Application of Ly93 in preparation of anti-epileptic seizure medicine

Abstract

The invention provides an application of Ly93 in preparation of an anti-epileptic seizure drug, and provides a new thought for an epileptic seizure treatment drug. Research results show that the Ly93 can significantly reduce the grade of epileptic seizure in a KA-induced mouse epileptic seizure model, can significantly reduce the duration of PTZ-induced epileptic seizure, can significantly inhibit neuroinflammation in epileptic seizure, and does not affect the weight of a mouse. Therefore, the Ly93 serving as the epileptic seizure treatment medicine is remarkable in effect, relatively good in safety and relatively small in toxic and side effects.

Description

Technical Field

[0001] The invention belongs to the field of development of epilepsy therapeutic drugs, and specifically relates to the application of Ly93 in the preparation of anti-epileptic seizure drugs. Background Art

[0002] Epilepsy is a common neurological disease characterized by abnormal neuronal discharges. Its clinical manifestations include varying degrees of convulsions, stupor, foaming at the mouth, and multiple comorbidities. Severe cases may lead to death. It is characterized by suddenness, spontaneity, and recurrence.

[0003] Recurrent epileptic seizures are the main clinical feature of epilepsy patients. Existing clinical intervention measures, including drug therapy, neurostimulation therapy, surgical treatment, and diet therapy, can exert anti-epileptic effects by suppressing seizure symptoms. However, these treatment measures often have defects such as liver toxicity, cognitive impairment, and poor compliance during clinical use. Therefore, it is urgent to explore new drugs for the treatment of epileptic seizures.

[0004] Ly93, CAS No.1883528-69-5, chemical formula C 21 H 20 N2O2, molecular weight 332.40, chemical structure as described in formula (I), is a new type of small molecule compound, widely used in preclinical experimental research, and has stable physical and chemical properties. After searching, no reports of Ly93 as a drug for treating epileptic seizures were found in the prior art.

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

[0006] In view of the shortcomings of existing treatments for epileptic seizures, the present invention aims to provide the use of Ly93 in the preparation of anti-epileptic seizure drugs.

[0007] The technical solution of the present invention is: The present invention provides application of Ly93 in preparing an anti-epileptic seizure drug.

[0008] The present invention provides a pharmaceutical composition, which comprises Ly93 and a conventional pharmaceutical carrier.

[0009] The conventional pharmaceutical carriers include dimethyl sulfoxide (DMSO), polyethylene glycol 400 (PEG400), Tween 80 (Tween80) and the like.

[0010] Furthermore, the pharmaceutical composition is a preparation for oral or parenteral administration.

[0011] Furthermore, the preparations include tablets, capsules, granules, pills, oral solutions, injections, transdermal preparations, sprays or aerosols, etc.

[0012] The examples of the present invention further indicate the use of Ly93 in the preparation of an anti-epileptic drug, wherein the drug has a dosage of 25 mg / kg-100 mg / kg, has a significant anti-epileptic effect, has good safety, and has less toxic side effects.

[0013] The present invention provides multiple evidences to support that Ly93 may be a new drug for controlling epileptic seizures. The specific evidences are as follows: In the epileptic seizure model induced by kainate (KA), we used 25mg / kg and 100mg / kg of Ly93 to intervene, and then used the Racine scoring standard to evaluate the epileptic seizures of mice. The experimental results showed that both doses of Ly93 could significantly reduce the epileptic seizure grade in the epileptic seizure model induced by KA, among which 100mg / kg of Ly93 had a more significant effect and could significantly reduce the number of epileptic seizures and the duration of epileptic seizures; at the same time, in the epileptic seizure model induced by pentylenetetrazol (PTZ), our experimental results showed that when Ly93 was at a dose of 100mg / kg, the latency of PTZ-induced epileptic seizures in mice was significantly prolonged, and both 25mg / kg and 100mg / kg doses could significantly reduce the duration of PTZ-induced epileptic seizures; further experimental results showed that in the epileptic seizure model induced by KA and PTZ, we used 25mg / kg and 100mg / kg of Ly93 to significantly reduce the duration of PTZ-induced epileptic seizures in mice. After intervention with Ly93, the changes in neuroinflammation-related indicators (including TNF-α, IL-1β and IL-6) were analyzed. The experimental results showed that Ly93 significantly inhibited neuroinflammation in epileptic seizures. Specifically, 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 the KA-induced epileptic seizure mouse model. In addition, we also analyzed the effect of Ly93 on the body weight of the KA-induced epileptic seizure mouse model. The experimental results showed that Ly93 did not affect the body weight of mice, indicating that Ly93 has good safety. Based on the above experimental results, Ly93 may be a new drug that effectively improves epileptic seizures and has great clinical application prospects.

[0014] Compared with the prior art, the advantages of the present invention are: The present invention provides the use of Ly93 in the preparation of a drug for treating epileptic seizures, providing a new idea for the drug for treating epileptic seizures.

[0015] Ly93 has good safety and less toxic side effects as a drug for treating epileptic seizures.

[0016] Ly93 is effective as a drug for treating epileptic seizures.

[0017] The detailed structure of the present invention is further described below in conjunction with the accompanying drawings and specific implementation methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a diagram showing the effect of Ly93 on the score of the KA-induced mouse epileptic seizure model, where: A. epileptic seizure score; B. number of epileptic seizures; C. duration of epileptic seizures.

[0019] Figure 2 This is a diagram showing the effect of Ly93 on the score of the PTZ-induced mouse epilepsy model, where: A. epilepsy onset latency; B. epilepsy seizure score; C. number of epilepsy seizures; D. epilepsy seizure duration.

[0020] Figure 3 The diagram shows the effect of Ly93 on neuroinflammation in the KA-induced mouse epileptic seizure model, including: A. Statistical analysis of TNF-α mRNA expression level; B. Statistical analysis of IL-1β mRNA expression level; C. Statistical analysis of IL-6 mRNA expression level.

[0021] Figure 4 The figure shows the effect of Ly93 on the body weight of KA-induced epileptic seizure model in mice, where: A. The body weight of mice after drug intervention; B. The change in body weight of mice before and after drug intervention. DETAILED DESCRIPTION

[0022] Example 1: Ly93 significantly improves the symptoms of KA-induced epileptic seizure model in mice

[0023] Experimental groups: control group, KA group, KA+25mg / kg Ly93 group, and KA+100mg / kg Ly93 group, 12 rats in each group; the control group was stereotactically injected with normal saline, the KA group was stereotactically injected with 250ng / μL KA prepared in normal saline, the KA+25mg / kgLy93 group and the KA+100mg / kg Ly93 group were stereotactically injected with 250ng / μL KA, and at the same time, they were intraperitoneally injected with 2.5mg / ml and 10mg / ml Ly93 prepared with 5% DMSO, 40% PEG400, 5% Tween80, and 50% normal saline, respectively, 2h in advance.

[0024] Seizure scoring: After the mice woke up, two researchers recorded the seizure grade, number of seizures, and duration of seizures for each mouse within 90 minutes after KA injection. The seizures of mice were evaluated according to the following Racine scoring criteria: Stage 0: no seizures; Stage 1: rhythmic twitching of the face and whiskers; Stage 2: regular nodding or no facial twitching; Stage 3: multi-limb myoclonus and spasm; Stage 4: uncontrolled falls; Stage 5: generalized tonic-clonic seizures with running and jumping; Stage 6: death.

[0025] Experimental results analysis: The experimental results of each group are as follows Figure 1 As shown, the experimental results showed that Ly93 significantly reduced the grade, number and duration of epileptic seizures induced by KA, suggesting that Ly93 has a good anti-epileptic effect.

[0026] Example 2: Ly93 significantly improves the symptoms of PTZ-induced epileptic seizure model in mice Experimental groups: control group, PTZ group, PTZ+25mg / kg Ly93 group, and PTZ+100mg / kg Ly93 group, 9 rats in each group; the control group was intraperitoneally injected with normal saline, the PTZ group was intraperitoneally injected with 14mg / ml PTZ prepared in normal saline at a dose of 70mg / kg, the PTZ+25mg / kg Ly93 group and the PTZ+100mg / kg Ly93 group were intraperitoneally injected with 14mg / ml PTZ and, 2h in advance, were intraperitoneally injected with 2.5mg / ml and 10mg / ml Ly93 respectively, which were made up of 5% DMSO, 40% PEG400, 5% Tween80, and 50% normal saline.

[0027] Seizure scoring: The seizure latency period is defined as the period from PTZ injection to the first seizure, and the seizure grade, number of seizures and duration of seizures of each mouse within 30 minutes after PTZ injection are recorded. The seizure scoring criteria are the same as those in Example 1.

[0028] Experimental results analysis: The experimental results of each group are as follows Figure 2 As shown, the experimental results showed that Ly93 significantly prolonged the latency of PTZ-induced epileptic seizures and significantly reduced the duration of PTZ-induced epileptic seizures. These results confirmed that Ly93 has a good anti-epileptic effect.

[0029] Example 3: Ly93 significantly inhibits neuroinflammation in a KA-induced mouse epileptic seizure model Experimental groups: control group, KA group, KA+25mg / kg Ly93 group, KA+100mg / kg Ly93 group, 6 mice each. The drug intervention method was the same as Example 1. Subsequently, the mouse hippocampal tissue was isolated, and mRNA of each group was extracted to detect neuroinflammation-related factors (including TNF-α, IL-1β and IL-6 mRNA).

[0030] RT-qPCR detection: After anesthesia, the mice in each group were decapitated and the brains were removed and placed on a culture dish containing pre-cooled saline. The hippocampal tissue was isolated and the expression of inflammatory marker molecules TNF-α, IL-1β, and IL-6 mRNA was detected by RT-qPCR. 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 are: (TNF-α Forward) 5'-CAGGGCGTGCCTATGTCTC -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 measured using 2 -△△Ct method to calculate.

[0031] Experimental results analysis: The analysis results of each group are as follows Figure 3As shown in AC, the expression levels of TNF-α, IL-1β, and IL-6 mRNA in the KA model were significantly increased compared with the normal control group, while the 25 mg / kg and 100 mg / kg Ly93 intervention groups reduced the expression levels of TNF-α, IL-1β, and IL-6 mRNA, indicating that Ly93 can more effectively inhibit KA-induced neuroinflammation in epileptic seizures.

[0032] Example 4: Ly93 does not affect the body weight of KA-induced epileptic mice Experimental groups: control group, KA group, KA+25mg / kg Ly93 group, and KA+100mg / kg Ly93 group, 8 mice in each group.

[0033] Body weight monitoring: Before and after drug intervention, the weight of each mouse in each group was measured using a weight scale, and the weight of each group of mice after drug administration and the weight change of each group of mice before and after drug intervention were counted.

[0034] Experimental results analysis: The experimental results are as follows Figure 4 As shown, our analysis found that 25 mg / kg and 100 mg / kg of Ly93 had no significant effect on the body weight of mice, indicating that Ly93 has good safety.

[0035] The above is a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, can make equivalent replacements or changes according to the technical solutions and concepts of the present invention, which 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.

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 include 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 1 or 2, characterized in that: The preparations include tablets, capsules, granules, pills, oral solutions, injections, transdermal preparations, sprays or aerosols, etc.

Images