Use of gsk484 in the preparation of a drug for resisting status epilepticus

Abstract

This invention provides the application of GSK484 in the preparation of anti-status epilepsy drugs, offering a new approach to the treatment of status epilepsy. The results of this invention show that GSK484 can significantly prolong the latency of Pilo-induced status epileptic seizures in mice, reduce the seizure severity in a Pilo-induced status epileptic model, and significantly reduce the duration of Pilo-induced status epileptic seizures. GSK484 can significantly inhibit neutrophil activation and extracellular trap formation in status epilepsy, and it does not affect mouse body weight. Therefore, GSK484 is an effective treatment for status epilepsy with good safety and minimal toxic side effects.

Description

Technical Field

[0001] This invention belongs to the field of drug development for the treatment of status epilepticus, specifically involving the application of GSK484 in the preparation of anti-status epileptic drugs. Background Technology

[0002] Status epilepticus (SE) is a severe neurological crisis associated with epilepsy, characterized by persistent or recurrent seizures without recovery of consciousness between seizures, and is associated with high rates of disability and mortality. 24%–43% of SE patients develop treatment-resistant status epilepticus (RSE), which has a short-term mortality rate three times that of non-refractory SE, and approximately 90% of RSE survivors eventually relapse. Therefore, early identification and timely intervention of SE are crucial for improving patient prognosis.

[0003] Current clinical interventions mainly include benzodiazepines, first- and second-line antiepileptic drugs, and anesthesia and sedation, all of which aim to stop epileptic seizures by inhibiting abnormal neuronal discharges. However, these treatments are often accompanied by respiratory depression, circulatory instability, liver and kidney toxicity, and a high incidence of recurrent seizures (RSE). Some patients do not respond well to existing treatments, and the prognosis remains unsatisfactory. Therefore, elucidating the pathogenesis of seizures (SE) and exploring safer and more effective novel treatment strategies are of significant clinical importance.

[0004] GSK484, CAS No.: 1652591-81-5, Chemical Formula: C 27 H 32 ClN5O3, with a molecular weight of 510.03, and its chemical structure as shown in Formula (I), is a small molecule compound developed by GlaxoSmithKline. A search of existing technologies has not revealed any reports on the use of GSK484 as a treatment for epileptic seizures or seizures.

[0005] Formula (1). Summary of the Invention

[0006] In view of the shortcomings of existing drugs for the treatment of status epilepticus, the present invention aims to provide the application of GSK484 in the preparation of anti-status epileptic drugs.

[0007] The technical solution of this invention is: This invention provides the application of GSK484 in the preparation of anti-status epilepsy drugs.

[0008] The present invention provides a pharmaceutical composition comprising GSK484 and a conventional pharmaceutical carrier.

[0009] Furthermore, the pharmaceutical carrier includes diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorbents, lubricants, etc., commonly used in the pharmaceutical field.

[0010] Furthermore, the pharmaceutical carrier is selected from one or more of starch, sucrose, cellulose derivatives, alginate, gelatin, kaolin, soap clay, talc, calcium stearate, magnesium stearate, polyethylene glycol, flavoring, sweetener, and flavoring agent.

[0011] Furthermore, the pharmaceutical composition is a formulation administered orally or non-orally.

[0012] Furthermore, the formulation includes tablets, capsules, granules, pellets, oral solutions, injections, transdermal preparations, sprays, or aerosols.

[0013] This invention also provides the application of GSK484 in the preparation of drugs that prolong the latency period of status epilepticus seizures.

[0014] This invention also provides the use of GSK484 in the preparation of drugs to reduce the duration of status epilepticus seizures.

[0015] This invention also provides the application of GSK484 in the preparation of drugs that inhibit neutrophil activation and neutrophil extracellular trap formation in status epilepticus.

[0016] The embodiments of the present invention further demonstrate that the application of GSK484 in the preparation of anti-status epilepsy drugs has a significant anti-epileptic effect, good safety, and few toxic side effects.

[0017] The embodiments of the present invention further indicate that the dosage of the drug is 4 mg / kg-8 mg / kg.

[0018] This invention provides multiple pieces of evidence supporting the possibility that GSK484 may be a novel drug for controlling seizures (SE). Specific evidence is as follows: In a pilocarpine-induced SE model in mice, we intervened with GSK484 and GSK199 respectively, and assessed seizure activity using the Racine scoring system. Generally, in a Pilo-induced SE model, a longer seizure latency indicates a better drug intervention effect. Experimental results showed that GSK484 at both 4 mg / kg and 8 mg / kg doses significantly prolonged the seizure latency and significantly shortened the duration of SE, suggesting a significant therapeutic advantage for GSK484, with 4 mg / kg GSK484 showing better efficacy. In contrast, the same dose of GSK199 showed no significant improvement. Furthermore, we analyzed the effects of the two drugs on neutrophil-related indicators in SE mice, including MPO content and CitH3 protein expression. The results showed that GSK484 significantly reduced MPO content in the hippocampus of mice and significantly downregulated CitH3 protein expression, suggesting that it can effectively inhibit neutrophil activation and the formation of extracellular neutrophil traps (NETs) during sepsis (SE). GSK199, however, showed no significant inhibitory effect on these indicators. Furthermore, we analyzed the effect of GSK484 on the body weight of SE mouse models. The results showed no significant change in mouse body weight after GSK484 intervention, indicating its good safety profile. Based on these results, GSK484 is a novel drug that effectively improves SE and has significant clinical application potential.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention provides the application of GSK484 in the preparation of drugs for the treatment of status epilepticus, and provides a new approach to the treatment of status epilepticus.

[0020] 2. GSK484 has good safety and few toxic side effects as a treatment for status epilepticus.

[0021] 3. GSK484 is a highly effective treatment for status epilepticus.

[0022] The detailed structure of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. Attached Figure Description

[0023] Figure 1 The graph shows the effect of GSK484 on the score of the Pilo-induced status epilepticus model in mice, where: A. Seizure latency; B. Seizure score; C. Seizure duration.

[0024] Figure 2The figure shows the effect of GSK484 on neutrophil activation and extracellular trap formation in a Pilo-induced mouse status epilepticus model, including: A. MPO content results; B. Representative protein bands of CitH3; C. Statistical analysis results of CitH3 protein expression.

[0025] Figure 3 The figure shows the effect of GSK484 on the body weight of a Pilo-induced status epilepticus mouse model. Detailed Implementation

[0026] Example 1: GSK484 significantly improved symptoms of a Pilo-induced mouse model of status epilepticus. Experimental groups: 10 animals each of the following groups: control group, Pilo group, Pilo + 4 mg / kg GSK484 group, Pilo + 8 mg / kg GSK484 group, Pilo + 4 mg / kg GSK199 group, and Pilo + 8 mg / kg GSK199 group. The control group received intraperitoneal injection of saline. The Pilo group received intraperitoneal injection of 350 mg / kg Pilo prepared with saline. The Pilo + 4 mg / kg GSK484 group and the Pilo + 8 mg / kg GSK484 group received intraperitoneal injection of 350 mg / kg Pilo, and simultaneously received pre-administered tail vein injections of 4 mg / kg and 8 mg / kg GSK484 prepared with saline, respectively. The Pilo + 4 mg / kg GSK199 group and the Pilo + 8 mg / kg GSK199 group received intraperitoneal injection of 350 mg / kg Pilo, and simultaneously received pre-administered tail vein injections of 4 mg / kg and 8 mg / kg GSK199 prepared with saline, respectively.

[0027] Seizure scoring: Seizure latency was defined as the time from Pilo injection to the onset of the first seizure. The seizure grade and duration were recorded for each mouse within 90 minutes after Pilo injection. Seizures in mice were assessed according to the following Racine scoring system: Stage 0: No seizures; Stage 1: Rhythmic twitching of the face and whiskers; Stage 2: Regular head nodding or twitching without facial tics; Stage 3: Myoclonus and spasms of multiple limbs; Stage 4: Uncontrolled falls; Stage 5: Generalized tonic-clonic seizures with running and jumping; Stage 6: Death.

[0028] Analysis of experimental results: The experimental results of each group are as follows Figure 1 As shown. Figure 1 A showed that, compared with the Pilo group, GSK484 significantly prolonged the latency of Pilo-induced SE attacks in mice, while GSK199 intervention did not show significant improvement. Figure 1 B shows that the SE seizure scores of all groups increased rapidly after modeling, while the GSK484 group showed a certain downward trend in the later stage of the seizure, and the GSK199 group was basically the same as the Pilo group. Figure 1 The results showed that GSK484 significantly shortened the duration of Pilo-induced status epilepticus (SE), and its effect was significantly better than that of GSK199. Overall, GSK484 has significant therapeutic advantages for Pilo-induced status epilepticus, manifested in prolonged seizure latency, reduced seizure severity, and shortened seizure duration, with 4 mg / kg GSK484 showing the best efficacy.

[0029] Example 2: GSK484 significantly inhibited neutrophil activation and neutrophil extracellular trap (NET) formation in a Pilo-induced mouse model of status epilepticus. Experimental groups: 4 mice each in the control group, Pilo group, Pilo + 4 mg / kg GSK199 group, and Pilo + 4 mg / kg GSK484 group (3 mice per group for Western blot detection of CitH3 protein expression). Drug intervention was the same as in Example 1. Subsequently, mouse hippocampal tissue was isolated for the detection of MPO levels and the NETs marker CitH3 protein expression.

[0030] MPO Level Detection: The MPO level in the hippocampus tissue of mice in each group was detected using a mouse myeloperoxidase (MPO) ELISA kit. After tissue collection, the tissue was washed with pre-cooled PBS (0.01 M, pH 7.4), and homogenized with PBS at a 1:9 ratio. The homogenate was then centrifuged at 5000×g for 10 min, and the supernatant was collected. Subsequently, the kit instructions were followed: 100 μL of standard or sample was added to each well, followed by incubation, washing, addition of biotinylated antibody, SA-HRP incubation, and color development. After terminating the reaction, the absorbance was measured at 450 nm, and the MPO content was calculated based on the standard curve. Figure 2 As shown in Figure A, the MPO level in the SE model was significantly higher than that in the normal control group, while the MPO level was significantly reduced in the 4 mg / kg GSK484 intervention group. Moreover, the reduction in MPO level was more significant than that in the same dose of GSK199, suggesting that GSK484 can more effectively inhibit neutrophil activation in SE mice.

[0031] Western Blot Analysis: 1. Extraction and concentration determination of hippocampal tissue proteins: Two small steel balls were added to a grinding tube, the tissue was placed in the grinding tube, peeled and weighed, and tissue lysis buffer (V) was added at a ratio of 15 μL / mg. RIPA :V Pro :V 磷A :V 磷B=100:1:1:1) Add lysis buffer to each grinding tube in sequence, grind 3 times in a grinder, centrifuge at 12,000 rpm for 10 min, then take 1 μL of supernatant and use BCA working solution (VA solution:VB solution = 50:1) to determine the concentration. Add 5*loading solution to the remaining supernatant at 1 / 4 of the volume, boil in a metal bath at 100℃ for 10 min, and vortex for 30 s; 2. Western blot experiment to detect the protein expression level in the hippocampus: Separate equal amounts of protein (20 μg) from different groups by gel electrophoresis and membrane transfer method, load the sample according to the loading amount calculated according to the measured protein concentration, electrophoresis at 80V for 30 min, then switch to 120V for 1 h, transfer membrane at 250mA for 1 h, then block the PVDF membrane with 5% skim milk for 1 h, cut the target band according to the molecular weight, and incubate overnight in a shaking incubator at 4℃ with primary antibody. The next day, the primary antibody was recovered, washed three times with TBST for 10 minutes each time, then incubated with the corresponding secondary antibody for 1 hour. The secondary antibody was recovered, washed three times with TBST for 10 minutes each time, and then developed. Western blot results are shown below. Figure 2 As shown in Figure B, CitH3 protein expression in the hippocampus of the Pilo group was significantly increased compared to the normal control group, suggesting a significant increase in NET formation after SE induction. Compared with the Pilo group, CitH3 protein expression was significantly decreased in the 4 mg / kg GSK484 intervention group, while there was no significant difference in CitH3 protein expression in the 4 mg / kg GSK199 group. Figure 2 C). The above results indicate that Pilo-induced status epilepticus significantly promotes the formation of NETs in the hippocampus, while GSK484 can significantly inhibit CitH3 protein expression, suggesting that it can effectively inhibit the formation of NETs in the hippocampus of SE mice; in contrast, the intervention effect of GSK199 is weak, with almost no significant inhibitory effect.

[0032] Example 3: GSK484 does not affect the body weight of Pilo-induced status epilepticus model mice. Experimental groups: control group, Pilo group, Pilo + 4 mg / kg GSK199 group, and Pilo + 4 mg / kg GSK484 group, with 10 mice in each group initially. During the experiment, 3 mice died in the Pilo group and 1 mouse died in the Pilo + 4 mg / kg GSK199 group. The final sample sizes for each group were: control group 10 mice, Pilo group 7 mice, Pilo + 4 mg / kg GSK199 group 9 mice, and Pilo + 4 mg / kg GSK484 group 10 mice.

[0033] Weight monitoring: After the drug intervention in each group was completed, the weight of each mouse in each group was measured using a scale, and the weight of each group of mice after drug intervention was recorded.

[0034] Analysis of experimental results: The experimental results are as follows Figure 3 As shown, there was no significant difference in overall mouse body weight among the groups. Compared with the control group, the Pilo group showed no significant change in body weight; compared with the Pilo group, the 4 mg / kg GSK199 group and the 4 mg / kg GSK484 group also showed no significant change in body weight. These results indicate that neither GSK199 nor GSK484 intervention had a significant effect on mouse body weight; combined with the aforementioned efficacy results, this suggests that 4 mg / kg GSK484 exerts its anti-SE effect while exhibiting good safety.

[0035] The above description is a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and concept of the present invention, should be covered within the scope of protection of the claims of the present invention.

Claims

1. Application of GSK484 in the preparation of anti-status epilepsy drugs.

2. The application according to claim 1, characterized in that, The drug is a pharmaceutical composition comprising GSK484 and a conventional pharmaceutical carrier.

3. The application according to claim 2, characterized in that, The pharmaceutical carriers include diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorbents, and lubricants commonly used in the pharmaceutical field.

4. The application according to claim 3, characterized in that, The pharmaceutical carrier is selected from one or more of the following: starch, sucrose, cellulose derivatives, alginate, gelatin, kaolin, soap clay, talc, calcium stearate, magnesium stearate, polyethylene glycol, flavoring, sweetener, and flavoring agent.

5. The application according to claim 1, characterized in that, The drug is a preparation administered orally or non-orally.

6. The application according to claim 5, characterized in that, The formulations include tablets, capsules, granules, pellets, oral solutions, injections, transdermal preparations, sprays, or aerosols.

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