Use of Ly93 in the preparation of antiepileptic drugs

Abstract

The present invention provides the use of Ly93 in the preparation of anti-seizure drugs, providing a new idea for seizure treatment drugs. The research results of the present invention show that Ly93 can significantly reduce the seizure grade in the KA-induced mouse seizure model, can significantly reduce the seizure duration induced by PTZ, Ly93 can significantly inhibit neuroinflammation during seizures, and Ly93 does not affect the body weight of mice. Therefore, Ly93 has significant effects as a seizure treatment drug, has good safety, and has small toxic and side effects.

Description

Technical Field

[0001] The present invention belongs to the field of developing drugs for treating epilepsy, and particularly relates to the application of Ly93 in the preparation of anti-epileptic drugs. Background Art

[0002] Epilepsy is a common neurological disease characterized by abnormal neuronal discharges, with clinical manifestations including convulsions of varying degrees, staring blankly, foaming at the mouth, and multiple comorbidities. In severe cases, it can lead to death. It is characterized by sudden onset, spontaneity, and recurrence.

[0003] Repeated epileptic seizures are the main clinical feature of epilepsy patients. Existing clinical intervention measures, including drug therapy, nerve stimulation therapy, surgical treatment, and dietary therapy, can all exert anti-epileptic effects by suppressing seizure symptoms. However, these treatment measures often have defects such as hepatotoxicity, cognitive impairment, and poor compliance during clinical use. Therefore, there is an urgent need to explore new drugs for treating epileptic seizures.

[0004] Ly93, CAS No.1883528-69-5, chemical formula C 21 H 20 N2O2, with a molecular weight of 332.40, and a chemical structural formula as shown in Formula (I), is a new type of small molecule compound that is widely used in pre-clinical experimental research and has stable physical and chemical properties. After retrieval, no reports on Ly93 as a drug for treating epileptic seizures have been found in the prior art.

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

[0006] Aiming at the deficiencies of existing treatments for epileptic seizures, the present invention aims to provide the application of Ly93 in the preparation of anti-epileptic drugs.

[0007] The technical solution of the present invention is as follows:

[0008] The present invention provides the application of Ly93 in the preparation of anti-epileptic drugs.

[0009] The present invention provides a pharmaceutical composition, which comprises Ly93 and a conventional pharmaceutical carrier.

[0010] The conventional pharmaceutical carrier includes, for example, dimethyl sulfoxide (DMSO), polyethylene glycol 400 (PEG400), Tween 80 (Tween80), etc.

[0011] Furthermore, the pharmaceutical composition is a preparation administered by oral or non-oral routes.

[0012] Furthermore, the preparation includes tablets, capsules, granules, dripping pills, oral solutions, injections, transdermal agents, sprays or aerosols, etc.

[0013] In the examples of the present invention, it is further suggested that the application of Ly93 in the preparation of anti-seizure drugs, the dose of the drug is 25 mg / kg - 100 mg / kg, and the anti-seizure effect is significant, with good safety and small toxic and side effects.

[0014] Multiple pieces of evidence in the present invention support that Ly93 may be a novel drug for controlling seizures. The specific evidence is as follows: In the kainic acid (abbreviated as KA)-induced mouse seizure model, after we intervened with 25 mg / kg and 100 mg / kg of Ly93, the Racine scoring standard was used to evaluate the seizure situation of the mice. The experimental results showed that both doses of Ly93 could significantly reduce the seizure grade in the KA-induced mouse seizure model. Among them, 100 mg / kg of Ly93 had a more significant effect and could significantly reduce the number of seizures and the duration of seizures; at the same time, in the pentylenetetrazole (abbreviated as PTZ)-induced mouse seizure model, our experimental results showed that when the dose of Ly93 was 100 mg / kg, the latency of PTZ-induced seizure mice was significantly prolonged, and both 25 mg / kg and 100 mg / kg doses could significantly reduce the duration of PTZ-induced seizures; further experimental results found that in the KA- and PTZ-induced seizure mouse models, after we intervened with 25 mg / kg and 100 mg / kg of Ly93, the changes in neuroinflammatory-related indicators (including TNF-α, IL-1β, and IL-6) were analyzed. The experimental results showed that Ly93 significantly inhibited neuroinflammation during seizures. Specifically, both 25 mg / kg and 100 mg / kg of Ly93 could significantly reduce the mRNA expression levels of TNF-α, IL-1β, and IL-6 in the hippocampus of KA-induced seizure mouse models; in addition, we also analyzed the effect of Ly93 on the body weight of KA-induced seizure mouse models. The experimental results showed that Ly93 did not affect the body weight of the mice, indicating that Ly93 has good safety. Based on the above multiple experimental results, Ly93 may be a novel drug that can effectively improve seizures and has great clinical application prospects.

[0015] Compared with the prior art, the advantages of the present invention are as follows:

[0016] The present invention provides the application of Ly93 in the preparation of drugs for treating seizures, providing a new idea for drugs for treating seizures.

[0017] Ly93, as a drug for treating seizures, has good safety and small toxic and side effects.

[0018] Ly93 is significantly effective as an anti-epileptic drug.

[0019] The following further describes the detailed structure of the present invention in conjunction with the accompanying drawings and specific embodiments. Description of the Drawings

[0020] Figure 1 Effect diagram of Ly93 on the scoring of KA-induced mouse epilepsy seizure model, where: A. Epilepsy seizure score; B. Number of epilepsy seizures; C. Duration of epilepsy seizures.

[0021] Figure 2 Effect diagram of Ly93 on the scoring of PTZ-induced mouse epilepsy seizure model, where: A. Latency of epilepsy seizures; B. Epilepsy seizure score; C. Number of epilepsy seizures; D. Duration of epilepsy seizures.

[0022] Figure 3 Effect diagram of Ly93 on neuroinflammation in KA-induced mouse epilepsy seizure model, where: A. Statistical analysis of mRNA expression level of TNF-α; B. Statistical analysis of mRNA expression level of IL-1β; C. Statistical analysis of mRNA expression level of IL-6.

[0023] Figure 4 Effect diagram of Ly93 on the body weight of KA-induced mouse epilepsy seizure model, where: A. Body weight of mice after drug intervention; B. Change value of body weight of mice before and after drug intervention. Specific Embodiments

[0024] Example 1: Ly93 significantly improves the symptoms of KA-induced mouse epilepsy seizure model

[0025] Experimental grouping: 12 mice in each of the control group, KA group, KA + 25 mg / kg Ly93 group, and KA + 100 mg / kg Ly93 group; the control group was stereotactically injected with normal saline, the KA group was stereotactically injected with 250 ng / μL KA prepared with normal saline, and the KA + 25 mg / kg Ly93 group and KA + 100 mg / kg Ly93 group were intraperitoneally injected with 2.5 mg / ml and 10 mg / ml Ly93 composed of 5% DMSO, 40% PEG400, 5% Tween 80, and 50% normal saline 2 h before stereotactic injection of 250 ng / μL KA.

[0026] Seizure scoring: After the mice regained consciousness, two researchers recorded the seizure grade, seizure frequency, and seizure duration of each mouse within 90 minutes after KA injection. The seizure of mice was evaluated according to the following Racine scoring criteria: Stage 0: No seizure; Stage 1: Rhythmic twitching of the face and whiskers; Stage 2: Regular nodding or facial twitching without; Stage 3: Multilateral myoclonus and spasm; Stage 4: Uncontrolled fall; Stage 5: Generalized tonic-clonic seizure with running and jumping; Stage 6: Death.

[0027] Analysis of experimental results: The experimental results of each group are as Figure 1 shown. The experimental results indicate that Ly93 significantly reduces the seizure grade, seizure frequency, and seizure duration induced by KA, suggesting that Ly93 has a good anti-seizure effect.

[0028] Example 2: Ly93 significantly improves the symptoms of a PTZ-induced mouse seizure model

[0029] Experimental grouping: There were 9 mice in each of the control group, PTZ group, PTZ + 25 mg / kg Ly93 group, and PTZ + 100 mg / kg Ly93 group; the control group was intraperitoneally injected with normal saline, the PTZ group was intraperitoneally injected with 14 mg / ml PTZ prepared with normal saline at a dose of 70 mg / kg, and the PTZ + 25 mg / kg Ly93 group and PTZ + 100 mg / kg Ly93 group were intraperitoneally injected with 2.5 mg / ml and 10 mg / ml Ly93 composed of 5% DMSO, 40% PEG400, 5% Tween80, and 50% normal saline 2 hours before intraperitoneally injecting 14 mg / ml PTZ.

[0030] Seizure scoring: The seizure latency was defined as the time period from PTZ injection to the first seizure, and the seizure grade, seizure frequency, and seizure duration of each mouse within 30 minutes after PTZ injection were recorded. The seizure scoring criteria were the same as in Example 1.

[0031] Analysis of experimental results: The experimental results of each group are as Figure 2 shown. The experimental results indicate that Ly93 significantly prolongs the seizure latency induced by PTZ and significantly reduces the seizure duration induced by PTZ, which confirms that Ly93 has a good anti-seizure effect.

[0032] Example 3: Ly93 significantly inhibits neuroinflammation in a KA-induced mouse seizure model

[0033] Experimental grouping: 6 mice in each of the control group, KA group, KA + 25 mg / kg Ly93 group, and KA + 100 mg / kg Ly93 group. The drug intervention method was the same as that in Example 1. Subsequently, the hippocampal tissues of the mice were isolated, and mRNA was extracted from each group for detection of neuroinflammatory-related factors (including TNF-α, IL-1β, and IL-6 mRNA).

[0034] RT-qPCR detection: After anesthesia, the heads of the mice in each group were cut off, and the brains were taken out and placed on a petri dish containing pre-cooled physiological saline. The hippocampal tissues were isolated, and RT-qPCR was used to detect the expression of the inflammatory marker molecules TNF-α, IL-1β, and IL-6 mRNA. The primers for TNF-α, IL-1β, IL-6, and the internal reference gene β-actin were from Primerbank (https: / / pga.mgh.harvard.edu / primerbank / ). Their sequences were as follows: (TNF-α Forward) 5’-CAGGCGGTGCCTATGTCTC -3’ (SEQ ID NO.1), (TNF-α Reverse) 5’-CGATCACCCCGAAGTTCAGTAG -3’ (SEQ ID NO.2); (IL-1β Forward) 5’-GAAATGCCACCTTTTGACAGTG -3’ (SEQ ID NO.3), (IL-1β Reverse) 5’-TGGATGCTCTCATCAGGACAG -3’ (SEQ ID NO.4); (IL-6 Forward) 5’-CAGAAGGAGTGGCTAAGGACCA -3’ (SEQ ID NO.5), (IL-6 Reverse) 5’-ACGCACTAGGTTTGCCGAGTAG -3’ (SEQ ID NO.6); (β-actin Forward) 5’-GTGACGTTGACATCCGTAAAGA -3’ (SEQ ID NO.7), (β-actin Reverse) 5’-GCCGGACTCATCGTACTCC-3’ (SEQ ID NO.8). The expression levels of TNF-α, IL-1β, and IL-6 mRNA in each group were calculated using the 2 -△△Ct method.

[0035] Analysis of experimental results: The analysis results of each group are as Figure 3As shown in A-C, the mRNA expression levels of TNF-α, IL-1β, and IL-6 in the KA model were significantly higher than those in the normal control group, while the 25 mg / kg and 100 mg / kg Ly93 intervention groups decreased the mRNA expression levels of TNF-α, IL-1β, and IL-6, suggesting that Ly93 can more effectively inhibit KA-induced epileptic seizure neuroinflammation.

[0036] Example 4: Ly93 does not affect the body weight of KA-induced epileptic mice

[0037] Experimental grouping: 8 mice in each of the control group, KA group, KA + 25 mg / kg Ly93 group, and KA + 100 mg / kg Ly93 group.

[0038] Body weight monitoring: Before and after drug intervention, the body weight of each mouse in each group was weighed using a weighing scale, and the body weight of each group of mice after drug intervention and the change value of the body weight of each group of mice before and after drug intervention were statistically analyzed.

[0039] Analysis of experimental results: As shown in Figure 4 We analyzed and found that neither 25 mg / kg nor 100 mg / kg of Ly93 had a significant effect on the body weight of mice, suggesting that Ly93 has good safety.

[0040] The above is the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and its concept of the present invention, makes equivalent substitutions or changes, and should be covered by the protection scope of the claims of the present invention.

Claims

1. Application of Ly93 in the preparation of anti-epileptic drugs. The chemical structure of Ly93 is shown in formula (I): Formula (I).

2. The use according to claim 1, characterized in that: The medicine is a pharmaceutical composition, which comprises Ly93 and a conventional pharmaceutical carrier.

3. The use according to claim 2, characterized in that: The conventional pharmaceutical carriers are dimethyl sulfoxide, polyethylene glycol 400 and Tween 80.

4. The use according to claim 1 or 2, characterized in that: The medicine is a preparation for oral or parenteral administration.

5. The use according to claim 4, characterized in that: The preparation is in the form of tablets, capsules, granules, pills, oral solutions, injections, transdermal preparations, sprays or aerosols.

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