Application of teruravone in preparation of drug for treating amyotrophic lateral sclerosis

Abstract

Disclosed is an application of Teruravone in the preparation of a drug for treating the amyotrophic lateral sclerosis disease. Teruravone can significantly increase the survival rate of TDP-43 M337V stably transfected cells and reduce the LDH leakage rate, and has a potency superior to that of Edaravone, indicating that Teruravone possesses stronger neuroprotective activity. Preliminary clinical trials indicate that after oral administration of Teruravone to an ALS patient for 3 months, the Norris Scale score and the neurofilament light chain protein level in the blood remained generally stable, and the decline in ALSFRS-R score was significantly slowed. Thus, the clinical effect of delaying the disease course of ALS is achieved, and no serious adverse effects were observed.

Description

Use of tereflunomide in preparation of drugs for treating amyotrophic lateral sclerosis TECHNICAL FIELD

[0001] The present application belongs to the field of pharmaceutical chemistry, and relates to use of tereflunomide in preparation of drugs for treating amyotrophic lateral sclerosis. BACKGROUND

[0002] Amyotrophic lateral sclerosis (ALS) is a complex multifactorial disease characterized by the loss of motor neurons, and its pathogenesis is not clear. ALS is the most common type of motor neuron disease, and is mainly manifested in progressive development of skeletal muscle weakness, atrophy, fasciculation, bulbar paralysis and pyramidal signs, which will gradually worsen with the development of the disease, and even affect the respiratory muscles, leading to dyspnea. Patients may also have symptoms such as subjective numbness, limb paralysis, weight loss, arrhythmia, and may also face emotional problems such as depression and anxiety.

[0003] ALS is a rare disease that cannot be cured so far, and the incidence rate is about 3 per 100,000. The peak of ALS usually occurs at the age of 45 or older, and the age of onset is becoming younger, and a small number of patients develop the disease at the age of 20 or so. The incidence rate of men is higher than that of women. Only a small part of ALS patients is related to heredity, and the pathogenesis of most patients is not clear. ALS can be treated by a series of methods, including nutrition management, respiratory support, psychological treatment and rehabilitation physiotherapy training. At present, only two marketed drugs, riluzole and edaravone, are used for treating ALS, and neither of them has achieved satisfactory clinical therapeutic effect. Therefore, it is urgent to find an effective therapeutic drug for ALS.

[0004] SUMMARY

[0005] Tereflunomide, chemical name: 2-methyl-5-imino-benzo[d][1,3]oxazino[5-b]pyrazole, has the structure shown in formula I.

[0006] Tereflunomide is a new compound (CN101508696A) designed and synthesized based on the lead compound edaravone through the molecular skeleton migration drug design method. Rat experiments show that tereflunomide can significantly improve the neurological deficit symptoms of cerebral ischemia-reperfusion animals in a dose-dependent manner, reduce the cerebral infarction area, reduce the degree of brain damage, reduce brain edema, and inhibit lipid peroxidation of damaged brain tissue. The results of clinical trials of stroke patients show that tereflunomide can significantly improve the symptoms of stroke, has good safety and tolerability, and no serious adverse events related to tereflunomide drugs have occurred during the clinical trial process.

[0007] The inventors found through cell research that terephthalyl acetic acid has no obvious influence on the morphology of ALS-related stable cells, and terephthalyl acetic acid has no obvious influence on the survival rate and lactate dehydrogenase (LDH) leakage rate of SOD1G93A stable cells, can significantly improve the survival rate of TDP-43M337V stable cells and reduce the LDH leakage rate, and the effect is better than that of edaravone, indicating that terephthalyl acetic acid has strong nerve cell protection activity, and terephthalyl acetic acid can be used as a new and effective drug for treating ALS patients.

[0008] Preliminary clinical trials show that after ALS patients orally take terephthalyl acetic acid for 3 months, the Norris scale score and the level of neurofilament light chain (NFL) in blood remain stable on the whole, the decrease of ALSFRS-R score is significantly slowed down, and the clinical effect of delaying the course of ALS has been achieved; and no serious adverse reactions are found.

[0009] The purpose of the present application is to provide the application of terephthalyl acetic acid in the preparation of a drug for treating amyotrophic lateral sclerosis (ALS).

[0010] The dosage form of the drug is oral preparation and injectable preparation.

[0011] The oral preparation is tablet, capsule and granule, and the injectable preparation is freeze-dried powder injection and spray-dried sterile powder injection. BRIEF DESCRIPTION OF DRAWINGS

[0012] Figure 1 is the influence of terephthalyl acetic acid on the morphology of hSOD1G93A stable cells.

[0013] Figure 2 is the influence of terephthalyl acetic acid on the morphology of TDP-43M337V stable cells.

[0014] Figure 3 is the monthly average change of ALSFRS-R before and after taking terephthalyl acetic acid.

[0015] Figure 4 is the change of modified Norris scale score before and after taking terephthalyl acetic acid.

[0016] Figure 5 is the change of blood NFL level before and after taking terephthalyl acetic acid. DETAILED DESCRIPTION

[0017] Example 1

[0018] Study on the protective effect of terephthalyl acetic acid on hSOD1G93A and TDP-43M337V stable cells

[0019] Drugs and reagents: Tereflunomide (No. CRM-A1901) was prepared with DMSO to a concentration of 0.01M Tereflunomide stock solution; Edaravone was prepared with DMSO to a concentration of 0.01M Edaravone stock solution; ATN-224 was prepared with DMSO to a concentration of 0.01M ATN-224 stock solution. According to the experimental needs, the above stock solutions were diluted with culture medium. CCK kit (Beijing Zhuangmeng Biological Technology Co., Ltd.); lactate dehydrogenase (LDH) kit (microplate method) (Nanjing Jiancheng Biological Engineering Institute).

[0020] Experimental method:

[0021] TDP-43M337V and SOD1G93A were transfected into NSC-34 cells to form TDP-43M337V stable transfection cells, and SOD1G93A was transfected into NSC-34 cells to form hSOD1G93A stable transfection cells, which are effective ways to screen ALS drugs.

[0022] Set up empty vector group (Empty), control group (Control), Edaravone group, ATN-224 group, and different doses of Tereflunomide group. The empty vector group cells are transfected with empty vector cells, and the other groups are stable transfection cells. Each stable transfection cell was inoculated in a 96-well plate with a seeding density of 5x10 4 μL, cultured for 24h, and the medium was discarded. The control group was added with 10μL fresh medium per well, the Edaravone group was added with 10μL Edaravone medium with a final concentration of 10μM per well, the ATN-224 group was added with 10μL ATN-224 medium with a final concentration of 10μM per well, and the different doses of Tereflunomide group was added with 10μL Tereflunomide medium with a final concentration of 0.1, 0.3, 1, 3, and 10μM per well, respectively. Continue to incubate for 24h, add 10μL CCK-8 solution per well, continue to incubate in the incubator for 1h, mix on the shaker, and detect the absorbance value at 450nm on the enzyme label instrument.

[0023] Lactate dehydrogenase (LDH) assay: according to the instructions of lactate dehydrogenase (LDH) kit.

[0024] Statistical method:

[0025] All results are expressed as means ± SEM. One-way ANOVA was used, combined with post hoc test (LSD) test to compare the differences between groups, and P<0.05 indicates significant difference.

[0026] Experimental results:

[0027] A1. Effects of Teri on the morphology of hSOD1 G93A stable cells

[0028] As shown in Figure 1, the cells transfected with empty vector were triangular or polygonal, with clear cell edges, firm adhesion, obvious protrusions, no abnormal interweaving into nets, no obvious swelling or deformation of the cell body, and rare dead cells floating in the supernatant.

[0029] Compared with the cells transfected with empty vector, the hSOD1 G93A stable cells had decreased adhesion ability, swollen and rounded cell bodies, reduced protrusions, shortened axons, and more dead cells floating in the supernatant.

[0030] Edaravone (10 μM), ATN-224 (10 μM), and Teri at various concentrations had no obvious effects on the morphology of hSOD1 G93A stable cells.

[0031] A2. Effects of Teri on the morphology of TDP-43 M337V stable cells

[0032] As shown in Figure 2, the cells transfected with empty vector were triangular or polygonal, with clear cell edges, firm adhesion, obvious protrusions, no abnormal interweaving into nets, no obvious swelling or deformation of the cell body, and rare dead cells floating in the supernatant.

[0033] Compared with the cells transfected with empty vector, the TDP-43 M337V stable cells had decreased adhesion ability, swollen and rounded cell bodies, reduced protrusions, shortened axons, and more dead cells floating in the supernatant.

[0034] Edaravone (10 μM) and Teri at various concentrations had no obvious effects on the morphology of TDP-43 M337V stable cells.

[0035] A3. Effects of Teri on the survival rate of hSOD1 G93A stable cells and TDP-43 M337V stable cells

[0036] To investigate whether Teri can improve ALS-related cell damage, the effects of Teri on the survival rate of hSOD1 G93A and TDP-43 M337V stable cells were determined, and the results are shown in Table 1.

[0037] Compared with the empty vector group, the survival rate of the control group hSOD1G93A stable cells was significantly reduced, indicating that the hSOD1G93A stable cells were damaged; compared with the control group, 10 μM of ATN-224 could significantly increase the survival rate of the hSOD1G93A stable cells, while 10 μM of edaravone and each concentration of tereflunomide had no significant effect on the survival rate of the hSOD1G93A stable cells. Compared with the empty vector group, the survival rate of the control group TDP-43M337V stable cells was significantly reduced, indicating that the TDP-43M337V stable cells were damaged; compared with the control group, 10 μM of edaravone could significantly increase the survival rate of the TDP-43M337V stable cells, and 0.3, 1 and 3 μM of tereflunomide also significantly increased the survival rate of the TDP-43M337V stable cells, suggesting that tereflunomide had a neuroprotective effect.

[0038] Table 1. Effect of tereflunomide on the survival rate of hSOD1G93A stable cells and TDP-43M337V stable cells Note: Data are expressed as mean ± SEM, n = 9; compared with the empty vector group, ## P < 0.01; compared with the control group, * P < 0.05, ** P < 0.01.

[0039] A4. Effect of tereflunomide on the LDH leakage rate of hSOD1G93A stable cells and TDP-43M337V stable cells

[0040] The effect of different concentrations of tereflunomide on the LDH leakage rate of cells is shown in Table 2. Compared with the empty vector group, the LDH leakage rate of the control group TDP-43M337V stable cells was significantly increased, indicating that the hSOD1G93A stable cells were damaged; compared with the control group, edaravone (10 μM), ATN-224 (10 μM) and each concentration of tereflunomide had no significant effect on the LDH leakage rate of the hSOD1G93A stable cells. Compared with the empty vector group, the LDH leakage rate of the control group TDP-43M337V stable cells was significantly increased, indicating that the TDP-43M337V stable cells were damaged; compared with the control group, 10 μM of edaravone could significantly reduce the LDH leakage rate of the TDP-43M337V stable cells, and 0.3 and 1 μM of tereflunomide also significantly reduced the LDH leakage rate of the TDP-43M337V stable cells, suggesting that tereflunomide had a neuroprotective effect.

[0041] Table 2. Effect of tereflunomide on the LDH leakage rate of hSOD1G93A stable cells and TDP-43M337V stable cells Note: Data are expressed as mean ± SEM, n = 6. Compared with the empty vector group,# P<0.05; compared with the control group, ## P<0.01, * P<0.05.

[0042] Example 2

[0043] Clinical studies have shown that the safety and tolerability of Teriparatide for injection in single dose of 480 mg, continuous administration of 590 mg within 24 hours, and continuous administration of 1550 mg within 72 hours in Chinese adult healthy subjects are good, and no unexpected, clinically significant safety problems have been found. Based on the good clinical safety evidence of Teriparatide, under the premise of full informed consent, 5 ALS patients (of which #000897 patients stopped taking the drug for 1 week due to other diseases) voluntarily tried Teriparatide, and the enrollment of subjects is shown in Table 3.

[0044] Table 3. Status of 5 subjects

[0045] This study fully complies with the requirements of clinical research ethics, all participants have signed the informed consent, the privacy and data security of the participants are fully protected, and the participants are ensured to withdraw from the study at any time.

[0046] Trial objectives:

[0047] To evaluate the efficacy and safety of Teriparatide in patients with amyotrophic lateral sclerosis (ALS).

[0048] Trial design:

[0049] ALS patients diagnosed (according to El Escorial criteria) aged 18-65 years old; exclusion criteria include disease duration less than 24 months, no use of edaravone in the past six months, and presence of clinically significant food and drug allergies or allergic reactions determined by the investigator.

[0050] Teriparatide preparation is oral disintegrating tablets (each tablet contains 60 mg of Teriparatide), and the subjects take twice a day, 1 hour before breakfast and dinner on an empty stomach with water, 1 tablet each time, and the drug intervention time is 3-6 months.

[0051] Follow-up is conducted every two weeks to record the progression of symptoms, side effects of drugs or treatments, etc.

[0052] The efficacy evaluation indexes include the revised ALS functional rating scale (ALSFRS-R) score, modified Norris scale score, nerve fiber light chain protein (NFL) blood level, and the subjective feeling of the subjects on the improvement of their own symptoms, including breathing, swallowing, movement, speech, etc. The subjects are instructed to record and report any adverse reactions related to the drug at any time. If serious side effects occur, the treatment is immediately stopped, and appropriate medical intervention is provided to the patients.

[0053] After 3 months of administration, the results of the subjects were preliminarily evaluated.

[0054] Results of the study:

[0055] 1. Functional and index evaluation

[0056] 1) Monthly change of ALSFRS-R before and after taking Teriparatide

[0057] Four subjects had comparable ALSFRS-R (AR) scores before and after taking the drug. As shown in Figure 3 and Table 4, the AR score of subject #000897 decreased sharply, and the monthly change of the AR scores of the other three subjects after taking the drug was reduced by 55%, 80%, and 63% respectively compared to before taking the drug.

[0058] Table 4. Monthly change of ALSFRS-R before and after taking Teriparatide

[0059] 2) Change of modified Norris scale score before and after taking Teriparatide

[0060] The modified Norris scale scores of the subjects are shown in Figure 4. The score of subject #000897 decreased rapidly, and the scores of the other subjects taking Teriparatide were generally stable.

[0061] 3) Change of blood NFL level before and after taking Teriparatide

[0062] The change of blood NFL level of the subjects before and after taking Teriparatide is shown in Figure 5. The NFL level of subject #000897 fluctuated greatly, and the blood NFL levels of the other four subjects were generally stable.

[0063] The preliminary clinical trial showed that the Norris scale scores and blood NFL levels of the patients were generally stable, and the decrease of ALSFRS-R scores was significantly slowed down. The overall course of ALS patients was stable, and the clinical effect of delaying the course of ALS had been achieved.

[0064] 2. Safety evaluation

[0065] No serious adverse events were reported in all subjects under the existing dosing amount and administration regimen, indicating that the dose and administration method obtained good safety within 3 months.

Claims

The application of teriella in preparing a drug for treating amyotrophic lateral sclerosis. Use according to claim 1, characterized in that The dosage form of the drug is oral preparation and injectable preparation. The pharmaceutical preparation according to claim 2, characterized in that The oral preparation is tablet, capsule and granule, and the injectable preparation is freeze-dried powder for injection and spray-dried sterile powder for injection.

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