Application of PI3Kalpha activator for treating amyotrophic lateral sclerosis

Abstract

The invention discloses an application of a PI3K alpha activator in treatment of amyotrophic lateral sclerosis. The invention relates to the field of biological medicine, and provides application of UCL-TRO-1938 or pharmaceutically acceptable salt, solvate, prodrug, stereoisomer, isotope variant or tautomer thereof in preparation of products for treating amyotrophic lateral sclerosis. Experiments prove that the UCL-TRO-1938 has remarkable effects of reducing apoptosis of motor neurons induced by iPSC (induced pluripotent stem cells) derived from a patient and reducing abnormal pathological changes of TDP-43; the UCL-TRO-1938 can significantly improve the movement dysfunction of SOD1-G93A model mice, and in addition, through pathological section, it is found that UCL-TRO-1938 can reduce the functions of glial cell hyperplasia, motor neuron apoptosis of the motor cortex and the anterior spinal cord and the like. Therefore, the UCL-TRO-1938 has a potential application value in the treatment of the patient suffering from amyotrophic lateral sclerosis.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to use of a PI3Kα activator for treating amyotrophic lateral sclerosis. Background Art

[0002] Amyotrophic lateral sclerosis (ALS), commonly known as "Lou Gehrig's disease", is a progressive, disabling neurodegenerative disease that mainly affects the motor neurons in the cerebral cortex and the motor cells in the anterior horns of the spinal cord. It progresses rapidly, with an average survival of 3-5 years after onset. The disease is characterized by selective degeneration of motor neurons, causing patients to gradually lose the ability to control voluntary muscles. ALS usually begins with limb muscle weakness and atrophy, then spreads to the whole body, eventually affecting breathing, swallowing, and language functions.

[0003] The etiology of ALS is complex and unknown. It is currently believed that it may involve genetic factors, environmental factors, and multiple pathological mechanisms in cells, including DNA damage, abnormal RNA metabolism, abnormal autophagy and protein homeostasis, mitochondrial dysfunction, neuroinflammation, cytoskeletal instability, and axonal transport dysfunction. About 5-10% of cases are familial ALS, among which the most common pathogenic genes are SOD1, C9orf72, and TDP-43. Most cases (90-95%) are sporadic, and their exact cause is still unclear.

[0004] Riluzole and edaravone are currently FDA-approved drugs for the treatment of ALS, but their effects are limited. Riluzole is the first approved ALS drug, and its main mechanism of action is to inhibit glutamate release and protect neurons, but it can only prolong the patient's survival by about 2-3 months, and its efficacy is limited. As the second FDA-approved drug for the treatment of ALS, edaravone's main mechanism of action is to scavenge free radicals and reduce oxidative stress. Although it can slow the rate of functional decline, its complex dosing regimen and potential side effects increase the difficulty of treatment. More importantly, neither of these two drugs can prevent the progression of the disease or cure ALS, and can only improve symptoms or delay the course of the disease to a limited extent.

[0005] Therefore, it is very necessary to find a new treatment strategy for ALS. Summary of the invention

[0006] The purpose of the present invention is to provide a use of a PI3Kα activator for treating amyotrophic lateral sclerosis.

[0007] In the first aspect, the present invention claims the use of UCL-TRO-1938 or its pharmaceutically acceptable salt, solvate, prodrug, stereoisomer, isotopic variant or tautomer in the preparation of a product for treating amyotrophic lateral sclerosis. UCL-TRO-1938 is a new small molecule compound and a potent allosteric activator of phosphatidylinositol 3-kinase α (PI3Kα). The product may be a medicine.

[0008] In a second aspect, the present invention claims the use of UCL-TRO-1938 or a pharmaceutically acceptable salt, solvate, prodrug, stereoisomer, isotope variant or tautomer thereof in the preparation of a product for improving motor dysfunction in an amyotrophic lateral sclerosis patient, wherein the product may be a medicine.

[0009] In a third aspect, the present invention claims the use of UCL-TRO-1938 or a pharmaceutically acceptable salt, solvate, prodrug, stereoisomer, isotopic variant or tautomer thereof in any of the following:

[0010] (A1) preparing a product for prolonging the survival of an organism suffering from amyotrophic lateral sclerosis;

[0011] (A2) preparing a product for delaying disease progression (as reflected by HLC score) in an amyotrophic lateral sclerosis patient;

[0012] (A3) preparing a product for delaying the decline of motor function in an organism suffering from amyotrophic lateral sclerosis;

[0013] (A4) preparing a product for slowing down the rate of weight loss in patients with amyotrophic lateral sclerosis;

[0014] (A5) preparing a product for improving gastrocnemius muscle atrophy in an amyotrophic lateral sclerosis patient;

[0015] (A6) preparing a product for reducing neuroinflammation in an amyotrophic lateral sclerosis patient;

[0016] (A7) preparing a product for reducing neuronal death in the motor cortex (layer VI) of an amyotrophic lateral sclerosis patient;

[0017] (A8) preparing a product for reducing the death of anterior horn motor neurons in the spinal cord of an amyotrophic lateral sclerosis patient;

[0018] (A9) Preparing a product for reducing the nuclear and cytoplasmic ectopic location and aggregation of TDP-43 in motor neurons of an amyotrophic lateral sclerosis patient.

[0019] Wherein, the product may be a medicine.

[0020] In a fourth aspect, the present invention claims the use of UCL-TRO-1938 or a pharmaceutically acceptable salt, solvate, prodrug, stereoisomer, isotopic variant or tautomer thereof in any of the following:

[0021] (B1) Reduce the abnormal pathological changes of TDP-43 in motor neurons derived from iPSCs of patients with amyotrophic lateral sclerosis in vitro;

[0022] (B2) Reduced apoptosis of motor neurons derived from iPSCs of patients with amyotrophic lateral sclerosis in vitro;

[0023] (B3) preparing a product for reducing abnormal pathological changes of TDP-43 in motor neuron cells induced by iPSCs from patients with amyotrophic lateral sclerosis;

[0024] (B4) Prepare a product for reducing apoptosis of motor neuron cells induced by iPSCs from patients with amyotrophic lateral sclerosis.

[0025] Wherein, the product may be a medicine.

[0026] In a fifth aspect, the present invention claims the use of UCL-TRO-1938 or a pharmaceutically acceptable salt, solvate, prodrug, stereoisomer, isotopic variant or tautomer thereof in any of the following:

[0027] (C1) Reversing the abnormal pathological changes of TDP-43 in motor neurons induced by sodium arsenite in vitro;

[0028] (C2) Reversal of sodium arsenite-induced apoptosis of motor neurons in vitro;

[0029] (C3) preparing a product for reversing the abnormal pathological changes of TDP-43 in motor neuron cells induced by sodium arsenite;

[0030] (C4) Preparing a product for reversing sodium arsenite-induced apoptosis of motor neuron cells.

[0031] In one embodiment of the present invention, the motor neuron cells are motor neurons induced and differentiated from induced pluripotent stem cells (iPS cells) derived from patients with amyotrophic lateral sclerosis. The product may be a medicine.

[0032] In a sixth aspect, the present invention claims a method for reversing abnormal pathological changes of TDP-43 in motor neuron cells induced by sodium arsenite in vitro.

[0033] The method claimed in the present invention for reversing the abnormal pathological changes of TDP-43 in motor neuron cells induced by sodium arsenite in vitro may include the following steps: treating the motor neuron cells with UCL-TRO-1938 or a pharmaceutically acceptable salt, solvate, prodrug, stereoisomer, isotopic variant or tautomer thereof at the same time or after treating the motor neuron cells with sodium arsenite in vitro.

[0034] In a seventh aspect, the present invention claims a method for reversing sodium arsenite-induced apoptosis of motor neuron cells in vitro.

[0035] The method for reversing sodium arsenite-induced apoptosis of motor neuron cells in vitro claimed in the present invention may include the following steps: treating the motor neuron cells with UCL-TRO-1938 or a pharmaceutically acceptable salt, solvate, prodrug, stereoisomer, isotopic variant or tautomer thereof simultaneously or after treating the motor neuron cells with sodium arsenite in vitro.

[0036] In one embodiment of the present invention, the motor neuron cells are motor neurons induced and differentiated from induced pluripotent stem cells (iPS cells) derived from amyotrophic lateral sclerosis patients.

[0037] In the above-mentioned related aspects, the diseased organism may be a human body, or an animal body, such as a mammal, specifically a mouse.

[0038] The term "prodrug" used in the present invention refers to any functional derivative (or precursor) of the compound UCL-TRO-1938 involved in the present invention, which generates the compound when applied to a biological system due to, for example, spontaneous chemical reactions, enzyme-catalyzed chemical reactions and / or metabolic chemical reactions. Compared with the resulting drug, the prodrug is usually inactive or less active, and can be used, for example, to improve the physiochemical properties of the drug, target the drug to specific tissues, improve the pharmacokinetic and pharmacokinetic properties of the drug and / or reduce side effects. Some common functional groups that are amenable to prodrug design include, but are not limited to, carboxyl, hydroxyl, amine, phosphate / phosphonate and carbonyl. Prodrugs typically obtained by modification of these groups include, but are not limited to, esters, carbonates, carbamates, amides and phosphates.

[0039] The term "derivative" as used in the present invention also includes metabolites of drugs, for example, molecules that are derived from (biochemical) modification or processing of the drug after administration to an organism, usually by a specialized enzymatic system, and that exhibit or retain the biological activity of the drug. It has been disclosed that metabolites are responsible for most of the therapeutic effects of the parent drug. In a specific embodiment, "metabolite" as used herein refers to a modified or processed drug that retains at least part of the activity of the parent drug.

[0040] Different routes of administration may require different forms of a compound. For example, certain salt forms may be more suitable for oral administration, while solvates may be more suitable for injection; certain variants (such as specific salt forms or crystalline forms) may be more stable during the production process or easier to store; isotopic variants may have application value in diagnostics or specific therapeutic applications.

[0041] The UCL-TRO-1938 involved in the present invention can be prepared into a variety of pharmaceutical dosage forms, including liquid dosage forms (such as solutions, suspensions, emulsions), semi-solid dosage forms (such as extracts) and solid dosage forms (such as powders, granules, tablets, capsules). Specifically, they can be as follows:

[0042] The drug may be contained in any suitable carrier material in any suitable amount and may be present in an amount of 1-99% of the total weight of the composition. The composition may be provided in a dosage form suitable for oral, parenteral (e.g., intravenous, intramuscular), rectal, dermal, nasal, vaginal, inhalant, dermal (patch) or ocular administration routes.

[0043] Thus, the composition can be in the form of, for example, tablets, capsules, pills, powders, granules, suspensions, emulsions, solutions, gels including hydrogels, pastes, ointments, creams, salves, infusions, isotonic delivery devices, suppositories, enemas, injections, implants, sprays or aerosols.

[0044] The UCL-TRO-1938 involved in the present invention can be prepared with a variety of pharmaceutical carriers and excipients, including sugars (such as lactose, glucose), polysaccharides (such as starch), gums (such as gum arabic), inorganic salts (such as calcium phosphate), proteins (such as gelatin), cellulose derivatives (such as microcrystalline cellulose), polymers (such as polyvinyl pyrrolidone), liquid carriers (such as water, syrup), preservatives (such as hydroxybenzoates) and other excipients (such as talc, magnesium stearate). Researchers can optimize the performance of the preparation according to the properties and therapeutic needs of specific drugs.

[0045] The administration routes of UCL-TRO-1938 involved in the present invention include various, especially non-oral administration, such as injection, topical administration, etc. Specifically, it includes oral, intravenous, intraperitoneal, subcutaneous, intradermal, intramuscular, topical administration, and administration into the spinal cord, spinal cavity, intraventricular, brain parenchyma and cranial cavity of the central nervous system.

[0046] In the present invention, the structural formula of UCL-TRO-1938 is as follows:

[0047]

[0048] The present invention has been verified through cell experiments of motor neurons (iMN) differentiated from induced pluripotent stem cells (iPSC) of ALS patients and animal experiments of ALS model mice (SOD1-G93A) that UCL-TRO-1938 has the efficacy of reducing apoptosis of iMN derived from patients and alleviating pathological ectopic and aggregation of TDP-43; at the same time, for SOD1-G93A model mice, it can improve TDP-43 ectopic and aggregation of MN in the anterior horn of the spinal cord, improve neuroinflammation, reduce apoptosis of large pyramidal cells in the motor cortex and MN in the anterior horn of the spinal cord, and can significantly improve motor dysfunction and prolong survival in behavior. These findings indicate that UCL-TRO-1938 has potential application value in the treatment of ALS. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 The survival curve of SOD1-G93A mice was prolonged after UCL-TRO-1938 treatment. *: p<0.05, ****: p<0.0001.

[0050] Figure 2 The tails of mice in each group were suspended and the ALSTDI neurological scores were obtained. A is a photo of the suspension; B is a quantitative graph of the scores of the whole group. *: p<0.05, ****: p<0.0001.

[0051] Figure 3 The figure shows the changes in motor function of 4.5-month-old SOD1-G93A mice (administered for four weeks). A is the grip strength of the forelimbs (corresponding to the grip test); B is the grip strength of the four limbs (corresponding to the grip test); C is the hanging endurance time (corresponding to the hanging test); D is the retention time on the rotating rod (corresponding to the rotating rod test). *: p<0.05, ***: p<0.001, ****: p<0.0001.

[0052] Figure 4The figure shows the changes in motor function of 2.5-4.5 month old SOD1-G93A mice (administered for 0-8 weeks). A is the grip strength of the forelimbs (corresponding to the grip test); B is the grip strength of the four limbs (corresponding to the grip test); C is the hanging endurance time (corresponding to the hanging test); D is the retention time on the rotating rod (corresponding to the rotating rod test). *: p<0.05, ***: p<0.001, ****: p<0.0001.

[0053] Figure 5 The weight changes of mice in different treatment groups. **: p<0.01, ****: p<0.0001.

[0054] Figure 6 This is a straight shot of the muscles of a 4.5-month-old SOD1-G93A mouse after sampling.

[0055] Figure 7 UCL-TRO-1938 was used to treat SOD1-G93A mutant ALS mice to improve neuroinflammation. A: Representative images show the number and status of reactive microglia in the lumbar spinal cord of mice (Iba1 labeled, 10 μm thick sections). B: Statistics of the number of reactive microglia in the anterior horn of the spinal cord. C: The number and status of activated astrocytes in the lumbar spinal cord of mice (GFAP labeled, 10 μm thick sections). D: Statistics of the number of activated astrocytes in the anterior column of the spinal cord. Data are expressed as mean ± SEM (6 mice per group), and statistical analysis was performed using Student's unpaired t-test. *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001, scale bar 100 μm.

[0056] Figure 8 UCL-TRO-1938 can significantly reduce the death of motor cortical neurons in ALS model mice. A: 10 μm thick mouse brain cryosections at layer VI of the motor cortex, DAPI, IBA1, and NeuN staining, and the number of neurons and microglial cell body area were detected. B: Statistics of neuron number. C: Statistics of microglial cell body area, data are expressed as mean ± SEM (3 mice per group), and Student's unpaired t-test was used for statistical analysis. ns: p>0.05, *: p<0.05, **: p<0.01, scale bar 20 μm.

[0057] Fig. 9The number of motor neurons in the lumbar spinal cord of mice is counted. A: 10 μm thick paraffin sections of the lumbar spinal cord of mice, DAPI and ChAT staining were used to detect the number of motor neurons. B: The number of motor neurons in the anterior horn of the spinal cord is counted. Data are expressed as mean ± SEM (6 mice per group), and statistical analysis was performed using Student's unpaired t-test. ****: p < 0.0001, scale bar 100 μm.

[0058] Fig.10 The distribution of TDP-43 in the lumbar motor neurons of the mouse spinal cord. A: 10 μm thick paraffin sections of the lumbar spinal cord of mice, DAPI, ChAT, TDP-43 staining to detect the distribution of TDP-43 in motor neurons. B: Distribution statistics of TDP-43 in the motor neurons in the anterior horn of the spinal cord. Data are expressed as mean ± SEM (6 mice per group), and statistical analysis was performed using Student's unpaired t-test. ns: p>0.05, *: p<0.05, ***: p<0.001, scale bar 5 μm.

[0059] Fig.11 Treatment of ALS patient-derived motor neurons with UCL-TRO-1938 improves abnormal pathological manifestations of TDP-43. A: iPSC-induced motor neurons, DAPI, TDP-43, MAP2 staining. Detection of the intracellular distribution of TDP-43. B: Distribution statistics of TDP-43 in iPSC-induced motor neurons. Two-way ANOVA with Sidak's multiple comparison test, *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001, scale bar 10μm.

[0060] Fig.12 Tunel apoptosis staining was used to observe the effect of UCL-TRO-1938 on apoptosis of patient-derived motor neurons. A: iPSC-induced motor neurons, FITC-Tunel staining. DAPI (blue) marks the nucleus; FITC-Tunel (green) marks apoptotic cells. B: Statistics of apoptotic signals in iPSC-induced motor neurons. Two-way ANOVA with Sidak's multiple comparison test, *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001, scale bar 100 μm. DETAILED DESCRIPTION

[0061] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0062] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0063] Example 1: Animal experiments verify the effect of UCL-TRO-1938 in treating amyotrophic lateral sclerosis

[0064] In the present invention, B6.Cg-Tg (SOD1 * G93A) 1Gur / J transgenic mice (abbreviated as SOD1-G93A, Jackson Laboratory, Stock No: 004435) are used as an experimental model of amyotrophic lateral sclerosis (ALS). This model is the most commonly used experimental animal model in ALS research. This model carries the human SOD1 gene G93A mutation and can reproduce the key histopathological features of sporadic and familial ALS patients. Experiments were conducted in a specific pathogen-free (SPF) environment. SOD1-G93A mice began to show hind limb tremor symptoms at about 100 days (3.5 months) of age, and then the condition rapidly worsened, with a median survival of 157.1 ± 9.3 days (5 months of age). Due to its high degree of simulation of the human ALS course, this model is currently widely used in preclinical trials for ALS pathogenesis research, potential treatment strategy evaluation, and new drug development, providing a reliable and widely recognized experimental platform for related research.

[0065] 1. UCL-TRO-1938 can prolong the survival of ALS model mice

[0066] 2.5-month-old SOD1-G93A male mice (n=24) were selected as experimental subjects. According to body weight, they were randomly divided into a vehicle control group (SOD1_Blk group, n=12) and a UCL-TRO-1938 administration group (SOD1_1938 group, n=12). The vehicle control group was given 0.1 ml / 10 g body weight of vehicle by oral gavage every day; the UCL-TRO-1938 administration group was given 10 mg / kg dose of UCL-TRO-1938 (dissolved in the vehicle, the administration volume was 0.1 ml / 10 g body weight) by oral gavage every day. Among them, the specific preparation method of the dosing solution is as follows: UCL-TRO-1938 is dissolved in DMSO to prepare a mother solution, and then diluted at a ratio of 5% DMSO mother solution: 95% corn oil (volume ratio), and fully mixed to obtain the final dosing solution. In the experiment, 2.5-month-old C57BL / 6J male mice with the same genetic background were set up as wild-type controls (WT group) (n=12).

[0067] The survival status of mice was observed and recorded daily during the experiment.

[0068] The experimental results are as follows Figure 1 As shown, it can be seen that the average lifespan of mice in the UCL-TRO-1938 group was 175 days, while that in the vehicle control group was 155 days, and the average lifespan of the group treated with the drug was extended by about 20 days (12.9%). In terms of median survival, the UCL-TRO-1938 group was 175 days, while that in the vehicle control group was 155 days, and the median survival of the group treated with the drug was extended by about 20 days, an increase of about 12.9%. These data indicate that UCL-TRO-1938 treatment can significantly prolong the lifespan and median survival of ALS model mice.

[0069] 2. UCL-TRO-1938 can significantly delay disease progression in ALS model mice

[0070] The disease progression of SOD1-G93A mice can be fully evaluated using the ALSTDI 0 to 4 neurological scoring system, which reflects the entire process of the disease from early to late stages. Scoring starts at 0, indicating that the hind limbs of the mouse can be fully extended and maintain posture when the tail is suspended; as the disease progresses, 1 point is manifested as weakness or tremor in hind limb extension, 2 points show abnormal walking and limb dragging, and 3 points show severe motor disorders; and finally 4 points means that the mouse cannot turn over on its own, which is considered the death of the mouse (reference: Duan, W., Guo, M., Yi, L. et al. The deletion of mutant SOD1 via CRISPR / Cas9 / sgRNA prolongs survival in an amyotrophic lateral sclerosis mouse model. Gene Ther 27, 157–169 (2020)).

[0071] 2.5-month-old SOD1-G93A male mice (n=24) were selected as experimental subjects. According to body weight, they were randomly divided into a vehicle control group (SOD1_Blk, n=12) and a UCL-TRO-1938 administration group (SOD1_1938, n=12). The vehicle control group was given 0.1 ml / 10 g body weight of vehicle by oral gavage every day; the UCL-TRO-1938 administration group was given 10 mg / kg dose of UCL-TRO-1938 (dissolved in the vehicle, the administration volume was 0.1 ml / 10 g body weight) by oral gavage every day. Among them, the specific preparation method of the dosing solution is as follows: UCL-TRO-1938 is dissolved in DMSO to prepare a mother solution, and then diluted at a ratio of 5% DMSO mother solution: 95% corn oil (volume ratio), and fully mixed to obtain the final dosing solution. In the experiment, 2.5-month-old C57BL / 6J male mice with the same genetic background were set up as wild-type controls (WT group) (n=12).

[0072] Neurological scores were assessed for each mouse every 2 weeks starting from the start of the experiment.

[0073] 4.5 months old results Figure 2 shown.

[0074] from Figure 2As can be seen from A, WT (wild-type) mice performed the best, with their hind limbs fully extended, meeting the standard of 0 points, indicating no disease symptoms; while the hind limbs of SOD1-G93A mice in the vehicle group (SOD1_Blk group) were obviously curled up and unable to fully extend, which may correspond to a level of 2-3 points, indicating that the disease has progressed to the middle and late stages and obvious movement disorders have occurred; the hind limb condition of SOD1-G93A mice in the UCL-TRO-1938-treated group (SOD1_1938 group) was between the WT group and the SOD1_Blk group. Although it was not fully extended, it was better than the SOD1_Blk group, corresponding to 1-2 points, indicating that disease progression was alleviated.

[0075] Figure 2 The bar graph in Figure B further quantifies this observation. The WT group had the lowest score, close to 0, confirming that wild-type mice had no disease symptoms; the SOD1_Blk group had the highest score, with an average of approximately 2.5 points, confirming that this group of mice had the most severe disease progression; the SOD1_1938 group had a score between the two, with an average of approximately 1.8 points, which was significantly lower than the SOD1_Blk group.

[0076] These results indicate that UCL-TRO-1938 treatment effectively slowed disease progression and improved motor function in SOD1-G93A mice, although not fully restored to wild-type levels, but significantly improved compared to the untreated SOD1_Blk group.

[0077] 3. UCL-TRO-1938 can significantly delay motor dysfunction in ALS model mice

[0078] 2.5-month-old SOD1-G93A male mice (n=24) were selected as experimental subjects. According to body weight, they were randomly divided into a vehicle control group (SOD1_Blk group, n=12) and a UCL-TRO-1938 administration group (SOD1_1938, n=12). The vehicle control group was given 0.1 ml / 10 g body weight of vehicle by oral gavage every day; the UCL-TRO-1938 administration group was given 10 mg / kg dose of UCL-TRO-1938 (dissolved in the vehicle, the administration volume was 0.1 ml / 10 g body weight) by oral gavage every day. Among them, the specific preparation method of the administration solution is as follows: UCL-TRO-1938 is dissolved in DMSO to prepare a mother solution, and then diluted at a ratio of 5% DMSO mother solution: 95% corn oil (volume ratio), and fully mixed to obtain the final administration solution. In the experiment, 2.5-month-old C57BL / 6J male mice with the same genetic background were set up as wild-type controls (WT group) (n=12).

[0079] During the experiment, the grip test, hanging test and rotating rod test were conducted every 2 weeks.

[0080] 1. Grip test

[0081] The gripping strength test is used to evaluate the limb strength of mice. SOD1-G93A model mice will experience a decrease in muscle strength when they become ill, while normal mice will show a stable and relatively high gripping strength value; the gripping strength value of diseased mice may gradually decrease as the disease progresses. Therefore, the effect of disease progression on muscle strength can be evaluated by measuring the maximum gripping strength of the mouse limbs.

[0082] The gripping device used in the gripping experiment consists of a counter and a gripping sensor plate. Before the experiment, ensure that the gripping plate of the measuring instrument is horizontal, and move the mouse into the experimental field to adapt for at least 30 minutes. Place the mouse on the gripping plate and let its limbs grasp firmly. Grab the mouse tail and gently pull it back horizontally to make the mouse detach from the gripping plate, and record the value of the measuring instrument. After the experiment, clean the gripping plate with 75% ethanol to remove the odor left by the mouse. For the same mouse, repeat the measurement 5 times, with an interval of 10 minutes between each measurement, and take the average of the 5 gripping values ​​for statistical analysis.

[0083] 2. Suspension experiment

[0084] The hanging experiment is used to evaluate the grasping strength of mice. SOD1-G93A model mice will experience decreased grasping strength and muscle endurance after the disease occurs. Normal mice are usually able to hang for a long time, and may often reach the critical value of 120 seconds; as the disease progresses, the hanging time of diseased mice may gradually shorten. Therefore, the effect of the disease on grasping ability and muscle endurance can be evaluated by measuring the length of time mice hang on the metal grid.

[0085] The suspension device used in the suspension experiment is 30 cm above the ground and consists of a metal grid with a gap of 0.5 cm. Before the experiment, the mice were moved into the experimental field to adapt for at least 30 minutes. The mouse was placed on the metal grid and gently shaken to let it grab the grid. Then the grid was turned over and the timing started until the mouse fell. The time the mouse stayed on the grid was recorded. 120 seconds was used as the critical value. The actual time was recorded if it was less than 120 seconds, and it was recorded as 120 seconds if it exceeded 120 seconds. After the experiment, the grid was cleaned with 75% ethanol to remove the odor left by the mouse. For the same mouse, the measurement was repeated 3 times, with an interval of 10 minutes between each measurement. The average of the 3 times was used for statistical analysis.

[0086] 3. Rotarod test

[0087] The rotarod test is used to evaluate the endurance and motor coordination of mice. SOD1-G93A model mice will have motor dysfunction when they are sick. Normal mice can usually stay on the rotarod for a long time, and may often approach or reach the critical value of 300 seconds; while the diseased mice may gradually decrease the time they stay on the rotarod as the disease progresses. Therefore, the motor coordination, sense of balance and overall endurance of animals can be evaluated by detecting the length of time mice stay on the accelerating rotating rod.

[0088] The rotating rod apparatus consists of a cylindrical rotating platform and a rotating rod (3.5 cm in diameter). Before the experiment, the mice were moved into the experimental field to adapt for at least 30 minutes. Two days before the formal test, all mice were placed on the rotating rod to adapt and trained 3 times a day at a constant speed (15 rpm), 300 seconds each time, and 30 minutes between each training. On the third day, the mice were placed on the rotating rod and the speed was set to accelerate uniformly from 1 rpm to 35 rpm (acceleration time 35 seconds) until the mice fell, and the time the mice stayed on the rotating rod was recorded. 300 seconds was used as the critical value, and the actual time was recorded if it was less than 300 seconds, and 300 seconds if it exceeded 300 seconds. After the experiment, the rotating rod was cleaned with 75% ethanol to remove the odor left by the mice. For the same mouse, the measurement was repeated 3 times, with an interval of 30 minutes between each measurement, and the average of the 3 times was taken for statistical analysis.

[0089] Together, these three experiments form a comprehensive motor function assessment system. As the disease progresses in SOD1-G93A model mice, it is expected that the performance in these experiments will gradually deteriorate. The difference from normal mice may not be obvious in the early stage, but over time, the difference will become more and more significant. By observing this gradual deterioration trend, researchers can evaluate the progression rate of the disease and the effectiveness of potential treatments.

[0090] 4. Results

[0091] The results are as follows Figure 3 and Figure 4 As shown:

[0092] Figure 3 Changes in motor function in 4.5-month-old SOD1-G93A mice (administered for four weeks).

[0093] Figure 4 Changes in motor function in 2.5-4.5 month old SOD1-G93A mice (dosing for 0-8 weeks).

[0094] It can be seen that in all tests, the WT group mice performed the best, the SOD1_Blk group mice performed the worst, and the SOD1_1938 group mice were between the two and significantly better than the SOD1_Blk group. This indicates that UCL-TRO-1938 treatment significantly improved the motor function of SOD1-G93A mice.

[0095] 4. UCL-TRO-1938 treatment can significantly slow down the weight loss rate of ALS model mice

[0096] Weight changes in SOD1-G93A mice are an important indicator for assessing the progression of amyotrophic lateral sclerosis (ALS). The weight gain of these transgenic mice in the early stages of life (about 60-90 days) is similar to that of wild-type mice, but begins to show significant changes after reaching peak weight around 90-100 days of age. Subsequently, with the appearance and progression of disease symptoms, the weight of SOD1-G93A mice begins to gradually decrease, especially after 120-140 days of age, when the rate of weight loss accelerates and may reach 20-30% or more of peak weight. In contrast, wild-type mice of the same age usually maintain a stable weight or continue to gain weight slowly.

[0097] 2.5-month-old SOD1-G93A male mice (n=24) were selected as experimental subjects. According to body weight, they were randomly divided into a vehicle control group (SOD1_Blk group) (n=12) and a UCL-TRO-1938 administration group (SOD1_1938 group) (n=12). The vehicle control group was given 0.1 ml / 10 g body weight of vehicle by oral gavage every day; the UCL-TRO-1938 administration group was given 10 mg / kg dose of UCL-TRO-1938 (dissolved in the vehicle, the administration volume was 0.1 ml / 10 g body weight) by oral gavage every day. Among them, the specific preparation method of the dosing solution is as follows: UCL-TRO-1938 is dissolved in DMSO to prepare a mother solution, and then diluted at a ratio of 5% DMSO mother solution: 95% corn oil (volume ratio), and fully mixed to obtain the final dosing solution. In the experiment, 2.5-month-old C57BL / 6J male mice with the same genetic background were set up as wild-type controls (WT group) (n=12).

[0098] Body weight was measured and recorded every 2 weeks during the experiment.

[0099] The results are as follows Figure 5As shown in the experimental results, the mice in the SOD1_1938 group showed a clear therapeutic effect throughout the experimental period (10 to 18 weeks). Compared with the SOD1_Blk group, the weight of the mice in the SOD1_1938 group continued to remain at a higher level, averaging about 26g, while the weight of the mice in the SOD1_Blk group was only 25-25.5g. The weight curve of the SOD1_1938 group showed better stability and less fluctuation. This weight advantage lasted from the beginning of the experiment to the end, confirming the long-term effectiveness of UCL-TRO-1938 in treating ALS.

[0100] These results clearly demonstrate that UCL-TRO-1938 treatment effectively alleviates SOD1 mutation-induced weight loss, providing strong supporting evidence for its use as a therapeutic strategy for ALS.

[0101] 5. UCL-TRO-1938 can significantly improve / delay gastrocnemius muscle atrophy in ALS model mice

[0102] Gastrocnemius muscle atrophy is one of the key indicators of ALS disease progression, directly reflecting the process of motor neuron degeneration and muscle denervation. In ALS model mice such as SOD1-G93A, this atrophy usually begins in the early stages of the disease and gradually worsens as the disease progresses. Studying gastrocnemius muscle atrophy not only provides important clues for understanding the pathogenesis of ALS, but also provides a reliable experimental basis for evaluating disease progression and testing potential treatments. As one of the main muscles of the lower limbs, changes in the gastrocnemius muscle directly reflect the core pathological characteristics of ALS, namely muscle atrophy caused by motor neuron degeneration, and therefore has important scientific value and clinical significance in ALS research.

[0103] 2.5-month-old SOD1-G93A male mice (n=24) were selected as experimental subjects. According to body weight, they were randomly divided into a vehicle control group (SOD1_Blk group, n=12) and a UCL-TRO-1938 administration group (SOD1_1938, n=12). The vehicle control group was given 0.1 ml / 10 g body weight of vehicle by oral gavage every day; the UCL-TRO-1938 administration group was given 10 mg / kg dose of UCL-TRO-1938 (dissolved in the vehicle, the administration volume was 0.1 ml / 10 g body weight) by oral gavage every day. Among them, the specific preparation method of the administration solution is as follows: UCL-TRO-1938 is dissolved in DMSO to prepare a mother solution, and then diluted at a ratio of 5% DMSO mother solution: 95% corn oil (volume ratio), and fully mixed to obtain the final administration solution. In the experiment, 2.5-month-old C57BL / 6J male mice with the same genetic background were set up as wild-type controls (WT group) (n=12).

[0104] To study gastrocnemius atrophy in SOD1-G93A model mice, the present invention adopts a standardized sampling and filming method. First, the mice were euthanized at 4.5 months of age and the hind limbs were disinfected. Then the skin was carefully peeled off and the entire gastrocnemius muscle was removed from the back of the calf. The removed muscle was placed on a white background with a standard scale next to it. It was photographed using a high-resolution camera.

[0105] Each sample (WT group, SOD1_Blk group, SOD1_1938 group) was photographed multiple times, keeping the settings and angles consistent. The most representative images were selected for comparison, and the sample weight was recorded and the size was measured. The whole process was maintained in a sterile environment and the samples were collected under the same conditions to ensure the comparability of the results.

[0106] The results are as follows Figure 6 As shown in the figure, the gastrocnemius muscle of the WT group mice was larger and appeared healthy pink, which was the normal control group. The gastrocnemius muscle of the SOD1_Blk group mice was significantly atrophied compared with the wild type, with a significantly smaller volume and darker color. The size of the gastrocnemius muscle of the SOD1_1938 group mice was between the WT group and the SOD1_Blk group, indicating that after treatment with UCL-TRO-1938, the gastrocnemius atrophy of the ALS model mice was improved to a certain extent.

[0107] 6. UCL-TRO-1938 can significantly reduce neuroinflammation in ALS model mice

[0108] Studies in recent years have shown that glial cells play a key role in the pathogenesis of ALS. In ALS patients and animal models, abnormal activation and proliferation of microglia and astrocytes is a significant pathological feature, a phenomenon known as "reactive gliosis." In ALS, reactive gliosis is often considered a double-edged sword because it often represents the occurrence of neurodegeneration. Initially, it may have a protective effect, but as the disease progresses, overactivated glial cells may release inflammatory factors and neurotoxic substances, accelerating neuronal death.

[0109] 2.5-month-old SOD1-G93A male mice (n=12) were selected as experimental subjects. According to body weight, they were randomly divided into a vehicle control group (SOD1_Blk group, n=6) and a UCL-TRO-1938 administration group (SOD1_1938, n=6). The vehicle control group was given 0.1 ml / 10 g body weight of vehicle by oral gavage every day; the UCL-TRO-1938 administration group was given 10 mg / kg dose of UCL-TRO-1938 (dissolved in the vehicle, the administration volume was 0.1 ml / 10 g body weight) by oral gavage every day. Among them, the specific preparation method of the administration solution is as follows: UCL-TRO-1938 is dissolved in DMSO to prepare a mother solution, and then diluted at a ratio of 5% DMSO mother solution: 95% corn oil (volume ratio), and fully mixed to obtain the final administration solution. In the experiment, 2.5-month-old C57BL / 6J male mice with the same genetic background were set up as wild-type controls (WT group, n=6).

[0110] To evaluate the effect of UCL-TRO-1938 treatment on glial proliferation in ALS model mice. Mice were euthanized at 4.5 months of age and 10 μm thick paraffin sections of the mouse spinal cord lumbar segment were used for labeling and quantitative analysis of microglia and astrocytes by immunofluorescence staining. The specific details are as follows: After the spinal cord lumbar segment of the above mice was obtained, the tissue was fixed with 4% paraformaldehyde (PFA) (4°C, 24 hours) and then embedded in paraffin. 10 μm thick sections were prepared using a microtome, dewaxed in xylene and rehydrated with graded ethanol, and antigen retrieval (citric acid or EDTA buffer may be used). The immunofluorescence staining process was as follows: 5% goat serum was blocked (room temperature, 1 hour), and primary antibodies: rabbit anti-IBA1 (Wako, 1:500) and mouse anti-GFAP (Millipore, 1:1000) were added, and incubated overnight at 4°C. The next day, secondary antibodies were added: Alexa Fluor 488-labeled anti-rabbit IgG and Alexa Fluor 594-labeled anti-mouse IgG (Invitrogen, 1:1000) and incubated at room temperature for 2 hours. Finally, the slides were mounted with Fluoromount-G (Southern Biotech) containing DAPI (1:5000, Sigma). After immunofluorescence staining, images were collected using a fluorescence microscope.

[0111] The results are as follows Figure 7 As shown, A and C show the immunofluorescence staining results of IBA1 and GFAP, respectively, and the scale bar is 100 μm. Image analysis software was used to analyze the immunofluorescence staining results of IBA1. + and GFAP + The cells were counted. B and D show the statistical results of cell counting in the anterior horn and anterior column of the spinal cord. The data are expressed as mean ± standard error (SEM).

[0112] The results showed that compared with the SOD1_Blk group, the number of microglia in the lumbar spinal cord of mice in the SOD1_1938-treated group was significantly reduced, and the number of astrocytes was also significantly reduced. Although the number of glial cells in the SOD1_1938 group was still higher than that in the WT group, the treatment effect was significant. These findings indicate that UCL-TRO-1938 can effectively inhibit the abnormal proliferation of glial cells associated with ALS.

[0113] VII. UCL-TRO-1938 can significantly reduce the death of motor cortical neurons in ALS model mice

[0114] The present invention evaluated the effects of UCL-TRO-1938 treatment on the number of neurons and microglial activation in layer VI of the motor cortex of ALS model mice.

[0115] 2.5-month-old SOD1-G93A male mice (n=6) were selected as experimental subjects. According to body weight, they were randomly divided into a vehicle control group (SOD1_Blk group, n=3) and a UCL-TRO-1938 administration group (SOD1_1938, n=3). The vehicle control group was given 0.1 ml / 10 g body weight of vehicle by oral gavage every day; the UCL-TRO-1938 administration group was given 10 mg / kg dose of UCL-TRO-1938 (dissolved in the vehicle, the administration volume was 0.1 ml / 10 g body weight) by oral gavage every day. Among them, the specific preparation method of the administration solution is as follows: UCL-TRO-1938 is dissolved in DMSO to prepare a mother solution, and then diluted at a ratio of 5% DMSO mother solution: 95% corn oil (volume ratio), and fully mixed to obtain the final administration solution. In the experiment, 2.5-month-old C57BL / 6J male mice with the same genetic background were set up as wild-type controls (WT group, n=3).

[0116] Mice were euthanized at 4.5 months of age and 10 μm thick frozen sections of mouse brain were analyzed by immunofluorescence staining. The tissues were fixed with 4% paraformaldehyde (PFA) (4°C, 24 hours) and then embedded in paraffin. 10 μm thick sections were prepared using a microtome, dewaxed in xylene and rehydrated with graded ethanol, and antigen retrieval (citric acid or EDTA buffer may be used). Immunofluorescence staining was performed using anti-NeuN antibody (1:500, Millipore) to label neurons and anti-IBA1 antibody (1:1000, Wako) to label microglia, incubated overnight at 4°C. The next day, the corresponding AlexaFluor fluorescent secondary antibody (1:1000, Invitrogen) was incubated for 2 hours at room temperature, and DAPI (1:10000) was used to stain the nucleus for 10 minutes. Images were acquired using a Zeiss LSM 880 confocal microscope, 40× oil objective, and z-stack interval of 1 μm. ImageJ software was used to analyze the NeuN in three random fields of view for each sample. + Count the cells and measure at least 50 IBA1 + The cell body area.

[0117] The results are as follows Figure 8 As shown:

[0118] Figure 8 A in the middle shows the results of immunofluorescence staining of layer VI of the motor cortex of three groups of mice: WT, SOD1_Blk, and SOD1_1938. DAPI (blue) marks the nucleus, NeuN (red) marks the neurons, and IBA1 (green) marks the microglia. The number of NeuN-positive cells in the SOD1_1938 group was significantly higher than that in the SOD1_Blk group, close to the level of the WT group.

[0119] Figure 8 Quantitative analysis in Figure B showed that the number of neurons in the SOD1_1938 group was significantly higher than that in the SOD1_Blk group (p<0.05), indicating that UCL-TRO-1938 administration can effectively slow down the neuronal loss caused by SOD1 mutation.

[0120] Figure 8 Center C shows the statistical results of microglial cell body area, which was significantly smaller in the SOD1_1938 group than in the SOD1_Blk group (p<0.01), close to the WT level, indicating that UCL-TRO-1938 administration can inhibit the abnormal activation of microglia.

[0121] Overall, UCL-TRO-1938 treatment significantly increased neuronal density and reduced microglial cell body area, indicating that UCL-TRO-1938 can effectively protect layer VI neurons in the motor cortex and inhibit the abnormal activation of microglia.

[0122] 8. UCL-TRO-1938 can significantly reduce the death of anterior horn motor neurons in the spinal cord of ALS model mice

[0123] 2.5-month-old SOD1-G93A male mice (n=12) were selected as experimental subjects. According to body weight, they were randomly divided into a vehicle control group (SOD1_Blk group, n=6) and a UCL-TRO-1938 administration group (SOD1_1938, n=6). The vehicle control group was given 0.1 ml / 10 g body weight of vehicle by oral gavage every day; the UCL-TRO-1938 administration group was given 10 mg / kg dose of UCL-TRO-1938 (dissolved in the vehicle, the administration volume was 0.1 ml / 10 g body weight) by oral gavage every day. Among them, the specific preparation method of the administration solution is as follows: UCL-TRO-1938 is dissolved in DMSO to prepare a mother solution, and then diluted at a ratio of 5% DMSO mother solution: 95% corn oil (volume ratio), and fully mixed to obtain the final administration solution. In the experiment, 2.5-month-old C57BL / 6J male mice with the same genetic background were set up as wild-type controls (WT group, n=6).

[0124] To evaluate the protective effect of UCL-TRO-1938 treatment on spinal cord anterior horn motor neurons in ALS model mice, mice were euthanized at 4.5 months of age and 10 μm thick paraffin sections of mouse spinal cord were analyzed by immunofluorescence staining. The tissues were fixed with 4% paraformaldehyde for 24 hours, dehydrated, transparent, and embedded in wax. The sections were dewaxed with xylene, rehydrated with gradient ethanol, and antigen retrieval was performed with 0.01 M citrate buffer (pH 6.0) (95°C, 20 minutes).

[0125] Immunofluorescence staining used anti-ChAT antibody (1:200, Millipore) to label motor neurons and incubated overnight at 4°C. The next day, Alexa Fluor 488-labeled anti-rabbit IgG secondary antibody (1:1000, Invitrogen) was used for incubation at room temperature for 2 hours, and DAPI (1:10000) was used to stain the nucleus for 10 minutes. Images were collected using a Zeiss LSM 880 confocal microscope, 20× objective, and z-stack interval of 1μm. ImageJ software was used to analyze ChAT in three random fields of view for each sample. + The cells were counted and the cell density per square millimeter was calculated.

[0126] The results are as follows Fig. 9 As shown, the results showed that the density of spinal anterior horn motor neurons in the SOD1_1938 group of mice was significantly increased compared with the SOD1_Blk group. This indicates that UCL-TRO-1938 treatment has a protective effect on spinal anterior horn motor neurons in ALS model mice.

[0127] IX. UCL-TRO-1938 can significantly reduce the nuclear and cytoplasmic ectopic localization and aggregation of TDP-43 in motor neurons of ALS model mice

[0128] The present invention evaluates the effect of UCL-TRO-1938 treatment on the distribution of TDP-43 in the anterior horn motor neurons of the spinal cord of ALS model mice. TDP-43 (TAR DNA-binding protein 43) is normally located in the nucleus and participates in RNA metabolism and gene expression regulation. In SOD1 mutant mice, TDP-43 was observed to transfer from the nucleus to the cytoplasm, which is highly consistent with the pathological characteristics of human ALS patients.

[0129] 2.5-month-old SOD1-G93A male mice (n=12) were selected as experimental subjects. According to body weight, they were randomly divided into a vehicle control group (SOD1_Blk group, n=6) and a UCL-TRO-1938 administration group (SOD1_1938, n=6). The vehicle control group was given 0.1 ml / 10 g body weight of vehicle by oral gavage every day; the UCL-TRO-1938 administration group was given 10 mg / kg dose of UCL-TRO-1938 (dissolved in the vehicle, the administration volume was 0.1 ml / 10 g body weight) by oral gavage every day. Among them, the specific preparation method of the administration solution is as follows: UCL-TRO-1938 is dissolved in DMSO to prepare a mother solution, and then diluted at a ratio of 5% DMSO mother solution: 95% corn oil (volume ratio), and fully mixed to obtain the final administration solution. In the experiment, 2.5-month-old C57BL / 6J male mice with the same genetic background were set up as wild-type controls (WT group, n=6).

[0130] Mice were euthanized at 4.5 months of age and 10 μm thick paraffin sections of the lumbar spinal cord were analyzed by immunofluorescence staining. The tissues were fixed with 4% paraformaldehyde for 24 hours, dehydrated, transparent, and embedded in wax. The sections were dewaxed with xylene, rehydrated with graded ethanol, and antigen retrieval was performed with 0.01 M citric acid buffer (pH 6.0) (95°C, 20 minutes).

[0131] Immunofluorescence staining used anti-ChAT antibody (1:200, Millipore) to label motor neurons and anti-TDP-43 antibody (1:500, ProteinTech), and incubated overnight at 4°C. The next day, Alexa Fluor 488-labeled anti-rabbit IgG and Alexa Fluor 594-labeled anti-mouse IgG secondary antibodies (both 1:1000, Invitrogen) were used for incubation at room temperature for 2 hours, and DAPI (1:10000) was used to stain the nucleus for 10 minutes. Images were collected using a Zeiss LSM 880 confocal microscope, 63× oil objective, and z-stack interval of 0.5μm. ImageJ software was used to analyze 10 randomly selected ChAT cells in each sample. + The nuclear-cytoplasmic fluorescence intensity ratio of TDP-43 was analyzed in the cells.

[0132] The results are as follows Fig.10 As shown, the results showed that the nuclear-cytoplasmic ratio of TDP-43 in the SOD1_1938-treated group was significantly increased compared with the SOD1_Blk group (p<0.05). These findings indicate that UCL-TRO-1938 can effectively improve the abnormal distribution of TDP-43 in motor neurons of ALS model mice and maintain its normal nuclear localization.

[0133] Based on the results of this example, it can be seen that the ALS model mice treated with UCL-TRO-1938 showed significant improvements in survival time, motor function, weight change and other aspects, and UCL-TRO-1938 showed a positive therapeutic effect.

[0134] Example 2: In vitro cell experiments verify the effect of UCL-TRO-1938 in treating amyotrophic lateral sclerosis

[0135] 1. UCL-TRO-1938 can significantly reduce abnormal pathological changes of TDP-43 in motor neurons induced by patient iPSCs

[0136] TDP-43 is an RNA-binding protein that is mainly localized in the cell nucleus. Under normal circumstances, it participates in a variety of RNA metabolic processes, including transcriptional regulation, splicing, mRNA stability and transport. Its correct nuclear localization is essential for maintaining the normal function of neurons. In the motor neurons of ALS patients, TDP-43 is often mislocalized and moves from the nucleus to the cytoplasm. This mislocalization may lead to two major problems: loss of function in the nucleus and acquisition of toxicity in the cytoplasm. Loss of function in the nucleus may affect normal RNA metabolism, while TDP-43 in the cytoplasm may form toxic protein aggregates.

[0137] Sodium arsenite (AS), as an oxidative stress inducer, can induce stress response and thus induce typical pathological manifestations of ALS, namely, nuclear-cytoplasmic translocation of TDP-43 and formation of abnormal aggregates.

[0138] The present invention utilizes the iPSC-induced differentiated motor neuron (iMN) model to evaluate the protective effect of UCL-TRO-1938 on motor neurons.

[0139] iPSCs were induced for neural development by the dual SMAD inhibition method. When the iPSC confluence reached about 80%, iPSCs were separated into small clumps using 1×ReleSR and inoculated into a culture dish containing 1×mTeSR and Y2763210μM). On the second day, mTeSR was replaced with the first stage culture medium (formula: basal culture medium is composed of 50% IMDM and 50% DMEM / F12, and the remaining additives are added on this basis, and 1% NEAA, 2% B27 supplement, 1% N2 supplement, 1% P / S, 20μM SB-431542, 0.1μM LDN-193189 and 3μM CHIR-99021 are added in sequence) to induce neural epithelium, and the medium is changed every day. On day 5-6, when the cell confluence reached about 80%, the cells were separated with 1×Accutase and replaced with the second-stage culture medium (formula: the first-stage culture medium plus 0.1μM RA, 1μM SAG and 10μM Y-27632) at 7.5×10 5 The cells were re-seeded at a density of 10 cells / well onto a matrigel-coated 6-well plate, Y-27632 was removed after 24 hours, and the cells were cultured for 2 generations with the second-stage medium to induce the generation of motor neuron precursors. From the fourth generation onwards, the third-stage medium (formula: the basal medium is composed of 50% IMDM and 50% DMEM / F12, and the remaining additives are added on this basis, and 1% NEAA, 2% B27 supplement, 1% N2 supplement, 1% P / S, 2.5μM DAPT, 0.1μM cAMP, 0.5μM retinoic acid, 0.1μM SAG, 200ng / ml ascorbic acid, 10ng / ml BDNF and 10ng / ml GDNF are added in sequence) were replaced every other day until the motor neurons matured.

[0140] For ALS patients (Patient) and healthy controls (Control), three subgroups were set up: Blank (administered with solvent reagent DMSO), AS treatment (0.5mM, 1h) and AS+UCL-TRO-1938 (10μM / 24h) treatment.

[0141] After treatment, cells were fixed with 4% PFA for 20 minutes, permeabilized with 0.03% Triton X-100 for 20 minutes, and blocked with 5% BSA for 1 hour. Immunofluorescence staining was performed with anti-TDP-43 antibody (Proteintech, 10782-2-AP, 1:500) and anti-MAP2 antibody (Proteintech, 67015-1-1g, 1:1000) at 4°C overnight, and the next day, Alexa Fluor 488-labeled anti-rabbit IgG and Alexa Fluor 594-labeled anti-mouse IgG (both Invitrogen, 1:1000) were incubated at room temperature for 2 hours, and DAPI (1:10000) was used to stain the nucleus for 5 minutes. Images were collected using a Zeiss LSM 880 confocal microscope, 63× oil objective, and z-stack interval of 0.5μm. TDP-43 nuclear-cytoplasmic fluorescence intensity ratio analysis was performed on 12 randomly selected cells in each group using ImageJ software.

[0142] The results are as follows Fig.11 As shown, the results showed that: after the treatment of vehicle reagent (Blank) and sodium arsenite stimulation (AS), TDP-43 nuclear-cytoplasmic heterotopia was observed in the induced motor neurons (iMN) of healthy controls (Control), and the treatment of UCL-TRO-1938 basically restored it; after the treatment of vehicle reagent (Blank) and sodium arsenite stimulation (AS), the iMN derived from ALS patients (Patient) showed obvious TDP-43 nuclear-cytoplasmic heterotopia, and the basal level of TDP-43 retention in the cytoplasm was higher than that of the control, indicating obvious disease characteristics. When the patient-derived iMN was stimulated with AS and UCL-TRO-1938 (1938) was added at the same time, TDP-43 nuclear entry could be restored.

[0143] 2. UCL-TRO-1938 can significantly reduce the apoptosis of motor neurons induced by patient iPSCs

[0144] The present invention uses the iPSC-induced motor neuron model to evaluate the effect of sodium arsenite (AS) on cell apoptosis and the potential protective effect of UCL-TRO-1938 compound. The induction of motor neurons is the same as above.

[0145] For ALS patients (Patient) and healthy controls (Control), three subgroups were set up: Blank (administered with solvent reagent DMSO), AS treatment (0.5mM, 1h) and AS+UCL-TRO-1938 (10μM / 24h) treatment.

[0146] After treatment, cells were stained with TUNEL. TUNEL staining was performed using the Servicebio Fluorescein (FITC) Tunel Cell Apoptosis Detection Kit according to the manufacturer's instructions. First, fix with 4% PFA at room temperature for 20 minutes, permeabilize with 0.03% Triton X-100 at room temperature for 20 minutes, add an appropriate amount of Equilibration Buffer to each sample to cover the entire sample area to be tested, and incubate at room temperature for 10 minutes. Mix Recombinant TdT enzyme: FITC-12-dUTP Labeling Mix: Equilibration Buffer = 1μL: 5μL: 50μL of TdT incubation buffer sufficient for all experiments, remove the balanced Equilibration Buffer as much as possible, then add an appropriate amount of TdT incubation buffer to each tissue sample, incubate at 37℃ in the dark for 1 hour, wash with PBS, and stain the nucleus with DAPI (1:1000) for 5 minutes. Images were collected using a Zeiss LSM 880 confocal microscope with a 20x lens. Image J software was used to analyze four randomly selected fields of view in each group, and the proportion of Tunel-positive cells to the total number of cells was calculated.

[0147] The results are as follows Fig.12 As shown, the results showed that after sodium arsenite stimulation (AS) was given to the induced motor neurons (iMN) of the healthy control (Control), the number of apoptotic cells increased significantly compared with the vehicle reagent (Blank), and the simultaneous administration of UCL-TRO-1938 could partially reduce neuronal apoptosis. Compared with the healthy control, the basal apoptosis level of iMN derived from ALS patients (Patient) was significantly increased; at the same time, after sodium arsenite stimulation (AS), the number of apoptotic cells increased significantly compared with the vehicle reagent (Blank), and the simultaneous administration of UCL-TRO-1938 could partially reduce neuronal apoptosis. This is consistent with the neuronal degeneration characteristics of ALS. UCL-TRO-1938 exhibits a strong neuroprotective effect and can effectively reduce cell apoptosis, and the effect is more significant in the disease group.

[0148] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. Use of UCL-TRO-1938 or a pharmaceutically acceptable salt, solvate, prodrug, stereoisomer, isotopic variant or tautomer thereof in the preparation of a product for treating amyotrophic lateral sclerosis.

2. Use of UCL-TRO-1938 or a pharmaceutically acceptable salt, solvate, prodrug, stereoisomer, isotope variant or tautomer thereof in the preparation of a product for improving motor dysfunction in an organism suffering from amyotrophic lateral sclerosis.

3. Use of UCL-TRO-1938 or a pharmaceutically acceptable salt, solvate, prodrug, stereoisomer, isotopic variant or tautomer thereof in any of the following: (A1) preparing a product for prolonging the survival of an organism suffering from amyotrophic lateral sclerosis; (A2) preparing a product for delaying disease progression in an organism suffering from amyotrophic lateral sclerosis; (A3) preparing a product for delaying the decline of motor function in an organism suffering from amyotrophic lateral sclerosis; (A4) preparing a product for slowing down the rate of weight loss in patients with amyotrophic lateral sclerosis; (A5) preparing a product for improving gastrocnemius muscle atrophy in an amyotrophic lateral sclerosis patient; (A6) preparing a product for reducing neuroinflammation in an amyotrophic lateral sclerosis patient; (A7) preparing a product for reducing the death of motor cortex neurons in an amyotrophic lateral sclerosis patient; (A8) preparing a product for reducing the death of anterior horn motor neurons in the spinal cord of an organism suffering from amyotrophic lateral sclerosis; (A9) Preparing a product for reducing the nuclear and cytoplasmic ectopic location and aggregation of TDP-43 in motor neurons of an amyotrophic lateral sclerosis patient.

4. Use of UCL-TRO-1938 or a pharmaceutically acceptable salt, solvate, prodrug, stereoisomer, isotopic variant or tautomer thereof in any of the following: (B1) Reduce the abnormal pathological changes of TDP-43 in motor neurons derived from iPSCs of patients with amyotrophic lateral sclerosis in vitro; (B2) Reduced apoptosis of motor neurons derived from iPSCs of patients with amyotrophic lateral sclerosis in vitro; (B3) preparing a product for reducing abnormal pathological changes of TDP-43 in motor neuron cells induced by iPSCs from patients with amyotrophic lateral sclerosis; (B4) Prepare a product for reducing apoptosis of motor neuron cells induced by iPSCs from patients with amyotrophic lateral sclerosis.

5. Use of UCL-TRO-1938 or a pharmaceutically acceptable salt, solvate, prodrug, stereoisomer, isotopic variant or tautomer thereof in any of the following: (C1) Reversing the abnormal pathological changes of TDP-43 in motor neurons induced by sodium arsenite in vitro; (C2) Reversal of sodium arsenite-induced apoptosis of motor neurons in vitro; (C3) preparing a product for reversing the abnormal pathological changes of TDP-43 in motor neuron cells induced by sodium arsenite; (C4) Preparing a product for reversing sodium arsenite-induced apoptosis of motor neuron cells.

6. The use according to claim 5, characterized in that: The motor neuron cells are motor neuron cells induced from iPSCs of patients with amyotrophic lateral sclerosis.

7. A method for reversing the abnormal pathological changes of TDP-43 in motor neuron cells induced by sodium arsenite in vitro, comprising the following steps: treating the motor neuron cells with UCL-TRO-1938 or a pharmaceutically acceptable salt, solvate, prodrug, stereoisomer, isotopic variant or tautomer thereof simultaneously or after treating the motor neuron cells with sodium arsenite in vitro.

8. A method for reversing apoptosis of motor neuron cells induced by sodium arsenite in vitro, comprising the following steps: treating the motor neuron cells with UCL-TRO-1938 or a pharmaceutically acceptable salt, solvate, prodrug, stereoisomer, isotopic variant or tautomer thereof simultaneously or after treating the motor neuron cells with sodium arsenite in vitro.

9. The method according to claim 8, characterized in that: The motor neuron cells are motor neuron cells induced from iPSCs of patients with amyotrophic lateral sclerosis.

Images