Pharmaceutical composition for treating tdp-43 proteinopathy and preparation method and application thereof

Abstract

This invention belongs to the field of pharmaceutical technology and discloses a pharmaceutical composition for treating TDP-43 proteinopathy, its preparation method, and its application. The pharmaceutical composition includes at least one class A compound and at least one class B compound; class A compound includes at least one selected from sodium aescinate, a sodium aescinate derivative, hederonium, or a hederonium derivative; class B compound includes at least one selected from dehydrated icariin, a dehydrated icariin derivative, diosmin, or a diosmin derivative. This pharmaceutical composition exhibits good therapeutic effects for TDP-43 proteinopathy.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and specifically relates to pharmaceutical compositions for treating TDP-43 protein disorders, their preparation methods, and applications. Background Technology

[0002] TDP-43 protein disorders are a collective term for a group of neurological diseases. The common pathological feature of these diseases is the formation of pathological protein inclusion bodies of transactive response DNA binding protein 43000 (TDP-43) in the affected area (Reference: Chen L. The important functional role of TDP-43 plays in amyotrophic lateralsclerosis-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 various aspects of mRNA transcription, splicing and stability, and RNA metabolism. Its activity regulation is manifested in its controlled shuttle between the nucleus and 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 found in the cell nucleus with almost no abnormal deposition. However, under pathological conditions, TDP-43 usually appears in large quantities as insoluble inclusion bodies or is located in large quantities in the cytoplasm. Some of it undergoes abnormal degradation, leading to loss of function and thus causing 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 patients with ALS, nearly 50% of patients with frontotemporal lobar degeneration (FTLD / FTD), and more than 30% of patients with Alzheimer's disease (AD) (References: Chen L. The important functional role of TDP-43 plays in amyotrophic lateralsclerosis-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 has been found in some patients with various other neurodegenerative diseases, including chronic traumatic encephalopathy (CTE), Lewybody 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 advancements in 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, Polvikoski T, 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 enormous challenges. Despite substantial investment, no effective drugs have yet been developed. The following section uses ALS (Amyotrophic Lateral Sclerosis), the most typical representative of TDP-43 proteinopathies, to illustrate its pathogenesis and drug development status.

[0007] Amyotrophic Lateral Sclerosis (ALS) can cause a combination of upper and lower motor neuron disease, affecting the muscles of the trunk, limbs, and face that it innervates. Patients gradually lose control of their muscles, leading to muscle atrophy. They usually die from respiratory failure 3 to 5 years after onset. The incidence rate is approximately 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 have familial gene mutations, such as TDP-43, SOD1, and FUS; while more than 90% are sporadic, meaning that no pathogenic gene has been identified, but their pathological features are highly correlated with the structural and functional abnormalities of the TDP-43 protein (Reference: EckRJ, 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. Among these TDP-43 mutations, the A315T mutation at the C-terminus has the most extensive functional impact, encompassing cytoplasmic localization, fragmentation, phosphorylation, ubiquitination, and insolubility. Therefore, its symptoms are relatively more severe than 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 A315Tmutant human TDP-43 expression improves functional deficits in a novelinducible transgenic mouse model of FTLD-TDP and ALS. Acta Neuropathol. 2015;130(5):661-78).

[0008] Most patients with ALS (Amyotrophic Lateral Sclerosis) exhibit abnormal accumulation and functional impairment of the TDP-43 protein in their nervous system, making ALS a typical example of TDP-43 protein disorders. Although drug development for ALS has been ongoing for many years, approved drugs have limited efficacy, and some have serious side effects, indicating that effective drugs for TDP-43 protein disorders caused by abnormal accumulation and functional impairment of the TDP-43 protein still lack effective treatments.

[0009] Riluzole was the first drug approved by the US FDA and the European Union for the treatment of ALS (Amyotrophic Lateral Sclerosis). Clinical studies have shown that riluzole treatment can reduce the loss of motor neurons, improve patient survival, and slow disease progression, but it cannot stop the progression of ALS, and patients' survival time is only increased by about 3 months. (References: 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 amytrophic lateral 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) is the next compound approved for the treatment of ALS (Amyotrophic Lateral Sclerosis) in several Asian countries, the United States, Canada, and Switzerland, following 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 studies have shown that edaravone has some effect in reducing motor decline and motor neuron degeneration, and slowing disease progression, but its efficacy is limited and unsatisfactory. In addition, this drug has strong side effects, and common adverse events include contusion, confusion, dysphagia, constipation, eczema, headache, bruising, gait disturbance, 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 efficacy for the treatment of TDP-43 proteinopathy. Summary of the Invention

[0012] The present invention aims to at least solve one of the technical problems existing in the prior art. To this end, the present invention proposes a pharmaceutical composition for treating TDP-43 proteinopathy, a method for its preparation, and its application. The pharmaceutical composition is effective in treating TDP-43 proteinopathy, particularly sporadic TDP-43 proteinopathy and familial TDP-43 proteinopathy with the TDP-43-A315T mutation.

[0013] Specifically, the pharmaceutical composition for treating TDP-43 proteinopathy includes at least one Class A compound and one Class B compound;

[0014] The Class A compounds include at least one of sodium aescinate, sodium aescinate derivatives, hederonein, or hederonein derivatives.

[0015] The Class B compounds include at least one of dehydrated icariin, dehydrated icariin derivatives, diosmin, or diosmin derivatives.

[0016] Preferably, the sodium aescinate derivative includes at least one of aescin, aescin A, aescin B, aescin C, aescin D, aescinogen, or protoaescinogen.

[0017] Sodium aescinate: CAS No. 20977-05-3, molecular formula C54H83NaO23. It is a white powder or crystalline powder with anti-inflammatory, anti-exudative, venous tone-increasing, blood circulation-improving, and brain function-correcting effects. It also has a significant protective effect against cerebral edema caused by carbon monoxide and other pollutants.

[0018] Escin: 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 84O 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 dehydrated icariin derivative includes at least one of icariin, icariin, or dehydrated icariin.

[0026] Anhydroicaritin: CAS No. 38226-86-7, molecular formula C 21 H 20 O6 is an isopentenyl 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 by regulating the 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] Dehydrated icariin (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, with the molecular formula C.28 H 32 O 15 .

[0031] Diosmin: CAS No. 520-27-4, molecular formula C 28 H 32 O 15 Chemical name: 7-{[6-oxo-(6-deoxy-α-L-mannose)-β-D-glucopyranose]oxy}-5-hydroxy-2-(3-hydroxy-4-methoxybenzene)-4H-L-benzopyran-4-one. It is a capillary protectant. Used to treat various symptoms related to venous and lymphatic insufficiency, as well as various symptoms of acute hemorrhoid flare-ups.

[0032] Preferably, the ivy saponin derivative includes at least one of Alpha-ivy saponin, Beta-ivyrin, ivy glycoside C, and ivy glycoside D.

[0033] Hederagenin: CAS No. 465-99-6, molecular formula C 30 H 48 O4. Ivy saponins are water-soluble pentacyclic triterpenoid saponins, which are pentacyclic triterpenoid compounds isolated from plants. They have many chemical properties, such as antispasmodic, anthelmintic, and cell growth inhibitory effects. They possess a wide range of pharmacological activities, including antitumor, anti-inflammatory, antidepressant, anti-neurodegenerative, anti-hyperlipidemia, antidiabetic, antileishmaniasis, and antiviral 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 Ci 41 H 66 O 11 .

[0036] Hederacoside C: CAS No. 14216-03-6, molecular formula 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 pharmaceutical composition comprises a binary composition consisting of one of a class A compound, including sodium aescinate and sodium aescinate derivatives (A1 subclass) and hederonium and hederonium derivatives (A2 subclass), and one of a class B compound, including icariin and icariin derivatives (B1 class) and diosmin and diosmin derivatives (B2 class), in a weight ratio of 1:20 to 20:1. Specifically, for example, ratios of 1:20, 1:10, 1:5, 1:1, 2:1, 5:1, 10:1, and 20:1.

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

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

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

[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 one of sodium aescinate and sodium aescinate derivatives, hederonium and hederonium derivatives, to one of diosmin and diosmin derivatives, and dehydrated icariin and dehydrated icariin derivatives is 1:10 to 10:1. Specifically, for example, it is 1:10, 1:8, 1:5, 1:1, 2:1, 5:1, 8:1, or 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, the ratios are 1:10, 1:8, 1:5, 1:1, 2:1, 5:1, 8:1, and 10:1.

[0046] Preferably, the pharmaceutical composition includes sodium aescinate or a sodium aescinate derivative, diosmin or a diosmin derivative, and an organic solvent.

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

[0048] Preferably, the pharmaceutical composition includes icariin or icariin derivatives, hederone and hederone derivatives, and an organic solvent.

[0049] A 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 includes the following steps:

[0051] The components are mixed to obtain the pharmaceutical composition. Furthermore, when taking the pharmaceutical composition of the present invention, patients may take the mixed pharmaceutical composition directly, or take the components of the pharmaceutical composition sequentially.

[0052] A third aspect of the present invention 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 drug for treating TDP-43 proteinopathy.

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

[0055] Preferably, the TDP-43 protein diseases include some types of diseases such as ALS, frontotemporal degeneration, Alzheimer's disease, chronic traumatic encephalopathy, Lewy body disease, Huntington's disease, argyrophilic cereal disease, and hippocampal sclerosis, one of the pathological features of these types being the detection of abnormal aggregation and pathological inclusion bodies of TDP-43 protein in the lesion 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 on 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 mutation, and patients with neurodegenerative diseases related to TDP-43 dysfunction.

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

[0059] (3) The pharmaceutical composition for treating TDP-43 proteinopathy according to the present invention comprises at least two of the following: sodium aescinate and sodium aescinate derivatives, dehydrated icariin and dehydrated icariin derivatives, diosmin and diosmin derivatives, and hederonium and hederonium derivatives. Through the specific combination of active pharmaceutical ingredients, the pharmaceutical composition achieves 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 sodium aescinate derivatives, dehydrated icariin and dehydrated icariin derivatives, and at least one of diosmin and diosmin derivatives, and hederamine and hederamine derivatives; or, the pharmaceutical composition comprises sodium aescinate or sodium aescinate derivatives, and dehydrated icariin or dehydrated icariin derivatives. Through a specific combination of active pharmaceutical ingredients, the pharmaceutical composition achieves a good therapeutic effect on TDP-43 protein disorders. TDP-43 protein disorders include patients with sporadic ALS, patients with TDP-43 gene A315T mutations, and patients with neurodegenerative diseases related to TDP-43 dysfunction.

[0061] (5) For diseases caused by the 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 significant effects. Other combinations, such as sodium aescinate derivatives with diosmin derivatives, sodium aescinate derivatives with icariin derivatives, ivy saponin derivatives with diosmin derivatives, and ivy saponin derivatives with dehydrated icariin derivatives, also have certain therapeutic effects. Sodium aescinate or diosmin alone also has a slight effect, but the effect is significantly less than the above-mentioned binary combinations 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 ivy saponin and dehydrated icariin have the most significant effects. Other combinations, such as sodium aescinate derivatives and diosmin derivatives, sodium aescinate derivatives and dehydrated icariin derivatives, and ivy saponin derivatives and diosmin derivatives, also have certain therapeutic effects. Ivy saponin alone and dehydrated icariin also have a slight effect, but their effects are significantly less than those of the combination of sodium aescinate and dehydrated icariin, and the combination of ivy saponin and dehydrated icariin.

[0063] (7) In Drosophila eye models with overexpression of wild-type and mutant TDP-43 A315T, phenotypic differences were observed between male and female flies, with male flies showing greater differences than female flies; differences also existed between wild-type and mutant TDP-43 overexpression. Furthermore, different drug combinations showed significant differences in rescue effects on male and female flies and on different types of TDP-43. Therefore, a strategy of providing differentiated treatment based on sex / degree of defect / cause is proposed. Attached Figure Description

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

[0065] Figure 2 The results of Example 1 show that the pharmaceutical composition of ivy saponin and dehydrated epimedium improved the pupal length defect caused by the expression of the TDP-43 mutant A315T in motor neurons;

[0066] Figure 3 This is the result of Example 2, which describes the effect of the pharmaceutical composition of ivy saponin and dehydrated epimedium on improving the motor neuron expression of the TDP-43 mutant A315T Drosophila larvae.

[0067] Figure 4 Example 3: The pharmaceutical composition of ivy saponin and dehydrated epimedium extract for the treatment of TDP-43 mutant A315T overexpression in the eye;

[0068] Figure 5 The results of Example 4, which describes the pharmaceutical composition hederagenin and dehydrated epimedium, show that it increases the number of neurons per unit area in female ALS mice.

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

[0070] Figure 7The results of the drug combination of sodium aescinate and icariin anhydride in Example 6 for treating the overexpression of the TDP-43 mutant A315T in the eye;

[0071] Figure 8 For the drug combination of sodium aescinate and diosmin in Example 7, and the results of the drug combination of sodium aescinate and icariin anhydride in improving the degree of necrotic black spots caused by the overexpression of TDP-43 wild-type in the eyes of male flies;

[0072] Figure 9 For the drug combination of sodium aescinate and diosmin in Example 8, and the results of the drug combination of sodium aescinate and icariin anhydride in treating female flies with overexpressed wild-type TDP-43 gene in the eyes at a culture temperature of 27°C;

[0073] Figure 10 For the results of the separate and combined administration of hederacoside (abbreviated as "Chang") and icariin anhydride (abbreviated as "Yin") in Example 1 of the comparative example in Drosophila with overexpressed TDP-43 mutant A315T in motor neurons;

[0074] Figure 11 For the results of the separate and combined administration of hederacoside (abbreviated as "Chang") and icariin anhydride (abbreviated as "Yin") in Example 2 of the comparative example in Drosophila with overexpressed TDP-43 mutant A315T in the eyes;

[0075] Figure 12 For the therapeutic effect of the separate and combined administration of sodium aescinate and diosmin on female flies with TDP-43 wild-type in Example 3 of the comparative example;

[0076] Figure 13 For the effect of the combination of hederagenin and icariin anhydride and the combined use of edaravone in Example 9;

[0077] Figure 14 For the effect that the combination of hederagenin and icariin anhydride is superior to riluzole in Example 4 of the comparative example. Detailed implementation manners

[0078] In order to make those skilled in the art more clearly understand the technical solutions described in the present invention, the following examples are listed for illustration. It should be noted that the following examples do not limit the scope of protection required by the present invention.

[0079] The raw materials, reagents or devices used in the following examples can be obtained from conventional commercial channels or can be obtained by known existing methods without special instructions.

[0080] Definition

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

[0082] Unless the context clearly indicates otherwise, the terms “a” and “an” as used herein include plural references. For example, reference to “a cell” includes multiple such cells and equivalents known to those skilled in the art, etc.

[0083] As used herein, the term "pharmaceutically acceptable" means that it can be administered to humans and / or other animals as subjects without producing excessive adverse reactions or side effects (such as toxicity, irritation, allergic reactions, etc.). The term "excipient" refers to auxiliary materials that coexist with the active ingredient in a pharmaceutical preparation without producing excessive adverse reactions or side effects, including carriers, osmotic pressure regulators, pH adjusters, diluents, disintegrants, excipients, solubilizers, stabilizers, preservatives, etc. The term "pharmaceuticalally acceptable excipient" refers to a highly safe excipient suitable for a specific pharmaceutical preparation and routinely used in pharmaceutical practice. The term "carrier" includes, but is not limited to, liposomes, liposomes, polymer micelles, nanostructured lipid carriers, solid lipid nanocarriers, mesoporous silica nanoparticles, etc.

[0084] As used herein, the term “pharmaceutically acceptable salt” may include alkali metal salts (e.g., sodium or potassium salts), alkaline earth metal salts (e.g., calcium or magnesium salts), and salts formed with suitable organic ligands (e.g., quaternary ammonium salts).

[0085] The preparation of the above-mentioned pharmaceutical composition involves selecting the corresponding active components (such as sodium aescinate and sodium aescinate derivatives, dehydrated icariin and dehydrated icariin derivatives, diosmin and diosmin derivatives, hederogen and hederogen derivatives) according to the designed concentration, dissolving them in water or an organic solvent, and then mixing them to obtain the final product.

[0086] To screen and verify the therapeutic effects of the above-mentioned drug combination, a Drosophila TDP-43 disease model was established. This involved overexpressing human TDP-43 wild-type or A315T mutant in specific tissues / organs / cells using the GA14-UAS system, inducing abnormal phenotypes / defects in Drosophila. Drosophila were cultured in an incubator at 25℃, 60% relative humidity, with 12 hours of light and 12 hours of darkness every 24 hours; a few experimental Drosophila were cultured at 27℃, with all other conditions remaining the same. The Drosophila diet consisted of a standard corn flour formula (each 1 liter of standard corn flour formula contained 68g corn flour, 43g maltose, 16g yeast powder, 9g soybean flour, 0.962g anhydrous calcium chloride, and 8g agar powder, boiled in distilled water and brought to a final volume, with 5mL propionic acid added as a preservative). The dissolved drug combination was added to the freshly prepared food (cooled to below 60℃) at the designed concentration and thoroughly mixed. The mixture was then dispensed into culture tubes, with an equal volume of solvent added to the food as a control group (no drug treatment). Drosophila hybridization was performed by selecting specific tissue / organ / cell-specific Gal4 virgin flies (maternal parent) and crossing them with UAS-hTDP-43-wt (wild type) or UAS-hTDP-43-A315T (mutant) (paternal parent). Each food / drug combination had at least three replicates, with each replicate containing five virgin flies and five male flies. The parental feed / drug combination tubes were changed every two days, for a total of two changes. Two days after the last feed change, the adult flies from both parents were removed. Depending on the experimental requirements, corresponding phenotypes / indicators were observed / detected during the larval, pupal, or adult stages of the offspring. The results were statistically analyzed and plotted using PrismGraphpad software.

[0087] The examples demonstrate the overexpression of wild-type or mutant strains in motor neurons and the eye, with phenotypic development and pupal stages and the degree of eye defects being examined, respectively. Based on the main pathological feature of TDP-43 disease—the mislocalization and aggregation of TDP-43 from the nucleus to the cytoplasm—overexpression of the wild-type human TDP-43 gene in the Drosophila eye causes a defective phenotype with varying degrees of necrotic black spots (none to the entire eye). Furthermore, the TDP-43 gene A315T mutation is also a common familial case; therefore, a model of Drosophila eye overexpression of the human TDP-43 mutant A315T was constructed, with a phenotype similar to the wild-type. It was also noted that in both models, the defect was more severe in male flies than in female flies. The area of ​​necrotic black spots in the eyes of the disease models was divided into three categories. Figure 1 The classification of necrotic black spots in the eyes of a fruit fly disease model and the control group are shown; among them, Figure 1 Image B corresponds to no (no dark spots), Image C corresponds to mild (the sum of the dark spot areas in both eyes is less than 1 / 4 of the area of ​​a single eye), and Image D corresponds to severe (the sum of the dark spot areas in both eyes is greater than or equal to 1 / 4 of the area of ​​a single eye); Image A is the control group (overexpressing red fluorescent protein RFP).

[0088] Example 1

[0089] Overexpression of the hTDP-43 mutant A315T by the OK371 driver in Drosophila motor neurons leads to lethality during the pupal stage, with the size of the pupae (measured by length) being significantly smaller than that of the control pupae, and only a small number developing to the late pupal stage. However, a small number of the pupae fed with the saponin yuan (abbreviated as "Chang") and icariside II (abbreviated as "Tuo Yin") drug combination can develop to the late pupal stage. More significantly, the size of the pupae (measured by length) shows a significant increase (P < 0.001), with an average increase of nearly 14%. The active ingredients (Chang, Tuo Yin) of the drug combination (the preparation method of the drug combination is to mix saponin yuan, icariside II with DMSO to obtain the drug combination, and the concentration of the active ingredients is controlled by the amount of saponin yuan and icariside II added) in the food are both 0.05 μg / mL. Since there are too few late pupae in the disease model without drug addition group (i.e., A315T, DMSO), only the length of the early pupae was statistically analyzed (the results are as shown in Figure 2 and Table 1).

[0090] Table 1

[0091]

[0092] Figure 2 The results show that the drug combination of saponin yuan and icariside II in Example 1 significantly improves the developmental defects (pupal length) caused by the expression of the TDP-43 mutant A315T in motor neurons. Figure 2 Figure A in it shows the comparison of pupae between the control, the disease model, and the drug addition group, Figure 2 Figure B in it (where "Relative length of pupae" represents the relative length of the pupae) is a statistical graph of the relative length of the pupae, and Table 1 is the analysis of the length statistical results, normalized to the RFP group without drug addition. Figure 2 In it, "Chang Tuo Yin" indicates that the composition contains saponin yuan and icariside II, and the numerical value after "Chang Tuo Yin" represents the corresponding concentration of the active ingredients (Chang, Tuo Yin). For example, "Chang Tuo Yin 0.05" means that in the drug combination, the concentrations of saponin yuan and icariside II in the food are both 0.05 μg / mL. "RFP, DMSO" means that the solvent in the food of the control group OK371 > RFP (OK371 drives the overexpression of RFP in motor neurons) Drosophila is DMSO. "A315T" means the disease model OK371 > A315T Drosophila, that is, OK371 drives the overexpression of TDP43 - A315T in motor neurons, producing the mutant TDP-43 protein at position A315. "A315T, Chang Tuo Yin 0.05" means that for OK371 > A315T, the concentrations of saponin yuan and icariside II in the food are both 0.05 μg / mL. Figure 2The B-plot analysis was performed using one-way ANOVA. "***" indicates a significant difference (P < 0.0005), and "**" indicates P < 0.001. In Table 1, "mean" represents the average relative length of pupae; "SD" represents the standard deviation, and N is the number of replicates. The length of at least 10 pupae was measured and averaged for each genotype / drug treatment in each replicate.

[0093] Example 2

[0094] A drug composition consisting of hederagenin (abbreviated as "Chang") and dehydrated icariin (abbreviated as "Deyin") (the drug composition is abbreviated as "Chang-Deyin", the solvent is DMSO, and the concentration of the active ingredient in the food is 0.05 μg / mL) significantly improved the motor ability of Drosophila larvae overexpressing the TDP-43 mutant A315T in their motor neurons. The motor ability of Drosophila larvae overexpressing the TDP-43 mutant A315T in their motor neurons was significantly worse than that of the control group expressing RFP. This was manifested in the fact that the time it took for the larvae to turn over to abdomen-up and abdomen-down was much longer than in the control group. However, the average turning time of the larvae was shortened by 40.6% by adding the drug composition consisting of hederagenin (abbreviated as "Chang") and dehydrated icariin (abbreviated as "Deyin") (the drug composition is abbreviated as "Chang-Deyin", the solvent is DMSO) to their food (results are shown in Figure 1). Figure 3 (as shown in Table 2).

[0095] Table 2

[0096]

[0097] Figure 3 The results of Example 2 show the improvement of motor ability in Drosophila overexpressing the TDP-43 mutant A315T in motor neurons by a pharmaceutical composition consisting of ivy saponin (abbreviated as ivy saponin) and dehydrated epimedium (abbreviated as epimedium) (the pharmaceutical composition is abbreviated as "ivy saponin" with a concentration of 0.05 μg / mL). Figure 3 The first image is a statistical chart, and Table 2 shows 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 it is a DMSO solvent control in the food of Drosophila with OK371 > RFP (OK371 drives the overexpression of RFP in motor neurons). "A315T, DMSO" indicates that it is a DMSO solvent in the food of Drosophila with OK371 > A315T (OK371 drives the overexpression of A315T in motor neurons). "A315T, Changtuoyin 0.05" indicates that the concentrations of hederagenin and anhydroicaritin in the food of Drosophila with OK371 > A315T are both 0.05 μg / mL. Figure 3 One-Way ANOVA test was used for the analysis in it. "**" 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 the turning-over times of at least 5 larvae for each genotype / drug treatment were measured and averaged for each repetition.

[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 It is the therapeutic result of the pharmaceutical composition of the combination of hederagenin and anhydroicaritin in Example 2 for female flies overexpressing the TDP-43 mutant A315T in the eyes. 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 indicates that the concentrations of hederagenin and anhydroicaritin in the food of the pharmaceutical composition are both 0.05 μg / mL. "A315T, DMSO" indicates 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" indicates 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 indicate 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 intragastric administration for 70 days, 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. Sections of 6 μm 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 the comparison of optical density, 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" indicates 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] A drug composition consisting of sodium aescinate (abbreviated as Aescin) and diosmin (abbreviated as Diosmin) (this drug composition is abbreviated as "Aescin-Diosmin"; the concentration of both active ingredients in food is 0.05 μg / mL, and the solvent is DMSO) significantly improved the degree of necrotic black spots induced by overexpression of TDP-43-wild type (wt) in the eyes of female flies. The proportion of no necrotic black spots increased by an average of 27 percentage points; the proportion of mild black spots decreased by an average of 23 percentage points; and the proportion of severe black spots decreased to 15.6% of the untreated group (results are shown in the figure). Figure 6 (As shown).

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

[0109] Example 6

[0110] A drug composition combining sodium aescinate (abbreviated as "aescin") and dehydrated icariin (abbreviated as "dehydrated icariin") showed significant therapeutic effects on female flies overexpressing the TDP-43 mutant A315T in the eyes. The drug composition (abbreviated as "aescin-dehydrated icariin", solvent: DMSO; concentration of active ingredient sodium aescinate: 0.001 μg / mL; concentration of dehydrated icariin: 0.005 μg / mL) treated with this drug composition increased the proportion of non-necrotic melasma by an average of nearly 16.1 percentage points; the proportion of severe melasma decreased by an average of 6.76 percentage points to 1.87%, approximately 21.7% of the untreated group; and the proportion of mild melasma decreased by an average of 9.27 percentage points (results are shown in the figure). Figure 7 (as shown in Table 4).

[0111] Table 4

[0112]

[0113] Figure 7 The results of treating female flies overexpressing the TDP-43 mutant A315T in the eyes with the pharmaceutical composition of sodium aescinate and dehydrated icariin, as described in Example 6, are as follows: Figure 7 It can be seen that the drug composition has a significant therapeutic effect. Figure 7 The chart is shown in Figure 4, and Table 4 shows the corresponding statistical data. Figure 7 In " Figure 7 ", "Qi 0.001 Tuoyin 0.005" indicates that the concentrations of sodium aescinate and icariin dehydrate in the food of the pharmaceutical composition are 0.001 μg / mL and 0.005 μg / mL respectively. "None", "Light", and "Heavy" are classifications of the black spots on the eyes of fruit flies by area. Figure 7 In " Figure 7 ", "n = 11, 49, 44" and "n = 27, 52, 25" respectively represent the number of fruit flies detected in each experimental repetition. In Table 4, "mean" is the average value, with the unit of %, SD is the standard deviation, and N is the number of experimental repetitions.

[0114] Example 7

[0115] The pharmaceutical compositions of the combination of sodium aescinate (abbreviated as Qi) and diosmin (abbreviated as Di), and the combination of sodium aescinate (abbreviated as Qi) and icariin dehydrate (abbreviated as Tuoyin) can significantly improve the degree of necrotic black spots generated by overexpression of TDP-43-wild type (wt) in the eyes of male flies. These pharmaceutical compositions are abbreviated as "QiDi" and "QiTuoyin". The solvent is DMSO, and the concentrations of the active ingredients in the two pharmaceutical compositions in the food are both 0.1 μg / mL and 0.05 μg / mL respectively. The effect of "QiDi" at 0.05 μg / mL is the most significant. The proportion of no necrotic black spots increases by an average of 13.82 percentage points, an increase of more than 0.69 times; the proportion of severe black spots decreases by an average of 22.8 percentage points, to only 4.3%, which is 15.9% of the non-drug group. Although many mild black spots turn into no black spots, due to a large number of severe black spots being converted into mild black spots, the change in the proportion of mild black spots is small. The effects of the two concentrations of "QiTuoyin", 0.1 μg / mL and 0.05 μg / mL, are the second. The proportion of severe black spots decreases significantly, to 8.73% and 9.13% respectively; the proportion of no black spots increases by 19.6 and 11 percentage points respectively, that is, an increase of more than 1 time and 0.55 times. In the group of "QiTuoyin" at 0.05 μg / mL concentration, the proportion of mild black spots increases because many severe black spots are converted into mild ones, and the decrease in the group of "QiTuoyin" at 0.1 μg / ml concentration is not obvious. The group of "QiDi" at 0.1 μg / ml also has a certain effect. The proportion of severe black spots decreases by 12.23 percentage points, and the proportion of no black spots increases by 8.63 percentage points (the results are as Figure 8 shown in Table 5).

[0116] Table 5

[0117]

[0118] Figure 8 This is the result of the improvement of the degree of necrotic black spots generated by overexpression of TDP-43-wild type (wt) in the eyes of male flies by the pharmaceutical compositions of the combination of sodium aescinate and diosmin, and the combination of sodium aescinate and icariin dehydrate in Example 7. Figure 8The chart is shown in Figure 5, and Table 5 shows the corresponding statistical data. Figure 8 "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-Dehydroepiandrosterone 0.1" indicates that the concentrations of sodium aescinate and dehydrated epimedium in the drug composition in food are both 0.1 μg / mL. "No drug added" indicates the solvent control. "None", "Light" and "Heavy" are classifications based on the area of ​​the black spots on the eyes of fruit flies. Figure 8 In Table 5, “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 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 drug compositions of sodium aescinate (abbreviated as "aescin") and diosmin (abbreviated as "di"), and sodium aescinate (abbreviated as "aescin") and dehydrated epimedium (abbreviated as "deep-inducing"), showed significant therapeutic effects on female flies. Overexpression of the TDP-43-wild-type (wt) gene in the eyes of female flies at a culture temperature of 27°C in this example, with expression levels higher than 25°C, significantly increased the severity of black spots. These drug compositions (abbreviated as "aescin-di" and "aescin-deep-inducing," respectively; DMSO as the solvent; and an active ingredient concentration of 0.05 μg / mL in the food) effectively improved black spot symptoms. Compared to the "untreated" group, the proportion of severe black spots decreased from 45.97% to 24.5%, a reduction of 52%; more than half of the severe black spots converted to mild black spots, thus increasing the proportion of mild black spots from 52.87% to 73.3%, an increase of over 20.43 percentage points; the proportion of no black spots also increased. Compared with the "no medication" group, the "Seven-Step Detoxification" combination showed a significant increase in the proportion of individuals without dark spots, rising from 1.13% to 13.85%, an increase of 11.26 times; the proportion of severe dark spots decreased by 8.8 percentage points, a reduction of nearly 20%; some mild dark spots transformed into no dark spots and some severe dark spots transformed into mild dark spots, therefore the change in the proportion of mild dark spots was not significant (results are as follows). Figure 9 (As shown).

[0121] Figure 9 The results of the drug composition of sodium aescinate and dehydrated epimedium in Example 8 on female flies cultured at 27°C are as follows: Figure 9 It can be seen that the drug composition has a significant therapeutic effect. Figure 9 This is a statistical chart. Figure 9"QiDi 0.05" in it indicates that the concentrations of sodium aescinate and diosmin in the food of the pharmaceutical composition are both 0.05 μg / mL. "QiTuoYin 0.05" indicates that the concentrations of sodium aescinate and anhydroicaritin in the food of the pharmaceutical composition are both 0.05 μg / mL. "Unmedicated" indicates the control. "None", "light", and "heavy" are classifications of the black spots on the eyes of fruit flies by area. Figure 9 "n = 29, 33, 27", "n = 34, 54, 44", and "n = 21, 22" in it represent the number of fruit flies detected in each experimental repetition.

[0122] Comparative Example 1

[0123] Overexpression of the TDP-43 mutant A315T in motor neurons leads to lethality during the pupal stage, and the pupal size is significantly smaller than that of the normal control group (see Example 1). The combination of hederagenin and anhydroicaritin with a concentration of 0.05 μg / mL in the food can partially rescue this phenotype. Using the drugs alone cannot achieve the effect of the combined use of hederagenin (abbreviated as "Chang") and anhydroicaritin (abbreviated as "TuoYin"), and there are significant differences (the results are as Figure 10 shown).

[0124] Figure 10 It is the results of using hederagenin (abbreviated as "Chang") and anhydroicaritin (abbreviated as "TuoYin") alone and in combination in Comparative Example 1. Figure 10 In Figure A in it, there are images of pupae of fruit flies overexpressing the hTDP-43 mutant A315T in motor neurons and fed with different drugs. Figure 10 In Figure B in it, there is a statistical chart, normalized to the drug combination group. Figure 10 In it, "A315T" represents overexpression of the hTDP-43 mutant A315T in motor neurons. "ChangTuoYin" indicates that the composition contains hederagenin and anhydroicaritin. "Chang" indicates hederagenin. "TuoYin" indicates anhydroicaritin. The number after the drug represents the drug concentration, with the unit of μg / mL. "ChangTuoYin 0.05" indicates that in the pharmaceutical composition, the concentrations of hederagenin and anhydroicaritin in the food are both 0.05 μg / mL. Figure 10 In Figure B in it, "n = 67", "n = 53", "n = 85", "n = 67", and "n = 73" respectively represent the number of pupae detected. One-Way ANOVA is used for analysis. "**" indicates a significant difference with P < 0.01, and "*" indicates a significant difference with P < 0.05.

[0125] Comparative Example 2

[0126] Under the same experimental conditions, using the drugs alone cannot achieve the effect of the combined use of hederagenin (abbreviated as "Chang") and anhydroicaritin (abbreviated as "TuoYin"). Here, it is for fruit flies with overexpression of TDP-43-A315T in the eyes.

[0127] Figure 11 The therapeutic effects of hederagein (abbreviated as hydatidin) and dehydrated epimedium (abbreviated as ephemeral) alone and in combination on TDP-43-A315T fruit flies were studied. Figure 11 It can be seen that using a single drug cannot achieve the same effect as using a combination of drugs. Figure 11 The chart is shown in Figure 6, and Table 6 shows the corresponding statistical data. Figure 11 The concentrations of ivy saponin and dehydrated icariin in food were both 0.05 μg / mL. "Chang 0.1" indicates that the concentration of ivy saponin in food was 0.1 μg / mL, "Chang 0.05" indicates that the concentration of ivy saponin in food was 0.05 μg / mL, "Dehydrated 0.1" indicates that the concentration of dehydrated icariin in food was 0.1 μg / mL, "Dehydrated 0.05" indicates that the concentration of dehydrated icariin in food was 0.05 μg / mL, "No drug added" indicates the control, and "None", "Light" and "Heavy" are classifications based on the area of ​​the black spots on the eyes of fruit flies. Figure 11 In Table 6, “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” represent the number of fruit flies in different experimental replicates. In Table 6, “mean” is the average, in %; SD is the standard deviation; and N is the number of experimental replicates.

[0128] Table 6

[0129]

[0130] Comparative Example 3

[0131] Under the same experimental conditions, the combined use of sodium aescinate (abbreviated as Aescin) and diosmin (abbreviated as Diosmin) did not achieve the same effect as the combined use of sodium aescinate (abbreviated as Aescin) and diosmin (abbreviated as Diosmin), as seen in the eyes of wild-type female flies that overexpressed TDP-43.

[0132] Figure 12 The therapeutic effects of sodium aescinate and diosmin, alone and in combination, on TDP-43-wild-type female flies were studied. Figure 12 It can be seen that using a single drug cannot achieve the same effect as using a combination of drugs. Figure 12 This is a statistical chart. Figure 12 "Seven-Di 0.05" indicates that the concentrations of sodium aescinate and diosmin in food are both 0.05 μg / mL; "Seven 0.1" indicates that the concentration of sodium aescinate in food is 0.1 μg / mL; "Di 0.1" indicates that the concentration of diosmin in food is 0.1 μg / mL; "None", "Light", and "Heavy" are classifications based on the area of ​​the black spots on the eyes of fruit flies. Figure 12In the text, “n=65,80”, “n=65,66”, “n=65,104”, and “n=28,51” represent the number of fruit flies in different experimental replicates.

[0133] Example 9

[0134] Adding edaravone to the binary combination of ivy saponin and dehydrated epimedium can enhance the therapeutic effect.

[0135] Overexpression of the TDP-43 mutant A315T in motor neurons led to pupal lethality, and the pupal size was significantly smaller than that of the normal control group (see Example 1). The combination of ivy saponins (abbreviated as ivy) and dehydrated epimedium (abbreviated as epimedium) in combination with edaravone (abbreviated as edaravone) was more effective than the binary combination, with significant differences.

[0136] Figure 13 The efficacy of combining ivy saponin and dehydrated epimedium with edaravone was studied. Figure 13 It can be seen that the combined drug therapy is more effective than the binary combination of ivy saponin and dehydrated epimedium. Figure 13 Figure A shows the TDP-43 mutant A315T overexpressed in motor neurons treated with different drugs. Figure 13 Figure B in the middle is a statistical graph, normalized to the DMSO control group. Figure 13 In the text, "A315T" indicates that the TDP-43 mutant A315T is overexpressed in motor neurons; "Changtuyin" indicates that the concentration of ivy saponin and dehydrated icariin in food is 0.05 μg / mL; "Changtuyin+Yida" indicates that the concentration of ivy saponin and dehydrated icariin in food is 0.05 μg / mL, and the concentration of edaravone is 0.04 μg / mL; and "DMSO" indicates the solvent control. Figure 13 In Figure B, “n=48”, “n=67”, and “n=52” represent the number of fruit flies tested. 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 model of the TDP-43 mutant A315T, the combination of hederamin and dehydrated icariin was superior to riluzole (e.g. Figure 14(As shown in Tables 7 and 8). Compared with the solvent (DMSO) control group: adding three different concentrations of riluzole to food, 0.04 μg / mL had no effect. The average percentages of "no", "mild", and "severe" dark spots in the eyes were 61.30%, 35.65%, and 3.05%, respectively, which were not significantly different from the control group's 60.30%, 35.18%, and 4.53%. The percentages of no dark spots with riluzole at concentrations of 1 μg / mL and 0.2 μg / mL were 65.68% and 66.30%, respectively, which were significantly different from the control group (P=0.012 and 0.008). The percentages of mild and severe dark spots were 30.25% and 30.93%, and 4.10% and 2.08%, respectively, which were not significantly different from the control group. Among the three concentrations of riluzole, 0.2 μg / mL was the optimal concentration.

[0139] The combination of ivy saponin and dehydrated epimedium resulted in 75.03% of patients without dark spots, an increase of 14.73 percentage points (24.4%) compared to the control, which was highly significant (P=0.00003). The proportion of mild dark spots was 22.45%, a decrease of 12.73 percentage points (36.2%) compared to the control, which was highly significant (P=0.0011). The proportion of 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 ivy saponin and dehydrated epimedium extract showed a significant advantage in the proportion of no dark spots and mild dark spots, with statistically significant differences (P values ​​of 0.00049 and 0.00170, respectively), while there was no significant difference in the proportion of severe dark spots.

[0141] Table 7

[0142]

[0143] Table 8

[0144]

[0145] Figure 14 This study compares the efficacy of the combination of ivy saponin and dehydrated icariin (hereinafter referred to as ivy saponin) with the marketed drug riluzole. On one hand, it shows that the ivy saponin and dehydrated icariin combination is more effective than the existing drug riluzole. On the other hand, since the usual dosage of riluzole in humans is 100 mg / day, by comparing the concentration ratio of ivy saponin and dehydrated icariin to the concentration of riluzole, the dosage of the binary combination drug of this invention used in human clinical trials can be estimated to be approximately 10 mg / day to 2000 mg / day. Preferably, the initial dosage for the ivy saponin and dehydrated icariin combination in the initial human trials is approximately 20 mg to 200 mg / day.

[0146] Figure 14 The statistical graph shows the effects of overexpressing the TDP-43 mutant A315T in the optic nerve under different drug treatments. Table 7 shows the corresponding statistical data, and Table 8 shows the results of the significance analysis. Figure 14 DMSO served as the solvent control group. "Riluzole 1", "Riluzole 0.2" and "Riluzole 0.04" indicate that the concentrations of riluzole in the food were 1 μg / mL, 0.2 μg / mL and 0.04 μg / mL, respectively. "Hydrogenin 0.25" indicates that the concentrations of the combination of hederagenin and dehydrated epimedium in the food were 0.25 μg / mL, respectively. "None", "Light" and "Heavy" are classifications based on the area of ​​the black spots on the eyes of fruit flies. Figure 14 In Table 7, “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 replicates. In Table 8, “mean” is the average, in %; SD is the standard deviation; N is the number of experimental replicates; and the variation represents the difference from 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. The use of the pharmaceutical composition in the preparation of a medicament for treating TDP-43 proteinopathy, characterized in that, The pharmaceutical composition includes at least one Class A compound and at least one Class B compound; The Class A compounds include at least one of sodium aescinate and hederone. The Class B compounds include at least one of dehydrated icariin and diosmin; The weight ratio of the Class A compound to the Class B compound is 1:1 or 1:

5.

2. The application according to claim 1, characterized in that, The pharmaceutical composition is used in combination with edaravone.

3. The application according to claim 2, characterized in that, The pharmaceutical composition also includes pharmaceutically acceptable excipients.

4. The application according to claim 2, characterized in that, The pharmaceutically acceptable excipients include at least one of pharmaceutical carriers, diluents, adjuvants, or excipients.

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