Use of small molecule compound 2-D08 in the preparation of a medicament for treating or preventing demyelinating diseases

Abstract

The present invention provides the use of the small molecule compound 2-D08 in the preparation of a drug for treating or preventing demyelinating diseases. As a small molecule compound, 2-D08 has a small molecular weight and is easily permeable through the blood-brain barrier to exert central pharmacodynamic effects. Moreover, the chemical synthesis method of 2-D08 is also very mature, which is conducive to reducing the preparation cost of drugs for treating demyelinating diseases, thereby alleviating the medical expenses of patients to a certain extent. 2-D08 can promote the regeneration of myelin sheaths of spinal cord axons, significantly improve the motor ability and nerve function recovery of mice, and is expected to become a disease-modifying treatment drug targeting the origin of brain damage in MS patients, with brain permeability and ion channel selectivity, and has very broad application prospects.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and particularly to a drug for treating and preventing demyelinating diseases. Background Art

[0002] Multiple Sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system. The disorder of the myelin sheath structure and function not only hinders the conduction of electrical signals of neuron axons, but also affects the nutrition and metabolism of neurons, thereby leading to neuron degeneration and death. The MS lesions involve regions such as cerebral white matter, spinal cord, optic nerve, brainstem and cerebellum, which will cause a series of neurological symptoms, such as cognitive impairment, limb numbness, ataxia, etc. Chronic demyelination and axonal degeneration are considered to be the main factors leading to permanent neurological dysfunction in MS patients. At present, there is no exact and effective treatment method for central demyelinating injury, and there is an urgent need to find an appropriate treatment strategy.

[0003] Oligodendrocytes are the myelin-forming cells of the central nervous system. The differentiation process includes three stages: oligodendrocyte precursor cells (OPCs), immature oligodendrocytes, and mature oligodendrocytes. Under normal physiological conditions, the number of oligodendrocyte precursor cells remains relatively stable. However, in demyelinating diseases, OPCs will undergo reactive proliferation and migrate to the damaged area, differentiating into mature oligodendrocytes to repair the myelin sheath. Therefore, OPCs are considered the main source cells for myelin regeneration. However, with the progression of demyelinating diseases, such as in the late stage of MS, the efficiency of spontaneous remyelination repair gradually decreases, resulting in irreversible neurological dysfunction. Therefore, in-depth understanding of the regulatory mechanisms of OPC proliferation and differentiation can provide new ideas and new strategies for intervening in the treatment of myelin regeneration in demyelinating diseases from the cellular source. Our previous experiments found that the expression of the inward rectifier potassium ion channel subtype 4.1 (Kir4.1) ion channel runs through the entire development process of oligodendrocyte lineage cells. In cerebral ischemia diseases, the defect of the Kir4.1 ion channel may be a key factor causing axonal demyelination in mice neurons. Therefore, it is suggested that the Kir4.1 ion channel may play an important role in promoting the differentiation of OPCs into oligodendrocytes, and thus promoting myelin formation and regeneration. Based on this, the inventor constructed a mouse demyelinating disease model, namely the experimental autoimmune encephalomyelitis (EAE) disease model. EAE well mimics the onset of MS in terms of pathology and clinical manifestations, and is a recognized experimental animal model for studying MS diseases. Given that oligodendrocytes are the key cell source for myelin formation, we therefore propose the hypothesis that the Kir4.1 ion channel in oligodendrocyte lineage cells may be a potential target for treating demyelinating diseases.

[0004] Small molecule compounds have the characteristics of low side effects, easy absorption, easy penetration through the blood-brain barrier, and wide sources compared with general macromolecular drugs. 2-D08 is a flavonoid drug that mainly acts as a small ubiquitin-like modifier protein (sumoylation, SUMO) inhibitor. SUMOylation modification is a multi-effect regulator that participates in regulating various processes such as cell differentiation, protein-protein interaction, and DNA repair. There is currently no literature report on the relationship between 2-D08 and demyelinating diseases and multiple sclerosis.

[0005]

[0006] Chemical structure formula of 2-D08 Summary of the invention

[0007] The object of the present invention is to provide the use of the small molecule compound 2-D08 or its salt in the preparation of a drug for treating or preventing demyelinating diseases, so as to solve the problems in the prior art.

[0008] To achieve the above object and other related objects, on the one hand, the present invention provides the use of the small molecule compound 2-D08 or its salt in the preparation of a drug for treating or preventing demyelinating diseases.

[0009] In some embodiments of the present invention, the demyelinating diseases include multiple sclerosis, neuromyelitis optica, Guillain-Barré syndrome, acute disseminated encephalomyelitis, multiple radiculoneuritis, ischemic encephalopathy, Alzheimer's disease.

[0010] In some embodiments of the present invention, 2-D08 or its salt in the drug is used as the sole active ingredient.

[0011] In some embodiments of the present invention, the effective dose of 2-D08 or its salt in the drug is 0.2-1 mg / kg.

[0012] In some embodiments of the present invention, the drug treats or prevents demyelinating diseases by activating the Kir4.1 ion channel.

[0013] On the other hand, the present invention provides the use of the small molecule compound 2-D08 or its salt in the preparation of a drug for activating the Kir4.1 ion channel, as a Kir4.1 ion channel opener.

[0014] In the present invention, the "small molecule compound 2-D08 or its salt" refers to the small molecule compound 2-D08 or a pharmaceutically acceptable salt of 2-D08, specifically referring to a salt formed by 2-D08 and an acid or a base that is suitable for use as a drug. The present invention provides a basis for lead compounds for the treatment of demyelinating diseases with 2-D08, 2-D08 salts, 2-D08 precursors or 2-D08 hydrates. Pharmaceutically acceptable salts include inorganic salts and organic salts. Among them, one type of salt is the salt formed by the compounds of the present invention and an acid. Acids suitable for forming salts include, but are not limited to: inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid; organic acids such as formic acid, acetic acid, trifluoroacetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, benzoic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, naphthalenesulfonic acid; and amino acids such as proline, phenylalanine, aspartic acid, glutamic acid. Another type of salt is the salt formed by the compounds of the present invention and a base. Bases suitable for forming salts include, but are not limited to: alkali metal salts (such as sodium salts or potassium salts), alkaline earth metal salts (such as magnesium salts or calcium salts), ammonium salts (such as lower alkanolammonium salts and other pharmaceutically acceptable amine salts), such as methylamine salts, ethylamine salts, propylamine salts, dimethylamine salts, trimethylamine salts, diethylamine salts, triethylamine salts, tert-butylamine salts, ethylenediamine salts, hydroxyethylamine salts, dihydroxyethylamine salts, trihydroxyethylamine salts, and amine salts formed by morpholine, piperazine, and lysine respectively.

[0015] In the present invention, "Demyelinating Diseases" refers to a neurological disease in which the loss of nerve myelin sheath is the main or initial pathological change, and the axons, cell bodies, and neuroglia are relatively less damaged, and it can occur in the central nervous system or the peripheral nervous system. Demyelinating diseases are divided into two types, namely, myelin formation disorder type and myelin destruction type. Demyelinating diseases of the myelin formation disorder type are caused by genetic metabolic defects in myelin formation, mainly including diseases such as leukodystrophy caused by abnormal myelin lipid metabolism, such as metachromatic leukodystrophy, spongy degeneration of white matter of the brain, and adrenoleukodystrophy. Demyelinating diseases of the myelin destruction type are acquired demyelinating diseases, and the causes include: ① immune-mediated, such as acute disseminated encephalomyelitis, multiple sclerosis, acute infectious polyneuritis (Guillain-Barré syndrome, GBS). ② viral infection, such as progressive multifocal leukoencephalopathy, subacute sclerosing panencephalitis. ③ nutritional disorders, such as combined system degeneration, central pontine myelinolysis. ④ hypoxia, such as delayed posthypoxic demyelinating encephalopathy, progressive subcortical ischemic encephalopathy. Generally, when diagnosing demyelinating diseases clinically, it mostly refers to immune-mediated demyelinating diseases, including multiple sclerosis, acute disseminated encephalomyelitis, multiple neuritis, etc.

[0016] In the present invention, 2-D08 or its salt in a drug is used as the sole active ingredient for treating or preventing demyelinating diseases, or 2-D08 or its salt can be used as the main active ingredient and combined with other drugs for treating or preventing demyelinating diseases.

[0017] In the present invention, "effective dose" refers to an effective amount for treating or alleviating the severity of one or more diseases mentioned in the present invention. The small molecule compound 2-D08 of the present invention can cause myelin regeneration and repair within a quite wide dose range, especially significantly within a very low dose range, such as an in vivo injection dose range of 0.2 - 1 mg / kg.

[0018] In the present invention, "potassium ion channel opener" refers to a reagent that can increase or upregulate the activity of potassium ion channels (specifically, Kir4.1 ion channels in the present invention). The activity of the Kir4.1 channel refers to the activity that allows potassium ions to pass through the cell membrane. The Kir4.1 channel regulates the potassium ion concentration in the extracellular fluid around nerve cells, transports excessive extracellular potassium ions to buffer the extracellular environment, and achieves the homeostasis balance of cytoplasmic potassium ions and the electrical activity level of neurons. In particular, it is found in the present invention that the Kir4.1 channel opener has a direct myelin repair function for neurons. The Kir4.1 ion channel opener includes compounds, complexes or mixtures that can increase the activity of the Kir4.1 channel, as well as preparations used in methods for enhancing Kir4.1 activity, etc.

[0019] In the present invention, using the Kir4.1 potassium ion channel protein as the screening target and a flavonoid small molecule compound library as the screening object, a candidate small molecule compound with strong affinity for the Kir4.1 potassium ion channel protein is screened out by the thermal shift analysis experiment of TSA. It is found that 2-D08 can be used as a potassium ion channel activator. By opening the Kir4.1 channel, it directly repairs the defect of the Kir4.1 channel expressed by OPC cells in the spinal cord of EAE mice, and then causes the regeneration and repair of the myelin sheath of neuronal axons. In vivo behavioral experiments and electrophysiological experiments both show that when EAE mice are given a low-dose injection of 2-D08 (1 mg / kg), the nerve and motor functions of EAE mice can be significantly repaired, and it is the preferred potential drug target for the treatment of MS diseases.

[0020] 2-D08 disclosed in the present invention is the first report of the application of this compound targeting potassium ion channels in the treatment field of demyelinating diseases. The inventors' previous studies found that in patients with acute ischemic stroke, the Kir4.1 ion channels in the ischemic infarct area of the cerebral cortex were severely damaged, and severe demyelination of axons occurred. Further evidence showed that the deletion of Kir4.1 channels in NG2 glial cells led to the loss of axonal myelin in a mouse model of cerebral ischemia. Treating ischemic mice with the natural plant extract luteolin, it was found that luteolin increased the Kir4.1 channel current in NG2 glial cells, thereby promoting axonal myelin regeneration, alleviating the infarct area, and ultimately improving motor function in a series of behavioral tests (Hong et al, eBioMedicine, 2023).

[0021] In the present invention, the inventors also applied luteolin to the treatment of EAE and found that luteolin could also inhibit the disease progression in mice and had a certain promoting effect on the repair of nerve function in mice. However, the effective dose of the drug used was larger than that of 2-D08 (5 mg / kg luteolin vs 1 mg / kg 2-D08). Moreover, in the experiment detecting the activation of Kir4.1 channel current, the percentage of Kir4.1 ion channels activated by 100 μM luteolin was 23%, while a similar effect could be achieved with 50 μM 2-D08. Therefore, it is suggested that 2-D08 is a compound with a stronger Kir4.1 channel activation effect than luteolin reported previously.

[0022] Chinese Patent CN106963755B discloses the application of the flavonoid pinocembrin in the preparation of drugs for the treatment of demyelinating diseases. This patented drug has significant structural differences from 2-D08. Its molecular formula is C 15 H 12 O4, with a molecular weight of 256.25. The hydroxyl groups in its structure are mainly on the A ring of the flavone. Compared with the molecular formula of 2-D08, which is C 15 H 10O5, with a molecular weight of 270.24, differs by one hydroxyl group, but all three hydroxyl groups of 2-D08 are on the B ring. Functionally, pinocembrin can also effectively improve and repair the disease state of EAE, but its drug dosage is 20 - 40 mg / kg, while the effective dosage of 2-D08 is 0.2 - 1 mg / kg. In terms of molecular mechanism, pinocembrin improves the differentiation of NG2 glial cells through the mTOR signaling pathway, and then achieves the effect of repairing myelin sheaths (Shao et al, Neurosci Bull, 2021), which is also completely different from the targeted Kir4.1 potassium channel of 2-D08. Especially, pinocembrin can only improve and partially repair the disease state of EAE mice at a dose as high as 20 mg / kg, which is 20 times higher than the effective dose of 2-D08. Therefore, 2-D08 is a brand-new compound targeting ion channels for the treatment of EAE, and its therapeutic effect is significantly better than that of pinocembrin at the same dose.

[0023] The advantages of the present invention are that the small molecule compound 2-D08 provided by the present invention can significantly relieve the onset process and severity of demyelinating diseases at a lower dose, and at the same time also has a significant effect of promoting myelin regeneration. In addition, as a small molecule compound, 2-D08 has a small molecular weight and is easy to cross the blood-brain barrier to exert central pharmacodynamic effects. Moreover, the chemical synthesis method of 2-D08 is very mature, which is conducive to reducing the preparation cost of demyelinating disease treatment drugs, thereby reducing the medical expenses of patients to a certain extent. 2-D08 can promote the regeneration of spinal cord axon myelin sheaths, significantly improve the motor ability and nerve function recovery of mice, and is expected to become a disease-modifying treatment drug targeting the origin of brain damage in MS patients, with brain permeability and ion channel selectivity, and has a very broad application prospect. Brief Description of the Drawings

[0024] Figure 1 It is the experimental result diagram of successfully constructing an EAE mouse model.

[0025] Figure 2 It is the experimental result diagram of the impaired function of Kir4.1 ion channels expressed by spinal cord NG2 glial cells in EAE.

[0026] Figure 3 It is the experimental result diagram of screening effective Kir4.1 channel openers.

[0027] Figure 4 It is the experimental result diagram that 2-D08 can effectively induce the opening of Kir4.1 potassium channels.

[0028] Figure 5 It is the experimental result diagram that 2-D08 can improve the myelin repair and motor ability of EAE mice.

[0029] Figure 6It is the experimental result graph showing that 2-D08 can effectively improve the neurological function score of EAE mice.

[0030] Figure 7 It is the experimental result graph showing the comparison of the therapeutic effects of the clinical drug Compound Borneol Tablets (4-AP) and 2-D08 on EAE mice.

[0031] Figure 8 It is the experimental result graph showing the comparison of the effects of luteolin and 2-D08 on the opening of potassium ion channels and the therapeutic effects on EAE mice.

[0032] Figure 9 It is the experimental result graph showing the comparison of the therapeutic effects of pinocembrin and 2-D08 on EAE mice. Detailed implementation manners

[0033] The following describes the technology of the present invention in detail in combination with specific implementation manners. It should be known that the following specific implementation manners are only used to help those skilled in the art understand the present invention, rather than limiting the present invention.

[0034] Through experiments, the present invention found that in the EAE mouse model, the Kir4.1 potassium ion channel expressed in glial cells is damaged, causing neuronal demyelination and nerve function injury. By opening agents of such potassium ion channels (for example: 2-D08), the channel function can be restored to normal, thereby repairing the damaged neuronal function. The Kir4.1 potassium ion channel opener 2-D08 of the present invention achieves the effect of restoring neuronal function through its action on glial cells.

[0035] The following describes in detail through examples the effect of a potassium ion channel opener drug taking 2-D08 as an example on the Kir4.1 potassium ion channel of glial cells and the repair of the nerve function of EAE demyelination-related diseases.

[0036] Example 1: Successfully constructing an EAE mouse model

[0037] Construct an EAE mouse model to detect the changes of the Kir4.1 channel in the disease state.

[0038] Male C57BL / 6 mice about 8 weeks old were used for model preparation, and the immunizing agent was MOG 33-35 protein, with a final concentration of 1 mg / ml, and injected subcutaneously at a dose of 0.3 ml / mouse, generally injecting 2 points behind the ears on both sides. The specific process of preparing the immunizing agent is as follows: Take an appropriate weight of MOG 33-35The protein was formulated into a solution with an initial concentration of 2 mg / ml using PBS, connected to a three-way tube with complete Freund's adjuvant in a volume ratio of 1:1, and repeatedly pushed back and forth to mix evenly. At this time, the mixture became milky white. After the immunization preparation was completed, a PTX solution with a final concentration of 1 ng / μl was prepared using physiological saline and intraperitoneally injected at a dose of 0.2 ml / animal. After 48 h, PTX was intraperitoneally injected again at the same concentration and dose. Figure 1 For the neurological function score and body weight change of EAE mice, Figure 1 A is the use of MOG 33-35 Protein-induced EAE mouse model schematic diagram. The clinical score of the mice reached the peak on the 20th day, accompanied by a decrease in the body weight of the mice. Figure 1 B Representative clinical manifestations of EAE mice (score 3, bilateral hind limb paralysis).

[0039] Example 2: Impaired function of Kir4.1 ion channels expressed by spinal cord NG2 glial cells in EAE

[0040] Using Pdgfrα-CreER TM ; mGFP fluorescence reporter transgenic mice. By using the whole-cell patch-clamp technique and immunoblotting (Western Blot) method, the changes in Kir4.1 channel current and the expression changes of Kir4.1 protein were analyzed under the EAE disease state. Figure 2 A is through immunohistofluorescence chemical experiments, showing that Pdgfrα-CreER TM ; In mGFP mice, GFP-positive cells have good co-localization with NG2 glial cells labeled with NG2 antibody. Figure 2 B is through the whole-cell patch-clamp technique, showing that EAE leads to a significant decrease in the macroscopic current of potassium channels and the current of Kir4.1 channel subtypes in spinal cord NG2 cells, accompanied by depolarization of the cell resting membrane potential and an increase in membrane impedance, suggesting impaired function of Kir4.1 channels in spinal cord NG2 glial cells. Figure 2 C is through the Western Blot method, demonstrating a significant decrease in Kir4.1 channel protein and myelin protein MBP in EAE mice. Figure 2 D are the Ture Gold myelin staining and MBP immunohistochemistry images of control group and EAE mice respectively, showing significant demyelination in EAE mice.

[0041] Example 3: Screening effective Kir4.1 channel openers

[0042] Kir4.1 potassium channel protein is used as the screening target, and the flavonoid small molecule compound drug library is used as the screening object. The thermal shift analysis experiment of TSA (Thermal Shift Assay) is used to screen out candidate small molecule compounds with strong affinity to Kir4.1 potassium channel protein. The specific steps are: the in vitro expressed Kir4.1 purified protein and dye complex are proportioned with each compound in a 384-well plate, and the dissolution curve of each group is detected by Roche LightCycler 480 machine, and the compound with the strongest affinity to Kir4.1 protein is screened and its inhibitory and open properties are verified. Through the screening of the existing natural small molecule library, the present invention selects 7 structural analogs of luteolin, a compound with myelin repair function discovered by the research group in the early stage, namely: kaempferol, genkwanin, lsorhamnetin, 7-Hydroxyflavone, eupatilin, 2-D08, luteolin. Figure 3 A is the binding ability of different small molecule compounds to Kir4.1 protein (except for those specially indicated, the concentration of other small molecules is 1 mM). The results show that 2-D08 exhibits a super strong ability to bind to Kir4.1 protein. Figure 3 B is a TSA experiment using different concentrations of 2-D08. The results show that the stability of 2-D08 binding to Kir4.1 is significantly dose-dependent.

[0043] Example 4: 2-D08 can effectively induce the opening of Kir4.1 potassium ion channel

[0044] Since the above experiment can only screen the affinity of the compound to the protein, the final binding efficiency with the protein is the inhibition or opening of the channel, which still needs further verification. HEK293 cells cultured in vitro were transfected with pLVX-CMV-KCNJ10-IRES-mCherry plasmid, and the rapid drug delivery system was used in the whole-cell patch clamp platform to detect the enhancement of Kir4.1 channel current by candidate compound 2-D08. The morphology of HEK293 cells transfected with KCNJ10 plasmid is as follows Figure 4 As shown. Further analysis of the increase in Kir4.1 current caused by administration of different doses of 2-D08 showed that at a concentration of 50μM, it caused a nearly 20% increase in Kir4.1 channel current (Holding potential: -140mV). This proves that 2-D08 can effectively serve as a Kir4.1 channel opener for the next step of intervention therapy research on EAE mice.

[0045] Example 5: 2-D08 can improve myelin repair and motor ability in EAE mice

[0046] To verify the effects of 2-D08 on the regeneration and repair as well as motor function of EAE mice, the EAE mice were randomly divided into a Sham group (n = 5), an EAE group (n = 10), and an EAE + 2-D08 group (n = 13). After modeling the 2-D08 group of mice, 2-D08 (1 mg / kg) was intraperitoneally injected into them every day, and at the same time, single-item behavioral indexes scoring and comprehensive neurological function scoring were carried out. Through a series of behavioral analyses on the mice: 1) motor evoked potential (MEP); 2) grid walking; 3) inclined plane test; 4) clinical neurological function scoring, etc., the recovery efficacy of 2-D08 on the motor function of mice was evaluated. The MEP electrophysiological experiment is a method for evaluating the function of spinal motor neurons in mice. The stronger the MEP, the more perfect the motor function of the mice; grid walking and inclined plane test are respectively used to detect the indexes of the ability of mice to control the placement of the hind paws and motor function. This experiment is used to evaluate the improvement of 2-D08 on the motor function of mice; the neurological function scoring of mice is used to observe and evaluate the onset of the disease and quantitatively evaluate the functional damage of neurological dysfunction in mice at different stages of the disease. Figure 5 A shows the effect of 2-D08 on the clinical neurological function scoring of EAE mice. The results show that 2-D08 significantly reduces the EAE score. Figure 5 B shows the spinal cord myelin pictures of the Sham group, EAE group, and EAE + 2-D08 group of mice taken by a transmission electron microscope at a magnification of 23000x, as well as the G-ratio fitting curve graph of the myelin sheath and the statistical analysis graph of the myelin sheath thickness. The results show that 2-D08 has an obvious promoting effect on the myelin sheath repair of EAE mice. Figure 5 C and 5D are the behavioral analyses of MEP, grid walking, and inclined plane test on mice, indicating that the motor functions of the mice treated with 2-D08 have been significantly improved.

[0047] Example 6: Evaluation of the effective dose of 2-D08 for improving the neurological function scoring of EAE mice

[0048] To further verify the effect of 2-D08 on the neurological function scoring of EAE mice, the EAE mice were evenly divided into a control group (n = 10), a 2-D08 0.2 mg / kg group (n = 13), and a 2-D08 1 mg / kg group (n = 13). 2-D08 was intraperitoneally injected into each group of EAE mice every day and clinical scoring was carried out. Figure 6Effect of 2-D08 on neurological function score of EAE mice. The results showed that 2-D08 could effectively improve the neurological function score of EAE mice in the low-dose group of 0.2 mg / kg, and could more significantly repair the neurological function of EAE mice in the 1 mg / kg dose group, suggesting that the effective dose range of 2-D08 for improving the neurological function of EAE mice was 0.2 - 1.0 mg / kg.

[0049] Example 7: Comparison of therapeutic effects of clinical drug Fupineng (4-AP) and 2-D08 on EAE mice

[0050] Aminopyridine sustained-release tablets (Fupineng, 4-AP) are the first and only drug globally approved for improving walking dysfunction in multiple sclerosis. The results of animal experiments showed that the therapeutic effect of 2-D08 on EAE was very consistent with that of 4-AP. The specific experimental procedure was as follows: On the 15th day after EAE mouse model establishment, EAE mice were evenly divided into a control group (n = 6), a 4-AP 1 mg / kg group (n = 11), and a 2-D08 1 mg / kg group (n = 11). Each group of mice was intraperitoneally injected with 2-D08 or 4-AP daily and subjected to behavioral experiments such as gridwalking, inclined plane test, and gait analysis. Figure 7 A is the comparison of clinical neurological function scores, Figure 7 B - C are the comparisons of grid walking and inclined plane behavioral analyses, Figure 7 D is the comparison of gait analysis behavioral analysis. The results showed that both the 4-AP group and the 2-D08 group had significantly improved the motor function of EAE mice at the same dose. * indicates that the difference between the 2-D08 group and the control group was significant, and # indicates that the difference between the 4-AP group and the control group was significant.

[0051] Example 8: Comparison of the effects of luteolin and 2-D08 on potassium channel opening and on the treatment of EAE mice

[0052] Luteolin can promote the myelin regeneration of axons in ischemic mice, relieve the infarct area, and improve motor function. The results of animal experiments showed that the therapeutic effect of 2-D08 was significantly better than that of luteolin. The specific experimental procedure was as follows: HEK293 cells cultured in vitro were transfected with the pLVX-CMV-KCNJ10-IRES-mCherry plasmid, and on the whole-cell patch clamp platform using a rapid drug delivery system, the enhancing changes of 2-D08 and luteolin on Kir4.1 channel current were detected. Figure 8 A and 8B are the increases in Kir4.1 channel current caused by different doses of luteolin and 2-D08 respectively. Figure 8C is a statistical result graph. It can be seen that the percentage of luteolin at 100 μM activating the Kir4.1 ion channel is 23%, while 2-D08 at 50 μM can achieve a similar effect. Figure 8 D shows the effects of 2-D08 and luteolin on the neurological function scores of EAE mice. The results indicate that compared with the luteolin (5 mg / kg) group, the 2-D08 group can improve the clinical scores and motor function repair of EAE mice at a lower dose (1 mg / kg).

[0053] Example 9: Comparison of the therapeutic effects of pinocembrin and 2-D08 on EAE mice

[0054] When the dose of pinocembrin is up to 20 mg / kg, it can improve and partially repair the disease state of EAE mice, which is 20 times higher than the effective dose of 2-D08. The specific experimental procedure is as follows: On the 15th day after the EAE mice were modeled, the EAE mice were evenly divided into a control group (n = 6), a pinocembrin 20 mg / kg group (n = 5), a pinocembrin 1 mg / kg group (n = 5), and a 2-D08 1 mg / kg group (n = 11). Each group of mice was intraperitoneally injected with 2-D08 or pinocembrin every day and subjected to grid walking and inclined plane test behavioral experiments. Figure 9 A is the comparison of clinical neurological function scores, Figure 9 B is the comparison of grid walking behavioral analysis, Figure 9 C is the comparison of inclined plane behavioral analysis. The results show that both 20 mg / kg pinocembrin and 1 mg / kg 2-D08 have significantly improved the motor function of EAE mice, while the 1 mg / kg pinocembrin group has no obvious improvement in the neurological function of EAE mice. It shows that the 2-D08 group can improve the clinical scores and motor function repair of EAE mice at a lower dose (1 mg / kg), and the effective dose is 20 times higher than that of pinocembrin. * indicates that the difference comparison between the 2-D08 group and the 1 mg / kg pinocembrin group is significantly significant, and # indicates that the difference comparison between the 20 mg / kg pinocembrin group and the 1 mg / kg pinocembrin group is significantly significant.

[0055] The above is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Use of the small molecule compound 2-D08 or its salt in the preparation of a drug, characterized in that, The drug treats or prevents multiple sclerosis.

2. Use of the small molecule compound 2-D08 or its salt according to claim 1 in the preparation of a drug, characterized in that, 2-D08 or its salt in the drug is used as the sole active ingredient.

3. Use of the small molecule compound 2-D08 or a salt thereof according to claim 1 in the preparation of a medicament, characterized in that, The effective dose of 2-D08 or its salt in the drug is 0.2 - 1 mg / kg.

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