2020; 14: 29).

[0011] Therefore, there is an urgent need for a new drug with better effect for treating TDP-43 proteinopathy. SUMMARY

[0012] The present application aims to at least solve one of the technical problems existing in the prior art. To this end, the present application provides a pharmaceutical composition for treating TDP-43 proteinopathy and a preparation method and application thereof. The pharmaceutical composition is used for treating TDP-43 proteinopathy, and has obvious therapeutic effect on sporadic TDP-43 proteinopathy and familial TDP-43 proteinopathy with TDP-43-A315T mutation.

[0013] Specifically, the pharmaceutical composition for treating TDP-43 proteinopathy comprises at least one of a class A compound and a class B compound;

[0014] The class A compound comprises at least one of sodium aescinate, a sodium aescinate derivative, hederagenin or a hederagenin derivative;

[0015] The class B compound comprises at least one of an anhydroicariside, an anhydroicariside derivative, diosmin or a diosmin derivative.

[0016] Preferably, the sodium aescinate derivative comprises at least one of aescin, aescin A, aescin B, aescin C, aescin D, hederagenin or protohederagenin.

[0017] Sodium Aescinate (CAS No. 20977-05-3, molecular formula C 54 H 83 NaO 23 It is in the form of white or crystalline powder, has the effects of anti-inflammatory, anti-exudation, increasing venous tension, improving blood circulation and correcting brain dysfunction, and has obvious protective effect on brain edema caused by carbon monoxide and the like.

[0018] Aescin (CAS No. 6805-41-0, molecular formula C 55 H 86 O 24 .

[0019] Aescin A (CAS No. 123748-68-5, molecular formula C 55 H 86 O 24 .

[0020] Aescin B (CAS No. 26339-90-2, molecular formula C 55 H 86 O 24 .

[0021] Aescin C: CAS No. 158732-55-9, molecular formula C 54 H 84 O 23 .

[0022] Aescin D: CAS No. 158800-83-0, molecular formula C 54 H 84 O 23 .

[0023] Aescigenin: CAS No. 17806-68-7, molecular formula C 30 H 48 O5.

[0024] Protoaescigenin: CAS No. 20853-07-0, molecular formula C 30 H 50 O6.

[0025] Preferably, the dehydroicaritin derivative comprises at least one of icaritin, icariin or dehydroicaritin.

[0026] Dehydroicaritin: CAS No. 38226-86-7, molecular formula C 21 H 20 O6. is a prenylflavonoid (3,5,7-trihydroxy-2-(4-methoxyphenyl)-8-(3-methylbut-2-enyl)-4H- chromen-4-one), is a natural compound with anticancer activity, possibly through the regulation of MAPK / ERK / JNK and JAK2 / STAT3 / AKT signaling pathways.

[0027] Icaritin: CAS No. 521-45-9, molecular formula C 21 H 22 O7.

[0028] Icariin: CAS No. 489-32-7, molecular formula C 33 H 40 O 15 .

[0029] Dehydroicaritin (baohuoside I, Icarisid II): CAS No. 113558-15-9, molecular formula C 27 H 30 O 10 .

[0030] Preferably, the Diosmin derivative includes Neodiosmin, CAS number 38665-01-9, molecular formula C 28 H 32 O 15 .

[0031] Diosmin: CAS number 520-27-4, molecular formula C 28 H 32 O 15 , chemical name 7-{[6-oxo-(6-deoxy-alpha-L-mannopyranosyl)-beta-D-glucopyranosyl]oxy}-5-hydroxy-2-(3-hydroxy-4-methoxyphenyl)-4H-L-benzopyran-4-one. Diosmin is a capillary-protective agent used for the treatment of various symptoms associated with venous and lymphatic insufficiency and for the treatment of various symptoms of acute hemorrhoidal attacks.

[0032] Preferably, the Hederagenin derivative includes at least one of Alpha-Hederin, Beta-Hederin, Hederacoside C, Hederaside D.

[0033] Hederagenin: CAS number 465-99-6, molecular formula C30H48O4. Hederagenin is a water-soluble pentacyclic triterpene saponin isolated from plants, which has many chemical properties such as antispasmodic, anthelmintic, and cytostatic. It has a wide range of pharmacological activities, including anti-tumor, anti-inflammatory, anti-depression, anti-neurodegenerative, anti-hyperlipidemia, anti-diabetic, anti-leishmaniasis, and anti-viral activities.

[0034] Alpha-Hederin: CAS number 27013-91-8, molecular formula C 41 H 66 O 12 .

[0035] Beta-Hederin: CAS number 35790-95-5, molecular formula C 41 H 66 O 11 .

[0036] Hederacoside C: CAS number 14216-03-6, molecular formula C 59 H 96 O 26 .

[0037] Hederaside D: CAS number 760961-03-3, molecular formula C53 H 86 O 22 .

[0038] Preferably, the weight ratio of the Class A compound to the Class B compound is 1:20 to 20:1.

[0039] Preferably, the pharmaceutical composition is a binary composition consisting of a Class A compound and a Class B compound in a weight ratio of 1:20 to 20:1; wherein the Class A compound comprises one of esculin sodium and esculin sodium derivatives (A1 subclass) and hederagenin and hederagenin derivatives (A2 subclass), and the Class B compound comprises one of aicar and aicar derivatives (B1), diosmin and diosmin derivatives (B2). Specifically, the weight ratio of the Class A compound to the Class B compound is, for example, 1:20, 1:10, 1:5, 1:1, 2:1, 5:1, 10:1, 20:1.

[0040] In some embodiments, the aforementioned Class A and / or Class B compounds can be used in combination with edaravone (MCI-186) for the treatment of TDP-43 diseases.

[0041] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable solvent and excipient.

[0042] Preferably, the pharmaceutically acceptable excipient comprises at least one of a pharmaceutical carrier, diluent, adjuvant or excipient.

[0043] Preferably, the solvent is selected from at least one of DMSO (dimethyl sulfoxide), ethanol, propylene glycol, glycerol.

[0044] Preferably, in the pharmaceutical composition, one of esculin sodium and esculin sodium derivatives, hederagenin and hederagenin derivatives, and one of diosmin and diosmin derivatives, aicar and aicar derivatives are included in a weight ratio of 1:10 to 10:1. Specifically, for example, 1:10, 1:8, 1:5, 1:1, 2:1, 5:1, 8:1, 10:1.

[0045] Preferably, in the pharmaceutical composition, one of esculin sodium or esculin sodium derivatives, and aicar or aicar derivatives are included in a weight ratio of 1:10 to 10:1. Specifically, for example, 1:10, 1:8, 1:5, 1:1, 2:1, 5:1, 8:1, 10:1.

[0046] Preferably, in the pharmaceutical composition, one of esculin sodium or esculin sodium derivatives, and diosmin or diosmin derivatives, an organic solvent are included.

[0047] Preferably, the pharmaceutical composition comprises sodium aescinate or sodium aescinate derivatives, and anhydrous icariin or anhydrous icariin derivatives, and an organic solvent.

[0048] Preferably, the pharmaceutical composition comprises anhydrous icariin or anhydrous icariin derivatives, and hederagenin and hederagenin derivatives, and an organic solvent.

[0049] The second aspect of the present application provides a preparation method of a pharmaceutical composition for treating TDP-43 proteinopathy.

[0050] Specifically, the preparation method of the pharmaceutical composition for treating TDP-43 proteinopathy comprises the following steps: mixing the components to obtain the pharmaceutical composition.

[0051] In addition, when taking the pharmaceutical composition of the present application, the patient can directly take the mixed pharmaceutical composition, or take the components of the pharmaceutical composition in sequence.

[0052] The third aspect of the present application provides the use of a pharmaceutical composition for treating TDP-43 proteinopathy.

[0053] The use of the above-mentioned pharmaceutical composition in the preparation of a medicament for treating TDP-43 proteinopathy.

[0054] Preferably, the TDP-43 proteinopathy includes sporadic TDP-43 proteinopathy and familial TDP-43 proteinopathy with TDP-43-A315T mutation.

[0055] Preferably, the TDP-43 proteinopathy includes some types of amyotrophic lateral sclerosis, frontotemporal lobar degeneration, Alzheimer's disease, chronic traumatic encephalopathy, Lewy body disease, Huntington's disease, argyrophilic grain disease, and hippocampal sclerosis, one of the pathological features of which is the detection of abnormal aggregation and pathological inclusion of TDP-43 protein in the affected area.

[0056] Compared with the prior art, the present application has the following beneficial effects:

[0057] (1) In view of the current situation that there is no effective drug for TDP-43 proteinopathy, the pharmaceutical composition of the present application has good therapeutic effect on TDP-43 proteinopathy. Here, the TDP-43 proteinopathy includes but is not limited to sporadic amyotrophic lateral sclerosis, and takes into account TDP-43 gene A315T mutation and TDP-43 function abnormality related neurodegenerative diseases.

[0058] (2) In some embodiments, the pharmaceutical composition of the present application shows a good synergistic effect, i.e. the effect of the binary composition is significantly greater than the effect of the single component. For example, for diseases caused by wild-type TDP-43 accumulation, the effect of sodium aescinate alone or diosmin alone is significantly less than the binary combination of sodium aescinate and diosmin.

[0059] (3) The pharmaceutical composition for treating TDP-43 proteinopathy of the present application comprises at least two of sodium aescinate and sodium aescinate derivatives, dehydroicariside and dehydroicariside derivatives, diosmin and diosmin derivatives, hederagenin and hederagenin derivatives. Through the combination of specific pharmaceutical active ingredients, the pharmaceutical composition has a good therapeutic effect on TDP-43 proteinopathy.

[0060] (4) The pharmaceutical composition of the present application comprises at least one of sodium aescinate and sodium aescinate derivatives, dehydroicariside and dehydroicariside derivatives, and at least one of diosmin and diosmin derivatives, hederagenin and hederagenin derivatives; or, the pharmaceutical composition comprises sodium aescinate or sodium aescinate derivatives, and dehydroicariside or dehydroicariside derivatives. Through the combination of specific pharmaceutical active ingredients, the pharmaceutical composition has a good therapeutic effect on TDP-43 proteinopathy. TDP-43 proteinopathy includes sporadic amyotrophic lateral sclerosis, and takes into account TDP-43 gene A315T mutation and TDP-43 dysfunction related neurodegenerative diseases.

[0061] (5) For diseases caused by wild-type TDP-43 accumulation, the combination of sodium aescinate and diosmin, the combination of sodium aescinate and dehydroicariside has the most obvious effect. The remaining combinations, such as sodium aescinate derivatives and diosmin derivatives, sodium aescinate derivatives and icariside derivatives, hederagenin derivatives and diosmin derivatives, hederagenin derivatives and dehydroicariside derivatives also have certain curative effect. Sodium aescinate or diosmin alone also has a slight effect, but the effect is significantly less than the binary combination of sodium aescinate and diosmin and the binary combination of sodium aescinate and dehydroicariside.

[0062] (6) For the disease caused by TDP-43 mutant TDP-43-A315T, the combination of sodium aescinate and dehydroicariside, the combination of hederagenin and dehydroicariside has the most obvious effect. The remaining combinations, such as sodium aescinate derivatives and diltia derivatives, sodium aescinate derivatives and dehydroicariside derivatives, hederagenin derivatives and diltia derivatives, also have certain curative effects. The use of hederagenin and dehydroicariside alone also has a slight effect, but its effect is obviously less than that of the combination of sodium aescinate and dehydroicariside and the combination of hederagenin and dehydroicariside.

[0063] (7) In the fruit fly eye overexpression model of TDP-43 wild type and mutant A315T, differences in phenotypes between female and male flies are observed, and the defects of male flies are more serious than those of female flies; there are also differences between overexpression of TDP-43 wild type and mutant A315T. And different drug combinations also have obvious differences in the rescue effect of female and male flies and different types of TDP-43. Therefore, the present application proposes a strategy for differential treatment based on gender, defect degree and etiology. BRIEF DESCRIPTION OF DRAWINGS

[0064] FIGS. 1A-1D show the classification of necrotic black spots in the eyes of the fruit fly disease model and the situation of the control group;

[0065] FIGS. 2A-2B are results of the drug combination of the combination of hederagenin and dehydroicariside of Example 1 to improve the pupa length defects caused by motor neurons expressing TDP-43 mutant A315T;

[0066] FIG. 3 is the result of the drug combination of the combination of hederagenin and dehydroicariside of Example 2 to improve the movement ability of fruit fly larvae expressing TDP-43 mutant A315T motor neurons;

[0067] FIG. 4 is the treatment result of the drug combination of the combination of hederagenin and dehydroicariside of Example 3 for overexpression of TDP-43 mutant A315T in the eyes;

[0068] FIGS. 5A-5C are results of the drug combination of the combination of hederagenin and dehydroicariside of Example 4 to increase the number of neurons per unit area of a mouse (female) with progressive sclerosis;

[0069] FIG. 6 is the result of the drug combination of the combination of sodium aescinate and diltia of Example 5 for overexpression of wild type TDP-43 in the eyes;

[0070] FIG. 7 is the result of the drug combination of the combination of sodium aescinate and dehydroicariside of Example 6 for overexpression of TDP-43 mutant A315T in the eyes;

[0071] Figure 8 is the result of improving the necrotic black spot produced by overexpression of TDP-43-wild type in the eyes of male flies by the combination of sodium aescinate and dioxadromarin in Example 7, and the combination of sodium aescinate and anhydroicaritin in the pharmaceutical composition;

[0072] Figure 9 is the result of using the combination of sodium aescinate and dioxadromarin in Example 8, the combination of sodium aescinate and anhydroicaritin in the pharmaceutical composition, on the overexpression of wild type TDP-43 gene in the eyes of female flies under the condition of incubation temperature of 27°C;

[0073] Figures 10A-10B are the results of using the combination of hederasaponin (abbreviated as heder) and anhydroicaritin (abbreviated as icar) alone and in combination in the overexpression of TDP-43 mutant A315T in motor neurons in fruit flies in Comparative Example 1;

[0074] Figure 11 is the result of using the combination of hederasaponin (abbreviated as heder) and anhydroicaritin (abbreviated as icar) alone and in combination in the overexpression of TDP-43 mutant A315T in the eyes of fruit flies in Comparative Example 2;

[0075] Figure 12 is the treatment effect of sodium aescinate and dioxadromarin alone and in combination on TDP-43-wild type in female flies in Comparative Example 3;

[0076] Figures 13A-13B are the effects of using the combination of hederagenin and anhydroicaritin in Example 9 in combination with edaravone;

[0077] Figure 14 is the effect of the combination of hederagenin and anhydroicaritin in Comparative Example 4 being superior to riluzole. DETAILED DESCRIPTION

[0078] In order to make the skilled person in the art more clearly understand the technical solutions described in the present application, the following examples are given for illustration. It should be pointed out that the following examples do not constitute a limitation on the scope of protection required by the present application.

[0079] The raw materials, reagents or devices used in the following examples, unless otherwise specified, can be obtained from conventional commercial channels, or can be obtained by existing known methods.

[0080] DEFINITIONS

[0081] Unless otherwise defined, all technical and scientific terms used in the present application have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. For the purposes of interpreting this specification, the following definitions will apply and, where appropriate and unless expressly stated to the contrary, singular forms will include the plural and vice versa.

[0082] The expressions "a" and "an" as used herein include plural references unless the context clearly dictates otherwise. For example, "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so on.

[0083] The term "pharmaceutically acceptable" as used herein means capable of being administered to a human and / or other animal as a subject without producing excessive adverse reactions or side effects (such as toxicity, irritation, allergic response, and the like) and the term "excipient" means an auxiliary material which is present in a pharmaceutical formulation together with an active ingredient and does not produce excessive adverse reactions or side effects, including carriers, osmotic pressure adjusting agents, pH adjusting agents, diluents, disintegrants, excipients, solubilizers, stabilizers, preservatives, and the like. The term "pharmaceutically acceptable excipient" means a high safety excipient which is suitable for a specific pharmaceutical formulation and is used in the pharmaceutical field in general. The term "carrier" includes, but is not limited to, liposomes, ethosomes, polymeric micelles, nanostructured lipid carriers, solid lipid nanoparticles, mesoporous silica nanoparticles, and the like.

[0084] The term "pharmaceutically acceptable salt" as used herein can include alkali metal salts (such as sodium or potassium salts), alkaline earth metal salts (such as calcium or magnesium salts), and salts formed with suitable organic ligands (such as quaternary ammonium salts).

[0085] The above pharmaceutical composition is prepared by dissolving the corresponding active ingredients (such as sodium aescinate and sodium aescinate derivatives, dehydrosphingosine and dehydrosphingosine derivatives, diosmine and diosmine derivatives, hederagenin and hederagenin derivatives) at a designed concentration in water or an organic solvent, and then mixing to obtain.

[0086] To screen and verify the therapeutic effect of the above drug combination, a fruit fly TDP-43 proteinopathy model was established, i.e. using the Gal4-UAS system to overexpress human TDP-43 wild type or A315T mutant in specific tissues / organs / cells to induce abnormal phenotypes / defects in fruit flies. Fruit flies were cultured in an incubator at 25°C, 60% relative humidity, 12 hours of light and 12 hours of darkness per 24 hours; a small number of experimental fruit flies were cultured at 27°C, with the same conditions. The fruit fly food was standard corn flour formula food (standard corn flour formula food contains 68 g of corn flour, 43 g of maltose, 16 g of yeast powder, 9 g of soybean powder, 0.962 g of anhydrous calcium chloride, and 8 g of agar powder per 1 liter, and distilled water is boiled to boiling, then the volume is adjusted, and 5 mL of propionic acid is added as a preservative), and the dissolved drug composition is added to the freshly prepared food cooled to below 60°C at the designed concentration, stirred evenly, and then divided into culture tubes. The same volume of solvent is added to the food as a control for the non-drug group. Fruit flies were crossed, and specific tissue organ / cell-specific Gal4 virgin flies (female parent) were crossed with UAS-hTDP-43-wt (wild type) or UAS-hTDP-43-A315T (mutant) (male parent), with 3 or more repeats for each food / drug group, and each repeat containing 5 virgin flies and 5 male flies. During the cross, the corresponding food / drug combination tube was replaced every two days, a total of two times, and the parent flies were removed after the last food change two days later. According to the experimental requirements, the corresponding phenotypes / indicators were observed / detected during the larval, pupal or adult stages of the offspring, and the results were analyzed and plotted by Prism Graphpad software after statistical analysis.

[0087] The examples describe the steps of overexpressing TDP-43 wild type or mutant in motor neurons and eyes, and detecting the phenotypes and the degree of eye defects during the development and pupal stages, respectively. Based on the main pathological features of TDP-43 proteinopathy, TDP-43 mislocalizes from the nucleus to the cytoplasm and forms aggregates, by overexpressing human TDP-43 wild type gene in fruit fly eyes, fruit fly eyes produce defective phenotypes with different degrees of necrotic black spots (none to whole eye). In addition, the A315T mutation of TDP-43 gene is also a common familial case, and accordingly a fruit fly eye model overexpressing human TDP-43 mutant A315T was constructed, which had similar phenotypes to the wild type. It is also noted that in both models, the defects of male flies are more severe than those of female flies. The area of necrotic black spots in the eyes of the disease model is divided into three categories, and FIG. 1A-FIG. 1D show the classification of necrotic black spots in the eyes of the fruit fly disease model and the control group; among them, FIG. 1B corresponds to none (no black spots), FIG. 1C corresponds to light (the sum of the black spot areas of both eyes is less than 1 / 4 of the area of a single eye), and FIG. 1D corresponds to heavy (the sum of the black spot areas of both eyes is greater than or equal to 1 / 4 of the area of a single eye); FIG. 1A is the control group (overexpressing red fluorescent protein RFP).

[0088] Example 1

[0089] OK371 drives overexpression of hTDP-43 mutant A315T in Drosophila motor neurons, resulting in pupal lethality, pupae significantly smaller in size (by length) than control pupae, and only a small amount of development to late pupae. Pupae fed the combination of hederagenin (abbreviated as hed) and dehydronodakenin (abbreviated as dehyd) pharmaceutical composition have a small amount of development to late pupae, and more significantly, a significant increase in pupae size (by length) (P < 0.001), with an average increase of nearly 14%. The active ingredients (hed, dehyd) of the pharmaceutical composition (the method of preparing the pharmaceutical composition is to mix hederagenin, dehydronodakenin and DMSO to obtain the pharmaceutical composition, and the concentration of active ingredients is controlled by the amount of hederagenin, dehydronodakenin added) in the food are both 0.05 μg / mL. Because the late pupae of the disease model without drug (i.e. A315T, DMSO) are too few, only the length of early pupae is counted (results shown in Figures 2A-2B and Table 1).

[0090] Table 1

[0091] Figures 2A-2B are results of Example 1 showing that the combination of hederagenin and dehydroicaritin significantly improved the developmental defects (pupa length) caused by TDP-43 mutant A315T in motor neurons. Figure 2A is a comparison of the pupae of the control, disease model, and drug-treated groups. Figure 2B is a bar graph of the relative length of the pupae (“Relative length of pupae” represents the relative length of the pupae). Table 1 is an analysis of the length statistics, normalized to the RFP, DMSO group. In Figures 2A-2B, “hede dehy” means the combination of hederagenin and dehydroicaritin, and the numbers after “hede dehy” represent the concentrations of the active ingredients (hede, dehy) in the combination. For example, “hede dehy 0.05” means that the concentration of hederagenin and dehydroicaritin in the combination was 0.05 μg / mL in the food. “RFP, DMSO” means the control group OK371 > RFP (OK371 drives RFP overexpression in motor neurons) flies were fed with food containing DMSO. “A315T” means the disease model OK371 > A315T flies, i.e., OK371 drives TDP43-A315T overexpression in motor neurons, resulting in a mutated TDP-43 protein at position A315. “A315T, hede dehy 0.05” means that the concentration of hederagenin and dehydroicaritin in the combination was 0.05 μg / mL in the food. Figure 2B was analyzed using One-Way ANOVA, and “***” means a significant difference P < 0.0005, and “**” means P < 0.001. In Table 1, “mean” means the average value of the relative length of the pupae, “SD” means the standard deviation, and N is the number of repeats. For each genotype / drug treatment, the length of at least 10 pupae was measured and the average value was calculated for each repeat.

[0092] Example 2

[0093] The combination of hederagenin and dehydroicaritin (the combination is referred to as “hede dehy”) at a concentration of 0.05 μg / mL in the food significantly improved the movement ability of Drosophila larvae overexpressing TDP-43 mutant A315T in motor neurons. The movement ability of the Drosophila larvae overexpressing TDP-43 mutant A315T in motor neurons was significantly worse than that of the control group expressing RFP, as the larvae were flipped to the abdomen up, and the time for the larvae to flip to the abdomen down was much longer than that of the control group. However, the time for the larvae to flip to the abdomen down was shortened by an average of 40.6% when the larvae were fed with food containing the combination of hederagenin and dehydroicaritin (the combination is referred to as “hede dehy”) at a concentration of 0.05 μg / mL in the food (results shown in Figure 3 and Table 2).

[0094] Table 2

[0095] Figure 3 is the result of the improvement of the locomotor ability of the fruit fly overexpressing TDP-43 mutant A315T in motor neurons by the pharmaceutical composition of Example 2 of combination of saponin genin of Hedera helix (abbreviated as “Hed”) and dehydrosiemen (abbreviated as “dehydrosi”) (the pharmaceutical composition is referred to as “Hed dehydrosi” for short, and the concentration is 0.05 μg / mL, respectively). Figure 3 is a statistical chart, and Table 2 is the corresponding statistical data. “Hed dehydrosi 0.05” in Figure 3 means that the concentration of the pharmaceutical composition of saponin genin of Hedera helix and dehydrosiemen in the food is 0.05 μg / mL, “RFP, DMSO” means the DMSO solvent control of OK371 > RFP (OK371 drives RFP to overexpress in motor neurons) fruit fly food, “A315T, DMSO” means the DMSO solvent of OK371 > A315T (OK371 drives A315T to overexpress in motor neurons) fruit fly food, “A315T, Hed dehydrosi 0.05” means that the concentration of saponin genin of Hedera helix and dehydrosiemen in the food of OK371 > A315T fruit fly is 0.05 μg / mL. The analysis in Figure 3 uses One-Way ANOVA test, and “**” in Figure 3 means P < 0.001; “Genotype Treatment” in Table 2 means genotype treatment, “Changed” means change, “mean” is the average time of turning over (seconds), SD is the standard deviation, and N is the number of repetitions. The turning over time of at least 5 larvae of each genotype / drug treatment is measured and the average value is calculated for each repetition.

[0096] Example 3

[0097] The pharmaceutical composition of combination of saponin genin of Hedera helix (abbreviated as “Hed”) and dehydrosiemen (abbreviated as “dehydrosi”) (the pharmaceutical composition is referred to as “Hed dehydrosi” for short, and the solvent is DMSO, and the concentration of the active ingredients of the pharmaceutical composition in the food is 0.05 μg / mL) has a good therapeutic effect on fruit flies overexpressing TDP-43 mutant A315T in the eyes. The proportion of severe black spots is reduced from 7.1% of the non-drug group to 2.17%, the proportion of non-necrotic black spots is increased by an average of 8 percentage points, and the proportion of mild black spots is reduced by an average of 3 percentage points (the results are shown in Figure 4 and Table 3).

[0098] Table 3

[0099] Figure 4 is the treatment result of the drug composition of the combination of saponin and dehydroicaritin of Example 2 on the fruit fly overexpressing TDP-43 mutant A315T in the eye. As can be seen from Figure 4, the drug composition has a better treatment effect. Figure 4 is a statistical chart, and Table 3 is the corresponding statistical data. “Saponin and dehydroicaritin 0.05” in Figure 4 means that the concentration of saponin and dehydroicaritin in the food is 0.05 μg / mL, “A315T, DMSO” means that the OK371 > A315T (OK371 drives A315T to overexpress in motor neurons) fruit fly food is DMSO solvent, “A315T, saponin and dehydroicaritin 0.05” means that the OK371 > A315T fruit fly food has a concentration of 0.05 μg / mL of saponin and dehydroicaritin, and “none”, “light” and “heavy” are the classifications of the fruit fly eye black spots according to the area. “n=71, 74, 79” and “n=76, 50, 78” in Figure 4 respectively represent the number of fruit flies detected in each experimental repeat; “mean” in Table 3 is the average, with % as the unit, SD is the standard deviation, and N is the number of experimental repeats.

[0100] Example 4

[0101] Female Prp-TDP43(A315T) were randomly divided into two groups, DMSO (n=4), combination of saponin (saponin) and dehydroicaritin (dehydroicaritin) (denoted as saponin / dehydroicaritin, n=5). The dosage of saponin used was 2 mg / kg / day, and the dosage of dehydroicaritin used was 10 mg / kg / day. After 70 days of intragastric administration, the female Prp-TDP43(A315T) mice were sacrificed by anesthesia according to the conventional process, the spinal cord at the lumbar enlargement was taken, fixed with formaldehyde, and embedded in paraffin. 6 micron sections were subjected to Nissl staining. C13220-01 NanoZoomer S360 digital slide scanner imaging, Indica Labs HALO AI for analysis of Nissl staining optical density and number of neurons per unit area of the anterior horn motor neurons. The optical density comparison used unpaired T test, and the number of neurons per unit area analysis used Mann Whitney test (as shown in Figures 5A-5C).

[0102] As can be seen from Figures 5A-5C (“Neurons” means neurons), the drug composition of the combination of saponin and dehydroicaritin has the effect of increasing the number of neurons per unit area of the mouse with progressive freezing.

[0103] Example 5: Fruit fly overexpressing TDP-43-wild type in the eye

[0104] The drug composition of sodium aescinate (abbreviated as QI) and dioxadromine (abbreviated as DI) combination (the drug composition is referred to as "QIDI"; the concentration of the two active ingredients in food is 0.05 μg / mL, and the solvent is DMSO) can significantly improve the degree of necrotic black spots in female flies overexpressing TDP-43-wild type (wt). The proportion of no necrotic black spots increased by an average of 27 percentage points; the mild black spots decreased by an average of 23 percentage points; and the severe black spots decreased to 15.6% of the non-drug group (as shown in FIG. 6).

[0105] FIG. 6 is the drug result of the drug composition of sodium aescinate and dioxadromine combination of Example 5. As can be seen from FIG. 6, the drug composition can significantly improve the degree of necrotic black spots in female flies overexpressing TDP-43-wild type (wt). "QIDI 0.05" in FIG. 6 means that the concentration of the drug composition of sodium aescinate and dioxadromine in food is 0.05 μg / mL, and "non-drug" means the control. "n = 80, 65" and "n = 65, 66" in FIG. 6 respectively represent the number of fruit flies detected in each experimental repeat.

[0106] Example 6

[0107] The drug composition of sodium aescinate (abbreviated as QI) and dioxadromine (abbreviated as DI) combination (the drug composition is referred to as "QIDI"; the drug composition in food, the concentration of the active ingredient sodium aescinate is 0.001 μg / mL, and the concentration of dioxadromine is 0.005 μg / mL) has obvious therapeutic effect on female flies overexpressing TDP-43 mutant A315T in the eyes. The proportion of no necrotic black spots treated by the drug composition of sodium aescinate and dioxadromine combination (the drug composition is referred to as "QIDI", the solvent is DMSO) increased by an average of nearly 16.1 percentage points; the severe black spots decreased by an average of 6.76 percentage points, and decreased to 1.87%, which is about 21.7% of the non-drug group; and the mild black spots decreased by an average of 9.27 percentage points (as shown in FIG. 7 and Table 4).

[0108] Table 4

[0109] Figure 7 shows the treatment results of the pharmaceutical composition of Example 6, aescin sodium and dehydroicaritin combination, on fruit flies overexpressing TDP-43 mutant A315T in the eyes. As can be seen from Figure 7, the pharmaceutical composition has a significant treatment effect. Figure 7 is a statistical chart, and Table 4 is the corresponding statistical data. In Figure 7, "aescin sodium and dehydroicaritin 0.001 and 0.005" means the concentrations of aescin sodium and dehydroicaritin in the food are 0.001 μg / mL and 0.005 μg / mL, respectively, and "none", "mild" and "severe" are the classifications of the fruit fly eye melanotic spots by area. In Figure 7, "n = 11, 49, 44" and "n = 27, 52, 25" represent the number of fruit flies detected in each experimental repeat, respectively; "mean" in Table 4 is the average, in %, SD is the standard deviation, and N is the number of experimental repeats.

[0110] Example 7

[0111] The pharmaceutical composition of aescin sodium (abbreviated as aescin) and diosmin (abbreviated as diosmin) combination and the pharmaceutical composition of aescin sodium (abbreviated as aescin) and dehydroicaritin (abbreviated as dehydroicaritin) combination can both significantly improve the degree of necrotic melanotic spots in fruit flies overexpressing TDP-43-wild type (wt) in the eyes. The pharmaceutical composition is referred to as "aescin and diosmin" and "aescin and dehydroicaritin" for short, the solvent is DMSO, and the concentrations of the active ingredients of the two pharmaceutical compositions in the food are 0.1 μg / mL and 0.05 μg / mL, respectively. The effect of 0.05 μg / mL "aescin and diosmin" is the most significant, with an average increase of 20.76 percentage points in the proportion of no melanotic spots, an increase of more than 1 fold; an average decrease of 22.8 percentage points in the proportion of severe melanotic spots, to only 4.3%, which is 15.9% of the non-drug group; although more mild melanotic spots are converted to no melanotic spots, the proportion of mild melanotic spots changes little because a large number of severe melanotic spots are converted to mild melanotic spots. The effects of 0.1 μg / mL and 0.05 μg / mL "aescin and dehydroicaritin" are less significant, with a significant decrease in the proportion of severe melanotic spots, which is 8.73% and 9.13%, respectively; an increase of 19.6 and 11 percentage points, i.e., an increase of more than 1 fold and 0.55 fold, respectively, in the proportion of no melanotic spots; the proportion of mild melanotic spots increases in the 0.05 μg / mL "aescin and dehydroicaritin" group due to the conversion of more severe melanotic spots to mild melanotic spots, and does not decrease significantly in the 0.1 μg / mL "aescin and dehydroicaritin" group. The 0.1 μg / mL "aescin and diosmin" group also has a certain effect, with a decrease of 12.23 percentage points in the proportion of severe melanotic spots and an increase of 8.63 percentage points in the proportion of no melanotic spots (results shown in Figure 8 and Table 5).

[0112] Table 5

[0113] Figure 8 shows the results of the drug composition of aescin sodium and doksan combined with aescin sodium and dehydronovan combined with the degree of improvement of the necrotic black spot caused by overexpression of TDP-43-wild type (wt) in the eyes of male flies. Figure 8 is a statistical chart, and Table 5 is the corresponding statistical data. In Figure 8, "Qidi 0.1" means that the concentration of aescin sodium and doksan in the drug composition is 0.1 μg / mL in food, "Qidetuni 0.1" means that the concentration of aescin sodium and dehydronovan in the drug composition is 0.1 μg / mL in food, "No drug" means the solvent control, and "none", "light" and "heavy" are the classification of the area of black spots in the eyes of fruit flies. In Figure 8, "n=47, 42, 32", "n=31, 30, 34", "n=22, 41, 28", "n=36, 31, 39" and "n=23, 15, 29" represent the number of fruit flies detected in each experimental repeat; "mean" in Table 5 is the average, with a unit of %, SD is the standard deviation, and N is the number of experimental repeats.

[0114] Example 8

[0115] The drug composition of aescin sodium (abbreviated as Qidi) and doksan (abbreviated as doksan) combined with aescin sodium (abbreviated as Qidi) and dehydronovan (abbreviated as dehydronovan) has a significant therapeutic effect on female flies. Overexpression of TDP-43-wild type (wt) gene in the eyes of female flies, the culture temperature of this example is 27℃, which is higher than 25℃, and the severity of black spots is significantly increased. The drug composition (the drug composition is abbreviated as "Qidi" and "Qidetuni" respectively; the solvent is DMSO, and the concentration of active ingredients in food is 0.05 μg / mL) can well improve the symptoms of black spots. Compared with the "no drug" group, the proportion of severe black spots in the "Qidi" combination group decreased by 52% from 45.97% to 24.5%; more than half of the severe black spots were converted to mild black spots, so the proportion of mild black spots increased by more than 20.43 percentage points from 52.87% to 73.3%; and the proportion of no black spots increased. The proportion of no black spots in the "Qidetuni" combination group increased significantly compared with the "no drug" group, from 1.13% to 13.85%, an increase of 11.26 times; severe black spots decreased by 8.8 percentage points, a decrease of nearly 20%; part of the mild black spots were converted to no black spots and part of the severe black spots were converted to mild, so the proportion of mild black spots did not change significantly (as shown in Figure 9).

[0116] Figure 9 is the result of the drug combination of Example 8, aescin sodium and dehydroicaritin, on female flies with a culture temperature of 27°C. As can be seen from Figure 9, the drug combination has a significant therapeutic effect. Figure 9 is a statistical chart. "Aescin 0.05" in Figure 9 means that the concentration of aescin sodium in the drug combination is 0.05 μg / mL, and the concentration of diosmin in the drug combination is also 0.05 μg / mL. "Aescin-dehydroicaritin 0.05" in Figure 9 means that the concentration of aescin sodium in the drug combination is 0.05 μg / mL, and the concentration of dehydroicaritin in the drug combination is also 0.05 μg / mL. "No drug" in Figure 9 means the control. "None", "light" and "heavy" are the classifications of the area of the black spots on the eyes of the fruit flies. "n=29, 33, 27", "n=34, 54, 44" and "n=21, 22" in Figure 9 represent the number of fruit flies detected in each experiment.

[0117] Comparative Example 1

[0118] Overexpression of TDP-43 mutant A315T in motor neurons results in pupal lethality and the size of pupae is significantly smaller than the normal control group (see Example 1). The combination of hederagenin and dehydroicaritin with a concentration of 0.05 μg / mL in food can partially rescue this phenotype. The effects of hederagenin (abbreviated as "hed") and dehydroicaritin (abbreviated as "deh") alone cannot reach the effect of the combination of hederagenin (abbreviated as "hed") and dehydroicaritin (abbreviated as "deh") (results shown in Figures 10A-10B).

[0119] Figures 10A-10B are the results of hederagenin (abbreviated as "hed") and dehydroicaritin (abbreviated as "deh") alone and in combination in Comparative Example 1. Figure 10A is an image of pupae fed with different drugs in fruit fly motor neurons overexpressing hTDP-43 mutant A315T, and Figure 10B is a statistical chart, normalized to the drug combination group. "A315T" in Figures 10A-10B represents overexpression of hTDP-43 mutant A315T in motor neurons, "hed-deh" means that the combination contains hederagenin and dehydroicaritin, "hed" means hederagenin, "deh" means dehydroicaritin, and the number after the drug represents the concentration of the drug in μg / mL. "hed-deh 0.05" in Figure 10B means that the concentration of hederagenin and dehydroicaritin in the drug combination is 0.05 μg / mL. "n=67", "n=53", "n=85", "n=67" and "n=73" in Figure 10B represent the number of pupae detected, and One-Way ANOVA was used for analysis. "**" means a significant difference P<0.01, and "*" means a significant difference P<0.05.

[0120] Comparative Example 2

[0121] Under the same experimental conditions, the individual use of saponin (abbreviated as saponin) and aescin (abbreviated as aescin) cannot achieve the effect of the combined use of saponin (abbreviated as saponin) and aescin (abbreviated as aescin), here in the eyes of fruit flies overexpressing TDP-43-A315T.

[0122] Figure 11 is the treatment effect of saponin (abbreviated as saponin) and aescin (abbreviated as aescin) alone and in combination on TDP-43-A315T fruit flies. As can be seen from Figure 11, the individual use cannot achieve the effect of the combined use. Figure 11 is a statistical chart, and Table 6 is the corresponding statistical data. In Figure 11, "saponin-aescin 0.05" means that the concentration of saponin and aescin in the food is 0.05 μg / mL, "saponin 0.1" means that the concentration of saponin in the food is 0.1 μg / mL, "saponin 0.05" means that the concentration of saponin in the food is 0.05 μg / mL, "aescin 0.1" means that the concentration of aescin in the food is 0.1 μg / mL, "aescin 0.05" means that the concentration of aescin in the food is 0.05 μg / mL, "no drug" means the control, and "none", "light" and "heavy" are the classification of fruit fly eye black spots by area. In Figure 11, "n=74, 71, 78", "n=76, 50, 78", "n=101, 111, 102", "n=75, 70, 87", "n=104, 63, 44" and "n=103, 74, 50" respectively represent the number of fruit flies in different experimental repeats. In Table 6, "mean" is the average, unit %, SD is the standard deviation, and N is the number of experimental repeats.

[0123] Table 6

[0124] Comparative Example 3

[0125] Under the same experimental conditions, the individual use of saponin (abbreviated as saponin) and aescin (abbreviated as aescin) cannot achieve the effect of the combined use of saponin (abbreviated as saponin) and aescin (abbreviated as aescin), here in the eyes of fruit flies overexpressing TDP-43-A315T.

[0126] Figure 12 is the effect of sodium aescinate and diosmin alone and in combination on TDP-43-wild type Drosophila treatment. As can be seen from Figure 12, the effect of the combination is superior to the effect of the individual drugs. Figure 12 is a statistical graph. In Figure 12, “aesc 0.05” means that the concentration of sodium aescinate and diosmin in the food is 0.05 μg / mL, “aesc 0.1” means that the concentration of sodium aescinate in the food is 0.1 μg / mL, “dios 0.1” means that the concentration of diosmin in the food is 0.1 μg / mL, and “none”, “light” and “heavy” are the classifications of the area of the black spots on the eyes of the fruit flies. In Figure 12, “n=65, 80”, “n=65, 66”, “n=65, 104” and “n=28, 51” respectively represent the number of fruit flies in different experimental repeats.

[0127] Example 9

[0128] The addition of edaravone to the binary combination of sarsasapogenin and dehydroicaritin can enhance the therapeutic effect.

[0129] Overexpression of TDP-43 mutant A315T in motor neurons leads to pupal lethality and the size of the pupae is significantly smaller than the normal control group (see Example 1). The effect of the combination of sarsasapogenin and dehydroicaritin combined with edaravone is superior to the binary combination and has a significant difference.

[0130] Figures 13A-13B are the effect of the combination of sarsasapogenin and dehydroicaritin combined with edaravone. As can be seen from Figures 13A-13B, the effect of the combination is superior to the binary combination of sarsasapogenin and dehydroicaritin. Figure 13A is the overexpression of TDP-43 mutant A315T in motor neurons treated with different drugs, and Figure 13B is a statistical graph, normalized to the DMSO control group. In Figures 13A-13B, “A315T” means overexpression of TDP-43 mutant A315T in motor neurons, “aesc dehyd + d” means that the concentration of sarsasapogenin and dehydroicaritin in the food is 0.05 μg / mL, “aesc dehyd + d + eda” means that the concentration of sarsasapogenin and dehydroicaritin in the food is 0.05 μg / mL and the concentration of edaravone is 0.04 μg / mL, and “DMSO” means the solvent control. In Figure 13B, “n=48”, “n=67” and “n=52” represent the number of fruit flies detected, and the analysis uses One-Way ANOVA test. “****” means a significant difference P<0.001, and “*” means a significant difference P<0.05.

[0131] Comparative Example 4

[0132] In the model of overexpression of TDP-43 mutant A315T in the fruit fly optic nerve, the effect of the combination of hederagenin and dehydroicaritin is better than that of riluzole (as shown in Figure 14 and Tables 7 and 8). Compared with the solvent (DMSO) control group: adding three different concentrations of riluzole in food, 0.04 μg / mL is ineffective, and the average proportion of eye black spots "none", "mild" and "severe" is 61.30%, 35.65% and 3.05%, respectively, which has no significant difference with the control group of 60.30%, 35.18% and 4.53%; the proportion of no black spots of riluzole with concentrations of 1 μg / mL and 0.2 μg / mL is 65.68% and 66.30%, respectively, which has significant difference compared with the control group (P = 0.012 and 0.008), and the proportion of mild black spots and severe black spots is 30.25%, 30.93% and 4.10% and 2.08%, respectively, which has no significant difference with the control group; 0.2 μg / mL of riluzole is the best among the three concentrations.

[0133] The proportion of no black spots of the combination of hederagenin and dehydroicaritin is 75.03%, which is increased by 14.73 percentage points on average compared with the control group, with an increase of 24.4%, which has a very significant difference (P = 0.00003), the average proportion of mild black spots is 22.45%, which is decreased by 12.73 percentage points on average compared with the control group, with a decrease of 36.2%, which has a very significant difference (P = 0.0011), and the proportion of severe black spots is 2.55%, which has no significant difference with the control group.

[0134] Compared with 0.2 μg / mL of riluzole, the combination of hederagenin and dehydroicaritin has a strong advantage in the proportion of no black spots and the proportion of mild black spots, and the significant difference P values are 0.00049 and 0.00170, respectively, and the proportion of severe black spots has no significant difference.

[0135] Table 7

[0136] Table 8

[0137] Figure 14 is a comparison of the effect of the combination of hederagenin and dehydroicaritin (referred to as hederagenin and dehydroicaritin) and the marketed drug riluzole. It can be seen that, on the one hand, the combination of hederagenin and dehydroicaritin has a better effect than the existing drug riluzole. On the other hand, the usual amount of riluzole currently used in the human body is 100 mg / day, and by comparing the ratio of the concentration of hederagenin and dehydroicaritin to the concentration of riluzole, it can be inferred that the amount of the binary combination drug of the present application used in human clinical experiments is approximately 10 mg / day-2000 mg / day. Preferably, the initial amount of the combination of hederagenin and dehydroicaritin for human experiment exploration is about 20 mg-200 mg / day.

[0138] Figure 14 is a statistical graph of the effect of overexpression of TDP-43 mutant A315T in optic nerves treated with different drugs, Table 7 is the corresponding statistical data, and Table 8 is the result of significance analysis of differences. In Figure 14, DMSO is the solvent control group, "Riluzole 1", "Riluzole 0.2" and "Riluzole 0.04" represent the concentration of riluzole in food as 1 μg / mL, 0.2 μg / mL and 0.04 μg / mL, respectively, "Anagalligenin 0.25" represents the concentration of anagalligenin and dehydroicariin in food as 0.25 μg / mL, respectively, and "None", "Light" and "Heavy" are the classification of the area of the black spot in the eyes of fruit flies. In Figure 14, "n=55, 83, 44, 56", "n=44, 51, 63, 59", "n=65, 38, 63, 82", "n=45, 44, 52, 88" and "n=29, 60, 57, 65" represent the number of fruit flies detected in different experimental repeats, respectively. In Table 7, "mean" is the average, with the unit of %, SD is the standard deviation, N is the number of experimental repeats, and the change represents the difference from the DMSO control. In Table 8, the data was subjected to significance analysis using Multiple t-tests, "****" represents a significant difference P<0.001, "***" represents a significant difference P<0.005, "**" represents a significant difference P<0.01, and "*" represents a significant difference P<0.05.

Claims

1. A pharmaceutical composition, characterized by, comprises at least one of a Class A compound and a Class B compound; the Class A compound comprises at least one of sodium aescinate, a sodium aescinate derivative, hederagenin, or a hederagenin derivative; the Class B compound comprises at least one of a dehydrosawainsonine, a dehydrosawainsonine derivative, diosmin, or a diosmin derivative.

2. The pharmaceutical composition of claim 1, wherein, the sodium aescinate derivative comprises at least one of aescin, aescin A, aescin B, aescin C, aescin D, hederagenin, or protohederagenin.

3. The pharmaceutical composition of claim 1, wherein, the dehydrosawainsonine derivative comprises at least one of icariin, icariin, or dehydrosicariin.

4. The pharmaceutical composition of claim 1, wherein, the diosmin derivative comprises neodiosmin.

5. The pharmaceutical composition of claim 1, wherein, the hederagenin derivative comprises at least one of Alpha-hederin, Beta-hederin, hederin C, hederin D.

6. The pharmaceutical composition of claim 1, wherein, a weight ratio of the Class A compound to the Class B compound is 1:20 to 20:

1.

7. The pharmaceutical composition according to any one of claims 1-6, characterized in that, the pharmaceutical composition can be used in combination with edaravone in the treatment.

8. The pharmaceutical composition of claim 7, wherein, the pharmaceutical composition further comprises a pharmaceutically acceptable solvent and an excipient.

9. The pharmaceutical composition of claim 7, wherein, the pharmaceutically acceptable excipient comprises at least one of a pharmaceutical carrier, a diluent, an adjuvant, or an excipient.

10. Use of the pharmaceutical composition of any one of claims 1-9 in the manufacture of a medicament for treating a TDP-43 proteinopathy.

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