Pharmaceutical composition for treating TDP-43 proteinosis as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medicines, and discloses a pharmaceutical composition for treating TDP-43 proteinosis as well as a preparation method and application of the pharmaceutical composition. The pharmaceutical composition comprises at least one compound A and at least one compound B, the compound A comprises at least one of sodium aescinate, a sodium aescinate derivative, hederagenin or a hederagenin derivative; and the compound B comprises at least one of anhydroicaritin, an anhydroicaritin derivative, diosmin or a diosmin derivative. The pharmaceutical composition has a good treatment effect on the TDP-43 proteinosis.

Description

Technical Field

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

[0002] TDP-43 proteinopathy is a general term for a group of various neurological diseases. The common pathological feature of these diseases is that the formation of pathological protein inclusions of transactive response DNA binding protein 43000 (TDP-43) protein can be detected in the diseased 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. Its activity regulation is reflected in its controlled shuttling between the cell nucleus and the cytoplasm (References: 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 normal human brain, TDP-43 is mainly present in the cell nucleus with almost no abnormal deposition. However, in pathological conditions, TDP-43 usually appears in large numbers as insoluble inclusion bodies or is located in the cytoplasm in large quantities. Some of them are abnormally degraded, resulting in loss of function, which leads to cytotoxicity and 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 have been 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 (References: Chen L. The important functional role of TDP-43 plays in amyotrophic lateral sclerosis-frontotemporaldementia. 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 has also been found in some patients with a variety of other neurodegenerative diseases, including chronic traumatic encephalopathy (CTE), Lewy body disease (LBD), Huntington's disease (HD), argyrophilic grain disease (AGD) and hippocampal sclerosis (Uchino, et al., 2015; de Boer, et al., 2020).

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

[0007] ALS can cause a combination of upper and lower motor neuron diseases, affecting the trunk, limbs, and head and facial muscles. Patients gradually lose control of their muscles, leading to muscle atrophy, and usually die of respiratory failure 3 to 5 years after onset. The prevalence is about 6 cases per 100,000 people, with men slightly more than women. (Reference: Lin, Cheng GC, Wu LY, Lai WY, LingTY, 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 caused by familial gene mutations, such as TDP-43, SOD1, FUS, etc.; while more than 90% are sporadic, that is, the pathogenic gene has not been identified, but their pathological characteristics are highly correlated with the structural and functional abnormalities of TDP-43 protein (Reference: Eck RJ, KraemerBC and Liachko NF. Regulation of TDP-43phosphorylation in aging and disease. GeroScience. 2021; 43: 1605–1614). There are at least 20 pathogenic mutations in the TDP-43 gene. Among these TDP-43 mutations, the A315T mutation located at the C-terminus has the most extensive functional effects, integrating cytoplasmic localization, fragmentation, phosphorylation and ubiquitination, and insolubility. Therefore, its symptoms are relatively severe compared with other mutations (Reference: Ke YD, van Hummel A, Stevens CH, Gladbach A, Ippati S, Bi M, Lee WS, Krüger 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 functional defects of TDP-43 protein are found in the nervous system of most ALS patients, so ALS is a typical representative of TDP-43 protein disease. Although people have been developing drugs for ALS for many years, the drugs that have been approved for marketing have little effect, and some drugs have serious side effects, indicating that there is still a lack of effective drugs for TDP-43 protein disease caused by abnormal accumulation and dysfunction of TDP-43 protein.

[0009] Riluzole is the first drug approved by the US FDA and the EU for the treatment of ALS. Clinical studies have shown that riluzole treatment can reduce the loss of motor neurons, improve the survival of patients, and slow the progression of the disease, but it cannot prevent the progression of ALS, and the survival of patients has only increased by about 3 months. (References: Bensimon G, Lacomblez L, Delumeau JC, Bejuit R, Truffinet P, Meininger VA study of riluzole in the treatment of advanced stage or elderly patients with amyotrophiclateral sclerosis. J Neurol. 2002; 249: 609-615; Doble A. The pharmacology and mechanism of action of riluzole. Neurology. 1996; 47: S233-41) Riluzole may cause side effects such as dizziness, fatigue, gastrointestinal symptoms and changes in liver function (References: 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 the next compound approved for the treatment of ALS in many Asian countries, the United States, Canada and Switzerland after 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). Multiple experimental studies have shown that edaravone has a certain effect in reducing motor decline and motor neuron degeneration and slowing down disease progression, but the efficacy is limited and is not satisfactory. In addition, the drug has strong side effects. Common adverse events include contusion, confusion, dysphagia, constipation, eczema, headache, bruising, gait disorders and allergic reactions (references: Cruz MP. Edaravone (Radicava): a novel neuroprotective agent for the treatment of amyotrophic lateral sclerosis. PT. 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 to provide a new drug with better effect for treating TDP-43 protein disease. Summary of the invention

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

[0013] Specifically, a 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 includes at least one of sodium aescinate, sodium aescinate derivatives, hederagenin or hederagenin derivatives;

[0015] The class B compound includes at least one of anhydroicariin, anhydroicariin derivatives, 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, aescinogenin or protoescinogenin.

[0017] Sodium Aescinate: CAS No. 20977-05-3, molecular formula C54H83NaO23. It is in the form of white powder or crystalline powder, and has the effects of anti-inflammatory, anti-exudation, increasing venous tension, improving blood circulation, and correcting brain dysfunction. It has a significant protective effect against brain edema caused by carbon monoxide.

[0018] Escin: CAS number is 6805-41-0, molecular formula is C 55 H 86 O 24 .

[0019] Aescin A: CAS number is 123748-68-5, molecular formula is 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 number is 158732-55-9, molecular formula is C 54 H 84 O23 .

[0022] Aescin D: CAS number is 158800-83-0, molecular formula is C 54 H 84 O 23 .

[0023] Aescigenin: CAS number is 17806-68-7, molecular formula is C 30 H 48 O5.

[0024] Protoaescigenin: CAS number is 20853-07-0, molecular formula is C 30 H 50 O6.

[0025] Preferably, the dehydrated icariin derivative includes at least one of icariin, icariin or dehydrated icariin.

[0026] Anhydroicaritin: CAS number is 38226-86-7, molecular formula is C 21 H 20 O6. It is a prenylated flavonoid compound (3,5,7-trihydroxy-2-(4-methoxyphenyl)-8-(3-methylbut-2-enyl)-4H-benzopyran-4-one), a natural compound with anticancer activity, possibly through regulating the MAPK / ERK / JNK and JAK2 / STAT3 / AKT signaling pathways.

[0027] Icaritin: CAS number is 521-45-9, molecular formula is C 21 H 22 O7.

[0028] Icariin: CAS number is 489-32-7, molecular formula is C 33 H 40 O 15 .

[0029] Dehydrated icariin (baohuoside I, Icarisid II): CAS number is 113558-15-9, molecular formula is C 27 H 30 O 10 .

[0030] Preferably, the diosmin derivatives include Neodiosmin, CAS No. 38665-01-9, and molecular formula C 28 H32 O 15 .

[0031] Diosmin: CAS number is 520-27-4, molecular formula is C 28 H 32 O 15 Chemical name: 7-{[6-oxy-(6-deoxy-α-L-mannose)-β-D-pyranose glucose]oxy}-5-hydroxy-2-(3-hydroxy-4-methoxyphenyl)-4H-L-benzopyran-4-one. It is a capillary protective drug. It is used to treat various symptoms related to venous and lymphatic insufficiency and various symptoms of acute hemorrhoid attacks.

[0032] Preferably, the hederagenin derivative includes at least one of Alpha-hederagenin, Beta-hederagenin, hederagenin C and hederagenin D.

[0033] Hederagenin: CAS number is 465-99-6, molecular formula is C 30 H 48 O4. Hederagenoside is a water-soluble pentacyclic triterpenoid saponin, which is a pentacyclic triterpenoid compound isolated from plants. It has many chemical properties, such as antispasmodic, anti-parasitic, and anti-cell growth inhibition. It has a wide range of pharmacological activities, including anti-tumor, anti-inflammatory, anti-depressant, anti-neurodegenerative, anti-hyperlipidemic, anti-diabetic, anti-leishmaniasis and anti-viral activities.

[0034] Alpha-Hederin: CAS No. 27013-91-8, molecular formula C 41 H 66 O 12 .

[0035] Beta-Hederin: CAS No. 35790-95-5, molecular formula C 41 H 66 O 11 .

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

[0037] Hederaside D: CAS No. 760961-03-3, molecular formula C 53 H 86 O 22 .

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

[0039] Preferably, the weight ratio of the two components in the binary composition of the pharmaceutical composition consisting of the A-class compound including sodium aescinate and sodium aescinate derivatives (A1 subclass) and hederagenin and hederagenin derivatives (A2 subclass), and the B-class compound including dehydrated icariin and dehydrated icariin derivatives (B1 class), diosmin and diosmin derivatives (B2 class) is 1:20 to 20:1. Specifically, 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 disease.

[0041] In some embodiments, the pharmaceutical composition further comprises pharmaceutically acceptable solvents and excipients.

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

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

[0044] Preferably, in the pharmaceutical composition, the weight ratio of sodium aescinate and its derivatives, hederagenin and its derivatives, to diosmin and its derivatives, anhydroicariin and its derivatives is 1:10 to 10:1. Specifically, for example, the weight ratio is 1:10, 1:8, 1:5, 1:1, 2:1, 5:1, 8:1, 10:1.

[0045] Preferably, the pharmaceutical composition comprises sodium aescinate or a sodium aescinate derivative and dehydrated icariin or a dehydrated icariin derivative 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, the pharmaceutical composition comprises sodium aescinate or a sodium aescinate derivative, diosmin or a diosmin derivative, and an organic solvent.

[0047] Preferably, the pharmaceutical composition comprises sodium aescinate or a sodium aescinate derivative, dehydrated icariin or a dehydrated icariin derivative, and an organic solvent.

[0048] Preferably, the pharmaceutical composition comprises dehydrated icariin or a dehydrated icariin derivative, hederagenin and a hederagenin derivative, and an organic solvent.

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

[0050] Specifically, a method for preparing a pharmaceutical composition for treating TDP-43 proteinopathy comprises the following steps:

[0051] The components are mixed to obtain the pharmaceutical composition. In addition, when taking the pharmaceutical composition of the present invention, the patient can directly take the mixed pharmaceutical composition, or take the components in the pharmaceutical composition one by one.

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

[0053] Application of the above pharmaceutical composition in the preparation of drugs for treating TDP-43 protein diseases.

[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 diseases such as ALS, 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 characteristics of these types is abnormal aggregation and pathological inclusion bodies of TDP-43 protein detected in the diseased area.

[0056] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0057] (1) In view of the current lack of effective drugs for TDP-43 protein diseases, the pharmaceutical composition of the present invention has a good therapeutic effect for TDP-43 protein diseases. TDP-43 protein diseases referred to herein include but are not limited to patients with sporadic ALS, patients with TDP-43 gene A315T mutations, and patients with neurodegenerative diseases related to TDP-43 dysfunction.

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

[0059] (3) The pharmaceutical composition for treating TDP-43 proteinopathy of the present invention comprises at least two of sodium aescinate and its derivatives, dehydrated icariin and its derivatives, diosmin and its derivatives, hederagenin and its derivatives. By combining specific active ingredients, the pharmaceutical composition has a good therapeutic effect on TDP-43 proteinopathy.

[0060] (4) The pharmaceutical composition of the present invention comprises at least one of sodium aescinate and its derivatives, dehydrated icariin and its derivatives, and at least one of diosmin and its derivatives, hederagenin and its derivatives; or, the pharmaceutical composition comprises sodium aescinate or its derivatives, and dehydrated icariin or its derivatives. Through the combination of specific active pharmaceutical ingredients, the pharmaceutical composition has a good therapeutic effect on TDP-43 proteinopathy. TDP-43 proteinopathy includes patients with sporadic ALS, patients with TDP-43 gene A315T mutation, and patients with neurodegenerative diseases related to TDP-43 dysfunction.

[0061] (5) For diseases caused by accumulation of wild-type TDP-43, the combination of sodium aescinate and diosmin, and the combination of sodium aescinate and dehydrated icariin have the most obvious effects. Other combinations, such as sodium aescinate derivatives and diosmin derivatives, sodium aescinate derivatives and icariin derivatives, hederagenine derivatives and diosmin derivatives, and hederagenine derivatives and dehydrated icariin derivatives, also have certain therapeutic effects. Sodium aescinate or diosmin alone also has a slight effect, but the effect is obviously not as good as the above-mentioned binary combination of sodium aescinate and diosmin and the binary combination of sodium aescinate and dehydrated icariin.

[0062] (6) For diseases caused by the TDP-43 mutant TDP-43-A315T, the combination of sodium aescinate and dehydrated icariin, and the combination of hederagenin and dehydrated icariin have the most obvious effects. Other combinations, such as sodium aescinate derivatives and diosmin derivatives, sodium aescinate derivatives and dehydrated icariin derivatives, and hederagenin derivatives and diosmin derivatives, also have certain therapeutic effects. Using hederagenin alone and dehydrated icariin also has a slight effect, but its effect is obviously not as good as the combination of sodium aescinate and dehydrated icariin and the combination of hederagenin and dehydrated icariin.

[0063] (7) In the model of overexpression of wild-type TDP-43 and mutant A315T in the fruit fly eye, phenotypic differences were observed between male and female flies, with male flies being more severe than female flies; there were also differences between overexpression of wild-type TDP-43 and mutant A315T. In addition, the rescue effects of different drug combinations on female and male flies and different types of TDP-43 were also significantly different. Therefore, a strategy of providing differentiated treatment based on sex / degree of defect / cause of disease is also proposed. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 The classification of necrotic black spots in the eyes of the fruit fly disease model and the control group;

[0065] Figure 2 The pharmaceutical composition of Example 1, which is a combination of hederagenin and dehydrated icariin, improves the pupal length defect caused by motor neurons expressing TDP-43 mutant A315T;

[0066] Figure 3 The results of Example 2 that the pharmaceutical composition of hederagenin and dehydrated icariin improves the motor ability of Drosophila larvae expressing TDP-43 mutant A315T in motor neurons;

[0067] Figure 4 The results of the pharmaceutical composition of Example 3 in combination with hederagenin and dehydrated icariin for the treatment of TDP-43 mutant A315T overexpressed in the eye;

[0068] Figure 5 The results of Example 4 showing that the pharmaceutical composition of hederagenin and dehydrated icariin increased the number of neurons per unit area in ALS mice (female);

[0069] Figure 6 The results of using the pharmaceutical composition of sodium aescinate and diosmin in Example 5 in eyes overexpressing wild-type TDP-43;

[0070] Figure 7 The results of the use of the pharmaceutical composition of sodium aescinate and dehydrated icariin in Example 6 on TDP-43 mutant A315T overexpressed in the eye;

[0071] Figure 8 The results of Example 7, in which the pharmaceutical composition of sodium aescinate and diosmin, and the pharmaceutical composition of sodium aescinate and dehydrated icariin, improve the degree of necrotic black spots produced by overexpression of TDP-43-wild type in the eyes of male flies;

[0072] Fig. 9The results of using the pharmaceutical composition of sodium aescinate and diosmin and the pharmaceutical composition of sodium aescinate and dehydrated icariin in Example 8 on female flies overexpressing the wild-type TDP-43 gene in the eyes cultured at 27°C;

[0073] Fig.10 The results of comparative example 1 are as follows: the use of hedera saponin (abbreviated as Chang) and dehydrated icariin (abbreviated as Yin) alone or in combination in Drosophila A315T, a mutant of motor neurons overexpressing TDP-43;

[0074] Fig.11 The results of comparative example 2, ivy saponin (abbreviated as Chang) and dehydrated icariin (abbreviated as Yin) used alone or in combination in Drosophila overexpressing TDP-43 mutant A315T in the eyes;

[0075] Fig.12 This is the therapeutic effect of sodium aescinate and diosmin alone and in combination on TDP-43-wild-type female flies in comparative example 3;

[0076] Fig.13 This is the effect of combining hederagenin and dehydrated icariin in combination with edaravone in Example 9;

[0077] Fig.14 This is comparative example 4, in which the combination of hederagenin and dehydrated icariin is superior to riluzole. DETAILED DESCRIPTION

[0078] In order to make the technical scheme of the present invention more clearly understood by those skilled in the art, the following embodiments are listed for illustration. It should be pointed out that the following embodiments do not limit the protection scope of the present invention.

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

[0080] definition

[0081] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly used in the field to which the present invention belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

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

[0083] The term "pharmaceutically acceptable" as used herein refers to an agent that can be applied to humans and / or other animals as subjects and does not produce excessive adverse reactions or side effects (such as toxicity, irritation, allergic reactions, etc.). The term "excipient" refers to an auxiliary material that is present in a pharmaceutical preparation at the same time as the active ingredient and does not produce excessive adverse reactions or side effects, including carriers, osmotic pressure regulators, pH regulators, diluents, disintegrants, excipients, solubilizers, stabilizers, preservatives, etc. The term "pharmaceutically acceptable excipient" refers to a high-safety excipient that is suitable for a specific pharmaceutical preparation and is routinely used in pharmacy. The term "carrier" includes, but is not limited to, liposomes, ethosomes, polymer micelles, nanostructured lipid carriers, solid lipid nanocarriers, mesoporous silica nanoparticles, etc.

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

[0085] The preparation of the above-mentioned pharmaceutical composition can be obtained by selecting corresponding active ingredients (such as sodium aescinate and sodium aescinate derivatives, dehydrated icariin and dehydrated icariin derivatives, diosmin and diosmin derivatives, hederagenin and hederagenin derivatives) according to the designed concentration, dissolving them in water or an organic solvent, and then mixing them.

[0086] In order to screen and verify the therapeutic effect of the above-mentioned drug combination, a Drosophila TDP-43 disease model was established, that is, the human TDP-43 wild type or A315T mutant was overexpressed in specific tissues, organs, and cells using the GAl4-UAS system to induce abnormal phenotypes / defects in Drosophila. Drosophila were cultured in an incubator at 25°C, 60% relative humidity, 12 hours of light and 12 hours of darkness every 24 hours; a few experimental Drosophila were cultured at 27°C, and the other conditions were the same. Drosophila food was a standard corn flour formula food (the standard corn flour formula food contained 68g corn flour, 43g maltose, 16g yeast powder, 9g soybean powder, 0.962g anhydrous calcium chloride, and 8g agar powder per liter, and distilled water was added to boil and constant volume, and 5mL propionic acid was added as a preservative.), the dissolved drug composition was added to the freshly prepared food cooled to below 60°C according to the designed concentration, stirred thoroughly, and dispensed into culture tubes, and the same volume of solvent was added to the food as a control for the undoped group. For fruit fly hybridization, select specific tissue / cell specific Gal4 virgin flies (female parent) and hybridize with UAS-hTDP-43-wt (wild type) or UAS-hTDP-43-A315T (mutant) (male parent), and repeat more than 3 times for each food / drug group, with 5 virgin flies and 5 male flies in each repeat; the parents of the hybridization change the corresponding food / drug combination tube every two days, for a total of two changes, and remove the adult flies of the parents two days after the last food change. According to the experimental requirements, observe / detect the corresponding phenotype / indicator in the offspring larvae, pupae or adult flies, and analyze and plot the result data through PrismGraphpad software after statistics.

[0087] The examples show that the wild type or mutant is overexpressed in motor neurons and eyes, and the phenotype of development and pupae and the degree of eye defects are detected respectively. Based on the fact that the main pathological feature of TDP-43 disease is the mislocalization of TDP-43 from the nucleus to the cytoplasm and the formation of aggregation characteristics, overexpression of the human TDP-43 wild-type gene in the eyes of fruit flies causes the eyes to produce defective phenotypes with varying degrees of necrotic black spots (none to the entire eye). In addition, the A315T mutation of the TDP-43 gene is also a common familial case. Based on this, a model of overexpression of the human TDP-43 mutant A315T in the eyes of fruit flies was constructed, and the phenotype was similar to that of the wild type. At the same time, it was noted that in both models, the defects of male flies were observed to be more serious than those of female flies. The area of ​​necrotic black spots produced in the eyes in the disease model was divided into three categories, Figure 1 The classification of necrotic black spots in the eyes of the fruit fly disease model and the control group; Figure 1 Figure B corresponds to none (no black spots), Figure C corresponds to mild (the sum of the black spot areas of the two eyes is less than 1 / 4 of the area of ​​a single eye), and Figure D corresponds to severe (the sum of the black spot areas of the two eyes is greater than or equal to 1 / 4 of the area of ​​a single eye); A is the control group (overexpression of red fluorescent protein RFP).

[0088] Example 1

[0089] OK371 drives overexpression of the hTDP-43 mutant A315T in Drosophila motor neurons, resulting in death in the pupal stage, and the size of the pupae (in length) was significantly smaller than that of the control pupae, and only a small number developed to the late pupal stage. However, a small number of pupae fed with the drug combination of hederagenin (abbreviated as Chang) and dehydrated icariin (abbreviated as Tuoyin) developed to late pupae, and the most obvious was that the size of the pupae (in length) increased significantly (P<0.001), with an average increase of nearly 14%. The drug combination (the preparation method of the drug combination is to take hederagenin, dehydrated icariin and DMSO to obtain a drug combination, and the concentration of the active ingredient is controlled by the amount of added hederagenin and dehydrated icariin) has an active ingredient (Chang, Tuoyin) concentration of 0.05μg / mL in the food. Because there were too few late pupae in the disease model without drug addition group (i.e. A315T, DMSO), only the length of the early pupae was counted (the results are shown in Figure 2 and Table 1).

[0090] Table 1

[0091]

[0092] Figure 2 The pharmaceutical composition of Example 1, which is a combination of hederagenin and dehydrated icariin, significantly improves the developmental defects (pupa length) caused by motor neurons expressing the TDP-43 mutant A315T. Figure 2 Figure A in the middle is a comparison of the pupae in the control group, the disease model group, and the drug-added group. Figure 2 Middle B ("Relative length of pupae" means the relative length of pupae) is the statistical graph of relative length of pupae, and Table 1 is the analysis of the length statistical results, normalized to the RFP untreated group. Figure 2 "Chang Tuo Yin" means that the composition contains hederagenin and dehydrated icariin, and the value after "Chang Tuo Yin" indicates the concentration of the active ingredients (Chang, Tuo Yin). For example, "Chang Tuo Yin 0.05" means that in the pharmaceutical composition, the concentrations of hederagenin and dehydrated icariin in the food are both 0.05 μg / mL, "RFP, DMSO" means that the food of the control group OK371>RFP (OK371 drives RFP to overexpress in motor neurons) fruit flies is DMSO solvent, "A315T" means the disease model OK371>A315T fruit flies, that is, OK371 drives TDP43-A315T to overexpress in motor neurons, producing a mutant TDP-43 protein at position A315, "A315T, Chang Tuo Yin 0.05" means that the concentrations of OK371>A315T hederagenin and dehydrated icariin in the food are both 0.05 μg / mL. Figure 2One-Way ANOVA was used for the analysis of Figure B. "***" indicates significant difference P<0.0005, and "**" indicates P<0.001. "Mean" in Table 1 indicates the mean of relative length of pupae; "SD" indicates standard deviation, and N is the number of repetitions. The length of at least 10 pupae was measured for each genotype / drug treatment in each repetition and averaged.

[0093] Example 2

[0094] The pharmaceutical composition of hederagenin (abbreviated as Chang) and dehydrated icariin (abbreviated as Tuoyin) (the pharmaceutical composition is referred to as "Chang Tuoyin", 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 significant improvement in the movement ability of fruit fly larvae that overexpress the TDP-43 mutant A315T in motor neurons. The movement ability of fruit fly larvae that overexpress the TDP-43 mutant A315T in motor neurons is significantly worse than that of the control group expressing RFP, as shown in that the time it takes for the larvae to flip over to the abdomen facing upward is much longer than that of the control group. When the pharmaceutical composition of hederagenin (abbreviated as Chang) and dehydrated icariin (abbreviated as Tuoyin) at a concentration of 0.05 μg / mL is added to the food (the pharmaceutical composition is referred to as "Chang Tuoyin", the solvent is DMSO), the turning time of the larvae is shortened by an average of 40.6% (the results are shown in the figure). Figure 3 and Table 2).

[0095] Table 2

[0096]

[0097] Figure 3 The results of the pharmaceutical composition of Example 2, which is a combination of hederagenin (abbreviated as Chang) and dehydrated icariin (abbreviated as Tuoyin) (the pharmaceutical composition is referred to as "Chang Tuoyin", with a concentration of 0.05 μg / mL respectively) on improving the motor ability of fruit flies overexpressing the TDP-43 mutant A315T in motor neurons. Figure 3 is a statistical chart, and Table 2 is the corresponding statistical data. Figure 3"Changtuoyin 0.05" in it indicates that the concentrations of hederagenin and anhydroicaritin in the food of the pharmaceutical composition are both 0.05 μg / mL. "RFP, DMSO" indicates that in the Drosophila food with OK371 > RFP (OK371 drives the overexpression of RFP in motor neurons), it is the DMSO solvent control. "A315T, DMSO" indicates that in the Drosophila food with OK371 > A315T (OK371 drives the overexpression of A315T in motor neurons), it is the DMSO solvent. "A315T, Changtuoyin 0.05" indicates that the concentrations of hederagenin and anhydroicaritin in the food of OK371 > A315T Drosophila are both 0.05 μg / mL. Figure 3 In the analysis, One-Way ANOVA was used for detection. "**" indicates P < 0.001; in Table 2, "Genotype Treatment" indicates genotype treatment, "Changed" indicates change, "mean" is the average turning-over time (seconds), SD is the standard deviation, N is the number of repetitions, and for each repetition, the turning-over times of at least 5 larvae for each genotype / drug treatment were measured and averaged.

[0098] Example 3

[0099] The pharmaceutical composition of the combination of hederagenin (abbreviated as Chang) and anhydroicaritin (abbreviated as Tuoyin) (this pharmaceutical composition is abbreviated as "Changtuoyin", the solvent is DMSO, and the concentration of the active ingredient of this pharmaceutical composition in the food is 0.05 μg / mL) has a good therapeutic effect on Drosophila overexpressing the TDP-43 mutant A315T in the eyes. The severe black spots decreased from 7.1% in the non-drug group to 2.17%; the proportion of non-necrotic black spots increased by an average of 8 percentage points; the mild black spots decreased by an average of 3 percentage points (the results are as Figure 4 shown in and Table 3).

[0100] Table 3

[0101]

[0102] Figure 4 For the treatment results of the female flies overexpressing the TDP-43 mutant A315T in the eyes with the pharmaceutical composition of the combination of hederagenin and anhydroicaritin in Example 2, from Figure 4 it can be seen that it has a good therapeutic effect. Figure 4 It is a statistical chart, and Table 3 is the corresponding statistical data. Figure 4"Changtuoyin 0.05" in it means that the concentrations of hederagenin and anhydroicaritin in the food of the pharmaceutical composition are both 0.05 μg / mL. "A315T, DMSO" means that the solvent in the food of Drosophila OK371>A315T (OK371 drives the overexpression of A315T in motor neurons) is DMSO. "A315T, Changtuoyin 0.05" means that the concentrations of hederagenin and anhydroicaritin in the food of Drosophila OK371>A315T are both 0.05 μg / mL. "None", "light" and "heavy" are classifications of the black spots on the eyes of Drosophila by area. Figure 4 "n = 71, 74, 79" and "n = 76, 50, 78" in it respectively represent the number of Drosophila detected in each experimental repetition; "mean" in Table 3 is the average value, with the unit of %, SD is the standard deviation, and N is the number of experimental repetitions.

[0103] Example 4

[0104] Female Prp-TDP43(A315T) was randomly divided into two groups, DMSO (n = 4), and the combination of hederagenin (Chang) and anhydroicaritin (Yin) (denoted as Chang / Yin, n = 5). The dosage of hederagenin was 2 mg / kg / day, and the dosage of anhydroicaritin was 10 mg / kg / day. After 70 days of intragastric administration, female Prp-TDP43(A315T) mice were anesthetized and sacrificed according to the conventional procedure. The spinal cord at the lumbar enlargement was taken, fixed with formaldehyde, and embedded in paraffin. 6-μm sections were stained with Nissl. Imaging was performed with a C13220-01 NanoZoomer S360 digital slide scanner, and the optical density of Nissl staining and the number of neurons per unit area in the anterior horn motor neurons were analyzed with Indica Labs HALO AI. Unpaired T-tests were used for optical density comparison, and the Mann Whitney test was used for the analysis of the number of neurons per unit area (the results are as Figure 5 shown).

[0105] From Figure 5 ("Neurons" represents neurons), it can be seen that the pharmaceutical composition of the combination of hederagenin and anhydroicaritin has the efficacy of increasing the number of neurons per unit area in amyotrophic lateral sclerosis mice.

[0106] Example 5: Female flies overexpressing TDP-43-wild type in the eyes

[0107] The drug composition of sodium aescinate (abbreviated as 7) and diosmin (abbreviated as DI) (the drug composition is referred to as "7DI"; the concentration of the two active ingredients in the food is 0.05μg / mL, and the solvent is DMSO) can significantly improve the degree of necrotic black spots in the eyes of female flies overexpressing TDP-43-wild type (wt). The proportion of non-necrotic black spots increased by an average of 27 percentage points; mild black spots decreased by an average of 23 percentage points; severe black spots decreased to 15.6% of the untreated group (the results are shown in Figure 6 shown).

[0108] Figure 6 The results of the use of the pharmaceutical composition of sodium aescinate and diosmin in Example 5 are as follows: Figure 6 It can be seen that the pharmaceutical composition can significantly improve the degree of necrotic black spots in the eyes of wild-type (wt) female flies overexpressing TDP-43. Figure 6 The “0.05 μg / mL” in the figure indicates that the concentrations of sodium aescinate and diosmin in the food of the drug composition are both 0.05 μg / mL, and “no drug added” indicates the control. Figure 6 "n=80, 65" and "n=65, 66" respectively indicate the number of fruit flies detected in each experimental repetition.

[0109] Example 6

[0110] The pharmaceutical composition of sodium aescinate (abbreviated as escin) and dehydrated icariin (abbreviated as de-icariin) has a significant therapeutic effect on female flies that overexpress the TDP-43 mutant A315T in their eyes. The proportion of non-necrotic black spots treated with the pharmaceutical composition of sodium aescinate and dehydrated icariin (the pharmaceutical composition is referred to as "seven de-icariin", the solvent is DMSO, the concentration of the active ingredient sodium aescinate in the food is 0.001μg / mL, and the concentration of dehydrated icariin is 0.005μg / mL) increased by nearly 16.1 percentage points on average; severe black spots decreased by an average of 6.76 percentage points to 1.87%, which is about 21.7% of the untreated group; mild black spots decreased by an average of 9.27 percentage points (the results are shown in Figure 2). Figure 7 and Table 4).

[0111] Table 4

[0112]

[0113] Figure 7 The results of the pharmaceutical composition of Example 6 combined with sodium aescinate and dehydrated icariin for the treatment of female flies overexpressing the TDP-43 mutant A315T in the eyes are shown in Table 1. Figure 7 It can be seen that the pharmaceutical composition has obvious therapeutic effect. Figure 7 is a statistical chart, and Table 4 is the corresponding statistical data. Figure 7 The "0.001 sodium aescinate and 0.005 dehydrated icariin" in the formula means that the concentrations of sodium aescinate and dehydrated icariin in the food of the pharmaceutical composition are 0.001 μg / mL and 0.005 μg / mL respectively. "None", "light" and "heavy" are the classifications of the black spots in the eyes of fruit flies according to their area. Figure 7 "n=11, 49, 44" and "n=27, 52, 25" in the figure respectively represent the number of fruit flies detected in each experimental repetition; "mean" in Table 4 is the mean in %, SD is the standard deviation, and N is the number of experimental repetitions.

[0114] Example 7

[0115] The pharmaceutical composition of sodium aescinate (abbreviated as escinate) and diosmin (abbreviated as diosmin), as well as the pharmaceutical composition of sodium aescinate (abbreviated as escinate) and dehydrated icariin (abbreviated as diosmin) can significantly improve the degree of necrotic black spots produced in the eyes of male flies overexpressing TDP-43-wild type (wt). The pharmaceutical composition is referred to as "seven diosmin" and "seven diosmin", the solvent is DMSO, and the concentrations of the active ingredients in the two pharmaceutical compositions in the food are 0.1μg / mL and 0.05μg / mL, respectively. 0.05μg / mL "seven diosmin" has the most significant effect, and the proportion of non-necrotic black spots increased by an average of 13.82 percentage points, which increased by more than 0.69 times; the proportion of severe black spots decreased by an average of 22.8 percentage points to only 4.3%, which is 15.9% of the group without drug addition; although more mild black spots were converted to non-black spots, the proportion of mild black spots changed little because a large number of severe black spots were converted to mild black spots. The two concentrations of "Qi Tuo Yin" 0.1μg / mL and 0.05μg / mL were the second most effective. The proportion of severe dark spots decreased significantly, to 8.73% and 9.13% respectively; the proportion of no dark spots increased by 19.6 and 11 percentage points, that is, more than 1 times and 0.55 times; the proportion of mild dark spots increased in the 0.05μg / mL concentration "Qi Tuo Yin" group because more severe dark spots were converted to mild ones, and the decrease in the 0.1μg / ml concentration "Qi Tuo Yin" group was not obvious. The 0.1μg / ml "Qi Di" group also had a certain effect, with the proportion of severe dark spots decreasing by 12.23 percentage points and the proportion of no dark spots increasing by 8.63 percentage points (the results are shown in Figure 8 and Table 5).

[0116] Table 5

[0117]

[0118] Figure 8 The pharmaceutical composition of Example 7, which is a combination of sodium aescinate and diosmin, and a pharmaceutical composition of a combination of sodium aescinate and dehydrated icariin, improve the degree of necrotic black spots produced by overexpressing TDP-43-wild type (wt) in the eyes of male flies. Figure 8is a statistical chart, and Table 5 is the corresponding statistical data. Figure 8 The "Seven-Di 0.1" indicates that the concentrations of sodium aescinate and diosmin in the drug composition in food are both 0.1 μg / mL, "Seven-To-Icaritin 0.1" indicates that the concentrations of sodium aescinate and dehydrated icariin in the drug composition in food are both 0.1 μg / mL, "No drug added" indicates solvent control, and "None", "Light" and "Heavy" are classifications of black spots in fruit fly eyes by area. Figure 8 "n=47, 42, 32", "n=31, 30, 34", "n=22, 41, 28", "n=36, 31, 39" and "n=23, 15, 29" in the figure represent the number of fruit flies detected in each experimental repetition; "mean" in Table 5 is the mean in %, SD is the standard deviation, and N is the number of experimental repetitions.

[0119] Example 8

[0120] The pharmaceutical composition of sodium aescinate (abbreviated as escin) and diosmin (abbreviated as diosmin), and the pharmaceutical composition of sodium aescinate (abbreviated as escin) and dehydrated icariin (abbreviated as diosmin) have obvious therapeutic effects on female flies. The TDP-43-wild type (wt) gene is overexpressed in the eyes of female flies. The culture temperature of this embodiment is 27°C, and its expression level is higher than 25°C, and the severity of black spots is significantly increased. The pharmaceutical composition (the pharmaceutical composition is abbreviated as "seven-di" and "seven-diosmin" respectively; the solvent is DMSO, and the concentration of active ingredients in the food is 0.05μg / mL) can well improve the symptoms of black spots. Compared with the "unmedicated" group, the proportion of severe black spots in the "seven-di" combination decreased from 45.97% to 24.5%, a decrease of 52%; more than half of the severe black spots were converted into mild black spots, so the proportion of mild black spots increased from 52.87% to 73.3%, an increase of more than 20.43 percentage points; the proportion of no black spots increased. Compared with the "no drug" group, the "Qi Tuo Yin" combination significantly increased the proportion of no dark spots, from 1.13% to 13.85%, an increase of 11.26 times; severe dark spots decreased by 8.8 percentage points, a decrease of nearly 20%; some mild dark spots were converted to no dark spots and some severe dark spots were converted to mild, so the proportion of mild dark spots did not change significantly (the results are shown in Fig. 9 shown).

[0121] Fig. 9 The results of the drug composition of sodium aescinate and dehydrated icariin in Example 8 on female flies cultured at 27°C are as follows: Fig. 9 It can be seen that the pharmaceutical composition has obvious therapeutic effect. Fig. 9 For statistical chart. Fig. 9The "Seven-Di 0.05" indicates that the concentrations of sodium aescinate and diosmin in food of the drug composition are both 0.05 μg / mL, "Seven-To-Icaritin 0.05" indicates that the concentrations of sodium aescinate and dehydrated icariin in food of the drug composition are both 0.05 μg / mL, "No medication" indicates the control, and "None", "Light" and "Heavy" are the classifications of the black spots on the fruit fly's eyes by area. Fig. 9 "n=29, 33, 27", "n=34, 54, 44" and "n=21, 22" indicate the number of fruit flies tested in each experimental repetition.

[0122] Comparative Example 1

[0123] Overexpression of the TDP-43 mutant A315T in motor neurons resulted in lethality in the pupal stage, and the pupal size was significantly smaller than that of the normal control group (see Example 1). The combination of hederagenin and dehydrated icariin at a concentration of 0.05 μg / mL in the food was able to partially rescue this phenotype. The effect of the combined use of hederagenin (abbreviated as Chang) and dehydrated icariin (abbreviated as Tuo Yin) could not be achieved by using either drug alone, with significant differences (the results are shown in Table 1). Fig.10 shown).

[0124] Fig.10 The results of comparative example 1 are as follows: single medication and combined medication results of hederagenin (abbreviated as Chang) and dehydrated icariin (abbreviated as Tuoyin). Fig.10 Figure A in the middle shows images of Drosophila pupae that overexpress hTDP-43 mutant A315T in motor neurons and are fed with different drugs. Fig.10 Figure B in the middle is a statistical chart, normalized to the drug combination group. Fig.10 "A315T" here represents the hTDP-43 mutant A315T overexpressed in motor neurons, "Chang Tuo Yin" means that the composition contains hederagenin and dehydrated icariin, "Chang" means hederagenin, "Tuo Yin" means dehydrated icariin, the number after the drug represents the drug concentration, in μg / mL, "Chang Tuo Yin 0.05" means that in the drug composition, the concentrations of hederagenin and dehydrated icariin in food are both 0.05 μg / mL. Fig.10 In Figure B, "n=67", "n=53", "n=85", "n=67" and "n=73" represent the number of pupae detected, respectively. One-Way ANOVA was used for analysis. "**" indicates significant difference P<0.01, and "*" indicates significant difference P<0.05.

[0125] Comparative Example 2

[0126] Under the same experimental conditions, single medication cannot achieve the same effect as combined medication of hederagenin (abbreviated as Chang) and dehydrated icariin (abbreviated as Tuoyin). Here, the fruit fly overexpresses TDP-43-A315T in the eyes.

[0127] Fig.11 The therapeutic effects of hederagenin (abbreviated as Chang) and dehydrated icariin (abbreviated as Tuoyin) alone and in combination on TDP-43-A315T in Drosophila were analyzed. Fig.11 It can be seen that the effect of combined medication cannot be achieved by taking the medication alone. Fig.11 is a statistical chart, and Table 6 is the corresponding statistical data. Fig.11 "Chang Tuo Yin 0.05" means that the concentrations of hederagenin and dehydrated icariin in food are both 0.05μg / mL, "Chang 0.1" means that the concentration of hederagenin in food is 0.1μg / mL, "Chang 0.05" means that the concentration of hederagenin in food is 0.05μg / mL, "Tuo Yin 0.1" means that the concentration of dehydrated icariin in food is 0.1μg / mL, "Tuo Yin 0.05" means that the concentration of dehydrated icariin in food is 0.05μg / mL, "No drug added" means the control, and "None", "Light" and "Heavy" are the classifications of the black spots in the fruit fly eyes by area. Fig.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 repetitions. "Mean" in Table 6 is the mean, the unit is %, SD is the standard deviation, and N is the number of experimental repetitions.

[0128] Table 6

[0129]

[0130] Comparative Example 3

[0131] Under the same experimental conditions, the effect of combined use of sodium aescinate (abbreviated as aescin) and diosmin (abbreviated as diosmin) could not be achieved by single drug use, here in female flies overexpressing wild-type TDP-43 in the eyes.

[0132] Fig.12 The therapeutic effects of sodium aescinate and diosmin alone and in combination on TDP-43-wild-type female flies are shown in Figure 2. Fig.12 It can be seen that the effect of combined medication cannot be achieved by taking the medication alone. Fig.12 For statistical chart. Fig.12 The "Seven-Di 0.05" means that the concentrations of sodium aescinate and diosmin in food are both 0.05μg / mL, "Seven-0.1" means that the concentration of sodium aescinate in food is 0.1μg / mL, "Di 0.1" means that the concentration of diosmin in food is 0.1μg / mL, and "None", "Light" and "Heavy" are the classifications of the black spots on the fruit fly's eyes according to the area. Fig.12"n=65,80", "n=65,66", "n=65,104" and "n=28,51" represent the number of fruit flies in different experimental repetitions.

[0133] Example 9

[0134] Adding edaravone to the binary combination of hederagenin plus anhydroicariin enhanced the therapeutic effect.

[0135] Overexpression of the TDP-43 mutant A315T in motor neurons resulted in lethality in the pupal stage, and the pupal size was significantly smaller than that of the normal control group (see Example 1). The effect of the combination of hedera saponin (abbreviated as Chang) and dehydrated icariin (abbreviated as Tuoyin) combined with edaravone (abbreviated as Ida) was better than the binary combination, with a significant difference.

[0136] Fig.13 The effect of the combination of hederagenin and dehydrated icariin and edaravone was investigated. Fig.13 It can be seen that the combined drug effect is better than the binary combination of hederagenin and dehydrated icariin. Fig.13 Figure A in the middle shows the overexpression of TDP-43 mutant A315T in motor neurons treated with different drugs. Fig.13 Figure B in the middle is a statistical chart, normalized to the DMSO control group. Fig.13 "A315T" in it means the TDP-43 mutant A315T is overexpressed in motor neurons, "Changtuyin" means the concentration of hederagenin and dehydrated icariin in food is 0.05 μg / mL, "Changtuyin+Ida" means the concentration of hederagenin and dehydrated icariin in food are both 0.05 μg / mL, and the concentration of edaravone is 0.04 μg / mL, and "DMSO" means solvent control. Fig.13 "n=48", "n=67" and "n=52" in Figure B indicate the number of fruit flies tested, and the analysis was performed using One-Way ANOVA. "****" indicates a significant difference P<0.001, and "*" indicates a significant difference P<0.05.

[0137] Comparative Example 4

[0138] In the Drosophila optic nerve overexpression TDP-43 mutant A315T model, the combination of hederagenin and anhydroicariin was superior to the effect of riluzole (e.g. Fig.14As shown in Table 7 and Table 8). Compared with the solvent (DMSO) control group: adding three different concentrations of riluzole to food, 0.04μg / mL was ineffective, and the average proportions of "none", "mild" and "severe" eye spots were 61.30%, 35.65% and 3.05%, respectively, which were not significantly different from the 60.30%, 35.18% and 4.53% of the control group; the proportions of no black spots with 1μg / mL and 0.2μg / mL riluzole were 65.68% and 66.30%, respectively, which were significantly different from the control group (P=0.012 and 0.008), and the proportions of mild black spots and severe black spots were 30.25%, 30.93%, 4.10% and 2.08%, respectively, which were not significantly different from the control group; 0.2μg / mL was the best among the three concentrations of riluzole.

[0139] The proportion of patients without dark spots in the combination of hederagenin and dehydrated icariin was 75.03%, which was an average increase of 14.73 percentage points compared with the control, with an increase of 24.4%, which was extremely significant (P=0.00003). The average proportion of patients with mild dark spots was 22.45%, which was a decrease of 12.73 percentage points compared with the control, with a decrease of 36.2%, which was very significant (P=0.0011). The proportion of patients with severe dark spots was 2.55%, which was not significantly different from the control.

[0140] Compared with 0.2 μg / mL riluzole, the combination of hederagenin and dehydrated icariin had a strong advantage in the proportion of no dark spots and mild dark spots, with significant difference P values ​​of 0.00049 and 0.00170, respectively, and there was no significant difference in the proportion of severe dark spots.

[0141] Table 7

[0142]

[0143] Table 8

[0144]

[0145] Fig.14 The present invention is a comparison of the effects of the combination of hederagenin and dehydrated icariin (abbreviated as Changtuyin) and the listed drug riluzole. On the one hand, it can be seen that the combination of hederagenin and dehydrated icariin is better than the existing drug riluzole. On the other hand, since the usual dosage of riluzole for human body is 100mg / day, it can be deduced that the dosage of the binary combination drug of the present invention used in human clinical trials is roughly 10mg / day-2000mg / day by comparing the concentration ratio of hederagenin and dehydrated icariin to the concentration ratio of riluzole. Preferably, the initial dosage for the combination of hederagenin and dehydrated icariin to start human trials is about 20mg-200mg / day.

[0146] Fig.14 Table 7 is a statistical diagram of the effect of overexpressing the TDP-43 mutant A315T in the optic nerve treated with different drugs, Table 7 is the corresponding statistical data, and Table 8 is the results of the significance analysis of the differences. Fig.14 DMSO was the solvent control group, "Riluzole 1", "Riluzole 0.2" and "Riluzole 0.04" indicated that the concentrations of riluzole in the food were 1 μg / mL, 0.2 μg / mL and 0.04 μg / mL, respectively, "Changdeyin 0.25" indicated that the concentrations of the combination of hederagenin and dehydrated icariin in the food were 0.25 μg / mL, respectively, and "None", "Light" and "Heavy" were the classifications of the black spots in the fruit fly eyes by area. Fig.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" respectively represent the number of fruit flies detected in different experimental repetitions. "mean" in Table 7 is the average, the unit is %, SD is the standard deviation, N is the number of experimental repetitions, and the change represents the difference with the DMSO control. The data in Table 8 were analyzed for significance using Multiple t-tests, "****" indicates a significant difference P < 0.001, "***" indicates a significant difference P < 0.005, "**" indicates a significant difference P < 0.01, and "*" indicates a significant difference P < 0.05.

Claims

1. A pharmaceutical composition, characterized in that Including at least one type of compound A and one type of compound B; The class A compound includes at least one of sodium aescinate, sodium aescinate derivatives, hederagenin or hederagenin derivatives; The class B compound includes at least one of anhydroicariin, anhydroicariin derivatives, diosmin or a diosmin derivative.

2. The pharmaceutical composition according to claim 1, characterized in that The sodium aescinate derivative comprises at least one of aescin, aescin A, aescin B, aescin C, aescin D, aescinogenin or protoescinogenin.

3. The pharmaceutical composition according to claim 1, characterized in that The dehydrated icariin derivative includes at least one of icariin, icariin or dehydrated icariin.

4. The pharmaceutical composition according to claim 1, characterized in that The diosmin derivatives include neodiosmin.

5. The pharmaceutical composition according to claim 1, characterized in that The hederagenin derivative includes at least one of Alpha-hederagenin, Beta-hederagenin, hederagenin C and hederagenin D.

6. The pharmaceutical composition according to claim 1, characterized in that The weight ratio of the type A compound to the type B compound is 1:20 to 20:

1.

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

8. The pharmaceutical composition according to claim 7, characterized in that The pharmaceutical composition also includes pharmaceutically acceptable solvents and excipients.

9. The pharmaceutical composition according to claim 7, characterized in that The pharmaceutically acceptable excipients include at least one of a pharmaceutical carrier, a diluent, an adjuvant or an excipient.

10. Use of the pharmaceutical composition according to any one of claims 1 to 9 in the preparation of a drug for treating TDP-43 proteinopathy.

Images