Medicine for preventing and treating Parkinson's disease and levodopa-induced dyskinesia in Parkinson's disease

Abstract

The invention belongs to the technical field of biological medicines, and in particular relates to a medicine for preventing and treating Parkinson's disease and levodopa-induced dyskinesia in Parkinson's disease, the active ingredient comprises a GHSR-1a bias agonist, and preferably further comprises clinical anti-Parkinson's disease medicines, such as mepanoba, levodopa and the like. According to the medicine provided by the invention, the abnormal rising level of nigra dopamine can be regulated and controlled by biased activation of a GHSR-1a receptor, so that the protection effect on dopaminergic neurons is achieved, and the risk of levodopa-induced dyskinesia in Parkinson patients after the levodopa is taken for a long time is reduced. The invention provides a new effective treatment approach for patients with Parkinson's disease, and provides an important theoretical basis for treating clinical Parkinson's disease and levodopa-induced dyskinesia in Parkinson's disease by using a GHSR-1a bias agonist.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to a drug for preventing and treating Parkinson's disease and levodopa-induced dyskinesia in Parkinson's disease. Background Art

[0002] Parkinson's disease is the second most common neurodegenerative disease, and its natural course is a gradual deterioration. Levodopa (L-DOPA), the "gold standard" for PD treatment, has always been its main therapeutic drug. L-DOPA can pass through the blood-brain barrier and decarboxylate to form dopamine. The supplementation of this neurotransmitter can temporarily relieve the behavioral abnormalities of PD, but because patients need to take medication for a long time to improve symptoms, and with the progression of the disease and the increase in treatment doses, most patients will experience L-DOPA-induced dyskinesia, which seriously affects the quality of life of patients and is an important cause of disability in PD patients. Almost all PD patients will suffer from motor complications within 15 to 20 years after diagnosis.

[0003] At present, continuous dopaminergic stimulation is the main concept for treating motor complications of PD. By adjusting the dosage and type of drugs, changing the method of administration, multi-target combined treatment and surgery, the motor complications of most patients can be controlled, but the treatment effect is limited and there are certain side effects. In view of this, it is necessary to provide an improved drug for the prevention and treatment of Parkinson's disease and levodopa-induced dyskinesia in Parkinson's disease to solve the above problems. Summary of the invention

[0004] The purpose of the present invention is to provide a drug for preventing and treating Parkinson's disease and levodopa-induced dyskinesia in Parkinson's disease, which regulates the abnormally elevated level of substantia nigra dopamine by preferentially activating GHSR-1a receptors, thereby exerting a protective effect on dopaminergic neurons, reducing the risk of levodopa-induced dyskinesia in Parkinson's patients who take levodopa for a long time, and providing a new and effective treatment approach for Parkinson's patients.

[0005] To achieve the above object, the present invention provides a drug for preventing and treating Parkinson's disease, wherein the active ingredient includes GHSR-1a biased agonist N8279, and the structural formula is as follows:

[0006]

[0007] Furthermore, the active ingredients also include Madopar and Levodopa.

[0008] Furthermore, the molar ratio of the N8279 to the Madopar or L-DOPA is 1:(1-100).

[0009] Furthermore, the mass content of the active ingredient in the medicine is 0.01% to 100%. The mass content of the active ingredient in the medicine is 0.1% to 10%.

[0010] Furthermore, the medicine also includes excipients, and the mass content of the excipients in the medicine is 0.01% to 99.9%.

[0011] Furthermore, the drug also includes excipients, which include one or more of disintegrants, solubilizers, emulsifiers, preservatives, flavoring agents, stabilizers, suspending agents, colorants, adhesives, fillers, and coating materials.

[0012] Furthermore, the dosage form of the drug is an oral dosage form, an injection dosage form or an inhalation dosage form.

[0013] Furthermore, the dosage of the drug is 50 mg / kg Tid.

[0014] In general, the above technical solution conceived by the present invention has the following technical advantages compared with the prior art:

[0015] The present invention provides a drug for preventing and treating Parkinson's disease and levodopa-induced dyskinesia. The drug and drug combination can be used to preferentially activate GHSR-1a receptors to regulate the abnormally elevated level of substantia nigra dopamine, thereby exerting a protective effect on Parkinson's disease dopaminergic neurons and achieving the purpose of treating Parkinson's disease and levodopa-induced dyskinesia. The present invention provides a new and effective treatment approach for Parkinson's disease patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The effect of 0.01-10 nM N8279 on N2a cell cytotoxicity;

[0017] Figure 2 The effect of 0.01-10 μM N8279 on N2a cell cytotoxicity;

[0018] Figure 3 The effect of N8279 on DA content in PC12 cells co-treated with L-DOPA and 6-OHDA;

[0019] Figure 4 The effect of N8279 on the voluntary movement ability of mice induced by MK-801;

[0020] Figure 5 The effect of N8279 on the voluntary movement ability of mice induced by MK-801;

[0021] Figure 6 The effect of N8279 on the TH protein content in the substantia nigra of mice induced by MK-801;

[0022] Figure 7 The effect of N8279 on the DAT protein content in the substantia nigra of mice induced by MK-801;

[0023] Figure 8 The effect of N8279 on MK-801-induced striatal dopamine and its metabolites in mice;

[0024] Fig. 9 The effect of N8279 on the AIM score of mice with L-dopa-induced dyskinesia;

[0025] Fig.10 The effect of N8279 on the voluntary movement ability of mice with L-dopa-induced dyskinesia. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0027] The following examples will verify the preventive and therapeutic effects of the drug and drug combination on Parkinson's disease and levodopa-induced dyskinesia through in vivo and in vitro experiments.

[0028] Example 1: Study on the toxic effect of N8279 on N2a cells

[0029] The cell viability was detected by Cell Counting Kit-8 (CCK-8).

[0030] Experimental methods:

[0031] (1) When the density of N2a cells in the cell culture flask reaches about 90%, N2a cells are plated at 5×10 4 The cells were inoculated into a 96-well plate at a density of 100 μL / mL, and 100 μL of culture medium was added to each well. The plate was placed at 37°C and 5% CO 2 The cells were cultured in a cell culture incubator and observed under a microscope. When the cell confluence in the plate reached 80% of the 96-well plate, different concentrations of N8279 solution (0, 0.01 nM, 0.03 nM, 0.1 nM, 0.3 nM, 1 nM, 3 nM, 10 nM, 0.1 μM, 1 μM, 10 μM) were added and cultured for 24 hrs.

[0032] (2) Add 10 μL of CCK-8 solution to each well and continue incubating in the incubator for 1-2 h in the dark.

[0033] (3) Use an ELISA reader to measure the absorbance at 450 nm.

[0034] (4) Calculate the cell survival rate: cell survival rate (%) = [A (experimental group) - A (blank control group)] / [A (control group) - A (blank control group)] × 100%.

[0035] Experimental results:

[0036] like Figure 1 and 2 As shown, N8279 at concentrations ranging from 0.01 nM to 10 μM had no toxicity to N2a cells.

[0037] Example 2: Study on the effect of N8279 on the dopamine elevation level of N2a cells

[0038] The ELISA (enzyme-linked immunosorbent assay) experiment was used to detect the cellular dopamine content at different treatment times.

[0039] Experimental methods:

[0040] (1) When the density of PC12 cells in the cell culture flask reaches about 90%, PC12 cells are plated at 1×10 5 The cells were inoculated into a 48-well plate at a density of 100 μL / mL, 300 μL of culture medium was added to each well, and the plate was placed at 37°C with 5% CO 2 The cells were cultured in a cell culture incubator. When the cell confluence in the plate reached 80% of the 96-well plate, 6-OHDA was added for treatment for 24 hrs, and then L-DOPA and N8279 were added for pretreatment for 30 min.

[0041] (2) The culture medium was discarded and the culture medium containing 6-OHDA, L-DOPA and N8279 was added again. The cells were treated for 10 min, 30 min, 60 min and 90 min respectively. The cell supernatant was aspirated and ELISA experiments were performed.

[0042] (3) The Rat DAELISA kit was equilibrated at room temperature for 20 min in advance, and standard wells and sample wells were set up. 50 μL of standard of different concentrations was added to the standard wells, 10 μL was added to the sample wells, and then 40 μL of sample diluent was added. Nothing was added to the blank wells.

[0043] (4) Except for the blank well, add 100 μL of the antibody to be tested labeled with HRP (horseradish peroxidase) to all other wells, seal the reaction wells with a sealing film, and incubate in a 37°C incubator for 60 min.

[0044] (5) Discard the liquid, pat dry with absorbent paper, fill each well with washing solution and let stand for 1 min, shake off the washing solution, pat dry, and repeat 5 times. Add 50 μL of substrate A and B to each well and incubate at 37°C in the dark for 15 min. Add 50 μL of stop solution to each well and measure the absorbance (OD) value at 450 nm within 15 min.

[0045] (6) Draw a standard curve and calculate the concentration value of each sample according to the curve equation.

[0046] Experimental results:

[0047] like Figure 3 , ELISA results showed that 6-OHDA could reduce the DA content of PC12 cells. After L-DOPA alone was given, the DA content of cells increased significantly, and after co-treatment with 6-OHDA, the DA content of cells decreased. After combined treatment with N8279, the DA content of cells decreased further, indicating that N8279 has the effect of reducing the abnormal increase of dopamine in PC12 cells.

[0048] Example 3: Study on the effect of N8279 on abnormal motor function of mice induced by MK801

[0049] MK-801-induced abnormal movement model in mice:

[0050] Eight-week-old C57BL / 6j male mice were used for modeling. They were pretreated with intraperitoneal injections of 5 mg / kg N8279 daily from day 1 to 7. Starting from day 8, 5 mg / kg N8279 was intraperitoneally injected daily. 1.5 hrs later, 0.25 mg / kg MK-801 was intraperitoneally injected for 4 consecutive days. On the last day, behavioral experiments (open field test) were performed and samples of the substantia nigra and striatum were collected 30 minutes after the injection of MK-801.

[0051] Open field test:

[0052] (1) The open field experiment was conducted in a low-light, low-noise environment, and the activities of the mice were recorded by a camera.

[0053] (2) The open field test requires an opaque acrylic box of 42 cm × 42 cm with an open top.

[0054] (3) Place the male mouse in the center of the open field and observe the total distance the mouse moves, the time it stays in the open field, and the average movement speed within 10 minutes. After each mouse experiment is completed, wipe the feces and urine in the box, and thoroughly clean the open field with 75% alcohol, and then wipe it dry with a paper towel or dry cloth before placing the next mouse.

[0055] (4) Analyze the changes in the total distance and average speed of mice in the open field.

[0056] Experimental results:

[0057] like Figure 4 and 5 The open field results showed that after treatment with MK-801 alone, the total movement distance, average speed, and center residence time of mice increased significantly. When combined with N8279 treatment, the total movement distance and average speed were significantly reduced compared with the MK-801 group, indicating that N8279 can antagonize the changes in mouse motor function induced by MK-801.

[0058] Example 4: Study on the effect of N8279 on the elevated level of dopamine in the substantia nigra of mice induced by MK801

[0059] 1. Western blotting was used to detect changes in the content of proteins related to dopamine synthesis, release, transport, and degradation in the substantia nigra, such as TH, VMAT2, DAT, and MAO-B.

[0060] Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

[0061] (1) 10% separation gel: Each gel requires 2 mL of separation gel buffer, 2.664 mL of 30% acrylamide, 80 μL APS, 3.2 μL TEMED, and 3.336 mL of double distilled water. Mix thoroughly and add to the gel plate. Add anhydrous ethanol to seal the plate and wait for it to solidify.

[0062] (2) 5% stacking gel: Each piece of gel requires 0.5 mL of stacking gel buffer, 0.34 mL of 30% acrylamide, 30 μL APS, 2 μL TEMED, and 1.14 mL of double distilled water. Pour out the anhydrous ethanol, add the mixed stacking gel, and quickly insert the comb teeth to wait for it to solidify.

[0063] (3) Protein separation: Add protein samples in sequence and run electrophoresis at a constant voltage of 80 V for the stacking gel and 130 V for the separation gel.

[0064] (4) Transfer: Place the sponge, filter paper, methanol-activated PVDF membrane, gel, and filter paper in the transfer clip in this order, and perform wet transfer at a constant current of 300 mA for 90 min.

[0065] (5) Block with 10% milk powder at room temperature for 2.5 hrs and incubate with primary antibody at 4°C for 8 hrs.

[0066] (6) Wash with TBST for 3 × 10 min, incubate with secondary antibody at room temperature for 1.5 hrs, and wash again with TBST buffer;

[0067] (7) Add developer solution and place in a developer for development.

[0068] (8) Use Image J analysis software to analyze the grayscale value of the bands.

[0069] Experimental results:

[0070] like Figure 6 and 7 The results of Western blotting showed that compared with the control group, after treatment with MK-801, the TH content showed an upward trend, while the DAT content showed no significant change. When MK-801 was treated with N8279 together, compared with the MK-801 group, the TH content was significantly decreased, while the DAT showed no significant change, indicating that N8279 can reduce the increase in TH protein expression in the substantia nigra of mice induced by MK-801, and play a major role in the dopamine synthesis process.

[0071] 2. High Performance Liquid Chromatography (HPLC)

[0072] (1) After the mice were anesthetized, their heads were quickly decapitated and their brains were removed. The bilateral striatums were quickly removed on ice.

[0073] (2) Place the removed striatum into a 1.5 mL EP tube and accurately weigh it, then add 80 μL of ice-cold sample pretreatment solution A.

[0074] (3) Grind in a tissue grinder for 10 seconds, twice. Place on ice for 1 hour. Centrifuge at 4°C, 12,000 rpm for 20 minutes. Carefully transfer 40 μL of supernatant into a new 15 mL EP tube, add 80 μL of ice-cold pretreatment solution B, then vortex to mix, and place on ice for 1 hour.

[0075] (4) Centrifuge at 12,000 rpm for 20 min at 4°C, remove the supernatant, and store it in a freezer at -80°C until testing.

[0076] Filter the sample 1-2 times before loading.

[0077] (5) Parameter setting: Set the flow rate to 0.5-1 μL / min and test each sample for 5-10 min.

[0078] (6) First test each standard and measure its retention time as a qualitative indicator, then test each group of samples.

[0079] (7) Finally, draw a standard curve: Draw a standard curve for the seven concentration gradient standards according to the detected concentrations to obtain the linear regression equation for each standard, and then calculate the sample concentration.

[0080] Experimental results:

[0081] like Figure 8The results showed that after treatment with MK-801 alone, the DA content in the mouse striatum increased significantly. When treated with N8279 in combination, the DA content in the mouse striatum tended to decrease compared with the MK-801 alone group, indicating that N8279 can partially antagonize the increase in dopamine content in the mouse striatum induced by MK-801.

[0082] Example 5: Study on the protective effect of N8279 on the levodopa-induced dyskinesia model

[0083] Dyskinesia modeling method:

[0084] Eight-week-old C57BL6 / j male mice were used for modeling and divided into control group, 6-OHDA group, 6-OHDA+L-DOPA group, and 6-OHDA+L-DOPA+N8279 group. 6-OHDA was injected with a final concentration of 3.6 mg / kg by stereotaxic brain injection, and the injection coordinates were anterior-posterior (AP): +1.00 mm, medial (ML): +2.10 mm, dorsal-ventral (DV): +00.30 mm; AP: +0.30, ML: +2.30 mm, DV: -2.90 mm. Three weeks after 6-OHDA injury, 15 mg / kg of L-DOPA and 12 mg / kg of benserazide hydrochloride were given by intraperitoneal injection every day for 3-4 weeks. In the last week, behavioral tests (AIM score, open field test) were performed 20 minutes after the last administration of L-DOPA and samples were collected.

[0085] AIM Rating:

[0086] In a 25 cm × 25 cm transparent acrylic box, cameras were installed on the side and bottom to observe the behavior of the mice and record the number and duration of various AIMs that occurred for each mouse.

[0087] According to the behavioral performance of mice, their involuntary motor behaviors were classified, such as abnormal movements of the mouth, limbs or trunk. (1) Forelimb AIM; (2) Axial AIM; (3) Orofacial AIM. After the rat PD dyskinesia model was successfully established, normal saline or drug treatment was continued for 21 days. AIM scores were performed on the 1st day and the 7th, 14th and 21st days. Starting from the administration of L-DOPA, the scores were observed for 1 minute every 20 minutes. The AIM score is the sum of the three scores. The highest AIM score is 108 points (scored 9 times, with a single highest score of 12 points).

[0088] Experimental results:

[0089] like Fig. 9 The results showed that the dyskinesia level of LID mice increased significantly at the 20th minute. After the combined administration of N8279, the dyskinesia level decreased significantly, which indicates that N8279 can reduce the dyskinesia level of LID mice.

[0090] Open field test:

[0091] (1) The open field experiment was conducted in a low-light, low-noise environment, and the activities of the mice were recorded by a camera.

[0092] (2) The open field test requires an opaque acrylic box of 42 cm × 42 cm with an open top.

[0093] (3) Place the male mouse in the center of the open field and observe the total distance the mouse moves, the time it stays in the open field, and the average movement speed within 10 minutes. After each mouse experiment is completed, wipe the feces and urine in the box, and thoroughly clean the open field with 75% alcohol, and then wipe it dry with a paper towel or dry cloth before placing the next mouse.

[0094] (4) Analyze the changes in the total distance and average speed of mice in the open field.

[0095] Experimental results:

[0096] like Fig.10 The open field results showed that after 6-OHDA treatment alone, the total movement distance and average speed of the mice were significantly reduced. When L-DOPA treatment was given, the total movement distance and average speed of the mice were significantly reduced, indicating that dyskinesia occurred 21 days after taking L-DOPA. When the mice were treated with N8279 in combination, the total movement distance and average speed were significantly increased compared with those of the LID group mice, indicating that N8279 can antagonize the changes in motor function of dyskinesia mice induced by L-DOPA.

[0097] In summary, the drug for preventing and treating Parkinson's disease provided by the present invention regulates the abnormally elevated level of substantia nigra dopamine by preferentially activating GHSR-1a receptors, thereby exerting a protective effect on dopaminergic neurons of Parkinson's disease, thereby achieving the purpose of treating Parkinson's disease and levodopa-induced dyskinesia.

[0098] It will be easily understood by those skilled in the art that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A drug for preventing and treating Parkinson's disease and levodopa-induced dyskinesia in Parkinson's disease, characterized in that: The active ingredient includes GHSR-1a biased agonist N8279, the structural formula of which is as follows:

2. The drug according to claim 1, characterized in that The active ingredients also include Madopar and Levodopa.

3. The drug according to claim 2, characterized in that The molar ratio of the N8279 to the Madopar or L-dopa is 1:(1-100).

4. The drug according to claim 1, characterized in that The mass content of the active ingredient in the medicine is 0.01% to 100%.

5. The drug according to claim 4, characterized in that The mass content of the active ingredient in the medicine is 0.1% to 10%.

6. The drug according to claim 1, characterized in that The medicine also includes auxiliary materials, and the mass content of the auxiliary materials in the medicine is 0.01% to 99.9%.

7. The drug according to claim 6, characterized in that The auxiliary materials include one or more of disintegrants, solubilizers, emulsifiers, preservatives, flavoring agents, stabilizers, suspending agents, colorants, adhesives, fillers, and coating materials.

8. The drug according to claim 1, characterized in that The dosage form of the drug is an oral dosage form, an injection dosage form or an inhalation dosage form.

9. The drug according to claim 1, characterized in that The dosage of the drug is 50 mg / kg Tid.

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