Application of PARP1 inhibitor in preparation of medicine for treating and / or relieving Friedel's ataxia

Abstract

The invention provides an application of a PARP1 inhibitor in preparation of a medicine for treating and / or relieving Friedel's ataxia, and belongs to the technical field of medicines. According to the application disclosed by the invention, B lymphocyte GM15850 and / or fibroblast GM03816 from a Friedel's Ataxia (FRDA) patient are / is taken as a research object, and the PARP1 inhibitor is verified to have the effects of remarkably improving cell proliferation and activity, and can be used for promoting the expression of cell mitochondria FXN protein and promoting the activity of a mitochondria compound; an FRDA mouse model YG8R is used as a research object, and it is verified that the PARP1 inhibitor can remarkably improve the movement function of mice and improve the FXN protein level of all tissues of the mice. The PARP1 inhibitor has the effect of treating and / or relieving FRDA, and can be applied to preparation of drugs for treating and / or relieving FRDA related diseases.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to the use of a PARP1 inhibitor in preparing a drug for treating and / or alleviating Friedreich's ataxia. Background Art

[0002] Friedreich's ataxia (FRDA) is a rare hereditary neurodegenerative disease, which is autosomal recessive and causes degeneration of the posterior spinal cord and cerebellum. The final pathological manifestation is mitochondrial dysfunction followed by degenerative changes in nerve and cardiomyocytes.

[0003] This disease has unique clinical features, such as childhood onset, progressive limb ataxia, accompanied by pyramidal tract signs, dysarthria, scoliosis, arched feet and heart damage. (1) The disease usually starts at the age of 4 to 15 years, occasionally in infants and after the age of 50. Both men and women can be affected. The first symptom is progressive gait ataxia, staggering gait, swaying from side to side, and easy to fall; within 2 years, ataxia of both upper limbs appears, showing clumsy movements and intention tremor; in this early stage, knee tendon reflexes and ankle reflexes disappear, cerebellar dysarthria or explosive speech occurs, and the reflexes of both upper limbs and both knee tendon reflexes in some patients can be preserved. The position and vibration sense of the joints of both lower limbs are impaired, and light touch, pain and temperature sense are usually not affected. Weakness of both lower limbs occurs later, which may be caused by upper or lower motor neuron damage, or both. (2) Patients usually have an extension plantar reflex within 5 years before the onset of symptoms. Weakness and atrophy of the medial foot muscles leading to cavus feet with claw toes are common physical signs and may also be isolated manifestations in unaffected family members. Progressive severe kyphoscoliosis can lead to functional disability and chronic restrictive lung disease. Cardiomyopathy can sometimes only be detected by echocardiography and can lead to congestive heart failure, which is the main cause of death. Other abnormalities include optic atrophy, nystagmus (mostly horizontal), paresthesia, tremor, hearing loss, vertigo, cramps, lower limb pain and diabetes. (3) Physical examination shows positive heel-knee-shin test and eyes-closed difficulty standing sign. Some cases have upper thoracic spinal deformity, some have optic atrophy, some have cavus feet, some have arrhythmias or heart murmurs, and a few have diabetes.

[0004] There is currently no cure for Friedreich's ataxia. In 2024, the United States approved the only drug Skyclarys that targets NRF2 to resist oxidative stress. Other treatments are mainly symptomatic, including physical therapy, rehabilitation training, use of walking aids, and medications to treat complications such as heart and diabetes. The course and severity of Friedreich's ataxia vary from person to person. Some patients may need a wheelchair in early adulthood, while others may still maintain a certain degree of independence at a later age. In summary, the efficacy of existing clinical strategies for the treatment of Friedreich's ataxia is not sufficient, and the prognosis of Friedreich's ataxia is poor, so further exploration of treatment strategies for Friedreich's ataxia is needed. Summary of the invention

[0005] In order to solve the problems existing in the prior art, the object of the present invention is to provide the use of a PARP1 inhibitor in the preparation of a drug for treating and / or alleviating Friedreich's ataxia and related diseases.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides the use of a PARP1 inhibitor in preparing a drug for treating and / or alleviating Friedreich's ataxia.

[0008] The present invention provides the use of a PARP1 inhibitor in preparing a drug for treating and / or alleviating related symptoms caused by Friedreich's ataxia.

[0009] Preferably, the drug has any of the following effects:

[0010] (1) Improve motor dysfunction;

[0011] (2) Improve hearing impairment and olfactory abnormalities;

[0012] (3) Improve cardiovascular system lesions;

[0013] (4) Improve endocrine system lesions;

[0014] (5) Improve bone deformities.

[0015] Preferably, the cardiovascular system lesions include myocardial hypertrophy and arrhythmia.

[0016] Preferably, the endocrine system lesions include impaired glucose tolerance, abnormal blood sugar and abnormal lipid metabolism.

[0017] Preferably, the skeletal deformity comprises scoliosis, kyphoscoliosis and pes cavus.

[0018] Preferably, the PARP1 inhibitors include AG14361, Olaparib and Saruparib.

[0019] Preferably, the active ingredient of the drug includes a PARP1 inhibitor.

[0020] Preferably, the drug further comprises a pharmaceutically acceptable carrier.

[0021] Preferably, the content of the PARP1 inhibitor in the drug is 50-100 wt %.

[0022] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0023] The present invention proposes for the first time the use of PARP1 inhibitors in the preparation of drugs for treating and / or alleviating Friedreich's ataxia and its related diseases. The B lymphocytes GM15850 and / or fibroblasts GM03816 of patients with Friedreich's ataxia are used as research objects, and it is verified that PARP1 inhibitors have the effect of significantly improving cell proliferation and activity, and can promote the expression of cell mitochondrial FXN protein and promote the activity of mitochondrial complexes; the Friedreich's ataxia mouse model YG8R is used as a research object, and it is verified that PARP1 inhibitors can increase the FXN protein level of various tissues of mice, and significantly improve the motor function and glucose and lipid metabolism level of mice. It shows that PARP1 inhibitors have a therapeutic and / or alleviating effect on Friedreich's ataxia, and can fundamentally treat Friedreich's ataxia by increasing the expression of mitochondrial FXN protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 : Results of cell proliferation ability test after administration of PARP1 inhibitor;

[0025] Figure 2 : Cell viability test results after PARP1 inhibitor administration;

[0026] Figure 3 : Results of cell FXN protein expression level detection after PARP1 inhibitor administration;

[0027] Figure 4 :The results of mitochondrial complex I activity detection after PARP1 inhibitor administration;

[0028] Figure 5 :JC-1 staining results of cell mitochondrial membrane potential after administration of PARP1 inhibitor;

[0029] Figure 6 :The results of protein expression of FXN in cell mitochondria after administration of PARP1 inhibitor;

[0030] Figure 7 : Results of wooden beam test in mice after administration of PARP1 inhibitor;

[0031] Figure 8 : Results of the mouse rotation fatigue test after administration of PARP1 inhibitors;

[0032] Fig. 9 : Fasting blood glucose of mice after administration of PARP1 inhibitor;

[0033] Fig.10 : Results of tissue FXN protein expression level detection after PARP1 inhibitor administration;

[0034] Fig.11 :H&E staining results of mouse myocardium after administration of PARP1 inhibitor;

[0035] Fig.12 :H&E staining results of mouse neural tissue after administration of PARP1 inhibitor;

[0036] Fig.13 : Nissl staining results of mouse neural tissue after administration of PARP1 inhibitor. DETAILED DESCRIPTION

[0037] The present invention provides the use of a PARP1 inhibitor in the preparation of a drug for treating and / or alleviating Friedreich's ataxia (FRDA); the use of a PARP1 inhibitor in the preparation of a drug for treating and / or alleviating the related symptoms caused by Friedreich's ataxia. PARP1 (poly ADP-ribose polymerase 1) inhibitors are a class of drugs targeting the PARP1 enzyme. The present invention uses GM15850 B lymphocytes and / or GM03816 fibroblasts of FRDA patients as research objects, and experimentally verifies that PARP1 inhibitors have the effect of significantly improving the proliferation and activity of FRDA-GM15850 and / or GM03816 cells, and can promote the expression of FXN protein in mitochondria of FRDA-GM15850 and / or GM03816 cells and promote the activity of mitochondrial complexes; at the same time, the present invention uses the FRDA mouse model YG8R as a research object, and experimentally verifies that PARP1 inhibitors can significantly improve the motor function of YG8R mice and increase the FXN protein levels in various tissues of YG8R mice. The results demonstrate that PARP1 inhibitors have a therapeutic and / or alleviating effect on FRDA and can be used to prepare drugs for treating and / or alleviating FRDA-related diseases.

[0038] Preferably, the drug of the present invention has any of the following effects:

[0039] (1) Improve motor dysfunction;

[0040] (2) Improve hearing impairment and olfactory abnormalities;

[0041] (3) Improve cardiovascular system lesions, including but not limited to myocardial hypertrophy and arrhythmia;

[0042] (4) Improve endocrine system diseases, including but not limited to impaired glucose tolerance, abnormal blood sugar (such as increased blood sugar, diabetes) and abnormal lipid metabolism;

[0043] (5) Improve skeletal deformities, including but not limited to scoliosis, kyphoscoliosis, and cavus feet.

[0044] The PARP1 inhibitor of the present invention can improve the incoordination of movement, abnormal cardiac tissue structure, elevated blood sugar and the like caused by Friedreich's ataxia, maintain / improve the number and morphology of Purkinje cells and neurons, and reduce the phenomenon of neurophagy in the cerebral cortex.

[0045] The PARP1 inhibitors of the present invention include but are not limited to AG14361, Olaparib and Saruparib.

[0046] The active ingredient of the drug of the present invention includes a PARP1 inhibitor, that is, the PARP1 inhibitor can be used as the only active ingredient, or the PARP1 inhibitor can be used in combination with other drugs. The content of the PARP1 inhibitor in the drug of the present invention is 50-100wt%, preferably 80-99wt%.

[0047] Preferably, the drug of the present invention further comprises a pharmaceutically acceptable carrier to ensure that the prepared drug is convenient for preparation and clinical application. Further preferably, the pharmaceutical excipient of the present invention is selected from at least one of a diluent, a wetting agent, a binder, a lubricant, a colorant, and a coating agent.

[0048] Preferably, the dosage form of the drug of the present invention is an oral preparation, which includes but is not limited to tablets, capsules, sustained-release preparations, granules, pills, injections, suspensions, dispersants, and syrups. The dosage form of the drug of the present invention is not limited thereto, and other achievable dosage forms are within the scope of protection of the present invention.

[0049] Preferably, when the drug prepared by the PARP1 inhibitor is used to treat FRDA patients, when the active ingredient of the drug is Olaparib, the recommended dosage of Olaparib is 100-600 mg, taken orally twice a day, with or without meals. For patients with moderate renal impairment (CLcr 31-50 mL / min), it is recommended to reduce the dose by 1 / 3 and take it orally twice a day; when the active ingredient of the drug is Saruparib, the clinically recommended dosage of Saruparib is 20-100 mg, taken orally once a day.

[0050] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0051] In a specific embodiment of the present invention, FRDA-GM15850 represents GM15850, a B lymphocyte cell line derived from a FRDA patient, purchased from Coriell; GM03816, a fibroblast cell derived from a FRDA patient, purchased from Coriell; GM14519 is a lymphocyte cell line derived from a normal person, purchased from Coriell; AG14361 is purchased from Aladdin (A125467-5 mg), Olaparib is purchased from Aladdin (O126162-250 mg), and Saruparib is purchased from Aladdin (S578989-10 mg); FRDA transgenic mice YG8R (#012253) and control mice Y47 (#024097) were purchased from Jackson Laboratory.

[0052] In the following examples, unless otherwise specified, all methods are conventional. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0053] Example 1

[0054] Detection of cell proliferation ability after administration of PARP1 inhibitor

[0055] FRDA-GM15850 cells were seeded in 6-well plates containing 1640 complete medium, with 5×10 cells per well. 6 After 24 hours, the cells were treated with PARP1 inhibitors AG14361 (14nM), Olaparib (10nM), and Saruparib (15nM), respectively. After 120 hours of treatment, the cells were collected, resuspended, stained with trypan blue, and counted. The FRDA-GM15850 control group was added with an equal volume of DMSO as the PARP1 inhibitor. The control GM14519 cells were inoculated in a 6-well plate containing 1640 complete medium and cultured at 5×10 6 cells were used as a blank control group of healthy human cells.

[0056] The results are as follows Figure 1As shown in the figure, GM14519 ctr represents the healthy human cell blank control group, GM15850 ctr represents the DMSO-treated patient cell control group, GM15850+AG14361 represents the AG14361-treated group, GM15850+Olaparib represents the Olaparib-treated group, and GM15850+Saruparib represents the Saruparib-treated group. The results show that PARP1 inhibitors can effectively improve the proliferation ability of FRDA-GM15850.

[0057] Example 2

[0058] Cell viability assay after PARP1 inhibitor administration

[0059] FRDA-GM15850 was inoculated into a 96-well plate containing 1640 complete medium, with 1×10 4 After 24 hours, the PARP1 inhibitors AG14361, Olaparib, and Saruparib were used to treat the cells, with concentrations of 14 nM, 10 nM, and 15 nM, respectively, and the treatment time was 120 hours. The FRDA-GM15850 control group was added with DMSO in an equal volume as the PARP1 inhibitor. GM14519 was inoculated into a 96-well plate containing 1640 complete medium and cultured at 1×10 4 Cells were used as blank control group. Washed once with 1×PBS, added with 10 μL CCK-8 working solution, and placed in a 37°C, 5% CO2 incubator for 4 hours. The absorbance was detected at 450 nm using an enzyme marker and the OD value was read.

[0060] The results are as follows Figure 2 As shown in the figure, GM14519 ctr represents the blank control group, GM15850 ctr represents the DMSO-treated control group, GM15850+AG14361 represents the AG14361-treated group, GM15850+Olaparib represents the Olaparib-treated group, and GM15850+Saruparib represents the Saruparib-treated group. The results showed that PARP1 inhibitors can effectively enhance the viability of FRDA-GM15850 cells.

[0061] Example 3

[0062] FXN protein expression level in cells after PARP1 inhibitor administration

[0063] FRDA-GM15850 were inoculated into 6-well plates containing 1640 complete medium, with 6×10 cells per well. 6After 24 hours, the cells were treated with PARP1 inhibitors AG14361 (14 nM), Olaparib (7.5 nM, 10 nM), and Saruparib (15 nM), respectively. After 120 hours of treatment, the cells were collected. The FRDA-GM15850 control group was added with DMSO in an equal volume as the PARP1 inhibitor. GM14519 was inoculated in a 6-well plate containing 1640 complete medium and cultured at 6×10 6 cells as the blank control group.

[0064] The protein expression level was detected by Western blotting method, and the operation steps were as follows: collect cells and centrifuge (1000rpm, 5min) to remove the supernatant, and wash with PBS. According to the cell quantity, add an appropriate amount of cell lysis solution, resuspend the cells, and place them on ice for 10min, shaking 2 to 3 times during the period. Centrifuge at 15000rpm, 4℃, for 10min. Transfer the supernatant after centrifugation to a new pre-cooled eppendorf (EP) tube, and determine the protein concentration with Bradford working solution to ensure that the loading amount of each sample is consistent, and add an appropriate amount of 5×SDS-loading buffer, heat at 100℃ for 5min to fully denature the protein. After the sample is briefly centrifuged, add the sample to the SDS-PAGE gel loading well with a microinjector, and perform electrophoresis at a voltage of 100V. After the electrophoresis is completed, remove the gel from the device, remove the concentrated gel, and transfer the protein on the separation gel to the NC membrane using an electrotransfer instrument under a steady current of 250mA. After the transfer, the membrane was placed in the Ponceau staining solution for 2 minutes, and the staining solution that was not bound to the protein was washed away with pH 5.5 acid water, and the stained NC membrane was photographed. Then the membrane was placed in Tris-PM containing 5% skim milk powder and blocked at room temperature for 1 hour. After the blocking was completed, an appropriately diluted primary antibody was added and incubated overnight at 4°C. After the incubation, the primary antibody was recovered and the membrane was washed with buffer Tris-PM for 10 minutes each time, 4 times. The membrane was incubated with an appropriately diluted secondary antibody at room temperature for 1 hour. After the incubation was completed, it was washed 4 times with Tris-PM for 10 minutes each time. The protein on the membrane was bound to the chemiluminescent substrate, exposed and photographed using the Western blot chemiluminescent imaging system.

[0065] The results are as follows Figure 3As shown in the figure, GM14519 DMSO represents the blank control group, GM15850 DMSO represents the DMSO-treated control group, GM15850AG14361 represents the AG14361-treated group, GM15850 Ola 7.5nM and GM15850 Ola10nM both represent the Olaparib-treated group, and GM15850 Saruparib represents the Saruparib-treated group. The results showed that PARP1 inhibitors increased the expression level of FXN in FRDA-GM15850 cells.

[0066] Example 4

[0067] Activity of mitochondrial complex I after PARP1 inhibitor administration

[0068] FRDA-GM15850 were inoculated into 6-well plates containing 1640 complete medium, with 6×10 cells per well. 6 After 24 hours, the cells were treated with PARP1 inhibitors AG14361 (14 nM), Olaparib (10 nM), and Saruparib (15 nM), respectively. After 120 hours of treatment, the cells were collected. The FRDA-GM15850 control group was added with DMSO in an equal volume as the PARP1 inhibitor. GM14519 was inoculated in a 6-well plate containing 1640 complete medium and cultured at 6×10 6 cells as the blank control group.

[0069] Collect cells and centrifuge (1000rpm, 5min) to remove supernatant, and wash once with PBS. According to the amount of cells, add an appropriate amount of cell lysis solution, resuspend the cells, place on ice for 10min, and shake 2 to 3 times during the period. After the cells are lysed, the determination of mitochondrial complex I activity is carried out strictly according to the instructions of the kit. The mitochondrial complex I activity detection kit was purchased from Suzhou Keming Biotechnology Co., Ltd.

[0070] The results are as follows Figure 4 As shown in the figure, GM14519 represents the blank control group, GM15850 represents the control group where GM15850 is not treated with PARP1 inhibitor, GM15850+AG14361 represents the AG14361-treated group, GM15850+Olaparib represents the Olaparib-treated group, and GM15850+Saruparib represents the Saruparib-treated group. The results showed that PARP1 inhibitor significantly increased the activity of FRDA-GM15850 mitochondrial complex I.

[0071] Example 5

[0072] JC-1 staining of mitochondrial membrane potential after PARP1 inhibitor administration

[0073] FRDA-GM15850 was inoculated into 6-well plates containing 1640 complete medium, with 1×10 5 After 24 hours, the cells were treated with PARP1 inhibitors AG14361 (14 nM), Olaparib (10 nM), and Saruparib (15 nM), respectively. After 120 hours of treatment, the cells were collected. The FRDA-GM15850 control group was added with DMSO in an equal volume as the PARP1 inhibitor.

[0074] Take 1.0×10 5 ~6.0×10 5 Cells were resuspended in 0.5 mL of cell culture medium containing serum and phenol red. 0.5 mL of JC-1 staining working solution was added and mixed by inverting several times. Incubated in a cell culture incubator at 37°C for 20 min. After incubation, centrifuged at 600g for 3-4 min at 4°C to pellet the cells. The supernatant was discarded and the cells were carefully not to be removed by aspiration. Washed twice with JC-1 staining buffer (1×): resuspended cells with 1 mL of JC-1 staining buffer (1×), centrifuged at 600g for 3-4 min at 4°C to pellet the cells and the supernatant was discarded; resuspended cells with 1 mL of JC-1 staining buffer (1×), centrifuged at 600g for 3-4 min at 4°C to pellet the cells and the supernatant was discarded. Resuspended with an appropriate amount of JC-1 staining buffer (1×) and observed with a fluorescence microscope or laser confocal microscope.

[0075] The results are as follows Figure 5 As shown in the figure, GM15850+DMSO represents the DMSO-treated control group, GM15850+AG14361 represents the AG14361-treated group, GM15850+Olaparib represents the Olaparib-treated group, and GM15850+Saruparib represents the Saruparib-treated group; green fluorescence indicates that JC-1 exists in the mitochondria as a monomer, and the membrane potential decreases, and red fluorescence indicates that JC-1 exists in the mitochondria as a polymer, and the mitochondrial membrane potential is normal. The results show that PARP1 inhibitors can effectively maintain the normal membrane potential of mitochondria and improve the abnormal mitochondrial membrane potential of FRDA-GM15850 cells.

[0076] Example 6

[0077] Co-localization staining of mitochondria and FXN in fibroblasts after administration of PARP1 inhibitor

[0078] Mitochondrial staining: The cells were spread on a sterile 6-well plate and FRDA-GM03816 (1×10 51640 cells) were inoculated in a 6-well plate containing 1640 complete medium. After 24 hours, the PARP1 inhibitors AG14361 (14 nM), Olaparib (10 nM), and Saruparib (15 nM) were used for treatment. After 120 hours of treatment, the cells were collected. The FRDA-GM03816 control group was added with DMSO in an equal volume as the PARP1 inhibitor.

[0079] Add 0.5 mL of HBSS-diluted MitoTracker to the treated cells. TM Red FM (dilution ratio 1:10000), shake gently to mix. Incubate in a cell culture incubator at 37℃ for 20min. After incubation, wash twice with HBSS. Take out the cell slide, turn it upside down on a glass slide, add DAPI containing anti-fluorescence quenching in advance to the glass slide, and observe with a fluorescence microscope or laser confocal microscope.

[0080] FXN staining: Dilute FXN antibody (rabbit antibody) with 3% bovine serum albumin (BSA) at a ratio of 1:500, add 0.5mL of diluted antibody to drug-treated cells, and incubate at 37°C for 60min. After incubation, wash twice with 1×PBS. Dilute secondary antibody (goat anti-rabbit, purchased from Jackson Laboratory) with 3% BSA at a ratio of 1:1000, add 0.5mL of diluted secondary antibody to primary antibody-treated cells, and incubate at 4°C for 60min. After incubation, wash twice with 1×PBS. Take out the cell slide, turn it upside down on the slide, add DAPI containing anti-fluorescence quenching in advance to the slide, and observe with a fluorescence microscope or laser confocal microscope.

[0081] The results are as follows Figure 6 As shown in the figure, GM03816 DMSO represents the DMSO-treated control group, GM03816AG14361 represents the AG14361-treated group, GM03816 Olaparib represents the Olaparib-treated group, and GM03816 Saruparib represents the Saruparib-treated group; green fluorescence represents the expression of FXN protein, and red fluorescence represents mitochondria. The results show that PARP1 inhibitors can effectively increase the expression of FXN, increase the mitochondrial localization of FXN, and correct the abnormal morphology of FRDA-GM03816 cells.

[0082] Example 7

[0083] Behavioral scores of mice after PARP1 inhibitor administration

[0084] 1. Treatment of mice: Intraperitoneal injection of PARP1 inhibitors, AG14361 at a concentration of 15 mg / kg / d, Olaparib at a concentration of 10 mg / kg / d, and Saruparib at a concentration of 10 mg / kg / d, all for 30 days, all drugs were dissolved in a mixture of DMSO: olive oil (1:9), and the control group was injected with an equal volume of DMSO: olive oil (1:9). The experiment involved n represents the number of mice in each group.

[0085] 2. Wooden beam experiment: The length of the wooden beam was 1m, and the width was 12mm and 22mm respectively. The time it took for the mice to pass through the wooden beam was recorded to evaluate the coordination ability. The mice received 3 trainings and repeated the test 4 times, with at least 5min between each test.

[0086] The results are as follows Figure 7 As shown in the figure, Y47 represents the control mouse Y47 (#024097) injected with DMSO, YG8R represents the FRDA transgenic mouse YG8R (#012253) injected with DMSO, YG8R+AG14361 represents the FRDA transgenic mouse YG8R (#012253) injected with AG14361, YG8R+Olaparib represents the FRDA transgenic mouse YG8R (#012253) injected with Olaparib, and YG8R+Saruparib represents the FRDA transgenic mouse YG8R (#012253) injected with Saruparib; all data are expressed as Mean±SEM, n=9. The results show that PARP1 inhibitors can effectively shorten the time it takes for mice to pass through the wooden beam and improve the coordination ability of mice.

[0087] 3. Rotarod fatigue test: A rotarod fatigue tester (BiAngsi Co. Ltd.) was used to evaluate motor coordination. At least four experiments were performed, with an interval of no less than 200 s between each test. The speed of the rotarod was gradually increased from 4 rpm to 400 rpm, and the maximum time was set to 400 s. The time when the mouse fell off the rotarod was recorded.

[0088] The results are as follows Figure 8As shown in the figure, Y47 represents the control mouse Y47 (#024097) injected with DMSO, YG8R represents the FRDA transgenic mouse YG8R (#012253) injected with DMSO, YG8R+AG14361 represents the FRDA transgenic mouse YG8R (#012253) injected with AG14361, YG8R+Olaparib represents the FRDA transgenic mouse YG8R (#012253) injected with Olaparib, and YG8R+Saruparib represents the FRDA transgenic mouse YG8R (#012253) injected with Saruparib; all data are expressed as Mean±SEM, n=9. The results show that PARP1 inhibitors can effectively prolong the time of mice on the rotating rod and improve the coordination ability of mice.

[0089] Example 8

[0090] Fasting blood glucose levels in mice after PARP1 inhibitor administration

[0091] Mice were treated with intraperitoneal injection of PARP1 inhibitors, AG14361 at a concentration of 15 mg / kg / d, Olaparib at a concentration of 10 mg / kg / d, and Saruparib at a concentration of 10 mg / kg / d, for 30 days. The drugs were dissolved in a mixture of DMSO: olive oil (1:9), and the control group was injected with an equal volume of DMSO: olive oil (1:9). The experiment involved n, which represents the number of mice in each group.

[0092] After the mice were fasted for 12 hours, blood was collected from the tail tip of the mice, and the blood glucose of the mice was tested and recorded using a Yuyue blood glucose meter. After the recording was completed, the mice were allowed to resume their diet.

[0093] The results are as follows Fig. 9 As shown in the figure, YG8R represents the FRDA transgenic mouse YG8R (#012253) injected with DMSO, YG8RAG14361 represents the FRDA transgenic mouse YG8R (#012253) injected with AG14361, YG8R Olaparib represents the FRDA transgenic mouse YG8R (#012253) injected with Olaparib, and YG8R Saruparib represents the FRDA transgenic mouse YG8R (#012253) injected with Saruparib. The results showed that the fasting blood glucose level of YG8R mice was significantly reduced after administration of PARP1 inhibitors.

[0094] Example 9

[0095] Tissue FXN protein expression levels after PARP1 inhibitor administration

[0096] The protein expression level was detected by Western blotting method. The operation steps are as follows:

[0097] Mice were treated with intraperitoneal injection of PARP1 inhibitors, AG14361 at a concentration of 15 mg / kg / d, Olaparib at a concentration of 10 mg / kg / d, and Saruparib at a concentration of 10 mg / kg / d, for 30 days. The drugs were dissolved in a mixture of DMSO: olive oil (1:9), and the control group was injected with an equal volume of DMSO: olive oil (1:9). The experiment involved n, which represents the number of mice in each group.

[0098] The brain, cerebellum, brainstem, hippocampus, and heart of the mouse central nervous system tissues were removed, and each tissue was completely lysed with tissue lysis buffer (Biyuntian P0013). The clear supernatant was taken and transferred to a new pre-cooled Eppendorf (EP) tube, and the protein concentration was determined with Bradford working solution to ensure that the loading amount of each sample was consistent. An appropriate amount of 5×SDS-loading buffer was added and heated at 100℃ for 5min to fully denature the protein. After the sample was briefly centrifuged, the sample was added to the SDS-PAGE gel loading well with a microinjector and electrophoresed at a voltage of 100V. After the electrophoresis was completed, the gel was removed from the device, the concentrated gel was removed, and the protein on the separation gel was transferred to the NC membrane using an electrotransfer instrument under a steady current of 250mA. After the transfer was completed, the membrane was placed in a Ponceau red staining solution for 2min, and the staining solution that was not bound to the protein was washed away with acid water of pH 5.5, and the stained NC membrane was photographed. Then, the membrane was placed in Tris-PM containing 5% skim milk powder and blocked at room temperature for 1 hour. After blocking, the primary antibody was added at an appropriate dilution and incubated at 4°C overnight. After incubation, the primary antibody was recovered and the membrane was washed with Tris-PM for 10 minutes each time, 4 times. The membrane was incubated with the secondary antibody at an appropriate dilution at room temperature for 1 hour. After incubation, it was washed 4 times with Tris-PM for 10 minutes each time. The protein on the membrane was bound to the chemiluminescent substrate, exposed and photographed using the Western blot chemiluminescent imaging system.

[0099] The results are as follows Fig.10As shown in the figure, Y47 vehicle represents the control mouse Y47 (#024097) injected with DMSO, Y47AG14361 represents the control mouse Y47 (#024097) injected with AG14361, YG8R vehicle represents the FRDA transgenic mouse YG8R (#012253) injected with DMSO, YG8RAG14361 represents the FRDA transgenic mouse YG8R (#012253) injected with AG14361, YG8R Olaparib represents the FRDA transgenic mouse YG8R (#012253) injected with Olaparib, and YG8R Saruparib represents the FRDA transgenic mouse YG8R (#012253) injected with Saruparib. The results showed that the expression level of FXN protein in various tissues of YG8R mice was significantly increased after administration of PARP1 inhibitors.

[0100] Example 10

[0101] H&E staining of mouse heart tissue after PARP1 inhibitor administration

[0102] Mice were treated with intraperitoneal injection of PARP1 inhibitors, AG14361 at a concentration of 15 mg / kg / d, for 30 days. The drugs were dissolved in a mixture of DMSO: olive oil (1:9). The control group was injected with an equal volume of DMSO: olive oil (1:9). The experiment involved n, which represents the number of mice in each group. The heart was sliced ​​in the sagittal plane and stained with H&E (hematoxylin and eosin dye) to observe the cardiac tissue structure.

[0103] The results are as follows Fig.11 As shown in the figure, Y47 vehicle represents the control mouse Y47 (#024097) injected with DMSO, Y47AG14361 represents the control mouse Y47 (#024097) injected with AG14361, YG8R vehicle represents the FRDA transgenic mouse YG8R (#012253) injected with DMSO, and YG8RAG14361 represents the FRDA transgenic mouse YG8R (#012253) injected with AG14361. The results showed that the cardiac tissue structure of YG8R mice was abnormal and the arrangement of myocardial cells was disordered. The administration of PARP1 inhibitor improved the myocardial morphology and arrangement structure of YG8R mice.

[0104] Embodiment 11

[0105] H&E staining of mouse neural tissue after PARP1 inhibitor administration

[0106] Mice were treated with intraperitoneal injection of PARP1 inhibitors, AG14361 at a concentration of 15 mg / kg / d, for 30 days. The drugs were dissolved in a mixture of DMSO: olive oil (1:9). The control group was injected with an equal volume of DMSO: olive oil (1:9). The experiment involved n, which represents the number of mice in each group. The sagittal section of the whole brain was sliced ​​and stained with H&E (hematoxylin and eosin dye) to observe Purkinje cells in the cerebellum (the largest neurons in the cerebellar cortex) and neurophagocytosis in the cerebral cortex.

[0107] The results are as follows Fig.12 As shown in the figure, Y47 represents the control mouse Y47 (#024097) injected with DMSO group, Y47+AG14361 represents the control mouse Y47 (#024097) injected with AG14361 group, YG8R represents the FRDA transgenic mouse YG8R (#012253) injected with DMSO group, and YG8R+AG14361 represents the FRDA transgenic mouse YG8R (#012253) injected with AG14361 group; cerebellum represents the cerebellum group, the black arrows point to normal Purkinje cells, and the white arrows point to atrophic neurons; Cerebral cortex represents the cerebral cortex group, and the arrows point to the phenomenon of neurophagy. The results showed that after administration, the number and morphology of Purkinje cells were significantly maintained intact, and the phenomenon of neurophagy in the mouse cerebral cortex was reduced.

[0108] Example 12

[0109] Nissl staining of mouse neural tissue after administration of PARP1 inhibitor

[0110] Mice were treated with intraperitoneal injection of PARP1 inhibitors, AG14361 at a concentration of 15 mg / kg / d, for 30 days. The drugs were dissolved in a mixture of DMSO: olive oil (1:9). The control group was injected with an equal volume of DMSO: olive oil (1:9). The experiment involved n, which represents the number of mice in each group. The sagittal section of the whole brain was sliced ​​and Nissl stained to observe the neurons in the cerebellum.

[0111] The results are as follows Fig.13 As shown in the figure, Y47 represents the control mouse Y47 (#024097) injected with DMSO, Y47+AG14361 represents the control mouse Y47 (#024097) injected with AG14361, YG8R represents the FRDA transgenic mouse YG8R (#012253) injected with DMSO, and YG8R+AG14361 represents the FRDA transgenic mouse YG8R (#012253) injected with AG14361. The results showed that the number and morphology of neuronal cells were significantly improved after administration.

[0112] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of a PARP1 inhibitor in the preparation of a drug for treating and / or alleviating Friedreich's ataxia.

2. Use of PARP1 inhibitors in the preparation of drugs for treating and / or alleviating symptoms associated with Friedreich's ataxia.

3. The use according to claim 1 or 2, characterized in that: The drug has any of the following effects: (1) Improve motor dysfunction; (2) Improve hearing impairment and olfactory abnormalities; (3) Improve cardiovascular system lesions; (4) Improve endocrine system lesions; (5) Improve bone deformities.

4. The use according to claim 3, characterized in that: The cardiovascular system lesions include myocardial hypertrophy and arrhythmia.

5. The use according to claim 3, characterized in that: The endocrine system lesions include abnormal glucose tolerance, abnormal blood sugar and abnormal lipid metabolism.

6. The use according to claim 3, characterized in that: The skeletal deformities include scoliosis, kyphoscoliosis, and pes cavus.

7. The use according to claim 1 or 2, characterized in that: The PARP1 inhibitors include AG14361, Olaparib and Saruparib.

8. The use according to claim 1 or 2, characterized in that: The active ingredient of the drug includes a PARP1 inhibitor.

9. The use according to claim 8, characterized in that: The medicament further includes a pharmaceutically acceptable carrier.

10. The use according to claim 8, characterized in that: The content of the PARP1 inhibitor in the medicine is 50-100 wt %.

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