New application of perampanel

Abstract

The invention provides application of perampanel (PER) in preparation of a medicine for preventing, treating and / or relieving retinal nerve injury induced by hyperglycemia. Experimental results show that when the PER is used for treating ganglion cells (RGC) damaged by high glucose, the death of the RGC can be reduced, and the death of the RGC of a diabetes mellitus (DM) mouse can be reduced, which indicates that the PER has a potential neuroprotective effect on the RGC exposed under the condition of hyperglycemia, and has the potential of preventing, treating and relieving the RGC damage induced by the hyperglycemia.

Description

Technical Field

[0001] The invention belongs to the technical field of new drug uses, and in particular relates to a new use of the anti-epileptic drug perampanel. Background Art

[0002] Perampanel (PER) is a highly selective and non-competitive AMPAR antagonist, a new anti-epileptic drug approved by the US FDA in 2012. It has good oral bioavailability and good pharmacokinetic properties. It is extensively metabolized in the liver, mainly by CYP3A4 and CYP3A5. The half-life in clinical studies ranges from 53 to 136 h, with an average half-life of 105 h, but the half-life can be shortened in the presence of strong inducers (such as carbamazepine). Its safety and tolerability in human use have been fully demonstrated.

[0003] As a highly selective AMPA receptor antagonist, PER inhibits glutamate-induced excessive neurotransmission by non-competitively binding to AMPA receptors on the postsynaptic membrane, thus exerting an anti-epileptic effect. PER has been approved in many countries for the adjunctive treatment and monotherapy of patients with focal epilepsy aged ≥4 years with or without secondary generalized epilepsy, and for the adjunctive treatment of patients with primary generalized tonic-clonic seizures aged ≥12 years. In addition, many other effects of PER have been discovered: Chen et al. showed through a rat traumatic brain injury (TBI) model experiment that PER attenuated TBI-induced brain edema, brain contusion volume and gross motor dysfunction, and improved cognitive function after TBI; studies have shown that the AMPA receptor antagonist PER can reduce traumatic brain injury through antioxidant and anti-inflammatory activities, and PER can regulate neuronal necrosis after acute traumatic injury of cortical neurons through Akt / GSK3β signaling; and studies have found that it protects neurovascular units from traumatic injury by regulating Sirt3. In 2022, Kawakita et al. also proposed that the AMPA receptor antagonist PER can reduce acute blood-brain barrier damage after subarachnoid hemorrhage in mice.

[0004] To date, the clinical application of PER is mainly focused on the nervous system, such as epilepsy, Parkinson's disease, amyotrophic lateral sclerosis, and restless legs syndrome.

[0005] AMPAR is an ionotropic glutamate receptor composed of GluA1-4 subunits. + , K + and Ca 2+It is an important regulatory channel of the nervous system and was listed as an important target receptor in the national "Major New Drug Creation" major science and technology project. It is not only closely related to mental and nervous system diseases, but also participates in the signal pathways of cardiovascular diseases, tumors, respiratory diseases, and endocrine system diseases. It has broad development prospects as a drug target.

[0006] Diabetic retinopathy (DR), as a severe form of diabetic microvascular complications, has become one of the major blinding eye diseases. The microvascular lesions of DR are not single vascular lesions, but lesions of the vascular nerve unit as a whole. The role of the regulation of vascular nerve units in the development of DR, especially in early damage, has been increasingly valued by researchers.

[0007] Retinal ganglion cells (RGCs) are affected in patients with diabetes, suggesting that neuronal components play an important role in the development and progression of DR. Glutamate is an excitatory neurotransmitter in the nervous system that induces excitotoxicity and neurodegeneration in the retina and brain under a variety of pathological conditions. Increased extracellular glutamate levels are considered to be the main cause of retinal neurodegeneration in patients with diabetes. Previous studies have shown that excess glutamate may upregulate vascular endothelial growth factor production in the diabetic retina, and this effect can be inhibited by memantine, a noncompetitive N-methyl-D-aspartate receptor (NMDAR) antagonist. However, NMDAR has been extensively studied and its pharmacological potential has reached its limit.

[0008] Researching new drugs to prevent and treat diabetic retinopathy is a current research focus. Summary of the invention

[0009] In view of this, the technical problem to be solved by the present invention is to provide a new use of perampanel, which can be applied to prevent, treat and alleviate hyperglycemia-induced retinal ganglion cell damage.

[0010] The present invention provides the use of perampanel in preparing a drug for preventing, treating and / or alleviating retinal nerve damage.

[0011] Preferably, said prevention, treatment and / or alleviation comprises protecting retinal ganglion cells.

[0012] Preferably, said prevention, treatment and / or alleviation comprises inhibiting the levels of AMPA receptors.

[0013] Preferably, said prevention, treatment and / or alleviation comprises reducing the death of retinal ganglion cells.

[0014] Preferably, said preventing, treating and / or alleviating comprises preventing a decrease in the amplitude of phNR waves.

[0015] Preferably, the dosage of perampanel for preventing, treating and / or alleviating the disease is 3 mg / kg (mouse).

[0016] The above dosage is the mouse dosage during the experiment, which can be converted to the human dosage based on the mouse dosage = 9.1 X (human dosage) mg / kg.

[0017] Preferably, the retinal nerve damage is retinal nerve damage induced by hyperglycemia.

[0018] The present invention is the first to use perampanel (PER) in a high-glucose damaged RGC model and a diabetic (DM) mouse model to verify its therapeutic effect.

[0019] The structural formula of the perampanel is as follows:

[0020] .

[0021] The present invention shifts the research focus of treating or preventing hyperglycemia-induced RGC damage to aminomethylpropionic acid receptors (AMPARs), whose antagonists have shown neuroprotective effects in vitro. Under physiological conditions, AMPARs are less permeable to calcium than NMDARs. However, in the absence of GluA2 subunits, AMPARs become permeable to calcium, which may enhance the toxicity of glutamate. Notably, previous studies have reported that GluA2 subunit protein expression is significantly reduced in the retina of diabetic rats induced by streptozotocin (STZ).

[0022] The present invention also provides a medicine for treating retinal nerve damage, comprising perampanel and auxiliary materials.

[0023] Preferably, the medicine also includes other medicines having the function of treating retinal nerve damage.

[0024] Compared with the prior art, the present invention provides the use of perampanel in the preparation of drugs for preventing, treating and / or alleviating hyperglycemia-induced retinal nerve damage. Experimental results show that PER treatment of high-sugar-damaged RGC cells can reduce the death of RGCs and the death of retinal ganglion cells in DM mice, which indicates that PER has a potential neuroprotective effect on retinal neurons exposed to hyperglycemia and has the potential to prevent, treat and alleviate hyperglycemia-induced retinal nerve damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Results of cell experiments;

[0026] Figure 2Schematic diagram of mouse modeling and drug treatment, as well as the blood sugar and body weight change curve of mice;

[0027] Figure 3 The results are from animal experiments. DETAILED DESCRIPTION

[0028] In order to further illustrate the present invention, the following is a detailed description in conjunction with the embodiments. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, rather than limiting the claims of the invention.

[0029] All raw materials of the present invention have no particular limitation on their sources, and can be purchased from the market or prepared according to conventional methods known to those skilled in the art.

[0030] Example 1

[0031] PER treated RGC cells injured by high glucose. The specific experimental procedures were as follows: R28 retinal progenitor cell line, an immortalized adherent retinal progenitor cell line, was obtained from the Department of Ophthalmology and Visual Science, Eye, Ear, Nose and Throat Hospital, Fudan University (Shanghai, China) and used for in vitro studies. R28 cells were cultured in Dulbecco's modified Eagle's medium containing 5 mM glucose, 10% fetal bovine serum, and 1% penicillin / streptomycin and cultured in an incubator at 37°C and 5% CO2. To simulate high glucose injury, cells were in medium containing 30 mM D-glucose (high glucose [HG]) for 72 h. For PER treatment, 30 µM PER was added before cells were exposed to high glucose.

[0032] AMPA receptor immunofluorescence staining: AMAPR (GluA) was labeled with immunofluorescence, and DAPI was used to label the cell nucleus. Figure 1 As shown in A and B, it can be seen that the expression of GluA (green fluorescence) in RGC cells increased under high glucose treatment, indicating that the expression of GluA increased under high glucose conditions.

[0033] PI / Calcein-AM dead and live cell staining: PI marks dead cells, Calcein-AM marks live cells, such as Figure 1 As shown in C and D, PI (red fluorescence) increased under high glucose, indicating that high glucose caused RGC cell death, while the addition of PER could reduce the death of RGC under high glucose.

[0034] The experimental results are as follows Figure 1 shown.

[0035] Figure 1 In the figure, Figure A is the immunofluorescence result, and Figure B is the immunofluorescence quantitative analysis; Figure C is the PI / Calcein-AM staining image, and Figure D is the fluorescence quantitative analysis image. It can be seen that RGC cell apoptosis increases under high glucose treatment.

[0036] Figure 1 The results showed that PER treatment of high-glucose damaged RGC cells could reduce RGC death.

[0037] Example 2 Animal Experiment

[0038] The experimental process is as follows: Construction of diabetic (DM) mouse model: adult male C57BL / 6 mice weighing 25-30g and aged 6-8 weeks were selected and fasted for 12-16 hours. Use 0.1 mol / L sodium citrate buffer with a pH of 4.5 to prepare streptozotocin (STZ) to a final concentration of 4.5 mg / ml. Prepare under light-proof conditions and use immediately after preparation. Intraperitoneal injection for 5 consecutive days at a dose of 45 mg / kg. One week after the injection, tail vein blood samples were collected to test fasting blood glucose levels. Mice with blood glucose levels reaching or exceeding 16.7 mmol / L were considered to have successfully established a diabetic model. The weight and blood glucose levels of mice were measured and recorded regularly every week. Experimental animals that did not meet the criteria for elevated blood glucose levels will be excluded from the study.

[0039] The experimental animals were divided into the following groups:

[0040] a. Control group: C57BL / 6 mice

[0041] b. DR group: STZ-induced DM mice

[0042] c. DR group + perampanel: STZ-induced DM mice + 3 mg / kg perampanel intraperitoneal injection

[0043] The experimental results are as follows Figure 2 shown.

[0044] Figure 2 This is a schematic diagram of mouse modeling and drug treatment. Figure A is the blood sugar change curve of mice, and Figure B is the weight change curve of mice. Figure 2 This showed that PER did not affect the blood sugar and body weight of mice.

[0045] Figure 3Figure 1 is an in vivo experimental diagram of PER protecting the structure and function of retinal ganglion cells. The upper row of Figure A is an OCT image, the lower row is a retinal section HE staining image, Figure B (retinal thickness statistics on OCT), Figure C (GCL+IPL thickness statistics on OCT), Figure E, Figure F, and Figure H are retinal section HE staining RGC thickness statistics, retinal flat sheet RGC count statistics, Figure H is a phNR amplitude statistics, Figure D is a retinal flat sheet RGC staining image, and Figure G is a phNR image. As can be seen from Figures AE, the retinal ganglion cell layer of DM mice is significantly thinned, indicating that PER treatment can reverse the loss of RGCs; as can be seen from Figures D and F, the number of RGCs in DM mice is reduced, indicating that PER treatment can prevent the loss of RGCs; as can be seen from Figures G and H, the phNR wave amplitude of diabetic mice is reduced, indicating that PER treatment can effectively prevent the reduction of phNR wave amplitude and protect the function of RGCs.

[0046] The above animal experiments showed that PER treatment could reduce the death of RGC cells in DM mice.

[0047] The above embodiments are only used to help understand the method and core idea of ​​the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

1. Use of perampanel in the preparation of drugs for preventing, treating and / or alleviating retinal nerve damage.

2. The use according to claim 1, characterized in that: The prevention, treatment and / or alleviation includes protecting retinal ganglion cells.

3. The use according to claim 1, characterized in that: The prevention, treatment and / or alleviation comprises inhibiting the levels of AMPA receptors.

4. The use according to claim 1, characterized in that: The prevention, treatment and / or alleviation includes reducing the death of retinal ganglion cells.

5. The use according to claim 1, characterized in that: The prevention, treatment and / or alleviation includes preventing a decrease in the amplitude of the phNR wave.

6. The use according to claim 1, characterized in that: The dose of perampanel was 3.0 mk / kg (mouse).

7. The use according to claim 1, characterized in that: The retinal nerve damage is retinal nerve damage induced by hyperglycemia.

8. A drug for treating retinal nerve damage, comprising perampanel and excipients.

9. The drug according to claim 8, characterized in that Other drugs that have the function of treating retinal nerve damage are also included.

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