CN118078816A

CN118078816A - Use of Gu Mei tinib in preparing medicine for promoting myelination and regeneration

Info

Publication number: CN118078816A
Application number: CN202211438288.1A
Authority: CN
Inventors: 请求不公布姓名
Original assignee: Tianjin Shuangshuo Pharmaceutical Technology Co ltd
Current assignee: Tianjin Shuangshuo Pharmaceutical Technology Co ltd
Priority date: 2022-11-16
Filing date: 2022-11-16
Publication date: 2024-05-28

Abstract

The application relates to an application of Gu Mei tinib in preparing a medicament for promoting myelination and regeneration, in particular to an application in preparing a medicament for treating multiple sclerosis, in particular to a recurrent progressive multiple sclerosis. The application proves that the siteritinib has the effects of promoting myelination and regeneration after being orally taken and relieving EAE diseases through a C57/BL6 mouse demyelination model and an SJL/J mouse EAE model. Can be used for treating recurrent progressive multiple sclerosis. The application widens the application range of the valley metinib, improves the market value of the valley metinib, and provides a new clinical medicine for demyelinating diseases such as multiple sclerosis and the like.

Description

Use of Gu Mei tinib in preparing medicine for promoting myelination and regeneration

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to application of valley metinib in preparation of medicines for promoting myelination and regeneration.

Background

Multiple sclerosis (multiple sclerosis, MS), a chronic autoimmune disease that occurs in the central nervous system (central nervous system, CNS) with inflammatory demyelination as the primary pathology. The specific etiology and pathogenesis of the disease are currently unknown, and the disease is currently widely considered to be a nervous system difficult and complicated disease caused by the combination of genetic background factors, virus infection factors, autoimmune factors, environmental factors and the like.

The MS lesion position affects multiple parts of the brain ventricle, brain, cerebellum, optic nerve, spinal cord and the like of the CNS, and has the characteristics of high disability rate, easy persistence, relapse, difficult treatment and the like. The main pathologies of the disease include CNS ash, massive loss of myelin sheath in white matter, inflammatory cell infiltration represented by the periphery of small blood vessels, microglial hyperplasia (ganglion formation), axonal degeneration, irreversible progressive neural function degeneration and the like. Paralysis, aphasia, vision disorder, urinary dysfunction, tremor, numbness of sensation, ataxia and the like are the main clinical manifestations.

Recurrent-remitting (RELAPSING REMITTING, RR), primary progressive (primary progressive, PP), secondary progressive (secondary progressive, SP) and progressive recurrent (PRIMARY RELAPSING, PR) are four common clinical courses. Among them, RR-MS encompasses 85% of patients clinically, most commonly.

At present, MS endangers 2.3 hundred million people worldwide, is more common to young people, and has the average age of onset of illness of 30 years, and is one of the main diseases disabled by young people, wherein the proportion of men and women is 1:2. The MS patients clinically manifest symptoms such as ataxia, general paralysis, limb weakness, vision disorder, general pain and the like, so that the life of the patients cannot be self-care. Meanwhile, the disease has the characteristics of high disability rate, difficult treatment, easy persistent recurrence and the like, and brings great burden to families and friends while seriously reducing the life quality of patients. In addition, with the increasing awareness of MS and the increasing progress of imaging detection means, the incidence of MS has been on the rise worldwide. MS onset starts to increase along with changes in national lifestyle and living environment, and onset ages gradually get younger, becoming a clinically common neurological disease.

The causes of genetic background, environmental factors, autoimmune response, virus infection and the like are closely related to the onset of MS, and it is currently acknowledged that MS is mainly an immune-mediated disease caused by the common influence of genetic factors and environmental factors. At the same time, cellular immunity and humoral immunity are involved in this pathological process, resulting in brain and spinal cord injury and lesions in areas of the CNS.

It is currently widely believed that the immune response to myelin antigens mediated by CD4 ⁺ T lymphocytes (helper T cells) is an important pathogenesis of MS, leading to damage and lesions such as T cell-dominated inflammatory cell infiltration edema, demyelination in the CNS. Autoimmune T cells are activated under genetic or environmental induction, and then infiltrate across the blood brain barrier (blood brain barrier, BBB) into the CNS, binding to target antigen presented by APCs, forming antigen presenting cells. They exert various immune effects by secreting large amounts of specific cytokine interactions, promoting or inhibiting the processes of disease development and progression. Thl cells were once thought to be the primary T cells pathogenic to MS. The differentiated and mature Thl cells can secrete cytokines such as tumor necrosis factor a (tumor necrosis factor-a, TNF-alpha), interferon gamma (IFN-gamma), tumor necrosis factor B, IL-12 and the like, and can worsen the inflammatory response symptoms of MS in the process of mediating cellular immunity. And secretion of relevant factors IL-1, IFN-gamma, TNF-alpha and the like of the important inflammatory diseases in the early stage of the disease regulate and control the disease progress of MS.

Th2 cells mainly participate in humoral immunity by secreting relevant regulatory cytokines such as IL-lO, IL-6, IL-4 and the like, so as to reduce inflammatory reaction and relieve symptoms and play a role in protection. It is widely believed that Thl/Th2 cell imbalance is also one of the mechanisms underlying MS pathogenesis and that, in remission, the manifestation is a shift in Thl to Th 2-like responses.

Unlike Thl and Th2 cells, thl7 cells are novel CD4 ⁺ T cells discovered by later research, and the differentiation and development regulation mechanisms are relatively independent, are characterized by secreting a large amount of IL-17 effector, can secrete cytokines such as TNF-alpha, IL-22, IL-21 and the like, and play an important role in the occurrence and development of MS.

Treg cells mainly play a negative regulatory role in the immune system and can control autoimmune reactivity in vivo. In summary, both Thl and Thl7 cells can independently cause the occurrence and development of MS, are considered to be the main pathogenic T cells, can cause autoimmune diseases in which myelin sheath is destroyed by inflammatory and degenerative processes, and have profound significance in discussing the roles of Thl and Thl7 cells and their abnormal expression in MS pathogenesis.

At present, the exact cause and pathogenesis of MS is not yet studied, and it is mostly thought that the causative cause of MS may be an abnormal autoimmune response to central myelin components of individuals carrying genetically predisposed genes under the effect of some exogenous factors such as the acquired environment of viral infection, trauma, etc.

In terms of viral infection, various virus antibodies with increased titers, such as EB virus and human herpesvirus-6, are detected in cerebrospinal fluid and serum of MS patients, and it is presumed that viral infection plays a role in pathogenesis of MS, but at present, viruses are not isolated in brain tissues of MS patients.

In terms of genetic factors, MS has obvious family tendency, and studies indicate that a plurality of micro-genes affect the gene susceptibility of MS together, and genes related to MS mainly include human leukocyte antigen (human leucocyte antigen, HLA) genes, T Cell Receptor (TCR) genes, myelin basic protein (myelin basic protein, MBP) genes, apolipoprotein E genes and the like, and the genes may affect each other.

In terms of environmental factors, living environments such as latitude, ultraviolet irradiation conditions and eating habits can influence the onset and recurrence of MS, which has a high incidence in cold areas at high latitude, while the incidence decreases as it gets closer to the equator, from which it is inferred that the amounts of vitamin D (vitamin D) and ultraviolet irradiation may be related to the onset of multiple sclerosis.

The data of the world health organization (World Health Organization, WHO) shows that in china, the age-standardized mortality rate caused by MS is 0.1/10 ten thousand, and although the morbidity and mortality rate level is not high, the population base of China is large, and moreover, the disease is more frequent in the middle-aged and the young, and is easy to recur and often causes sequelae with different degrees, so that the life quality of patients and families thereof is greatly influenced, and the society and families bear economic and mental burden.

For the treatment of MS, no medicine capable of thoroughly curing is available at present, and the medicine can only suspend the illness state, delay the recurrence rate and reduce inflammatory response. Currently, drugs such as immunomodulators, immunosuppressants and the like are mainly used in clinic.

The treatment of MS mainly comprises acute phase treatment, remission phase treatment, symptomatic treatment and rehabilitation treatment. In 1993, disease-modifying treatments (DMTs) were used to treat multiple sclerosis, DMTs aimed at controlling disease progression, promoting repair of nervous tissue, prolonging remission time, preventing or reducing functional disability, having objective efficacy and showing superiority in improving the course of MS disease, however, the therapeutic objective of no recurrent attacks and no progression of disability has not yet been achieved yet, and currently no commercially available disease-modifying therapeutic drugs developed at home and abroad have been marketed.

NMPA (National Medical Products Administration national food and drug administration) has been approved to introduce interferon beta-1 alpha (Interferons beta-1 alpha, INF beta-1 alpha) and interferon beta-1 b (Interferons beta-1 b, INF beta-1 b) in sequence, but the domestic use rate is not high due to the expensive treatment cost.

Glucocorticoid (corticosteroids) has immunoregulatory and anti-inflammatory effects. And thus are used to treat acute relapses of MS to accelerate disease recovery.

Remission treatment is mainly immunomodulating treatment to help delay disease progression, the mechanism of action of immunomodulating drug treatment MS is to widely inhibit autoreactive lymphocyte mediated immune responses, most of which have a significant effect on relapsing remitting multiple sclerosis (RELAPSING REMITTING MS, RRMS) where inflammatory demyelination is still in the primary stage, and the therapeutic goal of these drugs is to reduce recurrence frequency and the number of lesions of magnetic resonance imaging (Magnetic Resonance Imaging, abbreviated as MRI) (including new, enlarged and enhanced T2 lesions) and delay disease progression.

Among them, interferon is an endogenous protein involved in virus and cell-mediated immune responses, and is also a first-line drug for correction and treatment of MS diseases. The mechanism of action of interferon- β is primarily to protect the integrity of the blood brain barrier (Blood Brain Barrier, BBB) thereby restricting T cells from entering the CNS, regulating T cell and B cell function, and restoring cytokine balance. Common adverse effects of interferon-beta should be mainly influenza-like symptoms, depressed mood, elevated liver enzymes, abnormal thyroid function, leukopenia or anemia, injection site reactions, etc.

Glatiramer acetate (GLATIRAMER ACETATE, GA) because GA and MBP are structurally similar, GA and MBP competitively bind to binding sites on antigen presenting cells and thereby inhibit excessive inflammatory responses in the body; in addition, GA is capable of inducing Th1 cell to Th2 cell conversion, thereby promoting production of anti-inflammatory cytokines to suppress inflammatory response of MS. GA has similar curative effect to INF-beta in terms of reducing recurrence rate and MRI focus, is more prominent than INF-beta in terms of delaying brain atrophy, but has poor curative effect in terms of delaying the progression of disability. Common adverse reactions mainly include local injection site reactions (including pain, itching, induration after local injection, and the like) and post-injection reactions (including facial flushing, chest distress, palpitations, spontaneously alleviating dyspnea, and the like), and rare adverse reactions are lipoatrophy after long-term use.

Natalizumab (NA) NA reduces inflammatory responses by predominantly binding to integrin α4 thereby preventing lymphocytes from entering the CNS. The most interesting and serious adverse effect of NA is progressive multifocal leukoencephalopathy (progressive multifocal-leukoencephalopathy, PML), a serious, possibly fatal, opportunistic infection of the brain caused by JC virus, precisely because of the risk of PML, NA is only used as a second-line drug for multiple sclerosis treatment, for patients whose other drug treatments are ineffective or unable to tolerate the side effects of the first-line drug.

Fingolimod (Fingolimod), the first oral drug available for the treatment of multiple sclerosis. Fingolimod prevents the release of active T cells from the lymph nodes by binding to sphingosine-1-phosphate receptors (sphingosine-1-phosphate receptor, S1 PR) on the T cell surface, thereby restricting T cells into the CNS and achieving immunosuppressive effects. Because the fingolimod has poor selectivity and is easy to combine with other S1PR subtype to generate corresponding adverse reaction, the adverse reaction is wider, and the adverse reaction mainly comprises first dose bradycardia, atrioventricular block, herpesvirus infection, hypertension and the like, and the case report of PML is also presented.

In addition to fingolimod, teriflunomide and dimethyl fumarate have been approved for the treatment of MS, but oral drugs have been the second-line treatment of choice for MS because of the greater risk of serious side effects of the three drugs in early or mild MS diseases, the major safety issues of which are potential long-term immunosuppression, increasing the risk of serious infections, and affecting immune surveillance, increasing the risk of malignancy.

At present, an EAE model can be successfully constructed by selecting various animal strains such as rabbits, mice, guinea pigs, rats, rhesus monkeys and the like. However, due to the characteristics of rich background knowledge, stable disease occurrence, comprehensive research reagents, low cost, easy availability, convenient operation and the like of rodents, in recent years, related experimental research is carried out by using a large and small EAE model. Stable EAE was successfully induced by injecting purified myelin antigen fragments or CNS tissue homogenates pylon into sensitive animals either with complete Freund's adjuvant (complete freund adjuvant, CFA) emulsified antigen emulsifying agent or Pertussis Toxin (PTX). Myelin component sensitizing antigens including myelin oligodendrocyte glycoprotein (myelin oligodendrocyte glycoprotein, MOG), MBP, myelin lipoproteins (proteolipid protein, PLP) and the like are commonly used.

Therefore, different types of EAE models can be established through different types of myelin-related antigen polypeptide fragments or antigen-specific T cell transfer and other modes, for example, lewis rats can be induced into an acute EAE model by MBP polypeptide, the inflammatory reaction is extremely obvious, and the model can be mainly used for researching the immune pathogenesis of MS, wherein C57BL/6 mice can be immunized by MOG _35-55 peptide to establish a single-phase progressive EAE model, the illness is slower, but the illness is extremely stable; after the SJL/J female mice are immunized by PLP _l39-15l protein polypeptide, the EAE model which is successfully established is characterized in that the later disease can be automatically relieved for many times and acutely attacks for many times, and the model is stable due to the advantages of clear background, high sensitivity and the like, and the model highly simulates the clinical patients with MS of 85 percent, thus being the animal model which is most commonly used for researching relapse clinically.

Sea and biopharmaceuticals (via the subsidiary Shanghai and pharmaceutical industry) in cooperation with Shanghai pharmaceutical institute and Shanghai green cereal pharmaceutical industry, a selective c-Met inhibitor glumetinib (Chinese name Gu Mei tinib) oral tablet formulation is being developed for potential solid tumor treatment, including non-small cell lung cancer (NSCLC).

3 Months 2020, japan began phase I trials of advanced solid tumors.

In 2017, 9 months, china initiated the Ia/Ib phase trial against advanced solid tumors.

2021 For 3 months, an Ib/II phase test for treating non-small cell lung cancer by combining Gu Mei tenib with terlipressin Li Shan of Junzi bioscience was started in China.

5 Months 2020, a phase Ib/II trial of combination of bumitinib with octtinib was initiated against metastatic non-small cell lung cancer.

The combination of the sea and pharmaceutical industry and Jiangsu Xuantai pharmaceutical industry claims an NDA application of the medicine in China for 2 months in 2022.

Gu Mei Tinib is a highly selective c-Met kinase inhibitor with an IC50 value of 0.42nM. Glumetinib has antitumor activity and good safety. Chemical formula C ₂₁H₁₇N₉O₂ S, molecular weight 459.48, chemical name 6- (1-methyl-1H-pyrazol-4-yl) -1- [ [6- (1-methyl-1H-pyrazol-4-yl) imidazo [1,2-a ] pyridin-3-yl ] sulfonyl ] -1H-pyrazolo [4,3-b ] pyridine, structural formula is as follows:

chinese patent CN104230922B, description example 27, gives a process for the preparation of bumetanide as follows:

120 mg of the compound of example 24, 64 mg of 1-methyl-1H-pyrazole-4-boronic acid pinacol ester and 120 mg of potassium carbonate were placed in a microwave reaction tube, 5 ml of dioxane, 2.5 ml of ethanol and 2.5 ml of water were added to the microwave reaction tube, air was replaced three times, and 11.8 mg of 1,1' -bis (diphenylphosphino) ferrocene palladium (II) dichloride dichloromethane complex was added to the microwave tube under nitrogen protection, and then the microwave tube was sealed. The microwave tube is placed in a microwave reactor to react for 30 minutes at the temperature of 90 ℃ and the reaction is completed. The above reaction solution was poured into 15 ml of water, extracted three times with methylene chloride, and the organic layer was dried over anhydrous sodium sulfate and concentrated, and the objective compound was obtained by flash chromatography (m=23 mg, yield: 17.3%).

¹HNMR(400MHz,CDCl₃)δ9.17(dd,J＝1.5,0.9Hz,1H),8.85(d,J＝1.9Hz,1H),8.46(dd,J＝1.8,0.8Hz,1H),8.33(d,J＝0.8Hz,1H),8.28(s,1H),7.95(d,J＝0.4Hz,1H),7.86(s,1H),7.82(d,J＝0.6Hz,1H),7.74(s,1H),7.72(dd,J＝9.3,0.8Hz,1H),7.63(dd,J＝9.3,1.7Hz,1H),4.04(s,3H),4.00(s,3H).

There is no report in the prior art and literature that glutethimide is used for indications other than tumors.

Disclosure of Invention

Applicant has focused on the study of rare disease treatment drugs, and found that Gu Meiti ni has a significant alleviating effect on multiple sclerosis.

The application firstly discloses an application of Gu Mei tinib in preparing a medicament for treating multiple sclerosis.

The Gu Mei tenib has the chemical structure as follows:

the application further discloses an application of Gu Mei tinib in preparing medicines for promoting myelination and regeneration.

Further, the agent for promoting myelination and regeneration is used for treating multiple sclerosis.

Still further, the agent for promoting myelination and regeneration is used for treating relapsing progressive multiple sclerosis.

Gu Mei-tennib was administered in a CPZ-induced mouse demyelination model and an Experimental Autoimmune Encephalomyelitis (EAE) model to promote remyelination, alleviate symptoms, and delay disease progression. Thus, gu Mei tenib has the effects of promoting myelination and regeneration, and has the function of protecting brain injury and central nervous system demyelinating disease model. Therefore, gu Mei tinib can be used for preparing medicines for promoting myelination and regeneration, and can be used for treating demyelinating diseases such as multiple sclerosis and the like.

The Gu Mei tinib of the invention is a pharmaceutical composition prepared from Gu Mei tinib serving as an active ingredient and a conventional pharmaceutical carrier. The pharmaceutical composition can be oral dosage forms such as tablets, dispersible tablets, buccal tablets, orally disintegrating tablets, sustained release tablets, capsules, soft capsules, dripping pills, granules and the like.

The invention has the beneficial effects that the invention discloses the application of the valley metinib in preparing the medicament for promoting myelination and regeneration, and is particularly suitable for treating recurrent progressive multiple sclerosis. Not only widens the application range of the valley metinib and improves the market value of the valley metinib, but also provides a new clinical medicine for demyelinating diseases such as multiple sclerosis and the like.

Abbreviation table:

MS: multiple sclerosis, multiple sclerosis;

CNS: central nervous system, the central nervous system;

RR: RELAPSING REMITTING, relapse-remission;

PP: primary progressive, primary progressive form;

SP: secondary progressive, secondary progressive forms;

PR: PRIMARY RELAPSING, progress the recovery type;

BBB: blood brain barrier, blood brain barrier;

APC: anti-PRESENTING CELLS, antigen presenting cells;

TNF- α: tumor necrosis factor-a, tumor necrosis factor a;

IFN-. Gamma.: interferon-gamma, interferon gamma;

Infβ -1α and: interferons β -1α, interferon β -1α;

INFβ -1b: interferons beta-1 b, interferon beta-1 b;

IL-1: interlukin-1, interleukin-1;

IL-6: interlukin-6, interleukin-6;

IL-4: interlukin-4, interleukin-4;

IL-lO: interlukin-10, interleukin-10;

IL-12: interlukin-12, interleukin-12;

IL-22: interlukin-22, interleukin-22;

IL-21: interlukin-21, interleukin-21;

HLA: human leucocyte antigen, human leukocyte antigens;

TCR: t cell receptor;

MBP: myelin basic protein myelin basic protein;

WHO: world Health Organization, world health organization;

DMTs: treatment of disease correction;

NMPA: national Medical Products Administration, national food and drug administration;

MRI: magnetic Resonance Imaging magnetic resonance imaging;

GA: GLATIRAMER ACETATE glatiramer acetate;

NA: natalizumab, natalizumab;

PML: progressive multifocal-leukoencephalopathy, progressive multifocal leukoencephalopathy;

s1PR: sphingosine-1-phosphate receptor, sphingosine-1-phosphate receptor;

CFA: complete freund adjuvant, complete Freund's adjuvant;

PTX: pertussis toxin;

MOG: myelin oligodendrocyte glycoprotein myelin oligodendrocyte glycoprotein;

PLP: proteolipid protein, MBP and myelin lipoproteins;

NSCLC: non-SMALL CELL lung cancer, non-small cell lung cancer;

c-Met: c-MESENCHYMAL-EPITHELIAL TRANSITION FACTOR, interstitial epidermal transforming factor;

OPC: oligodendrocyte Precursor Cell oligodendrocyte precursor cells;

And (3) OL: oligoderIdrocyte oligodendrocytes;

EAE: experimental Autoimmune Encephalomyelitis, experimental autoimmune encephalomyelitis;

CPZ: cuprizone, bicyclohexanoyl dihydrazone;

CMC-Na: carboxymethyl, cellulose Sodium, sodium carboxymethyl Cellulose;

MET: methylprednisolone methylprednisolone;

vitamin D: vitamin D;

corticosteroids: glucocorticoids;

Findolimod: fingolimod;

glumetinib: gu Mei Tinib;

Detailed Description

The present invention will be further described in detail by the following examples, which are illustrative of the present invention but are not intended to limit the present invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.

Example 1 Remyelination of Gumetinib in model animals

1.1 Preparation of a bicyclohexanoyl dihydrazone (CPZ) demyelinating animal model:

Male C57/BL6 mice at 6 weeks of age were randomly grouped by body weight after 1 week of adaptive feeding. The normal group was fed with normal feed throughout the course, and the model group and the dosing group were fed with 0.2% CPZ feed for 6 weeks to create demyelination models, 7 per group. Normal diet was resumed at week 7 and week 8 with simultaneous administration, gastric administration, once daily.

1.2 Preparation of drug samples

Sodium carboxymethylcellulose (CMC-Na) was formulated as a 0.05% solution with physiological saline. According to the dosage of mice, a proper amount of Gumeitinib and Methylprednisolone (MET) are added into O.05% CMC-Na solution to form suspension, and the suspension is preserved at 4 ℃ in dark place.

1.3 Experimental groups and dosing mice were divided into a normal control group, a model group, a Gu Mei low-dose group (2 mg/kg/d), a Gu Mei medium-dose group (10 mg/kg/d), a Gu Mei high-dose group (20 mg/kg/d) and a Methylprednisolone (MET) 1 mg/kg/d.

1.4 Water maze test (1-6 days after administration)

The activity of the mice is detected by a real-time monitoring system of the water maze of the mice. The water maze consists of a cylindrical water pool and a computer image acquisition and analysis system. The diameter of the cylindrical pool is 1200mm, and the height is 500mm. During the test, titanium dioxide is added into the tank and stirred uniformly to form a white background, and the water temperature is kept at 23+/-1 ℃. The periphery of the water tank is surrounded by a cloth curtain, no lamplight is directly irradiated in the water tank, the reference objects around the environment are fixed, and 4 quadrants are marked in 4 different directions of the water tank.

The experiment is divided into a positioning navigation stage and a space exploration stage.

And (3) in the navigation stage, the platform is placed at a position 1-2cm below the water surface for 5 days, and training is carried out for 1 time per day for a fixed time, and two quadrants are formed each time. Before and after each training, the platform is adapted to 10s, the detection time is 90s, if the platform is found in the time range and stays on the platform for 5s, the platform searching is calculated to be successful, the escape latency (unit: s), the swimming track, the total range, the average speed, the speed of passing through the position of the original platform and the like are recorded, if the platform is still not found for more than 90s, the mouse is guided to the platform by an experimenter, stays for 15s and then moves out of the pool, so that the experimenter knows and is familiar with the surrounding environment of the platform.

And in the space exploration stage, removing the platform, putting the animal into water from the diagonal quadrant of the original platform quadrant to the pool wall, monitoring the behavior track of the animal within 90 seconds, and recording the swimming track, the swimming distance in the target quadrant, the average speed, the speed of passing through the position of the original platform and the number of times of passing through the platform. The test data are as follows:

table 1 escape latency 5 days after dosing (mean±sd, n=7) for each group of test animals

Group of

Dosage (mg/kg/d)

Day1(s)

Day2(s)

Day3(s)

Day4(s)

Day5(s)

Normal control

/

78.1±11.7

75.7±14.1

55.4±16.1

38.3±14.3

25.3±7.1

Model group

/

88.8±16.3

85.3±12.5

78.8±4.9

75.1±12.3

65.9±12.4

Gu Mei Tinib Low dose group

2

83.6±2.8

78.7±11.9

72.3±8.6

54.3±15.4

45.4±7.9

Gu Mei Medium dose group of Tinib

10

78.7±14.0

72.9±15.4

65.6±15.1

50.4±6.2

32.4±12.2

Gu Mei high dose group of tenib

20

76.3±14.2

69.1±5.4

55.7±3.8

46.2±3.2

28.2±10.9

MET

1

89.5±8.2

83.1±13.2

76.8±16.9

72.1±2.5

63.5±16.6

。

The test results show that. In the first 5 days of positioning voyage, the escape latency of each group of mice tended to decrease with the extension of the training period. The early learning process and experimental preparation proved successful. On this basis, the escape latency is significantly reduced (P <0.01, P < O.01) in the Gu Mei th day, 1, 2, 3, 4, 5 th day, low, medium, and high dose groups of Gu Mei th day compared to the model group.

Training to day 5, the average escape time of the model group was 65.9.+ -. 12.4s, and that of Gu Mei tinib low, medium, and high dose groups was 45.4.+ -. 7.9s, 32.4.+ -. 12.2s, and 28.2.+ -. 10.9s, respectively. Gu Mei the escape latency was reduced for each dose group compared to the model group (P <0.01, P < o.01, P < 0.01). There was no statistical difference in escape latency for the first 5 days of MET compared to model group.

Table 2 exercise status at 5 and 6 days after administration (mean±sd, n=7) for each group of test animals

The swimming distance in the target quadrant on day 5 of the normal group was 52.8+ -8.1 cm, the swimming distance in the target quadrant on day 5 of the model group was 25.8+ -9.6 cm, and the model group had a statistical difference (P < 0.01) compared with the normal group. Gu Mei the swimming distance in the target quadrant on day 5 of the low, medium and high dose groups was 33.8+ -9.4 cm, 41.7+ -5.1 cm, 45.8+ -8.3 cm, with statistical differences (P <0.01 ) compared to the model group. Whereas MET groups were not statistically different from model groups.

The percent swimming time in the target quadrant on day 5 of the normal group was 69.0±19.9%, the percent swimming time in the target quadrant on day 5 of the model group was 33.1±14.4%, and the model group had a statistical difference (P < 0.01) compared to the normal group. The percent swimming time in the target quadrant on day 5 of Gu Mei low, medium, and high groups was 56.5±5.3%,62.3±19.3%, and 62.8±15.5% were statistically different from the model group (P <0.01 ). Whereas MET groups were not statistically different from model groups.

In the space exploration experiment, after each group of mice are trained for 5 days, the water platform is removed, the mice are launched opposite to the target quadrant, and the influence of Gu Meiti Ni on the space exploration ability of the mice is explored by taking the number of times of the mice penetrating the platform and the swimming distance in the target quadrant as indexes. The swimming distance in the target quadrant on day 6 of the normal group was 425.5.+ -. 163.5cm, the swimming distance in the target quadrant on day 6 of the model group was 205.2.+ -. 154.4cm, and the model group had a statistical difference (P < 0.01) compared to the normal group. Gu Mei the swimming distance in the target quadrant on day 6 of the low, medium and high dose groups was 356.1.+ -. 114.1cm, 378.2.+ -. 196.9cm, 388.9.+ -. 184.8cm, with statistical differences (P < O.01, P < O.01, P < O.01) compared to the model group. Whereas MET groups were not statistically different from model groups.

Within 90s, the number of times of crossing the normal group mice is 3.5+/-0.4 times, the number of times of crossing the model group mice is 1.8+/-1.1 times, and the model group has statistical difference (P < O.01) compared with the normal group. Gu Mei mice in low, medium and high dose groups were crossed 2.6.+ -. 1.2 times, 3.2.+ -. 0.7 times, 3.5.+ -. 1.1 times, and were statistically different from the model groups (P < O.01, P < O.01, P < 0.01). There was no statistical difference between MET group compared to model group.

This experiment demonstrates that Gu Mei low, medium, and high dose groups of tennib can significantly improve cognitive dysfunction in CPZ-induced demyelinated model mice compared to model groups.

1.5 Rotating rod (after water maze test)

Following the water maze test, the improvement effect of Gu Meiti Ni on CPZ-induced demyelination induced dyskinesia was examined using a rotating rod test. The training phase was 2 days after each group of mice completed the water maze test, the mice were trained twice daily, and the rotational speed was slowly increased from 5rpm to 10rpm for 300s. In the third day, the rotating speed of the rotating rod is slowly increased from 5rpm to 40rpm at the highest rotating speed, the total rotating time is 300s, the mice drop from the rotating rod or the rotating rod is held for 2 weeks to be the experiment ending time, and the maintaining time of the mice staying on the rotating rod is recorded. Each mouse was tested 3 times, and the average value of the residence time of the mice was taken as an index of the motor balance ability of the mice.

1.6 Elevated plus maze (rotating rod after test)

The present experiment uses the contradictory conflict between the mouse's heuristic for the new heterogeneous environment and the fear formation for the highly suspended open arm to investigate the anxiety state of the mice. The device consists of four arms (30X 5 cm) 40cm above the ground. Two of the arms are blocked by a wall 15cm high (called the closed arm), and the other two arms without a shield are called the open arms. The mice were placed facing the open arm in the middle and were left alone in the maze for 5min without being disturbed. After each mouse completes the test, the excrement is wiped clean by 75% ethanol, so that the interference to the test of the next mouse is avoided. The track of the mice in the arms was recorded with an Any-maze behavior tracking software, the number of times and time of entering the four arms respectively.

The elevated plus maze test was used to examine the improvement of anxiety mood caused by CPZ-induced demyelination by bumtinib. The number of times and residence time of the mice entering the open arm and the closed arm within 10min are recorded, and the residence time percentage and the number of times of entering the open arm of each group of mice are calculated to be used as indexes for judging anxiety.

1.7 Rotating rod and elevated plus maze test results

Table 3 performance of each group of test animals on rotating bars and overhead plus maze after dosing (mean±sd, n=7

The rotating rod test result shows that: normal mice had an average on-stick time of 303.5.9 s and model mice had an average on-stick time of 208.9+ -54.4 s, which was significantly reduced (P < o.01) compared to normal mice, indicating that CPZ may cause dysfunctional motor balance in mice. The average rod time of Gu Mei tinib low, medium and high dose groups is 238.5 +/-56.6 s, 254.3+/-53.4 s and 288.7+/-41.7 s respectively, compared with a model group, the effect of improving the movement balance dysfunction caused by the CPZ of the mice (P <0.01, P <0.01 and P < 0.01) is obviously improved, and the effect is obviously improved along with the increase of the dose. MET group was not statistically different (P > 0.05) compared to model group.

The results of the overhead plus maze test show that: the percentage of the times that the mice of the normal group enter the arm opening is 25.3+/-6.1%, the percentage of the times that the mice of the model group enter the arm opening is 11.2+/-8.6%, compared with the normal mice, the percentage of the times that the mice of the model group enter the arm opening is obviously reduced (P < 0.01), the percentage of the times that the mice of the Gu Mei-low, medium and high dose groups enter the arm opening is 15.2+/-2.8%, 21.2+/-6.7%, 23.3+/-2.7%, and compared with the model groups, the anxiety emotion (P < O.01, P < O.01, P < O.01) of the mice can be obviously relieved by Gu Mei-low, medium and high doses. The percentage of times MET group mice entered the open arm was not statistically different from the model group.

The time percentage of the mice in the normal group entering the arm is 6.3+/-1.7%, the time percentage of the mice in the model group entering the arm is 1.5+/-2.1%, compared with the normal mice in the model group, the time percentage of the mice in the model group entering the arm is obviously reduced (P < O.01), the time percentages of the mice in the Gu Mei low, medium and high dose groups entering the arm are respectively 2.7+/-1.5%, 3.4+/-3.1% and 5.3+/-1.4%, compared with the model group, the time percentage of the mice in each dose group of Gu Mei tinib can be increased, and the difference is obvious (P < O.01, P < O.01 and P < O.01). Gu Mei the low, medium and high doses of tinib significantly alleviate anxiety (P < O.01, P < O.01, P < O.01) in mice. The percentage of time that MET group mice entered the open arm was not statistically different from the model group.

Based on the above experiments, it is proved that the low, medium and high dose groups of the valley metinib can effectively improve the cognition, movement and anxiety dysfunction of cpz-induced demyelination model mice, and further play a certain role in treating demyelinating diseases.

Example 2 Effect of Gumetinib on EAE model mice

2.1 EAE model building

2.1.1 Preparation of antigen emulsifier:

Myelin basic protein (myelin basic protein, MBP) was dissolved in physiological saline to a final concentration of 0.4 mg/ml; CFA solution containing 2mg/ml of heat-inactivated Mycobacterium tuberculosis H37Ra is gently blown and mixed in a dark place. The MBP protein solution and complete Freund's adjuvant (complete freund adjuvant, CFA) are fully mixed according to the proportion of 1:l (volume ratio), meanwhile, a homogenizer is started for emulsification, each time the homogenizer rotates for 3min, the homogenizer is stopped for 1min until the white antigen emulsion is completely dripped into water, and if the white antigen emulsion is not diffused on the water surface, the complete emulsification is proved, and the preparation is successful. The antigen emulsifier is prepared by operating in ice bath in the whole process, and is prepared in an on-site way.

2.1.2 Model preparation:

SPF-class female SJL/J mice, 6-8 weeks old, weighing 20-22g, 42. The experimental group mice were subcutaneously injected with 0.2mL of antigen emulsifier at 4 points on the back (both sides of the spinal column) and were intraperitoneally injected with 0.5 μg/mouse pertussis toxin on day 1 and day 2, respectively; the mice in the blank group were each injected with physiological saline.

2.2 Experimental grouping:

The test mice were divided into a normal control group, a model group, gu Mei low (2 mg/kg/d), medium (10 mg/kg/d), high (20 mg/kg/d) and Methylprednisolone (MET) 1mg/kg/d doses. 7 animals per group, after 14 days of molding, were subjected to drug intervention 1 time a day.

2.3 EAE mouse medicine and treatment scheme

2.3.1 Preparation of drug samples,

The samples were prepared as in section 1.2, as follows:

Sodium carboxymethyl cellulose is formulated as a 0.05% solution in normal saline. According to the dosage of mice, a proper amount of Gumeitinib and Methylprednisolone (MET) are added into O.05% CMC-Na solution, and the mixture is stirred to form a suspension, and the suspension is preserved at 4 ℃ in dark.

2.3.2 Doses administered

The intragastric administration was continuously started daily on day 14 (the point of onset of mice) after the molding, the administration period was 21 days, the administration dose was Gu Mei low dose 2mg/kg/d for tennib, 10mg/kg/d for Gu Mei medium dose, 20mg/kg/d for Gu Mei high dose for tennib, and 1mg/kg/d for MET. The normal and model groups were given equal volumes of 0.05% cmc-Na solution.

During the administration period, feed and drinking water are normally supplied. The mice were observed and recorded daily for clinical manifestations, weight changes, neurological scores (Kono's scores), etc., from the day of modeling to the time of treatment end experimental observation.

2.4 Observations after dosing

Mice started to develop disease around the 10 th day of modeling, with a disease incidence of 100%. Normal mice have bright hair, powerful tail bending and good mental state; the model-building mice mainly show reduced feeding, dry fur, unsmooth fur, listlessness, obvious nervous system signs such as ataxia, tail paralysis, eye hemorrhage, paralysis of both hind limbs, quadriplegia, urinary disorder and the like, and serious morbidity and even dying.

Mice were weighed at a fixed time daily morning and scored for neurological function using KONO's scoring. Scoring criteria:

0 minutes, no symptoms;

1, the tail loses tension;

2 minutes, weak hindlimb force;

3, hind limb paralysis;

4, hind limb paralysis and forelimb paralysis;

5 minutes, dying or dying.

Symptoms were scored as + -0.5 if they were between the two scores. At the end of dosing, kono's scoring and weight monitoring were performed on the test mice and the data are as follows:

Table 4 Kono's score and body weight monitoring 35 days after model dosing (mean±sd, n=7) for each group of test animals

Group of	Dosage (mg/kg/d)	Kono's score	Body weight (g)
				Blank control	/	0	22.6±1.4
Model group	/	3.3±1.3	17.2±1.4
				Gu Mei Tinib Low dose group	2	1.7±1.2	18.6±0.7
Gu Mei Medium dose group of Tinib	10	1.6±0.9	19.3±0.6
				Gu Mei high dose group of tenib	20	1.2±0.5	21.7±0.6
MET	1	2.1±0.9	17.8±0.2

。

Clinical neurological function Kono's score after end of dosing, the neurological function score of EAE model mice was 3.3±1.3, whereas the score of Gu Mei tenib-dosed low, medium and high dose groups were 1.7± 1.2,1.6 ± 0.9,1.2 ±0.5, and the score of met group was 2.1±0.9, respectively. From the above data, gu Mei low, medium, high dose and MET groups were able to significantly reduce clinical function scores (P <0.01 ) compared to EAE model group mice. However Gu Mei the effect of the low, medium and high dose groups of tenib was superior to the MET group.

Body weight monitoring after the end of dosing, the average body weight of mice in the EAE model group was 17.2+ -1.4 g compared to the body weight of mice in the normal group (22.6+ -1.4 g). Whereas Gu Mei was low, medium and high doses with an average body weight of 18.6.+ -. 0.7g, 19.3.+ -. 0.6g, 21.7.+ -. 0.6g and an average body weight of 17.8.+ -. 0.2g for the MET group.

Based on the above results, gu Mei low, medium, and high doses of tennib can alleviate weight loss in mice with EAE models (P < o.01, P < 0.01), but MET group did not alleviate weight loss in mice with EAE models. There were statistical differences in the weight loss of mice in Gu Mei-tennib dose groups compared to model groups (P < o.01 ).

Based on the relieving effect of Gu Mei on the neurological scores and weight loss of SJL/JEAE model mice in low, medium and high dose groups of tennib, it can be concluded that Gu Mei tennib can effectively relieve recurrent progressive multiple sclerosis.

While the foregoing disclosure and examples have described the basic principles and features of the present application and the advantages of the present application, those skilled in the art will recognize that the present application is not limited to the embodiments described above, but rather that the embodiments and descriptions described are merely of the best modes for carrying out the present application, and that various changes and modifications may be made in the present application without departing from the spirit and scope of the present application, i.e., in terms of a treatment for multiple sclerosis, and in particular for relapsing progressive multiple sclerosis, with a use of Gumeitinib or a pharmaceutically acceptable salt, hydrate or other analogue thereof, alone or in combination with other medicaments, within the scope of the claimed invention, which is defined in the appended claims and their equivalents.

Claims

1. Use of Gu Mei tinib for the preparation of a medicament for promoting myelination and regeneration, characterized in that the chemical structure of Gu Mei tinib is as follows:

2. the use of claim 1, wherein the medicament is for the treatment of multiple sclerosis.

3. The use of claim 2, wherein the multiple sclerosis is relapsing progressive multiple sclerosis.

4. The use according to claim 1, wherein the medicament is a pharmaceutical composition made of Gu Mei tinib as active ingredient together with a conventional pharmaceutically acceptable carrier.

5. The use according to claim 4, wherein the pharmaceutical composition is in the form of a tablet, dispersible tablet, buccal tablet, orally disintegrating tablet, sustained release tablet, capsule, soft capsule, dripping pill or granule.