CN102406648A - Application of imatinib mesylate in preparation of anti-Parkinson's disease medicine - Google Patents

Abstract

The invention discloses an application of imatinib mesylate in preparing drugs for resisting Parkinson's disease. The application of imatinib mesylate (STI571) in the preparation of medicaments for treating neurodegenerative diseases, such as Parkinson's disease, is within the scope of the invention. Experiments prove that: the STI571 continuous administration can obviously improve the MPTP-induced motor dysfunction; can improve cognitive dysfunction of mice. STI571 can obviously improve MPTP-induced dopamine neuron loss, STI571 can inhibit rotenone-induced rat CA1 and DG region nerve cell apoptosis reaction, and STI571 can obviously inhibit rotenone-induced primary substantia nigra neuron apoptosis and Lewy corpuscle protein expression, and inhibit phosphorylation activation of PKC and Akt. STI571 is used as a specific inhibitor of c-Ab1, can resist PD formation, improve behavioral abnormality and cognitive dysfunction in PD, can be used as a new candidate medicine for resisting neurodegenerative diseases such as Parkinson's disease and the like, has good application prospect, and greatly expands the clinical indications of STI 571.

Description

Application of imatinib mesylate in preparation of anti-Parkinson's disease medicine

technical field

The invention relates to the application of imatinib mesylate in the preparation of medicaments for treating Parkinson's disease.

Background technique

Imatinib mesylate (STI571), the chemical name is 4-(4-methyl-1-piperazine)methyl-N-4-methyl-3-4-(3-pyridine)-2-pyrimidine Aminophenyl-aniline mesylate is currently mainly used in the treatment of chronic myelogenous leukemia in foreign countries. STI571 can significantly inhibit the proliferation of leukemia cells, arrest cells in G0/G1 phase, and have obvious induction of apoptosis. Inactivates the ubiquitous Abelson tyrosine kinase (Abl).

At present, there is no literature report on other functions of imatinib mesylate.

Contents of the invention

The object of the present invention is to provide the application of imatinib mesylate (STI571) in the preparation of medicines for treating neurodegenerative diseases.

In the application of imatinib mesylate provided by the present invention in the preparation of medicines for treating neurodegenerative diseases, the aforementioned neurodegenerative diseases may specifically be Parkinson's disease.

The aforementioned Parkinson's disease may be experimental Parkinson's disease induced by a mitochondrial electron transport chain inhibitor.

Further, the aforementioned mitochondrial electron transport chain inhibitor is 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or rotenone.

The application of imatinib mesylate in the preparation of products for motor dysfunction and/or cognitive dysfunction also falls within the protection scope of the present invention.

The application of imatinib mesylate in the preparation of preparations for inhibiting neuronal apoptosis also falls within the protection scope of the present invention.

Furthermore, the above-mentioned inhibition of nerve cell apoptosis can be achieved by inhibiting the phosphorylation of the T183 site of the neuron MST1 protein; the amino acid sequence of the neuron MST1 protein is shown in Sequence 1 in the sequence listing.

Specifically, the above-mentioned nerve cells are nerve cells in CA1 and/or DG regions of mice.

The apoptotic response of the above-mentioned neurons is a symptom induced by MPTP or rotenone.

The application of imatinib mesylate in the preparation of drugs for improving the loss of dopamine neurons also falls within the protection scope of the present invention.

The aforementioned loss of dopamine neurons is a symptom of MPTP or rotenone-induced formation.

Experiments have shown that continuous administration of STI571 for one week or two weeks can significantly improve the motor dysfunction induced by MPTP, which is manifested as significantly prolonging the time of turning the rod; STI571 can also significantly improve the decrease in spontaneous activity induced by MPTP. Compared with the MPTP model group, the spontaneous Both movement distance and average speed were significantly increased, thereby improving MPTP-induced motor dysfunction. Therefore, STI571 can improve the motor dysfunction in mice. Through positioning navigation test and space exploration test, it was found that STI571 could improve the cognitive dysfunction of mice. Further mechanism analysis showed that: STI571 can significantly improve MPTP-induced loss of dopamine neurons, STI571 inhibits rotenone-induced neuronal apoptosis in rat CA1 and DG regions, and STI571 can significantly inhibit rotenone-induced apoptosis of primary substantia nigra neurons And Lewy body protein expression, inhibit the phosphorylation activation of PKC and Akt. Therefore, as a specific inhibitor of c-Ab1, STI571 can effectively fight against the formation of PD, improve behavioral abnormalities and cognitive dysfunction in PD, and may be used as a candidate drug for neurodegenerative diseases such as Parkinson's disease, and has good potential. The application prospect has greatly expanded the clinical indications of STI571.

Description of drawings

Figure 1 is the effect of STI571 on the expression of p-MST1-T183 protein in rats with stereotaxic administration of rotenone (3h-3d) in CA1 and DG regions, A and B are 3, 6, 12, 24, respectively. The staining results of CA1 and DG areas of rats at 48 and 72 hours; C is the average optical density of neuronal p-MST1-T183 protein, which is the quantitative result of DG area in Figure B; it is 3, 6, 12, 24, 48 , 72 hours of results.

Figure 2 is the effect of STI571 on inhibiting the apoptosis response of nerve cells in the CA1 and DG regions of rats subjected to stereotaxic administration of rotenone (24-72h). Statistical results at each time point; B is the TUNEL staining result of rat CA1 area at 24, 48, and 72 hours; C is the TUNEL staining result of DG area.

Figure 3 is the rotary rod experiment, A, B and C are the test results of three days, one week and two weeks of continuous administration of STI571 respectively; ^** p<0.01 vs control, ##p<0.01 vs MPTP.

Figure 4 is the automatic movement experiment. The two figures in A are the results of the distance and average speed of spontaneous activities from left to right, respectively, and B is the representative spontaneous activity trajectory; ^** p＜0.01 vs control, ##p＜0.01 vs MPTP.

Figure 5 is the hidden platform experiment, A is the absconding latency period; B is the sailing distance to find the hidden platform; C is the average swimming speed, D is the swimming distance in the quadrant where the platform is located, and E is the residence time; ^** p<0.01 vs control, ## p<0.01 vs MPTP.

Figure 6 shows the space exploration experiment, A is the number of times crossing the platform; B is the time required to find the original platform location, C is the navigation distance required to find the original platform location; D is the swimming distance in the quadrant where the platform is located, and E is the residence time ; ^** p<0.01 vs control, ##p<0.01 vs MPTP.

Figure 7 is the visual platform experiment, A is the latency to find the platform, B is the swimming distance, C is the average swimming speed of mice, D is the swimming distance in the quadrant where the platform is located, and E is the residence time, ^* p<0.05 vs control, # #p<0.01 vs MPTP.

Figure 8 is the representative swimming trajectory in the directional navigation experiment, space exploration experiment and visual platform experiment, A is the swimming trajectory of the first experiment of the directional navigation experiment; B is the swimming trajectory of the last experiment of the directional navigation experiment; C is the space exploration The swimming trajectory of the experiment; D the swimming trajectory of the visual platform experiment.

Figure 9 is the representative search platform trajectory category (A) and proportion of MPTP (20mg.kg-1, i.p, 2 weeks) model group and STI571 (30mg.kg-1, i.p, 2 weeks) treatment group in the directional navigation experiment (B).

Figure 10 is the loss of tyrosine hydroxylase (TH) positive neurons (A, the left picture is the result of TH immunohistochemistry in the substantia nigra, and the right picture is the average optical density of TH positive neurons); increased stria nigra The expression of TH protein in the body part (B), in the figure Hippo is the hippocampus, Cortex is the cortex, Striatum is the striatum, N is normal; M is MPTP; S is STI571.

Figure 11 shows the protein expression of cleavage-capase 3; inhibits the protein expression of α-synuclein; inhibits the protein expression of p-PKC and p-Akt; Normal control group, rotenone model group and STI571 treatment group in the neuron PD-like cell model experiment.

Among them: DMSO, Rotenone and STI571+Rotenone in Figure 1 and Figure 2 are the normal control group, rotenone model group and STI571 treatment group in the experiment of STI571 inhibiting the apoptosis response of rat CA1 and DG area neurons induced by rotenone, respectively. In Fig. 3-Fig. 10, control, MPTP and STI571 are normal control group, rotenone model group and STI571 treatment group in experiments related to MPTP-induced chronic PD model in c57BL/6J mice.

Detailed ways

The present invention will be further described below in conjunction with specific examples, but the present invention is not limited to the following examples.

In the following examples, unless otherwise specified, all are conventional methods.

Embodiment 1, imatinib mesylate (STI571) treats Parkinson's disease

1. STI571 can significantly improve MPTP-induced motor dysfunction in mice with chronic Parkinson's disease

Parkinson's disease is mainly due to the pathological changes in the cells located in the substantia nigra of the midbrain, the synthesis of dopamine is reduced, and the excitatory effect of acetylcholine is relatively enhanced. Its incidence rate ranks first among neurodegenerative diseases in the elderly. The main behavioral manifestations are slowness and lack of movement, muscle rigidity, resting tremor, and postural instability.

1. Modeling

Eight-week-old c57BL/6J mice were randomly divided into 3 groups, 10 mice in each group, administered intraperitoneally in the following manner, once a day, for two consecutive weeks. [1] control group: normal saline; [2] MPTP group: MPTP (20mg/kg, dissolved in normal saline); [3] STI571+MPTP group: intraperitoneal injection of STI571 (30mg/kg, dissolved in normal saline) for one hour After that, give MPTP (20mg/kg, dissolved in normal saline); Brain Dis 25:177-183).

Chronic Parkinson's disease was induced with MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) to c57BL/6J mice (purchased from Weitong Lihua Company) according to the above method ( Parkinson's disease, PD) model.

2. Grouping

The following three groups of c57BL/6J mice were subjected to the rotarod test and the spontaneous locomotor test:

Normal control group: daily intraperitoneal injection of normal saline (0.1ml/10g);

MPTP model group: according to the dose of 20mg/kg body weight, MPTP was injected intraperitoneally into mice for 14 consecutive days;

STI571 treatment group: STI571 was injected intraperitoneally into mice at a dose of 30 mg/kg body weight, and MPTP was injected intraperitoneally into mice at a dose of 20 mg/kg body weight one hour later for 14 consecutive days.

3. Rotorod

Rotating rod experiment: After the mouse starts to crawl on the rotating rod, it automatically counts the time, and when it falls from the rotating rod, it can be monitored by an infrared device, and the timing is automatically stopped and the time for the mouse to move on the rotating rod is displayed. The experiment was performed three days, one week and two weeks after the 14-day period of administration of ST1571 or MPTP. The experiment adopted a rotational speed of 40 revolutions per minute.

The results are shown in Figure 3. Continuous administration of STI571 for one week or two weeks can significantly improve MPTP-induced motor dysfunction, manifested as a significant prolongation of the rod rotation time.

4. Locomoter activity test

Spontaneous movement experiment: the mice were placed in the center of a spontaneous movement monitor (round, 50 cm in diameter), and the environment was kept quiet. There are multiple beams of infrared rays passing through the spontaneous movement monitor, and the infrared beams can be detected and counted when the animals are active. After 2 minutes of pre-adaptation, the number of spontaneous movements of the mice within 10 minutes was recorded. The experiment was performed two weeks after ST1571 administration.

The results are shown in Figure 4. On the chronic PD model of c57BL/6J mice induced by MPTP, STI571 can also significantly improve the decrease of spontaneous activity induced by MPTP. Compared with the MPTP model group, the spontaneous activity distance and average speed were significantly increased, Thereby ameliorating MPTP-induced motor dysfunction.

2. STI571 can significantly improve MPTP-induced cognitive dysfunction in mice with chronic Parkinson's disease

The Morris water maze was designed by the British psychologist Morris in the early 1980s and applied to the study of the brain mechanism of learning and memory. It is currently the most reliable and effective method for testing animal behavior and memory functions. This device can observe and record the time required for animals to search for targets after entering the water, the strategies they adopt, and their swimming trajectories, which can help analyze and infer the unit's learning, memory, spatial orientation, and cognitive functions. It is widely used in basic and applied research in the field of neurobiology. The Morris water maze includes a circular pool filled with water, a platform hidden under the water, and a set of automatic image acquisition and processing systems (cameras, monitors, and analysis software, etc.). The more classic Morris water maze test program mainly includes two parts: place navigation and spatial probe trail.

1. Positioning navigation test (hidden platform test)

Experimental procedure: for 7 days, the rats (normal control group, MPTP model group and STI571 treatment group in step 1) were put into the water several times from 3 water entry points facing the pool wall every day, and the places they found hidden under the water surface were recorded. The platform time is the escape latency (escape latency, EL). What is detected is that mice learn to find hidden platforms with fixed positions during repeated training, and form stable spatial cognition, which is formed by processing spatial information. The position of the platform has nothing to do with the position and state of the mouse. It is a kind of reference cognition with the different self as the reference point, and the memory formed is a kind of spatial reference memory.

The results are shown in Figure 5. On the chronic PD model of c57BL/6J mice induced by MPTP, in the hidden platform experiment, the spatial learning ability of MPTP-induced mice was significantly reduced, which was manifested by a significant prolongation of the ambush latency (Figure 5A); finding the hidden The platform sailing distance (Fig. 5B) was significantly prolonged; the average swimming speed (Fig. 5C) was significantly reduced; the swimming distance (Fig. 5D) and residence time (Fig. 5E) in the quadrant of the platform were significantly shortened. After two weeks of continuous administration, STI571 can significantly improve the above spatial learning and memory impairment.

2. Space exploration experiment

Experimental procedure: remove the platform after the above-mentioned positioning and navigation test, and then choose a water entry point to put the mice (normal control group, MPTP model group and STI571 treatment group) into the pool, and record their swimming trajectory within a certain period of time. Investigate the ability of the mice to maintain the spatial position memory of the original platform. There are many parameters available for analysis in the test, including the swimming distance in each quadrant, the ratio of the swimming distance in the original platform quadrant to the total distance, the number of crossing platforms, and the percentage of swimming distance in the middle and outer rings.

The results are shown in Figure 6. On the chronic PD model of c57BL/6J mice induced by MPTP, in the spatial exploration experiment, the spatial memory ability of MPTP-induced mice was significantly reduced, and the number of crossing platforms (Figure 6A) was significantly reduced; The sailing distance required to find the original platform location (Fig. 6B, 6C) was significantly prolonged; the swimming distance (Fig. 6D) and residence time (Fig. 6E) in the quadrant of the platform were significantly shortened. After two weeks of continuous administration of STI571, the above-mentioned spatial memory impairment can be significantly improved.

3. Visual platform experiment

Experimental procedure: Compared with the hidden platform experiment, only the platform under the water surface is transferred to the water surface, and the rest of the steps are exactly the same.

The results are shown in Figure 7. On the chronic PD model of c57BL/6J mice induced by MPTP, in the visual platform experiment, the mice in the MPTP and STI571 treatment groups showed a higher latency to find the platform (Figure 7A) and swimming distance (Figure 7B). ) and the swimming distance in the quadrant where the platform is located (Fig. 7D) and residence time (Fig. 7E), there was no significant difference, only the average swimming speed was reduced by MPTP, which was relieved by STI571 (Fig. 7C).

4. The effect of STI571 on the trajectory of PD mice in directional navigation, space exploration and visual platform experiments

In the above positioning navigation test, on the chronic PD model of c57BL/6J mice induced by MPTP, the swimming routes of the mice in the MPTP model group were significantly increased in the orientation navigation test, and the swimming routes to the platform could be significantly reduced after two weeks of STI571 treatment (Fig. 8A) ; Compared with the mice on the second day of training, the swimming routes of each group were significantly improved after training for 7 days (Fig. 8B).

In the above-mentioned space exploration and visual platform experiments, the swimming trajectories of each group were similar to those in the directional navigation experiments (Fig. 8C, 8D).

5. Analysis of the trajectory category of STI571 in the directional navigation experiment

In the above directional navigation experiment, on the MPTP-induced chronic PD model of c57BL/6J mice, the representative search platform trajectory categories (Figure 9A) and proportions (Figure 9B) of the MPTP MPTP model group and the STI571 treatment group in the directional navigation experiment. Mice in the MPTPMPTP model group mainly used random (Random) swimming trajectories, the normal control group mainly used linear (Beeline) swimming trajectories, and the STI571 treatment group mainly used tropism (Tropism) swimming trajectories.

In summary, STI571 can effectively improve motor and cognitive dysfunction in PD.

Embodiment 2, mechanism research

1. STI571 can significantly improve MPTP-induced loss of dopamine neurons

Experimental procedure: Take two mice from each of the normal control group, MPTP model group and STI571 treatment group and fix them with 4% paraformaldehyde first. After the perfusion is completed, take out the mouse brain and put it in 4% paraformaldehyde for further fixation. On day 3, after the fixation was completed, frozen sections were made for the substantia nigra. The prepared frozen sections were immunostained with TH antibody (1:100, sigma, T1299), and the average optical density of TH positive neurons was counted using image pro plus software for the staining results. After the rest of the mice were killed by neck dislocation, the hippocampus, cortex, and striatum proteins were taken and lysed with RIPA (5ul/1g protein) on ice for 30 minutes, ultrasonicated for 9s/9s three times, and then centrifuged for 45 minutes, and the supernatant lysate was stored at -80°C. In subsequent tissue western blot experiments, TH antibody (1:5000, sigma, T1299) and secondary antibody (anti-mouse IgG; (1:10000), GE healthcare, Lot386066) were used to detect the expression of TH in tissue samples.

The results are shown in Figure 10. In the MPTP-induced chronic PD model of c57BL/6J mice, STI571 can significantly inhibit the loss of MPTP-induced tyrosine hydroxylase (TH)-positive neurons, that is, inhibit the loss of DA-ergic neurons during PD. STI571 can also improve MPTP-induced reduction of TH protein expression in the nigrostriatum (Fig. 10B).

2. STI571 inhibits rotenone-induced neuronal apoptosis in rat CA1 and DG regions

1. Modeling

Get SD rats with a size of 180-200g and divide them into three groups, and administer them in the DG area of the rats by the method of brain stereotaxic injection in the following manner. Brain tissues were perfused and fixed to make paraffin sections of the hippocampus. [1] control group: DMSO 4ul; [2] rotenone group: Rotenone 4ul (2.5ug/ul, dissolved in DMSO); [3] STI571+MPTP group: STI571 4ul (20mM, dissolved in saline) + Rotenone 4ul ( 2.5ug/ul, soluble in DMSO) (Gonzalo I.Cancinol, Enrique M.Toledo, Nancy R.Leall, DiegoE.Hernandezl, L.Fernanda Yévenesl, Nibaldo C(2008) STI571 prevents apoptosis, tauphosphorylation and behavioral Alzheimer's impairments induced 's beta-amyloid deposits Brain 131:2425-42).

According to the above method, the apoptosis response of rat CA1 and DG area nerve cells induced by rotenone in SD rats (purchasable from Victoria Lihua Co., Ltd.).

2. Grouping

Normal control group: DMSO 4ul

MPTP model group: Rotenone 4ul (2.5ug/ul, dissolved in DMSO)

STI571 treatment group: STI571 4ul (20mM, dissolved in normal saline) + Rotenone 4ul (2.5ug/ul,

Soluble in DMSO)

1. Expression of p-MST1-T183 protein

Stereotaxic localization of the CA1 and DG regions of the SD rat brain, administration of rotenone (rotenone, 10 μg, 3-72h) with a microinjection pump, and STI571 (42mg, 3-72h) at the same time, with p-Mst1-T183 antibody (1:100 , Cell Signaltechnology), and secondary antibody (anti-Rabbit IgG; (1:200), GE healthcare) for DAB (Zhongshan Jinqiao) immunohistochemical staining to observe the expression of p-MST1-T183 protein in CA1 and DG neurons Change. For the staining results, the image pro plus software was used for quantitative analysis of the average optical density. The results showed that STI571 had a significant inhibitory effect on neuronal injury in CA1 and DG regions induced by rotenone treatment for 6h-3d, inhibited the protein expression of p-MST1-T183, that is, inhibited the phosphorylation of the T183 site of neuronal MST1 protein, thereby Inhibit neuronal apoptosis response (Figure 1).

2. Observation of cell morphology

At 24h, 48h, and 72h of the above stereotaxic administration, TUNEL staining (ApopTag Flurescein In SituApoptosis Detection Kit S7110, CHEMICON) was used to observe the apoptotic response of nerve cells.

The number of Hoechest stained (blue) cells and the number of TUNEL kit stained (green) cells were counted using image pro plus software, and the ratio of the latter to the former was the percentage of apoptosis. The results are shown in Figure 2, STI571 can significantly inhibit the apoptotic response of nerve cells in the CA1 region induced by rotenone treatment for 24 hours; inhibit the apoptosis response of nerve cells in the DG region induced by rotenone treatment for 24-72 hours (Figure 2).

3. STI571 can significantly inhibit the apoptosis of primary substantia nigra neurons and the expression of Lewy body protein induced by rotenone, and inhibit the phosphorylation and activation of PKC and Akt

1. Experimental steps

Primary culture of substantia nigra neurons from 12-day SD fetal mice (Thomas Fath1, Yazi D Ke2, Peter Gunning1, Ju¨rgen Go¨tz2 & Lars M Ittner (2009) Primary support cultures of hippocampal and substantia nigra neurons NATURE PROTOCOLS 4: 78-85), after 3 days of differentiation, they were divided into three groups and treated with drugs according to the following methods. [1] control group: no drug treatment [2] rotenone group: Rotenone (final concentration 5uM) treatment for 30min; [3] STI571+MPTP group: STI571 (final concentration 5uM) pretreatment for one hour, add Rotenone (final concentration 5uM) ) for 30 min; the cells were collected, lysed with RIPA, and centrifuged to save the supernatant.

In the PD-like cell model of SD rat primary substantia nigra neurons induced by rotenone (Rotenone), STI571 can significantly inhibit the expression of cleavage-capase 3 induced by rotenone; inhibit the expression of Lewy body protein α-synuclein ; Inhibit the protein expression of p-PKC and p-Akt, the antibodies involved are anti-cleavage-capase3 (cell signal), anti-α-synuclein (ABCOM), anti-p-PKC (santa), anti- p-Akt (cell signal) and secondary antibodies anti-mouse and anti-Rabbit IgG (GEhealth) (Figure 11).

Therefore, as a specific inhibitor of c-Ab1, STI571 can effectively fight against the formation of PD, improve behavioral abnormalities and cognitive dysfunction in PD, and may be used as a candidate drug for neurodegenerative diseases such as Parkinson's disease, and has good potential. The application prospect has greatly expanded the clinical indications of STI571.

Claims (8)

1. Application of imatinib mesylate in the preparation of medicines for the treatment of neurodegenerative diseases. 2. The application according to claim 1, characterized in that: the neurodegenerative disease is Parkinson's disease. 3. Application of imatinib mesylate in the preparation of products for motor dysfunction and/or cognitive dysfunction. 4. Application of imatinib mesylate in the preparation of preparations for inhibiting neuronal apoptosis. 5. The application according to claim 4, characterized in that: the inhibition of nerve cell apoptosis is achieved by inhibiting the phosphorylation of the neuron MST1 protein T183 site; the amino acid sequence of the neuron MST1 protein is as follows: Shown in sequence 1 in the sequence listing. 6. The application as claimed in claim 4 or 5, characterized in that: said nerve cell apoptosis response is induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine or rotenone formed symptoms. 7. The use of imatinib mesylate in the preparation of drugs for improving the loss of dopamine neurons. 8. The application as claimed in claim 7, characterized in that: the loss of dopamine neurons is a symptom induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine or rotenone .

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