TDP1 and eltrombopag in the treatment of myotonic dystrophy type 2

Abstract

The present invention relates to the use of TDP1 and eltrombopag in the treatment of myotonic dystrophy type 2. The present invention uses a fruit fly model to identify that TDP1 knockdown can effectively rescue neurodegeneration in the CCTG repeat fruit fly model, reduce pigment production block, cell death and ommatidium fusion, and improve motor defects. At the same time, in vitro screening using small molecules found that eltrombopag can be used as a TDP1 inhibitor and can significantly reduce disease cytotoxicity. The present invention provides a new target and pharmacological substance for CCTG repeat expansion diseases in myotonic dystrophy type 2, and expands the medical use of eltrombopag. The present invention provides a new drug with clinical application prospects for the treatment of CCTG repeat expansion diseases in myotonic dystrophy type 2.

Description

Technical Field

[0001] The present invention relates to applications of TDP1 and eltrombopag in treating myotonic dystrophy type 2, and in particular to applications of eltrombopag in preparing TDP1 inhibitors and drugs for treating myotonic dystrophy type 2, belonging to the field of biomedicine. Background Art

[0002] Myotonic dystrophy (DM) is one of the most common dominantly inherited neuromuscular diseases in adults. Symptoms of DM are characterized by muscle stiffness, progressive weakness, and muscle wasting, often affecting multiple tissues. It can also cause cardiac dysfunction, insulin resistance, excessive sleepiness, intellectual disability, and cognitive impairment. DM2 is divided into type 1 and type 2, depending on the underlying cause. Type 2 (DM2) is typically caused by a CCTG repeat expansion within the first intron of the zinc finger protein 9 gene (ZNF9). Tyrosyl-DNA phosphodiesterase 1 (TDP1) hydrolyzes the bond between topoisomerase 1 (TOP1) and the 3' phosphate group of DNA. To remove supercoiled structures formed during DNA replication and transcription, TOP1 catalyzes transient single-strand DNA breaks. TDP1 hydrolyzes the phosphodiester bond between TOP1 and DNA, removing TOP1 and thus repairing the break. No studies have shown its therapeutic or preventive effects in CCTG duplication disorders, particularly myotonic dystrophy type 2.

[0003] Eltrombopag is a non-peptide agonist of thrombopoietin receptor (TpoR), and its chemical structure is shown in Formula 1. Its molecular formula is C 25 H 22 N4O4, with a molecular weight of 442.5. Eltrombopag is primarily used to treat idiopathic immune thrombocytopenia. There are currently no reports of its use as a TDP1 inhibitor or a drug for the treatment of CCTG repeat expansion in myotonic dystrophy type 2.

[0004] Summary of the Invention

[0005] In response to the deficiencies of the prior art, the present invention provides the use of eltrombopag in the preparation of TDP1 inhibitors and drugs for treating myotonic dystrophy type 2.

[0006] In order to solve the above technical problems, the technical solutions of the present invention are as follows:

[0007] TDP1 as a therapeutic target for myotonic dystrophy type 2.

[0008] Use of TDP1 as a target in developing or designing drugs for preventing or treating myotonic dystrophy type 2.

[0009] Use of a substance capable of inhibiting TDP1 gene expression or TDP1 protein activity in the preparation of a product for preventing or treating myotonic dystrophy type 2.

[0010] Furthermore, the substance capable of inhibiting TDP1 gene expression includes but is not limited to TDP1 siRNA or gene editing tools that affect TDP1 expression.

[0011] Optionally, the affecting TDP1 expression comprises knocking out, replacing or modifying several bases in TDP1.

[0012] Furthermore, the substance capable of inhibiting the activity of TDP1 protein includes a compound capable of inhibiting the activity of TDP1 protein.

[0013] Use of a TDP1 inhibitor in preparing a medicament for preventing or treating myotonic dystrophy type 2.

[0014] Use of eltrombopag in the preparation of TDP1 inhibitors.

[0015] Use of eltrombopag in the preparation of a medicament for preventing or treating myotonic dystrophy type 2.

[0016] Optionally, the drug further comprises other drugs for treating myotonic dystrophy type 2 and / or pharmaceutically acceptable excipients.

[0017] Furthermore, TDP1 inhibitors can treat CCTG repeat expansion diseases in myotonic dystrophy type 2 by regulating CCTG repeat sequence contraction.

[0018] Furthermore, the symptoms of myotonic dystrophy type 2 are abnormalities of the human nervous system including one or more of cognitive dysfunction, sleep disorders, tremors, paralysis, and the like.

[0019] Furthermore, the myotonic dystrophy type 2 is also manifested as one or more of muscle atrophy, pain, stiffness and progressive weakness in the human muscle system.

[0020] The present invention can help overcome the current lack of effective therapeutic drugs to treat CCTG repeat expansion diseases caused by myotonic dystrophy type 2.

[0021] The present invention discloses the use of a small molecule compound, eltrombopag, in the preparation of a drug for preventing or treating myotonic dystrophy type 2. The present invention proposes for the first time that eltrombopag can significantly downregulate TDP1 protein activity in a Drosophila model of CCTG repeat expansion disease, thereby reducing the number of CCTG repeats and alleviating neurodegeneration caused by repeat expansion. During administration, no significant toxicity was shown to wild-type fruit flies. Eltrombopag is an FDA-approved small molecule compound that easily crosses the blood-brain barrier to reach the brain and exert its effects. Therefore, eltrombopag can be made into a drug for the prevention or treatment of myotonic dystrophy type 2, with promising prospects for clinical application.

[0022] The present invention uses the small molecule compound eltrombopag to test its inhibitory effect on TDP1 protein activity in vitro, finding that it can significantly inhibit TDP1 enzyme activity. The compound was then formulated into a drug culture medium and used to culture a Drosophila model of myotonic dystrophy type 2 (MD2) with a CCTG repeat expansion disease. Eltrombopag was found to inhibit multiple disease phenotypes in the MD2 Drosophila model. Using a small molecule compound inhibitor of TDP1, eltrombopag can suppress neurodegeneration and improve motor deficits in the MD2 Drosophila model with a CCTG repeat expansion disease, potentially treating MD2 with a CCTG repeat expansion disease.

[0023] The present invention utilizes eltrombopag, a small molecule compound screened using a fruit fly model. By inhibiting TDP1 protein activity, it can effectively rescue neurodegeneration in fruit flies with CCTG repeat expansion disease caused by myotonic dystrophy type 2, reduce pigment production block, cell death, and ommatidium fusion, and improve motor defects in the CCTG repeat fruit fly model, without showing significant toxicity during administration. The present invention provides new pharmacological substances and targets for the prevention or treatment of CCTG repeat expansion disease caused by myotonic dystrophy type 2, broadening the medical uses of eltrombopag. The present invention provides a new drug and drug target with clinical application prospects for the treatment of CCTG repeat expansion disease caused by myotonic dystrophy type 2. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of the hybridization scheme of Drosophila used in the relevant embodiments of the present invention.

[0025] Figure 2 This is a diagram showing the effects of TDP1 genetic knockdown on compound eye neurodegeneration in the CCTG repeat Drosophila model. Figure 2 A is GMR>CCTG 16 ,GMR>CCTG16 +TDP1 RNAi,GMR>CCTG 720 and GMR>CCTG 720 +TDP1 RNAi genotype Drosophila compound eye light microscope image, Figure 2 B is a scoring chart for the severity of neurodegeneration in Drosophila compound eyes.

[0026] Figure 3 This is a diagram showing how TDP1 knockdown affects the motility of Drosophila in the CCTG repeat Drosophila model. Figure 3 A is Mef2>CCTG 16 and Mef2>CCTG 720 Movement diagram of genotype fruit flies. Figure 3 B is Mef2>CCTG 720 group and Mef2>CCTG 720 Figure 3. Movement status of +TDP1 RNAi genotype fruit flies.

[0027] Figure 4 This is a graph showing the effect of TDP1 knockdown on CCTG repeat size in a Drosophila model. Figure 4 A is Mef2>CCTG 720 and Mef2>CCTG 720 Agilent chip electrophoresis gel image of the chest DNA of +TDP1 RNAi genotype fruit flies after PCR amplification. Figure 4 B is the average length of CCTG repeats for each genotype.

[0028] Figure 5 This is the effect diagram of Eltrombopag's inhibition of TDP1 enzyme activity in vitro. Figure 5 A is the fluorescence intensity graph of TDP1 protein incubated with substrate under the action of DMSO or 80μM Eltrombopag, Figure 5 B is the relative activity of TDP1 protein incubated with substrate under the action of 0, 5, 10, 15, 25, 35, 50, and 100 μg / mL Eltrombopag (with 0 μM as 100%), and the statistical IC50 is 14.12 μg / mL.

[0029] Figure 6 Eltrombopag affects the CCTG repeats in Drosophila, where the fhos gene is a downstream gene affected by myotonic dystrophy type 2. The mature spliced ​​version of the normal fhos gene contains exon 10, but the CCTG repeats cause the spliced ​​version to not contain exon 10. Figure 6 A is Mef2>CCTG 16 、Mef2>CCTG 720 and Mef2>CCTG720 PCR gel images of fruit flies in the +Eltrombopag treatment group with and without exon 10 sheared copies, Figure 6 B is the grayscale analysis result of the gel image.

[0030] Figure 7 Figure 3: Eltrombopag affects CCTG repeats in Drosophila nuclear foci using a cy3-CAGG probe and Mef2>CCTG. 16 、Mef2>CCTG 720 and Mef2>CCTG 720 The body wall muscles of third-instar larvae of the +TDP1 RNAi genotype were hybridized to detect the accumulation of CCUG toxic RNA. Figure 7 A is the result of in situ hybridization observed by laser confocal microscope. Figure 7 B is the statistical diagram of the average aggregation area of ​​CCUG toxic RNA (according to Figure 7 The CY3-CAGG related image in A was obtained by calculation).

[0031] Figure 8 Figure 1. Eltrombopag rescues CCTG repeat muscle degeneration in Drosophila. Figure 8 A is for Mef2>CCTG 16 、Mef2>CCTG 720 and Mef2>CCTG 720 The results of paraffin section and HE staining of the chest muscles of Drosophila in the +Eltrombopag treatment group. Figure 8 B is the average muscle area calculated based on the staining results.

[0032] Figure 9 Figure 1. Eltrombopag rescues CCTG repeat neurodegeneration in Drosophila compound eyes. Figure 9 A is GMR>CCTG 16 、GMR>CCTG 720 and GMR>CCTG 720 Light microscopy image of the Drosophila compound eyes in the +Eltrombopag treatment group. Figure 9 B is a scoring chart for the severity of neurodegeneration in Drosophila compound eyes.

[0033] Figure 10 Figure 1 shows that eltrombopag rescues CCTG repeat motility in Drosophila. Figure 10 A is Mef2>

[0034] CCTG 16 、Mef2>CCTG 720 group and Mef2>CCTG 720 Figure 3. Movement of fruit flies in the +Eltrombopag-treated group. Figure 10 B is a diagram showing the relative movement activity of fruit flies based on the movement status diagram. DETAILED DESCRIPTION

[0035] The present invention will be described in detail below with reference to the embodiments. It should be noted that the embodiments and features of the embodiments of the present invention can be combined with each other without conflict.

[0036] The present invention will be further described in detail below with reference to examples, but the embodiments of the present invention are not limited thereto.

[0037] Drosophila strains:

[0038] Mef2-GAL4 tool flies (which can drive UAS-gene expression specifically in muscle), GMR-GAL4 tool flies (which can drive UAS-gene expression specifically in the compound eye), TDP1 RNAi flies, and DB flies were purchased from the Bloomington Drosophila Stock Center in the United States. DB flies are tool flies that possess four balancers on each of the four chromosomes in two pairs of chromosomes 2 and 3. These four balancers can each impart four distinct, easily observable phenotypes and prevent chromosome recombination.

[0039] CCTG repeat Drosophila model, namely UAS-CCTG 16 and UAS-CCTG 720 Drosophila, gifted by Professor Nancy M. Bonini of the University of Pennsylvania.

[0040] Drosophila were fed a cornmeal-soybean meal-yeast medium prepared according to the BDSC Cornmeal Food recipe recommended by the Bloomington Drosophila Stock Center website, and cultured and crossed at a constant temperature of 25°C.

[0041] Hybridization protocol:

[0042] A: Mef2-GAL4 or GMR-GAL4 female flies (virgin flies) were collected within 8 hours of eclosion and incubated with UAS-CCTG 16 、UAS-CCTG 720 The male fruit flies and TDP1 RNAi male fruit flies were placed in the same glass tube. The male and female fruit flies would mate and produce offspring after about 12 hours. After about 10 days of culture, the offspring would emerge as adults. The offspring were collected and Mef2>CCTG was obtained. 16 、Mef2>CCTG 720 、GMR>CCTG 16 、GMR>CCTG 720and GMR>TDP1 RNAi genotype flies.

[0043] B: Collect Mef2-GAL4 female fruit flies (virgin flies) within 8 hours of eclosion and incubate with UAS-CCTG 720 Male fruit flies were hybridized, and F1 generation virgin flies were collected and hybridized with DB fruit flies. A single F2 generation virgin fly was hybridized with DB fruit flies. The GAL4 sequence and CCTG repeat flanking sequence of the offspring were amplified by PCR to identify whether they were recombinant fruit flies, and the F3 generation Mef2>CCTG was obtained. 720 Genotype flies for crossing with TDP1 RNAi to obtain the F4 generation Mef2>CCTG 720 +TDP1 RNAi flies.

[0044] C: DB female fruit flies (virgin flies) were collected within 8 hours of eclosion and incubated with GMR-GAL4 and UAS-CCTG. 720 The male fruit flies were hybridized, and the F1 generation fruit flies were collected. The two F1 generation fruit flies were hybridized, and the F2 generation GMR>CCTG was obtained through the balancer. 720 Genotype flies for crossing with TDP1 RNAi to obtain F3 generation GMR>CCTG 720 +TDP1 RNAi flies.

[0045] For detailed hybridization protocols, see Figure 1 A, B, C, “—” above and below represent the genotypes of the two homologous chromosomes, “;” before and after represent the genotypes of chromosomes 2 and 3, “+” represents the wild type (no GAL4 or gene on both or one of the homologous chromosomes), and “X” represents hybridization.

[0046] Experimental reagents:

[0047] Preparation of drug culture medium: Add 500 μL of corn flour-soybean flour-yeast medium, 1 μL of drug (eltrombopag concentration is 10 mM, solvent is DMSO), and 2 μL of apple green dye (to indicate drug mixing) to a 5 mL centrifuge tube. Vortex mix thoroughly, cool, and store at 4°C. The final eltrombopag concentration is approximately 20 μM.

[0048] Preparation of non-drug culture medium: 500 μL corn flour-yeast culture medium, 1 μL DMSO, and 2 μL apple green pigment (to indicate whether the drug is mixed) are added to a 5 mL centrifuge tube, mixed using a vortex mixer, and stored at 4°C after cooling.

[0049] DMSO was purchased from Sigma-Aldrich, product number D2438.

[0050] The kit used for high GC-PCR was TaKaRa LA from Takara. with GC Buffer, catalog number RR02BG.

[0051] High Sensitivity DNA kit was purchased from Agilent, catalog number 5067-4626.

[0052] DNA and RNA extraction kits were purchased from Qingke Company, with product numbers TSP201-200 and TSP413.

[0053] The reverse transcription kit was purchased from Novozymes, catalog number R312-02.

[0054] TDP1 protein was purchased from abcam, catalog number ab131921.

[0055] DNA fragments and substrates were synthesized by Bioscientific Research Institute. Tris-HCl, KCl, DTT, EDTA, Triton X-100, and 4% PFA were purchased from Bioscientific Research Institute with catalog numbers B548127-0500, A501159-0500, A300862-0005, B540625-0005, A110694-0100, and E672002-0500, respectively.

[0056] Paraffin was purchased from Bio-Technology Co., Ltd. with the catalog number A601888-0500, and the hematoxylin and eosin staining kit was purchased from Bio-Technology Co., Ltd. with the catalog number C0105S.

[0057] The instruments and equipment are shown in Table 1:

[0058] Table 1

[0059]

[0060]

[0061] Example 1: Knockdown of TDP1 rescues the compound eye degeneration phenotype in the CCTG duplication Drosophila model

[0062] GMR group>CCTG 16 :GMR>CCTG were obtained as described in the hybridization protocol 16 For Drosophila, male Drosophila were collected and anesthetized on a CO2 ventilation plate. The compound eye phenotype of male Drosophila (n ≥ 100) on the 7th day after eclosion was observed and photographed under a light microscope.

[0063] GMR>TDP1 RNAi group: flies with TDP1 knocked down in the nervous system by GMR>TDP1 RNAi were raised in the culture medium (crossing was performed using the above-mentioned hybridization protocol, the same below);

[0064] GMR>CCTG 720 Group: GMR>CCTG raised in the culture medium 720 Drosophila expressing CCTG toxic repeats in the nervous system;

[0065] GMR>CCTG 720 +TDP1 RNAi group: flies with TDP1 knocked down in the nervous system and expressing CCTG toxic repeats were raised in the culture medium;

[0066] The above genotypes were also photographed using a scanning electron microscope to photograph the compound eyes of the fruit flies. The scores were based on the affected areas of four indicators: reduced compound eye area, loss of pigmentation, scab spots (necrosis of visual neurons), and retinal collapse. Normal score was 0, when the affected area was 0-15%, it was 10 points, when the affected area was 15-40%, it was 20 points, when the affected area was 40-65%, it was 30 points, and when it was above 65%, it was 40 points. The final score was the sum of the scores of the four indicators. The higher the score, the deeper the degree of degeneration of the fruit fly compound eyes. Figure 2 As shown, TDP1 knockdown significantly rescued the phenotype of compound eye pigment loss and scarring in CCTG-replicated flies.

[0067] Example 2: Effect of TDP1 knockdown on locomotor activity in the CCTG repetitive Drosophila model

[0068] After the corresponding fruit flies are placed in the fruit fly movement ability detection system, the system automatically monitors the fruit fly activity behavior for the detection of fruit fly movement ability, active frequency and circadian rhythm. Specifically, the target genotype offspring male fruit flies (n≥30) on the 15th day after eclosion are collected, and one fruit fly is placed in each glass tube containing culture medium. The movement ability is monitored by the fruit fly movement ability detection system. There is a laser beam in the middle of the glass tube. The system automatically counts the number of fruit flies passing through. The total movement data, that is, the number of times the fruit fly passes through the middle of the glass tube, is recorded every 30 minutes. The monitoring is carried out from the 15th day after eclosion to the 25th day and beyond. Among them, the target genotypes include Mef2>CCTG 16 ,Mef2>CCTG 720 and Mef2>CCTG 720 +TDP1 RNAi (obtained by the above hybridization scheme, the same below), and the experiment was repeated more than three times for each genotype. The number of times flies of different genotypes passed through the center of the glass tube within 10 days from the 15th day after eclosion to the 25th day was counted. The activity of the Mef2-GAL4 genotype was set as 100%, and the relative activity of the other genotypes was calculated based on this.

[0069] like Figure 3 As shown, TDP1 knockdown could significantly improve Mef2>CCTG 720CCTG expression in the nervous system 720 Caused by crawling difficulties.

[0070] Example 3: Effect of TDP1 knockdown on CCTG repeat number in the Drosophila model

[0071] After DNA was extracted from adult Drosophila thoracic muscles, CCTG repeat fragments were amplified using Takara's High GC-PCR kit, and repeat number reduction was detected using an Agilent High Sensitivity DNA chip. Specifically, male Drosophila offspring of the target genotype (n=5) were collected on day 15 of eclosion, and thoracic muscle tissue was excised. Target DNA was extracted using the DNA extraction kit according to the instructions provided. After High GC-PCR amplification using LA Taq enzyme, fragment length and distribution were analyzed using the High Sensitivity DNA kit on a 2100 Bioanalyzer.

[0072] like Figure 4 As shown, TDP1 knockdown can significantly reduce the CCTG in the Drosophila thorax. 720 Repeat segment length.

[0073] Example 4: Inhibitory effect of eltrombopag on TDP1 enzyme activity in vitro

[0074] Synthesize 5'-FAM-AAAGCAGGC TTC AAC GCAACTGTG AAG ATC GCT TGG GTG CGT TGAAGC CTG CTT T-BHQ1-3' as a TDP1 enzyme substrate. Add 20 ng of TDP1 protein purchased from abcam, DMSO as a control or a small molecule inhibitor to a final concentration of 80 μM, and a DNA substrate to a final concentration of 0.5 μM. The remaining reaction buffer consists of 20 mM Tris-HCl pH 8, 100 mM KCl, 10 mM DTT, 10 mM EDTA, and 0.05% Triton X-100 in a total volume of 25 μl. Incubate at 37°C in a microplate reader for 1 h.

[0075] like Figure 5 As shown, fluorescence detection found that Eltrombopag can inhibit the activity of TDP1 protein and is an effective inhibitor of TDP1 protein.

[0076] Example 5: Eltrombopag rescues aberrant alternative splicing of CCTG repeats in a Drosophila model

[0077] Myotonic dystrophy is primarily caused by toxic RNA affecting the splicing of regulatory proteins, leading to abnormal selective splicing of a series of downstream genes. RNA was extracted from the thorax of Drosophila adults using an RNA extraction kit according to the manufacturer's instructions. RNA was then reverse-transcribed into cDNA using a reverse transcription kit. Exon 10 and the surrounding region of the fhos gene were amplified by PCR, and the relative amounts of transcripts containing and excluding exon 10 were analyzed by agarose gel electrophoresis.

[0078] Mef2>CCTG 16 Group: Obtain target genotype flies as described in the hybridization protocol and raise Mef2>CCTG flies in the non-drug medium. 16 Drosophila until 15 days after eclosion;

[0079] Mef2>CCTG 720 Group: Obtain target genotype flies as described in the hybridization protocol and raise Mef2>CCTG flies in the non-drug medium. 720 Drosophila until 15 days after eclosion;

[0080] Mef2>CCTG 720 +Eltrombopag group: Obtain target genotype fruit flies as described in the hybridization protocol and raise Mef2>CCTG in the drug medium. 720 Drosophila until 15 days after eclosion;

[0081] On the 15th day after eclosion, male fruit flies (n=10) from the above groups were collected, and the chest muscle tissue was cut. RNA was extracted using the RNA extraction kit according to the instructions of the kit, and reverse transcribed into cDNA using a reverse transcription kit. Exon 10 and the surrounding region of the fhos gene were amplified by PCR, and the relative amounts of transcripts containing and not containing exon 10 were analyzed by agarose gel electrophoresis. Then, the selective splicing of fhos was investigated. Figure 6 As shown, eltrombopag was fed to DM2 model flies, Mef2>CCTG 16 In normal Drosophila, most of the transcripts are +exon 10, Mef2>CCTG 720 The abnormal group generally contains transcripts of -exon 10, and Eltrombopag can effectively alleviate the selective splicing abnormality of the Drosophila fhos gene.

[0082] Example 6: Eltrombopag rescues toxic RNA aggregation in the CCTG repeat Drosophila model

[0083] Myotonic dystrophy type 2 patients develop toxic RNA aggregates in the nuclei of muscle cells, which are a good indicator of disease severity. Genotypes were as described in Example 5. Third-instar larvae of the target genotype were dissected and fixed with 4% PFA. Cy5-CAGGCAGGCAGGCAGGCAGG-Cy5 probe and DAPI dye were used to detect Mef2>CCTG. 16 、Mef2>CCTG 720 and Mef2>CCTG 720 The thorax muscles of the larvae in the +Eltrombopag group were incubated overnight, the probe and dye were washed away with PBS solution, and fluorescence was detected under a laser microscope.

[0084] like Figure 7 As shown, eltrombopag can effectively rescue the accumulation of toxic CCTG repeat RNA in the nuclei of Drosophila muscle cells.

[0085] Example 7: Eltrombopag rescues muscle degeneration in the CCTG repeat Drosophila model

[0086] Male fruit flies were collected 15 days after eclosion, and paraffin sections of the thoracic muscles were prepared and stained with hematoxylin and eosin. Muscle size was observed under a microscope. Male fruit flies of the target genotype described in Example 5 were selected and fixed in a 4% PFA solution and dehydrated in anhydrous ethanol solution. In a hybridization oven set at 65°C, the fruit flies were placed in melted paraffin solution for 1 hour for paraffinization. The paraffin blocks were then cooled to room temperature, sectioned, and stained using a hematoxylin and eosin staining kit. Muscle area was then observed and counted under a microscope.

[0087] like Figure 8 As shown, eltrombopag can effectively restore the chest muscle atrophy phenotype of the CCTG duplication Drosophila model and rescue symptoms such as muscle area reduction.

[0088] Example 8: Eltrombopag can effectively rescue compound eye degeneration in the CCTG repeat Drosophila model

[0089] As in Example 1, GMR>CCTG 7 days after eclosion was selected. 16 Group, GMR>CCTG 720 Group and GMR>CCTG 720Male fruit flies in the +Eltrombopag group were collected and anesthetized in a CO2-ventilated plate. The compound eye phenotype of male flies (n ≥ 100) on day 7 after eclosion was observed and photographed under a stereomicroscope using a light microscope. Severity was scored based on the affected area of ​​four indicators: reduced compound eye area, depigmentation, scabby spots (optical neuron necrosis), and retinal collapse. A score of 0 was assigned to normal, 10 to 15% of the affected area, 20 to 15% of the affected area, 30 to 40% of the affected area, and 40 to 65% of the affected area. The final score was calculated by summing the scores for these four indicators; higher scores indicate more advanced compound eye degeneration.

[0090] like Figure 9 As shown, eltrombopag can effectively rescue compound eye degeneration in the CCTG repeat Drosophila model.

[0091] Example 9: Eltrombopag can effectively rescue the motor activity of the CCTG repeat Drosophila model

[0092] As in Example 2, Mef2>CCTG 15-25 days after eclosion 16 group, Mef2>CCTG 720 group and Mef2>CCTG 720 The motility and activity frequency of fruit flies in the +Eltrombopag group were tested. Specifically, male fruit flies of the 15th day after eclosion (n≥30) were collected, and one fruit fly was placed in each glass tube containing culture medium. The motility was monitored by the fruit fly motility detection system, and the movement data was recorded every 30 minutes. The data were monitored from the 15th day after eclosion to the 25th day and beyond. The number of times fruit flies of different genotypes passed through the center of the glass tube within 10 days from the 15th day after eclosion to the 25th day after eclosion was counted. Mef2>CCTG 16 The activity of the genotype was set as 100%, and the relative activity of the other genotypes was calculated based on this.

[0093] like Figure 10 As shown, eltrombopag can effectively rescue the locomotor activity of the CCTG repeat Drosophila model.

[0094] The contents illustrated in the above embodiments should be understood as these embodiments are only used to more clearly illustrate the present invention, and are not used to limit the scope of the present invention. After reading the present invention, various equivalent modifications of the present invention by those skilled in the art shall fall within the scope defined by the claims attached to this application.

Claims

1. Use of eltrombopag in the preparation of a medicament for preventing or treating myotonic dystrophy type 2.

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