CN115501344A

CN115501344A - Application of iron chelating agent

Info

Publication number: CN115501344A
Application number: CN202211200489.8A
Authority: CN
Inventors: 邱景富; 陈承志; 邹镇; 段鑫昊; 田鑫; 肖飞; 邱宇
Original assignee: Chongqing Medical University
Current assignee: Chongqing Medical University
Priority date: 2022-09-29
Filing date: 2022-09-29
Publication date: 2022-12-23

Abstract

The invention provides application of an iron chelator in preparation of a medicament for treating epilepsy. The invention discovers that the iron chelator can inhibit the level of iron homeostasis genes, improve the phenomenon of iron deposition induced by epilepsy, slow down the severity of epileptic seizure, improve the survival rate of epileptic seizure, prolong the latency period of epileptic seizure, reduce the frequency and duration of epileptic seizure, and the like. The invention discloses the inhibition effect of deferasirox on epileptic seizure, not only provides a new application for old medicines, but also more importantly discloses a new idea for an epileptogenesis mechanism.

Description

Application of iron chelating agent

Technical Field

The invention relates to the technical field of biology, in particular to application of an iron chelating agent.

Background

Iron is a trace element necessary for human body, and has important significance for maintaining various neurophysiological functions of organism. The latest research shows that the iron content in the human body is just like a double-edged sword, namely the iron has positive promotion effect on improving the nerve conduction and the cognitive function development of the children at the early stage of the brain development of the human body; however, an imbalance in iron homeostasis caused by excessive iron intake with aging can induce a range of neurological disorders such as epilepsy and even involve the development of neurological tumors (Th born et al, 2018).

Because the pathogenesis of epilepsy is not completely clear, the antiepileptic drugs used at present mainly control clinical symptoms, about one third of patients can progress to intractable epilepsy, and the treatment effect is not ideal. Secondly, antiepileptic drugs have relatively many toxic and side effects, and many epileptics have poor compliance and high incidence of status epilepticus. The method is particularly important for the detailed research of epileptic pathogenesis and the exploration of antiepileptic drugs.

Currently, three clinically used iron chelators are Deferiprone (DFP), deferoxamine (DFO) and Deferasirox (DFX), and are mainly used for treating iron overload of thalassemia patients after blood transfusion. Deferasirox is a novel oral chelator that mediates iron storage by selectively binding the iron form of iron, a achiral tridentate ligand of ferric iron (i.e., three polar interaction sites per molecule); two desferrites form a complex Fe- [ deferasirox with a trivalent iron] ₂ It is a specific, highly selective iron chelator that does not cause zinc or copper excretion. In 2005 the FDA approved oral iron chelator, deferasirox, as a first line therapy for transfusion-associated iron overload in the clinic, to treat iron overload after transfusion in thalassemia patients.

Therefore, it is very necessary to develop a new application of an iron chelator.

Disclosure of Invention

In view of this, the technical problem to be solved by the present invention is to provide an application in the preparation of a medicament for treating epilepsy.

The invention provides application of an iron chelator in the preparation of a medicament for the treatment of epilepsy.

Preferably, said treating epilepsy comprises inhibiting iron homeostasis gene levels.

Preferably, the iron homeostatic genes are Fth1 and Iscu.

Preferably, said treatment of epilepsy comprises ameliorating epilepsy-induced iron deposition.

Preferably, the treatment of epilepsy comprises reducing the severity of seizures, increasing seizure survival, extending seizure latency, reducing seizure frequency and duration.

Preferably, the treatment of epilepsy comprises reducing the frequency of minute excitatory currents or reducing the frequency of action potentials.

Preferably, the iron chelator is deferasirox.

Preferably, the dosage of the deferasirox for treating the epilepsy is 125mg/kg.

Preferably, the epilepsy is epilepsy caused by pentaerythrite and/or hydrargyrum.

The invention provides a medicament for treating epilepsy, which comprises an iron chelator.

Compared with the prior art, the invention provides the application of the iron chelator in preparing the medicine for treating epilepsy. The invention discovers that the iron chelator can inhibit the level of iron homeostatic genes, improve the phenomenon of iron deposition induced by epilepsy, slow down the severity of epileptic seizure, improve the survival rate of epileptic seizure, prolong the latent period of epileptic seizure, reduce the frequency and duration of epileptic seizure and the like. The invention discloses the inhibition effect of deferasirox on epileptic seizure, which not only provides a new application for old medicines, but also more importantly provides a new idea for disclosing an epileptic occurrence mechanism.

Drawings

FIG. 1 is a graph of the measurement of iron content in mouse tissues and patients according to example 1 of the present invention; wherein, figure 1 shows that the iron content of the brain tissue of the AKA epilepsy model mouse is obviously increased; FIG. 1B shows that the iron content in brain tissue of epileptic patient is increased significantly; FIG. 1C direct treatment of DFX did not significantly affect the background iron content of the tissues;

FIG. 2 shows the measurement of iron content and iron homeostasis genes in mouse tissues after the deferasirox treatment of example 1 and example 2 of the present invention; wherein, figure 2A deferasirox can alter the expression of tissue iron homeostasis genes; figure 2B deferasirox significantly reduces tissue iron content;

FIG. 3 is a mouse epileptic behavioral assay of examples 3 and 4 of the present invention; wherein, fig. 3A PTZ epilepsy model behavioral assay shows that DFX has the effect of reducing the seizure level and the trend of increasing PTZ seizure survival rate; fig. 3B KA behavioral assessment that DFX is effective in extending the time to first SRS (spontaneous recurrent seizures) and reducing the total number of SRS;

FIG. 4 shows that the field potential of mice measured by deferasirox treatment according to example 5 of the present invention can reduce the number of spontaneous epileptic seizures and seizure time;

FIG. 5 patch clamp assay results of example 6 of the present invention; among them, deferasirox of fig. 5A, 5B, and 5C can decrease the frequency of mEPSCs (minimal excitatory postsynaptic current).

Detailed Description

The invention provides application of an iron chelating agent, and a person skilled in the art can use the content to reference the text and appropriately modify the process parameters to realize the purpose. It is expressly intended that all such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the scope of the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

Deferasirox (Exjade, nova), formerly known as ICL670 and CGP 72 670, is an N-substituted bis-hydroxyphenyltriazole iron chelator prepared in a two-step process from salicylic acid. Its chemical name is 4- [3, 5-bis (2-hydroxyphenyl) -1H-1,2, 4-triazol-1-yl ] benzoic acid.

Deferasirox is a trigeminal chelator that mediates iron storage by selectively binding to the iron (Fe 3 +) form. Trigeminal chelators require two ligand molecules to bind one iron atom. In contrast, deferoxamine is a hexadentate (1. The efficiency of chelation, e.g. the proportion of excreted iron to iron which can theoretically be bound, is very high, varying from 15% to 27%. In rats, deferasirox is four to five times as efficient in promoting hepatic iron excretion as deferoxamine.

The oral bioavailability of deferasirox is 70%, and the bioavailability of deferasirox is improved to different degrees when the deferasirox is taken with a meal. It is absorbed rapidly, peaks in plasma concentration within the median of 1.5 to 4 hours after oral administration, and reaches steady state concentrations within 3 days. About 99% of deferasirox binds to proteins, mainly to serum albumin, and cannot be replaced in vitro by warfarin, digoxin or diazepam. The drug is mainly glucuronidated and undergoes minimal (8%) oxidative metabolism by the cytochrome P-450 (CYP) system. The elimination half-life of 7 deferasirox is 8 to 16 hours, and the deferasirox can be taken once a day.

Experiments prove that the iron chelator plays an important role in inhibiting the iron homeostatic gene level.

Specifically, the iron homeostatic genes are Fth1 and Iscu.

The result of the iron homeostasis related gene RT-PCR shows that the iron homeostasis gene is changed in intestinal tissues, but the Fth1 and Iscu genes in the cortex are changed, which prompts the iron homeostasis gene to regulate the brain iron content.

The invention provides the use of an iron chelator for the preparation of a medicament for ameliorating epileptic-induced iron deposition.

Experiments prove that the iron content measurement result shows that deferasirox can effectively play an iron chelation effect in an epilepsy model, so that the phenomenon of iron deposition induced by epilepsy is improved.

In particular, the treatment of epilepsy of the present invention includes reducing the severity of seizures, increasing seizure survival, extending seizure latency, and reducing the frequency and duration of seizures.

Experiments prove that the severity of epileptic seizure can be effectively relieved by the deferasirox treatment.

Experiments prove that the frequency and the total duration of SLEs collected by epileptic mice can be obviously reduced by treating deferasirox, and the deferasirox can effectively slow down the epileptic mouse attack.

Experiments prove that the PTZ epilepsy model shows that DFX has the effect of reducing the epileptic seizure grade and the trend of improving the PTZ epileptic seizure survival rate.

DFX is effective in prolonging the time to first SRS (spontaneous recurrent seizures) and reducing the total number of SRS.

The invention also discloses application of the iron chelator in preparing a medicament for treating and reducing the frequency of micro excitatory current or the frequency of action potential.

Experiments prove that the frequency of micro excitatory current can be obviously reduced by the deferasirox treatment, the amplitude of the tiny excitatory current is not influenced, the frequency of action potential can also be reduced, and the deferasirox treatment can inhibit epileptic seizure by influencing pre-excitatory synaptic transmission.

The iron chelator of the invention is deferasirox.

The inventor constructs a pentaerythrite and hydramic acid epilepsy model mouse in an earlier stage, uses deferasirox for intervention treatment, and finally proves that deferasirox can effectively improve the disease development of epilepsy through epilepsy ethology and related index detection.

The dose of the deferasirox for treating epilepsy is 125mg/kg.

The iron chelator of the invention is deferasirox.

According to the present invention, the dosage form of the drug includes one or more of injection preparation, oral preparation and spray preparation. The pharmaceutically acceptable excipients are not limited in the present invention and are well known to those skilled in the art.

The invention provides application of an iron chelator in the preparation of a medicament for the treatment of epilepsy. The invention discovers that the iron chelator can inhibit the level of iron homeostasis genes, improve the phenomenon of iron deposition induced by epilepsy, slow down the severity of epileptic seizure, improve the survival rate of epileptic seizure, prolong the latency period of epileptic seizure, reduce the frequency and duration of epileptic seizure, and the like. The invention discloses the inhibition effect of deferasirox on epileptic seizure, not only provides a new application for old medicines, but also more importantly provides a new idea for disclosing an epileptogenesis mechanism.

To further illustrate the present invention, the following examples are provided to describe in detail the use of an iron chelator provided herein.

A125 mg DFX dispersible tablet purchased from Nowa company is dissolved in 12.5mL sterilized normal saline, and is prepared into a uniformly distributed suspension after ultrasonic mixing, and the suspension is perfused for 2 times for 4 weeks in a dosage of 2.5 mg/capsule for 3 days.

Example 1 establishment of mouse KA-induced TLE epilepsy model

Taking an adult male pathogen-free (SPF) grade C57BL/6J mouse and a pentobarbital sodium intraperitoneal injection anesthetized mouse, fixing the incisors of the mouse by using an adapter after tail pinching reflection disappears, slightly pressing an incisor clamp cross rod, placing the mouse on a stereo positioning instrument, and adjusting the position of the adapter to enable an ear rod to smoothly enter the auditory canal of the mouse. And adjusting the ear rod to enable the left and right scales to be leveled, keeping the head of the mouse at the central position of the U-shaped opening of the positioning instrument, screwing the ear rod, and simultaneously screwing the portal clamp screw to fix the head of the mouse, wherein the brain of the mouse is in a horizontal position by visual inspection. The gauze stained with normal saline was placed on the mouse eyes to prevent the eyes from drying. Cutting off hair of the head by using surgical scissors, exposing the scalp, longitudinally cutting the scalp after sterilizing by using iodophor, dipping hydrogen peroxide by using a cotton swab to completely remove periosteum, exposing front and back fontanels, and simultaneously clearly observing the intersection point of a sagittal suture and a coronal suture, namely a Bregma point, adjusting the coordinate of a positioning instrument by taking the point as a three-dimensional coordinate origin, and taking the coordinates of ML (1.5 mm), AP (-2.0 mm) and DG (-1.5 mm) as injection sites of the CA1 region of the hippocampus. After positioning is finished, a skull drill is used for slightly grinding a skull drill hole at an injection site, a micro-injector with the maximum range of 0.5 mu L is used for sucking 1.0nmol KA dissolved in 50 mu L of physiological saline, slow injection is carried out for 3min, and a needle is left for 5min to prevent backflow of the KA. After the injection, the microinjector was slowly pulled out, and after the skull of the mouse was sterilized, the scalp of the mouse was sutured with surgical sutures. The control group was injected with the same amount of physiological saline by the same method.

Determination of tissue iron content

(1) The kit was taken out of the refrigerator to equilibrate to room temperature for at least 20min before the experiment began.

(2) Preparing 2mg/L of iron standard application liquid and an iron color reagent according to the kit instruction, and paying attention to light shielding in the whole process.

(3) Accurately weighing the weight of the tissue to be detected, and putting the weighed tissue into a 5mL sterilized EP tube according to the weight (g): volume (mL) =1 ratio of 9 volume of physiological saline was added, and the tissue was homogenized on ice sufficiently using a homogenizer. Then placing the mixture in a centrifuge at 2500r/min for centrifugation for 10min, and sucking the supernatant into a new EP tube to be tested.

(4) Adding a sample to be tested and an iron color developing agent according to a certain amount according to the instruction, and simultaneously preparing a blank tube and a standard tube which are required correspondingly.

(5) Adding the reagent, shaking, mixing, boiling in water bath for 5min, cooling with flowing water, centrifuging at 3500r/min in centrifuge, and centrifuging for 10min.

(6) And taking the supernatant, planning the layout, adding the supernatant into a 96-well plate, adjusting the wavelength to 520nm, adjusting the double distilled water to zero, and measuring the absorbance OD value of each tube.

(7) And substituting OD values of all the tubes into an instruction calculation formula, and leveling by using the protein concentration of the sample to calculate the tissue iron content of the specific sample.

The protein concentration of the sample was determined by BCA protein concentration assay kit according to the following method:

(1) The kit was removed from the freezer and allowed to equilibrate at room temperature for at least 20min prior to assay.

(2) And taking out the protein standard in the kit, mixing the protein standard preparation liquid into the protein standard, uniformly mixing to obtain a 25mg/mL protein standard solution, diluting the solution into a protein standard application liquid with a final concentration of 0.5mg/mL, wherein the diluent is the same as the solution of the sample.

(3) The number of samples and protein standards required to be added is counted and the BCA configuration liquid is removed, as per BCA reagent a: BCA reagent B was 50: the proportion of 1 is prepared into BCA working liquid, and the BCA working liquid is stored in a dark place after being uniformly shaken and mixed.

(4) And taking out the 96-well plate, planning a sample adding layout, adding the protein standard application solution into the well plate according to the volume of 0, 1,2,4, 8, 12, 16 and 20 mu L, and simultaneously adding the sample diluent to make up the volume of each well to 20 mu L so as to ensure that the final concentration of the protein standard is 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4 and 0.5mg/ml.

(5) The prepared samples to be tested are diluted at least 5 times in concentration and added into a pore plate, each pore volume is also 20 mu L, and at least 1 sample adding pore is prepared for each sample.

(6) Adding the prepared BCA working solution into each hole, incubating 200 mu L of each hole in the dark at 37 ℃ for 30min, and taking out the place at 562nm of an enzyme labeling instrument to measure the absorbance of each hole.

(7) And (3) making a standard curve according to the absorbance of the protein standard and the corresponding concentration, bringing the absorbance of the sample into the standard curve, and calculating the actual protein concentration of the sample to be detected according to the diluted concentration.

And (4) analyzing results: the difference of the iron content of the intestinal tissue and the brain tissue has statistical significance, the iron content of the temporal lobe cortex tissue of an epileptic patient is also increased, and the fact that the epilepsy has an iron aggregation phenomenon in animals or people is proved. While at the level of low background iron content, deferasirox treatment does not affect the iron content.

Results are shown in FIG. 1, which is a graph of iron content measurements in mouse tissues and patients; wherein, FIG. 1A shows that the content of iron in the brain tissue of a mouse with a KA epilepsy model is obviously increased; FIG. 1B shows that the iron content in brain tissue of epileptic patients is increased significantly; FIG. 1C shows that DFX treatment directly did not significantly affect the background level of iron in the tissue.

Example 2

Establishment of mouse KA-induced TLE epilepsy model

Taking adult male pathogen-free (SPF) grade C57BL/6J mice and pentobarbital sodium intraperitoneal injection anesthetized mice, fixing the incisors of the mice by using an adapter after tail pinching reflection disappears, slightly pressing an incisor clamp cross rod, placing the mice on a stereo positioning instrument, and adjusting the position of the adapter to ensure that an ear rod can smoothly enter the auditory canals of the mice. And adjusting the ear rod to enable the left and right scales to be leveled, keeping the head of the mouse at the central position of the U-shaped opening of the positioning instrument, screwing the ear rod, and simultaneously screwing the portal clamp screw to fix the head of the mouse, wherein the brain of the mouse is in a horizontal position by visual inspection. The gauze stained with physiological saline is placed on the eyes of the mice to prevent the eyes from drying. Cutting off hair of the head by using surgical scissors, exposing the scalp, longitudinally cutting the scalp after sterilizing by using iodophor, dipping hydrogen peroxide by using a cotton swab to completely remove periosteum, exposing front and back fontanels, and simultaneously clearly observing the intersection point of a sagittal suture and a coronal suture, namely a Bregma point, adjusting the coordinate of a positioning instrument by taking the point as a three-dimensional coordinate origin, and taking the coordinates of ML (1.5 mm), AP (-2.0 mm) and DG (-1.5 mm) as injection sites of the CA1 region of the hippocampus. After positioning is finished, a skull drill is used for slightly grinding a skull drill hole at an injection site, a micro-injector with the maximum range of 0.5 mu L is used for sucking 1.0nmol KA dissolved in 50 mu L of physiological saline, slow injection is carried out for 3min, and a needle is left for 5min to prevent backflow of the KA. After the injection was completed, the microinjector was slowly pulled out, and after the skull of the mouse was sterilized, the scalp of the mouse was sutured using surgical sutures. The control group was injected with the same amount of physiological saline by the same method.

2.1 extraction of RNA

(1) An appropriate amount of mouse cortex, hippocampus, and intestinal tissue was removed from a-80 ℃ freezer, approximately 50mg of tissue was weighed into 5mL EP tubes, 1mL TRIzol reagent was added to each tube, and lysis was performed on ice using a high speed homogenizer until no visible tissue particles were visible.

(2) The homogenate was transferred to a 1.5mL EP tube and centrifuged at 12000r for 10min at 4 ℃.

(3) The supernatant was transferred to a new EP tube, 200. Mu.L of chloroform was added thereto, and the mixture was inverted and mixed. Standing at room temperature for 5min, and centrifuging at 12000r for 10min at 4 deg.C.

(4) After centrifugation, the mixture was divided into three layers, and the aqueous phase liquid in the uppermost layer was carefully sucked up, about 250. Mu.L, transferred into a new EP tube, followed by addition of an equal amount of isopropyl alcohol, mixing, standing at room temperature for 10min, and then centrifuged at 12000r for 10min at 4 ℃.

(5) Carefully discard the supernatant, add 75% ethanol in DEPC water, mix gently, and then centrifuge at 12000r for 10min at 4 ℃.

(6) The ethanol was discarded, the remaining precipitate was RNA, which was left to stand at room temperature for 3min with the lid open, and 30. Mu.L of enzyme-free DEPC water was added to dissolve the RNA therein.

(7) And detecting the purity and concentration of the RNA, wherein if the concentration is more than 500 ng/mu L and the ratio of OD260/OD280 is between 1.8 and 2.0, the concentration purity of the extracted RNA reaches the standard, and the RNA can be used for subsequent experiments.

2.2 reverse transcription of RNA into cDNA

According to the reaction system in Table 2, each reagent and RNA were added to 200. Mu.L of enzyme-free EP tube. The total amount is 20. Mu.L, and the amount of RNA in the reverse transcription system is ensured to be 1ug. The reverse transcription conditions were: 37 ℃,15min → 85 ℃,5s → 4 ℃ and infinity. After completion of reverse transcription, the sample was taken out and stored at-20 ℃.

TABLE 2 reverse transcription reaction System

Table 2 The reverse transcription system

RealTime PCR reaction

Preparing a reaction system according to the table 4, arranging 3 compound holes on each sample, adding the prepared system into eight enzyme-free rows of tubes, wherein each hole is 10 mu L, mixing uniformly, centrifuging and then placing into a PCR instrument for amplification.

The amplification conditions were 95 ℃ pre-denaturation for 3min → (95 ℃ denaturation 10s → 60 ℃ annealing, 30 s), 40 cycles → 72 ℃ extension for 30s.

And calculating the relative RNA expression amount of 2-delta Ct according to the cycle time CT value of each hole.

TABLE 3 configuration of qPCR reaction System (10. Mu.L)

Table 3 Configuration of qPCR reaction system(10μL)

The results are shown in FIG. 2, and FIG. 2 shows the measurement of iron content and iron-homeostasis gene of mouse tissue after deferasirox treatment. Among them, fig. 2A shows that deferasirox can alter the expression of tissue iron homeostasis genes. As can be seen in FIG. 2B, deferasirox significantly reduced the tissue iron content

And (4) analyzing results:

The iron content determination result shows that deferasirox can effectively exert an iron chelation effect in an epilepsy model, so that the phenomenon of iron deposition induced by epilepsy is improved.

Example 3 pentaerythrityl model for epileptic ignition

The partial experiment of the pentaerythrite ignition model is divided into two groups, a Vehicle group and a DFX group, and the test is performed by respectively intragastric normal saline and DFX every day. Two groups of mice are intraperitoneally injected with 35mg/kg of pentylenetetrazol once every other day (every 48 h), the injection time is fixed at the same time interval in the morning, 30min is observed after one cage is injected, and the attack grade of the mice is recorded. A total of 15 injections were given, with seizure ratings recorded, and body weights re-weighed after each 5 injections, with seizure ratings referenced to Racine scoring standards. If the attack of III or more is occurred for 3 times or more continuously, the establishment of the model is determined to be successful. After the experiment is finished, the attack grade and the death rate of each group of mice are counted.

Fig. 3A behavioral determination of PTZ epilepsy model indicates that DFX has the effect of decreasing seizure grade, and a tendency to increase PTZ seizure survival rate. Fig. 3B KA behavioral assessment DFX was effective in extending the time to first SRS (spontaneous recurrent seizures) and reducing the total number of SRS.

Example 4 mouse marinic acid-induced TLE epileptic model establishment and behavioral observations

Taking adult male pathogen-free (SPF) grade C57BL/6J mice and pentobarbital sodium intraperitoneal injection anesthetized mice, fixing the incisors of the mice by using an adapter after tail pinching reflection disappears, slightly pressing an incisor clamp cross rod, placing the mice on a stereo positioning instrument, and adjusting the position of the adapter to ensure that an ear rod can smoothly enter the auditory canals of the mice. And adjusting the ear rod to enable the left and right scales to be leveled, keeping the head of the mouse at the central position of the U-shaped opening of the positioning instrument, screwing the ear rod, and simultaneously screwing the portal clamp screw to fix the head of the mouse, wherein the brain of the mouse is in a horizontal position by visual inspection. The gauze stained with physiological saline is placed on the eyes of the mice to prevent the eyes from drying. Cutting off hair of head with surgical scissors, exposing scalp, disinfecting with iodophor, cutting off scalp longitudinally, dipping hydrogen peroxide with cotton swab to completely remove periosteum, exposing front and back fontanelle, and clearly observing the intersection point of sagittal suture and coronal suture, i.e. Bregma point, adjusting coordinate of position finder with the point as three-dimensional coordinate origin, and taking ML (1.5 mm), AP (-2.0 mm) and DG (-1.5 mm) coordinates as injection site of Hippocampus CA1 region. After positioning is finished, a skull drill is used for slightly grinding a skull drill hole at an injection site, a micro-injector with the maximum range of 0.5 mu L is used for sucking 1.0nmol KA dissolved in 50 mu L of physiological saline, slow injection is carried out for 3min, and a needle is left for 5min to prevent backflow of the KA. After the injection was completed, the microinjector was slowly pulled out, and after the skull of the mouse was sterilized, the scalp of the mouse was sutured using surgical sutures. The control group was injected with the same amount of physiological saline by the same method. And (3) after the modeling is finished, waiting for the mouse to wake up, observing the seizure condition of the mouse, and determining that the model is successfully established when SRSs above the IV level appear. The mice were placed in a rearing cage and placed under continuous video surveillance for 1 month. During the observation, the seizure condition of the mice is recorded, and the grade of the seizure is judged by adopting a Racine scoring standard.

And (4) analyzing results:

FIG. 4 is a mouse field potential measurement; deferasirox treatment can reduce the number and time of spontaneous seizures. The behavioral observation result shows that the latent period of the epilepsy attack of the mice has statistical significance and different attack grades compared with the control group in the deferasirox group under the monitoring state. Deferasirox treatment was shown to be effective in reducing the severity of seizures.

Example 5 measurement of Local Field Potential (LFP)

After the video behavioural monitoring is finished, a KA epilepsy model mouse is taken, a pentobarbital sodium is injected into the abdominal cavity to anaesthetize the mouse, the mouse is fixed on a stereotaxic apparatus after sufficient anaesthetization, the scalp at the suture position before cutting is cut, and the previous KA injection site in the CA1 area can be clearly seen after iodophor disinfection. A burr hole was drilled at this site as the LFP monitoring site, and compression with a sterile cotton swab was noted to reduce bleeding. Holes are drilled at the approximately symmetrical positions on the left side, and a fixing screw with the specification of 1.0 multiplied by 2mm is screwed in to prevent the electrode in the subsequent experiment from falling off. A symmetrical position of the skull on two sides of the frontal lobe is drilled, and two screws with the specification of 1.0 multiplied by 6mm are screwed as reference electrodes to be connected with the ground wire. Then, a piece of customized iron sheet is prevented between the screws, self-setting dental cement with proper proportion is prepared and smeared between the skull, the iron sheet and the screws are fixed, the monitoring site is exposed in the process, and after the cement is set, a small cotton wool absorbs iodophor to prevent infection at the opening of the monitoring site. After the installation was completed, the mice were placed on a heating pad and ready to begin LFP recording after the mice fully awakened.

The mouse is fixed on the LFP recording table through the installed special iron sheet, so that the disturbance of the mouse on the monitoring is prevented. The signal connector is in butt joint with the recording electrode, an electric signal amplifier is started, a cotton swab is dipped in water to wet the monitoring site, the recording electrode is deeply inserted into the monitoring site to the hippocampus, a MAP data acquisition system is used for continuously monitoring for 30min after a base line is stable, then, neuroexplor software is used for checking the LFP recording condition, and the energy spectrum and the electroencephalogram change condition are analyzed.

And (4) analyzing results:

the frequency and the total duration of SLEs collected within 30min of an epileptic mouse can be obviously reduced by treating the deferasirox, and the deferasirox is proved to be capable of effectively relieving the epileptic mouse attack.

Example 6 Whole cell patch clamp recordings

6.1. Arrangement of related liquids

(1) Magnesium-free Artificial cerebrospinal fluid (ACSF) was prepared according to the following formulation.

Magnesium-free artificial cerebrospinal fluid formula

Stirring and mixing uniformly after the preparation is finished, matching the content of 95 percent of O2+5 percent of CO2 with mixed gas according to the formula, continuously introducing for 45min, adjusting the pH to 7.30-7.40 after the oxygenation is finished, and adjusting the osmotic pressure to Osm300-310 and then placing at 4 ℃ for later use.

(2) Preparing brain slice liquid:

brain slice preparation

Stirring uniformly by magnetic force, introducing the mixed gas for 45min, adjusting pH to 7.30-7.40, and adjusting Osm to 300-320 by osmotic pressure.

(3) Electrode internal liquid formulation for recording Action Potential (AP):

formula of electrode internal liquid with action potential

After preparation, the mixture is stirred uniformly, the pH is adjusted to 7.2, and the osmotic pressure is adjusted to Osm 275-290.

(4) Electrode internal liquid formulation for recording minimal excitatory postsynaptic currents (mEPSCs)

Stirring well after the preparation, introducing mixed gas for 45min, adjusting pH to 7.30-7.40, and adjusting osmotic pressure Osm to 275-290.

(5) Electrode internal liquid formulation for recording micro inhibitory post-synaptic currents (mIPSCs)

Formula for recording mIPSCs electrode internal solution

And (3) uniformly mixing the solution after the preparation is finished, introducing mixed gas for 45min, and adjusting according to the following conditions: PH value

7.30-7.40, and the osmotic pressure Osm is 270-280.

2.6.2 preparation of in vitro brain tablets

After the DFX intervention treatment was completed, mice from the treated and control groups were taken for whole cell patch clamp experiments. Precooling the prepared slice liquid in advance to form an ice-water mixture state, continuously inputting mixed gas (95% + O2+5% + CO2), placing the magnesium-free artificial cerebrospinal fluid in a constant-temperature water bath, and simultaneously introducing the same mixed gas to a saturated state. Selecting experimental mice, injecting sodium pentobarbital into abdominal cavity, fixing mice after complete anesthesia, cutting chest skin to expose heart, perfusing precooled slice liquid through heart at uniform speed by using syringe needle, rapidly cutting head and taking out brain after perfusion, and pre-treating to 95% O ₂ With 5% of CO ₂ The mixture was saturated with 0 ℃ ACSF and incubated at room temperature for 2min. Brain tissue was then transferred to ice dishes, and the brain was dissected along the coronal axis using a clean blade, and the cerebellum, brainstem, prefrontal parts, and excess cortex were trimmed away. After being filtered and dried by filter paper, the mixture is transferred to a square agar fixed objective table, slowly placed in a water bath containing precooled slices, and introduced with mixed gas of the components. And (4) slicing by adopting a vibrating microtome after adjusting parameters, wherein the slicing thickness is about 300 mu m. And (3) smoothly transferring the cut brain slices into artificial cerebrospinal fluid, and incubating for 1h at the temperature of 35 +/-1 ℃ for later use for subsequent operation.

2.6.3 Whole cell patch clamp recordings

When the recording is started, the recording channel is flushed with ultrapure water, then the prepared ASCF is introduced, the mixed gas is continuously introduced, and the flow rate of the recording liquid is controlled to be 1-2 mL/min. The electrode drawing instrument is prepared, and the brain slice is carefully transferred into a recording bath tank to fix the brain slice to prevent the brain slice from moving. Using an upright microscope to search the CA1 area of the hippocampus under a low power microscope, injecting prepared electrode solution into a glass electrode, positioning to a pyramidal cell to be recorded, switching a high power microscope, controlling an objective lens to slowly fall below the liquid level, and clearly seeing that the glass electrode is positioned at the center of a visual field. Slowly regulating the glass electrode to descend to the surface of the brain slice, regulating the micro-controller to slowly apply positive pressure to the glass electrode to enable the glass electrode to be connected to the surface of a cell, observing that the electrode and the surface of a cell membrane form a concave shape, and then removing the positive pressure and applying negative pressure to complete high-resistance sealing and membrane breaking operations. Subsequently, in the current clamp mode, magnesium-free artificial cerebrospinal fluid was perfused and the Action Potential (AP) of the target cells was recorded at resting potential. The voltage was controlled at-70 mV in voltage clamp mode and the mini excitatory postsynaptic currents (mini excitatory postsynaptic currents-mEPSC) were recorded as mini inhibitory postsynaptic currents (mini inhibitory postsynaptic currents-mIPSC). 1 μ M TTX is added in the process to inhibit sodium ion channels, and 100 μ M PTX is added in the mEPSC process to achieve the effect of inhibiting GABAA receptors; 20 μ M DNQX and 40 μ M APV were added during mIPSC recordings to inhibit AMPA and NMDA receptors, respectively. And (4) importing the data to the step of finally analyzing the frequency and the amplitude of each record index by using Clampfit10.3 software.

FIG. 5 shows patch clamp assay results; figures 5A, 5B, 5C deferasirox decreases the frequency of mEPSCs (micro excitatory postsynaptic current).

And (4) analyzing results:

from patch clamp result analysis, it can be seen that deferasirox treatment can significantly reduce the frequency of micro excitatory current, has no influence on the amplitude, and can also reduce the frequency of action potential, confirming that deferasirox treatment can inhibit epileptic seizure by influencing pre-excitatory synaptic transmission.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and amendments can be made without departing from the principle of the present invention, and these modifications and amendments should also be considered as the protection scope of the present invention.

Claims

1. Use of an iron chelator for the manufacture of a medicament for the treatment of epilepsy.

2. The use of claim 1, wherein the treatment of epilepsy comprises inhibiting iron homeostasis gene levels.

3. The use of claim 1, wherein the iron homeostasis genes are Fth1 and Iscu.

4. The use according to claim 1, wherein the treatment of epilepsy comprises ameliorating epilepsy-induced iron deposition.

5. The use of claim 1, wherein the treatment of epilepsy comprises reducing the severity of seizures, increasing seizure survival rate, extending seizure latency, reducing seizure frequency and duration.

6. The use of claim 1, wherein the treatment of epilepsy comprises reducing the frequency of minimal excitatory currents or reducing the frequency of action potentials.

7. Use according to any one of claims 1 to 6, wherein the iron chelator is deferasirox.

8. The use according to claim 7, wherein the dose of deferasirox for treating epilepsy is 125mg/kg.

9. Use according to any one of claims 1 to 6, wherein the epilepsy is epilepsy induced by pentaerythrityl and/or hydramic acid.

10. A medicament for the treatment of epilepsy, comprising an iron chelator.