Application of β-aminopropionitrile in preparing a preparation for reversing lamotrigine resistance

Abstract

Application of β-aminopropionitrile in the preparation of a formulation for reversing lamotrigine resistance. The present invention belongs to the field of development of therapeutic drugs for lamotrigine resistance. The present invention provides the application of β-aminopropionitrile (abbreviated as BAPN) in the preparation of a formulation for reversing lamotrigine (abbreviated as LTG) resistance, providing a new idea for finding therapeutic drugs for LTG resistance. BAPN has better safety and smaller toxic and side effects as a formulation for reversing LTG resistance, and the effect of BAPN as a formulation for reversing LTG resistance is more significant.

Description

Technical Field

[0001] The invention belongs to the field of development of therapeutic drugs for lamotrigine resistance, and particularly relates to application of beta-aminopropionitrile in preparing a preparation for reversing lamotrigine resistance. Background Art

[0002] Lysyl oxidase (Lox) was previously considered an important collagenase that remodels the extracellular matrix. However, recent research results from our research group have found that Lox can aggravate epilepsy-induced brain damage as a new type of neuronal ferroptosis regulator. In addition, in animal models of epilepsy, inhibiting Lox can block ferroptosis and reduce neuronal damage, thereby reducing epileptic seizures.

[0003] β-aminopropionitrile (BAPN) is a classic Lox inhibitor that can effectively inhibit Lox activity. In models of myocardial fibrosis and chronic heart failure, BAPN significantly inhibited Lox-mediated collagen cross-linking, thereby reducing myocardial fibrosis. In animal and cell models of gastric cancer and breast cancer, BAPN inhibited Lox, thereby blocking the invasion and migration of cancer cells and inhibiting tumor growth. In a clinical trial of scleroderma, BAPN was used for 67 consecutive days without significant toxic side effects.

[0004] Reversing the resistance of antiepileptic drugs is a major problem in the field of epilepsy treatment. The pathogenesis of chronic or drug-resistant epilepsy is far from being clearly explained. Lamotrigine (LTG) is a common second-generation antiepileptic drug. Compared with first-generation antiepileptic drugs, LTG has less effect on drug-metabolizing enzymes, high safety, low teratogenicity, and low risk of fetal neurological and intellectual developmental disorders after use by pregnant women. It is the first choice for the treatment of focal and generalized epilepsy in women of childbearing age and children. As a mood stabilizer, LTG is also the best choice for patients with epilepsy and depression. However, the efficacy of LTG is limited. In generalized epilepsy, LTG is less effective in controlling epileptic seizures than first-generation antiepileptic drugs sodium valproate and carbamazepine. In addition, as the duration of medication increases, some patients who were initially effective develop resistance to lamotrigine. About 20% of patients have epilepsy relapses within two years and eventually give up monotherapy due to poor control. The probability of epileptic seizure remission in patients with monotherapy resistance is significantly reduced, and most of them become patients with refractory epilepsy, and lifelong epileptic seizures may require surgical treatment. In addition, combination drug regimens increase the risk of patients developing neuropsychiatric disorders and other side effects, further increasing the disease burden on patients.

[0005] The prior art related to lamotrigine resistance includes: CN02811350.0 discloses a combination comprising a P-glycoprotein (P-gp) inhibitor and an antiepileptic drug selected from phenytoin (5,5-diphenyl-2,4-imidazolidinedione), carbamazepine, lamotrigine, gabapentin, oxcarbazepine, sodium valproate, and topiramate, and its use in preventing, delaying the progression of, or treating diseases, especially epilepsy.

[0006] Finding new drugs for reversing lamotrigine resistance is of great significance. Summary of the Invention

[0007] Aiming at the deficiencies of the existing treatment of lamotrigine, the present invention aims to provide the application of β-aminopropionitrile in the preparation of a preparation for reversing lamotrigine resistance, which has better safety and smaller toxic and side effects.

[0008] The technical solution of the present invention is:

[0009] The present invention provides the application of β-aminopropionitrile in the preparation of a preparation for reversing lamotrigine resistance.

[0010] Further, the lamotrigine resistance preparation comprises a resistance preparation against lamotrigine and a conventional pharmaceutical carrier.

[0011] Further, the lamotrigine resistance preparation includes preparations administered by oral or non-oral routes.

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

[0013] The present invention identifies BAPN as a novel drug for reversing LTG resistance. The specific evidence is as follows: After establishing an LTG-resistant mouse model, qPCR and WB techniques were used to detect the mRNA and protein expression levels of Lox in different brain regions (including the hippocampus, cerebellum, frontal cortex, and piriform cortex) of LTG-resistant and LTG-sensitive mouse models. It was found that the mRNA expression of Lox in the hippocampus and cerebellum of the LTG-resistant mouse model was significantly increased, and the protein expression of Lox in the hippocampus was significantly increased, suggesting that the increased expression of hippocampal Lox may be related to LTG resistance; further experimental results showed that in the LTG-resistant mouse model, we intervened with sodium valproate (abbreviated as VPA) and BAPN respectively. Among them, VPA, a well-known anti-epileptic drug with significant efficacy, was used as a control, and the Racine score and EEG were used to evaluate its effect on reversing LTG resistance. The experimental results showed that 100 mg / kg BAPN significantly reversed the LTG resistance effect; we further analyzed the effect of BAPN on ferroptosis-related indicators (including the mRNA expression of PTGS2 and GPX4) in the LTG-resistant mouse model. The experimental results showed that BAPN inhibited ferroptosis in the LTG-resistant model to a certain extent. The specific evidence is that BAPN inhibited the expression of PTGS2 mRNA in the hippocampus of the LTG-resistant mouse model and increased the expression of GPX4 mRNA in the hippocampus of the LTG-resistant mouse model; in addition, we also analyzed the effect of BAPN on the body weight of the LTG-resistant mouse model. The experimental results showed that BAPN did not affect the body weight of the LTG-resistant mice, suggesting that BAPN has good safety. Based on the above multiple research results, BAPN may be a novel drug for effectively reversing LTG resistance and has great clinical application prospects.

[0014] Compared with the prior art, the advantages of the present invention are as follows:

[0015] The present invention provides the application of BAPN in the preparation of a preparation for reversing LTG resistance, providing a new idea for drugs for treating LTG resistance.

[0016] BAPN as a preparation for reversing LTG resistance has better safety and smaller toxic and side effects.

[0017] BAPN as a preparation for reversing LTG resistance has a more significant effect.

[0018] The following further explains and illustrates the detailed structure of the present invention in conjunction with the accompanying drawings and specific embodiments. Description of the Drawings

[0019] Figure 1 It is a graph of the protein expression results of mRNA of Lox in different brain regions (including the hippocampus, cerebellum, frontal cortex, and piriform cortex) of LTG-resistant mice, where Figure 1A is the result of qPCR experiments, including the mRNA expression of Lox in the hippocampus, cerebellum, frontal cortex, and piriform cortex of LTG-resistant and LTG-sensitive mice. Figure 1 B-C are the results of WB experiments, including representative WB images of Lox in the hippocampus, cerebellum, frontal cortex, and piriform cortex of LTG-resistant and LTG-sensitive mice ( Figure 1 B) and statistical graphs ( Figure 1 C).

[0020] Figure 2 This is a graph showing the effect of BAPN on reversing the LTG-resistant mouse model. Among them, A-B are statistical graphs of Racine score for baseline before BAPN or VPA administration and seizures after LTG administration, including seizure scores ( Figure 2 A), seizure durations ( Figure 2 B); C is the statistical result of electroencephalogram, which is the duration of afterdischarge stimulated before and after BAPN or VPA administration at baseline and after 15 mg / kg LTG administration.

[0021] Figure 3 This is a graph showing the effect of BAPN on ferroptosis in the LTG-resistant mouse model. Among them, Figure 3 A is a statistical graph of PTGS2 mRNA expression; Figure 3 B is a statistical graph of GPX4 mRNA expression.

[0022] Figure 4 This is a graph showing the effect of BAPN on the body weight of the LTG-resistant mouse model. Specific implementation methods

[0023] Experimental grouping: The experiment was divided into 2 groups, namely the LTG-sensitive group (n = 6) and the LTG-resistant group (n = 6). Electroencephalogram recording electrode wires were implanted in all mice at the position with coordinates AP: -2.0 mm, ML: -1.8 mm, DV: -2.3 mm, and electrostimulating electrode wires were implanted at the position with coordinates AP: -2.9 mm; ML: +3.0 mm; DV: -3.0 mm. After 7 days of recovery, electrostimulation was carried out in groups. Among them, the LTG-sensitive group was intraperitoneally injected with 0.5% CMC-Na during electrostimulation ignition, and the LTG-resistant group was intraperitoneally injected with 1 mg / mL LTG prepared with 0.5% CMC-Na at a dose of 5 mg / kg three times a day during ignition to induce resistance. Each mouse was given six electrostimulations a day, with a half-hour interval between each stimulation. After three consecutive electrostimulations, a 5-level seizure indicated complete ignition. After complete ignition, both groups were subjected to a reactivity test with 15 mg / kg LTG.

[0024] qPCR experiment: The hippocampus, cerebellum, frontal cortex, and piriform cortex tissues of LTG-sensitive and LTG-resistant mice were isolated. Tissue mRNA was extracted, and qPCR experiments were performed to detect the mRNA expression of Lox. The experimental results showed that, as Figure 1 shown, the mRNA expression of Lox in the hippocampus and cerebellum regions of the LTG-resistant group was significantly increased compared with that of the LTG-sensitive group, and there were no significant differences in other regions.

[0025] WB experiment: The hippocampus, cerebellum, frontal cortex, and piriform cortex tissues of LTG-sensitive and LTG-resistant mice were taken. Tissue proteins were extracted, and WB experiments were performed to detect the protein expression of Lox. The experimental results showed that, as Figure 1 shown, the protein expression level of Lox in the hippocampus region of the LTG-resistant group was significantly increased compared with that of the LTG-sensitive group. The within-group differences in the protein expression of Lox in the cerebellum region were large, and the results were not significantly different. The protein expression of Lox in the frontal cortex and piriform cortex showed a downward trend but no statistical difference.

[0026] Experimental grouping: The experiment was divided into 4 groups, namely the LTG-sensitive group (n = 6), the LTG-resistant group (n = 7), the LTG-resistant + 100 mg / kg VPA group (n = 8), and the LTG-resistant + 100 mg / kg BAPN group (n = 8). Among them, VPA, a recognized anti-epileptic drug, was used as a control drug. The LTG-sensitive group was intraperitoneally injected with 0.5% CMC-Na during electroshock kindling. The remaining groups were intraperitoneally injected with 1 mg / mL LTG prepared with 0.5% CMC-Na at a dose of 5 mg / kg during kindling to induce resistance. After complete kindling, a reactivity test of 15 mg / kg LTG was performed on each group. After the test, the LTG-sensitive group and the LTG-resistant group were intraperitoneally injected with normal saline for 14 consecutive days. The LTG-resistant + 100 mg / kg VPA group was intraperitoneally injected with 20 mg / mL VPA prepared with normal saline at a dose of 100 mg / kg for 14 consecutive days. The LTG-resistant + 100 mg / kg BAPN group was intraperitoneally injected with 20 mg / mL BAPN prepared with normal saline at a dose of 100 mg / kg for 14 consecutive days.

[0027] LTG reactivity experiment: The fully ignited mice were placed in a transparent observation container, and the electroencephalogram monitoring and electrical stimulation circuits were connected. The seizure grade, seizure duration, and electroencephalogram within 2 minutes after electrical stimulation were recorded. Taking this seizure as the baseline seizure condition after electrical stimulation, 3 mg / mL LTG prepared with 0.5% CMC-Na was intraperitoneally injected at a dose of 15 mg / kg three hours later. One hour after administration, the circuit was connected and electrical stimulation was performed, and the seizure grade, seizure duration, and electroencephalogram within 2 minutes after electrical stimulation were recorded. The baseline seizure condition of each mouse was recorded once before administration for 14 days, and electrical stimulation was performed after three administrations of 15 mg / kg LTG, with each interval being 72 hours. The average seizure grade, average seizure duration, and average value of the afterdischarge duration recorded in the electroencephalogram during the baseline stimulation and three LTG administration tests were used as statistical indicators. The baseline seizure condition was recorded once after 14 days of administration, and electrical stimulation was performed after one administration of 15 mg / kg LTG. The average seizure grade, average seizure duration, and average value of the afterdischarge duration recorded in the electroencephalogram during the baseline stimulation and LTG administration test were used as statistical indicators. The experimental results showed that, as Figure 2 shown, after intraperitoneal injection of 15 mg / kg LTG before administration, the three indicators of seizure severity in the LTG-sensitive group: seizure grade, seizure duration, and afterdischarge duration decreased, and there were significant differences compared with other groups. After 14 days, there were no obvious differences in seizure severity among groups during baseline stimulation, but after administration of 15 mg / kg LTG, only in the LTG-resistant + 100 mg / kg BAPN group, there were obvious differences in seizure grade, seizure duration, and afterdischarge duration compared with the LTG-resistant group. In the LTG-sensitive group and the LTG-resistant + 100 mg / kg VPA group, some indicators were lower than those in the LTG-resistant group, but there were no significant differences.

[0028] Experimental grouping: The experiment was divided into 4 groups, namely the LTG-sensitive group (n = 4), the LTG-resistant group (n = 4), the LTG-resistant + 100 mg / kg VPA group (n = 4), and the LTG-resistant + 100 mg / kg BAPN group (n = 4); after the 15 mg / kg LTG reactivity test, the LTG-sensitive group and the LTG-resistant group were intraperitoneally injected with normal saline continuously for 14 days, the LTG-resistant + 100 mg / kg VPA group was intraperitoneally injected with 20 mg / mL VPA prepared with normal saline at a dose of 100 mg / kg continuously for 14 days, and the LTG-resistant + 100 mg / kg BAPN group was intraperitoneally injected with 20 mg / mL BAPN prepared with normal saline at a dose of 100 mg / kg continuously for 14 days.

[0029] Detection of ferroptosis-related indicators: The hippocampal tissues of the LTG-sensitive group, LTG-resistant group, LTG-resistant + 100 mg / kg VPA group, and LTG-resistant + 100 mg / kg BAPN group were taken, and tissue mRNA was extracted according to the steps in Experimental Example 1. The mRNA expressions of PTGS2 and GPX4 in the hippocampal tissues of mice were detected respectively. The experimental results showed that, as Figure 3 shown, the PTGS2 mRNA in the hippocampus of the LTG-resistant group was significantly increased and the mRNA expression of GPX4 was significantly decreased, while the PTGS2 mRNA in the LTG-resistant + 100 mg / kg BAPN group was significantly decreased and the GPX4 mRNA was significantly increased compared with the LTG-resistant group.

[0030] Experimental grouping: The experiment was divided into 4 groups, namely the LTG-sensitive group (n = 6), the LTG-resistant group (n = 7), the LTG-resistant + 100 mg / kg VPA group (n = 8), and the LTG-resistant + 100 mg / kg BAPN group (n = 8); after the 15 mg / kg LTG reactivity test, the LTG-sensitive group and the LTG-resistant group were intraperitoneally injected with normal saline for 14 consecutive days, the LTG-resistant + 100 mg / kg VPA group was intraperitoneally injected with 20 mg / mL VPA prepared with normal saline at a dose of 100 mg / kg for 14 consecutive days, and the LTG-resistant + 100 mg / kg BAPN group was intraperitoneally injected with 20 mg / mL BAPN prepared with normal saline at a dose of 100 mg / kg for 14 consecutive days.

[0031] Body weight monitoring: Taking body weight as the primary evaluation index for drug safety, the body weight data of mice on the 1st, 4th, 7th, and 14th days after drug administration were recorded. Using the body weight and body weight change value of mice as statistical indicators, the experimental results showed that, as Figure 4 shown, the body weight changes in the LTG-sensitive group and the LTG-resistant + 100 mg / kg BAPN group were relatively small. The body weights of the LTG-resistant group and the LTG-resistant + 100 mg / kg VPA group showed a downward trend.

[0032] 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 all should be covered by the protection scope of the claims of the present invention.

Claims

1. Use of β-aminopropionitrile in the preparation of a preparation for reversing lamotrigine resistance, wherein the lamotrigine resistance preparation is used for the treatment of epilepsy.

2. The use according to claim 1, characterized in that, the lamotrigine resistance preparation is a preparation administered by an oral or non-oral route.

3. The use according to claim 1, characterized in that, the lamotrigine resistance preparation is a tablet, capsule, granule, dripping pill, oral solution, injection, transdermal agent, spray or aerosol.

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