JP7544372B2 - Sodium channel binding agents and pharmaceutical compositions - Google Patents

Description

The present invention relates to a sodium channel binding agent and a pharmaceutical composition.

Dravet syndrome is an intractable epilepsy that occurs in infancy, and is a drug-resistant epilepsy for which existing antiepileptic drugs are ineffective. Dravet syndrome is also said to be caused by an abnormality in the gene (SCN1A) of the voltage-dependent sodium channel Na _v 1.1 (see, for example, Non-Patent Document 1). For example, Patent Document 1 describes a method of administering fenfluramine to treat Dravet syndrome. However, it is said that the use of fenfluramine is associated with the development of cardiac fibrosis and pulmonary hypertension. Patent Document 2 also proposes a triazole derivative as a voltage-dependent sodium channel Na _v 1.1 blocker.

Special table 2016-518387 publication Special table 2019-512015 publication

Higurashi N, et al., Mol Brain. 2013;6:19.

The pathology of Dravet syndrome is believed to be caused by the loss of function of the sodium channel Na _v 1.1 in inhibitory nerves. Therefore, the present invention aims to provide a selective sodium channel binding agent that is believed to be effective in treating Dravet syndrome, and a pharmaceutical composition containing the same.

The first aspect is a sodium channel binding agent which is represented by the following formula (I) or (II) and selectively binds to the sodium channel Na _v 1.1.

In the formula, ^X11 and ^X12 each independently represent an imino group or a sulfur atom. ^Y11 to ^Y14 each independently represent a nitrogen atom or a carbon atom having an aromatic group which may have a substituent. ^R11 to ^R18 each independently represent a hydrogen atom, a halogen atom, a hydrocarbon group having 1 to 12 carbon atoms which may have a substituent, a hydrocarbonoxy group having 1 to 12 carbon atoms which may have a substituent, or an aromatic group which may have a substituent, or at least one pair of substituents selected from the group consisting of ^R11 and ^R12 , ^R13 and ^R14 , ^R15 and ^R16 , and ^R17 and ^R18 are linked to each other to represent a condensed aromatic ring group which may have a substituent. ^Ar21 to ^Ar28 each independently represent an aromatic group which may have a substituent. ^R21 to ^R24 each independently represent a halogen atom, a hydrocarbon group having 1 to 12 carbon atoms, or a hydrocarbonoxy group having 1 to 12 carbon atoms. Each n independently represents a number from 0 to 3.

Y ¹¹ to Y ¹⁴ in formula (I) may be carbon atoms having an aromatic group which may have a substituent. The substituent in the aromatic group may be at least one selected from the group consisting of a hydroxyl group, a halogen atom, a hydrocarbon group having 1 to 18 carbon atoms which may have a substituent, a hydrocarbonoxy group having 1 to 12 carbon atoms which may have a substituent, a trialkylammonium group having an alkyl group having 1 to 12 carbon atoms, a dialkylsulfonium group having an alkyl group having 1 to 12 carbon atoms, and an aromatic sulfanyl group. The aromatic group may be at least one selected from the group consisting of a phenyl group, a naphthyl group, a pyridyl group, a pyridinium group, a quinolyl group, and a quinolinium group.

The second aspect is a pharmaceutical composition comprising a compound represented by the above formula (I) or (II) or a pharma- ceutical acceptable salt thereof.

The third aspect is a screening method for selecting a compound that selectively binds to a specific sodium channel among sodium channels having multiple subtypes. The screening method includes a compound group preparation step of preparing a group of candidate compounds, a binding ability evaluation step of evaluating the binding ability value of the candidate compounds included in the group of candidate compounds for a specific subtype of a sodium channel, a selection step of selecting a candidate compound whose binding ability value for the specific subtype is greater than its binding ability value for other subtypes, and an action evaluation step of evaluating the selected candidate compounds in vitro.

The present invention provides a selective sodium channel binding agent and a pharmaceutical composition containing the same.

FIG. 1 shows functional attenuation in inhibitory neurons from Dravet syndrome patients. FIG. 1 shows functional recovery by spider venom in inhibitory neurons from Dravet syndrome patients. FIG. 1 shows functional recovery by sodium channel binding agents in inhibitory neurons derived from Dravet syndrome patients. FIG. 1 shows functional recovery by sodium channel binding agents in inhibitory neurons derived from Dravet syndrome patients. FIG. 1 shows changes in firing frequency when a compound according to a comparative example was added to inhibitory neurons derived from a patient with Dravet syndrome. FIG. 1 shows changes in firing frequency when a compound according to a comparative example was added to inhibitory neurons derived from a patient with Dravet syndrome.

In this specification, the term "process" includes not only an independent process, but also a process that cannot be clearly distinguished from other processes, as long as the intended purpose of the process is achieved. Furthermore, the content of each component in a composition means the total amount of the multiple substances present in the composition when multiple substances corresponding to each component are present in the composition, unless otherwise specified. Hereinafter, embodiments of the present invention will be described in detail. However, the embodiments shown below are examples of sodium channel binding agents and pharmaceutical compositions for realizing the technical concept of the present invention, and the present invention is not limited to the sodium channel binding agents and pharmaceutical compositions shown below.

Sodium Channel Binding Agent The sodium channel binding agent contains at least one compound selected from the group consisting of a porphyrin derivative represented by the following formula (I) and a spirobifluorene derivative represented by the following formula (II), and selectively binds to the sodium channel Na _v 1.1.

Sodium channels are ion channels that allow sodium ions to pass through with high selectivity. Voltage-dependent sodium channels (Na _v channels) are known as sodium channels present in nerve cells and the like. Subtypes of voltage-dependent sodium channels are known, Na _v 1.1 to Na _v 1.9, and their distribution in vivo is different. The sodium channel binding agent of this embodiment selectively binds to Na _v 1.1 among voltage-dependent sodium channels. This makes it possible to effectively suppress epilepsy in Dravet syndrome. In addition, since the agent selectively binds to Na _v 1.1, it is possible to suppress side effects.

In formula (I), Y ¹¹ to Y ¹⁴ each independently represent a carbon atom having an aromatic group which may have a substituent, or a nitrogen atom. That is, the sodium channel binding agent may be a porphyrin compound represented by the following formula (Ia) or an azaporphyrin compound represented by the following formula (Ib).

In the formula, ^X11 and ^X12 each independently represent an imino group or a sulfur atom. ^Ar11 to ^Ar14 each independently represent an aromatic group which may have a substituent. ^R11 to ^R18 each independently represent a hydrogen atom, a halogen atom, a hydrocarbon group having 1 to 12 carbon atoms which may have a substituent, a hydrocarbonoxy group having 1 to 12 carbon atoms which may have a substituent, or an aromatic group which may have a substituent, or at least one pair of substituents selected from the group consisting of ^R11 and ^R12 , ^R13 and ^R14 , ^R15 and ^R16 , and R17 and ^R18 are linked to each other to represent a condensed aromatic ring group which may have a substituent. ^Ar21 to ^Ar28 each independently represent an aromatic group which may have a substituent. ^R21 to ^R24 each independently represent a halogen atom, a hydrocarbon group having 1 to 12 carbon atoms, or a hydrocarbonoxy group having 1 to 12 carbon ^atoms . Each n independently represents a number from 0 to 3.

Examples of the substituent in the aromatic group include a hydroxyl group, an amino group which may be substituted with a hydrocarbon group having 1 to 8 carbon atoms, a cyano group, a nitro group, a carboxy group, a carbamoyl group, a formyl group, a sulfanyl group, a halogen atom, a hydrocarbon group having 1 to 18 carbon atoms which may have a substituent, a hydrocarbonoxy group having 1 to 12 carbon atoms which may have a substituent, a trialkylammonium group having an alkyl group having 1 to 12 carbon atoms, a dialkylsulfonium group having an alkyl group having 1 to 12 carbon atoms, an aromatic sulfanyl group, and the like. The substituent may be at least one type selected from the group consisting of these, or may be a substituent formed by combining two or more types selected from the group consisting of these.

Examples of aromatic groups include phenyl, naphthyl, pyridyl, pyridinium, quinolyl, and quinolinium groups.

Examples of the substituents in the hydrocarbon group and the hydrocarbonoxy group include a hydroxyl group, an amino group which may be substituted with a hydrocarbon group having 1 to 8 carbon atoms, a cyano group, a nitro group, a carboxy group, a carbamoyl group, a formyl group, a sulfanyl group, a halogen atom, a hydrocarbonoxy group having 1 to 12 carbon atoms, a trialkylammonium group having an alkyl group having 1 to 12 carbon atoms, a dialkylsulfonium group having an alkyl group having 1 to 12 carbon atoms, an aromatic sulfanyl group, and the like. The substituents may be at least one type selected from the group consisting of these, or may be a combination of two or more types selected from the group consisting of these.

Examples of the hydrocarbon group include an alkyl group, an alkenyl group, an alkynyl group, an aryl group, and an arylalkyl group. The hydrocarbon group may be at least one type selected from the group consisting of these groups, or may be a hydrocarbon group formed by combining two or more types selected from the group consisting of these groups.

The compound represented by formula (I) may be in the form of a salt. When the compound represented by formula (I) is in the form of a salt, it may be a pharma- ceutically acceptable salt as described below. The compound represented by formula (I) may also have a ligand containing a metal at its center. Examples of the metal contained in the ligand include iron, cobalt, manganese, and magnesium. The ligand containing a metal includes a metal ion and a metal oxide.

Specific examples of compounds represented by formula (I) or (II) are listed below, but the present invention is not limited thereto. The compounds represented by formula (I) or (II) include compounds in which the nitrogen atom is protonated, deprotonated, and compounds in which a metal or metal ion such as iron, cobalt, manganese, or magnesium is coordinated to a porphyrin skeleton or an azaporphyrin skeleton. Furthermore, the compounds represented by formula (I) or (II) also include derivatives derived from the following exemplary compounds as seed candidate compounds.

The compounds represented by formula (I) or (II) can be synthesized by synthetic methods commonly used in the field of organic chemistry.

The compound represented by formula (I) or (II) may be a compound selected by the screening method described below. The screening method may be a method for selecting a compound that selectively binds to a specific sodium channel among sodium channels having multiple subtypes, and may include a compound group preparation step of preparing a candidate compound group, a binding ability evaluation step of evaluating the binding ability value of the candidate compounds included in the candidate compound group for a specific subtype of the sodium channel, and a selection step of selecting a candidate compound whose binding ability value with the specific subtype is greater than its binding ability value with other subtypes. The screening method may further include an action evaluation step of evaluating the selected candidate compounds in vitro.

In the compound group preparation step, a candidate compound group to be subjected to the screening method is prepared. The candidate compounds included in the candidate compound group may be, for example, compounds selected from commercially available compounds, or may be compounds registered in any compound database. The compound group preparation step may include, for example, selecting candidate compounds from the compounds registered in the compound database in accordance with the selection criteria described below. Selection criteria for selecting candidate compounds include the molecular weight of the compound, the topological polar surface area, and the predicted BBB activity value. By selecting candidate compounds based on the selection criteria, the number of compounds included in the candidate compound group can be reduced, and screening can be performed more efficiently.

The molecular weight of the compound used as the selection criterion may be selected, for example, by referring to the molecular weight of a known compound known to specifically bind to the target sodium channel. An example of a compound that specifically binds to Na _v 1.1 is δ-theraphotoxin Hm1a, a venom derived from the poisonous spider Togo starburst baboon (Heteroscodra maculata). δ-theraphotoxin is known to bind to the extracellular domain (EC) of Na _v 1.1 and specifically delay inactivation of the Na _v 1.1 channel.

Topological polar surface area (TPSA) is an index that corresponds to the area value of the polar portion of a molecular surface, and is one of the indices used to predict the transport properties of drugs. In general, the area value (PSA) of the polar region of a molecular surface is calculated based on the three-dimensional structure of the molecule, but TPSA is a value that is approximately calculated without information on the three-dimensional structure. TPSA can be calculated, for example, using Molecular Operating Environment (MOE; Fujitsu Kyushu Systems and MOLSIS Co., Ltd.).

The predicted BBB activity value is one of the indices for evaluating the permeability through the blood-brain barrier. The predicted BBB activity value of a compound is calculated, for example, based on quantitative structure-activity relationship (QSAR). The predicted BBB activity value can be calculated, for example, using MOE.

In the binding ability evaluation step, the binding ability value of the candidate compound included in the candidate compound group is evaluated for a specific subtype of a sodium channel, for example, Na _v 1.1. The binding ability evaluation step may include, for example, a first conformation calculation step of calculating the three-dimensional structure of the candidate compound, a second conformation calculation step of calculating the three-dimensional structure of the sodium channel, a binding ability value calculation step of performing a docking simulation between the candidate compound and the sodium channel, and a preliminary selection step of selecting the candidate compound based on the binding ability value.

In the first stable conformation calculation step, the conformation of the candidate compound is calculated. The conformation calculation can be performed by known methods such as molecular orbital method and molecular force field calculation. The conformation calculation of the candidate compound can be performed, for example, using MOE. In the second conformation calculation step, the conformation of a specific sodium channel is calculated. For example, a three-dimensional structure obtained from an existing protein database can be applied as the initial coordinate for the conformation calculation. In addition, the conformation calculation can be performed by known methods such as molecular orbital method and molecular force field calculation. The conformation calculation of the sodium channel can be performed, for example, using MOE.

In the binding ability value calculation step, a docking simulation between the candidate compound and the sodium channel is performed to calculate the binding ability value. In the docking simulation, a plurality of binding sites between the candidate compound and the sodium channel are searched for based on the conformation of the prepared candidate compound and the conformation of the sodium channel. Next, various positions of the candidate compound at each binding site are determined, and each position is scored to calculate the binding ability value (S value). The binding site of the candidate compound to the sodium channel is selected from the extracellular domain of the sodium channel, and may be, for example, the binding site of δ-teraphotoxin to Na _v 1.1.

In the preliminary selection step, candidate compounds are selected based on the calculated binding ability value. The selected candidate compounds have a predetermined binding ability value and are highly capable of binding to a specific sodium channel.

In the selection step, a candidate compound having a binding ability value for a specific subtype that is greater than its binding ability value for other subtypes is selected from the group of candidate compounds selected in the preliminary selection step. The binding ability value for the specific subtype is calculated using the binding ability value calculated in the binding ability evaluation step. The binding ability values of the candidate compounds for other subtypes are calculated in the same manner as the binding ability value for the specific subtype.

When there are a plurality of other subtypes, the selection step may include a plurality of selection steps. That is, the binding ability value of a candidate compound to a certain subtype (e.g., Na _v 1.2) other than a specific subtype (e.g., Na _v 1.1) is calculated, and when the binding ability value to a specific subtype is greater than the calculated binding ability value to a certain subtype, the candidate compound is selected, and only the selected candidate compound may be subjected to comparison with the binding ability value to the next subtype (e.g., Na _v 1.3). By repeating such comparison of binding ability values and selection of a candidate compound, a candidate compound having a binding ability value to a specific subtype greater than the binding ability values to other subtypes may be selected.

In the action evaluation step, the selected candidate compounds are evaluated in vitro to further select candidate compounds having an action. The in vitro evaluation method can be carried out, for example, by evaluating the action of the candidate compound by an electrophysiological method using nerve cells having a mutation in a gene encoding a specific subtype of sodium channel. The nerve cells used for the evaluation may be derived from an iPS cell line derived from a subject having a specific disease. In this case, nerve cells induced from an iPS cell line in which the gene mutation has been repaired can be used as a control for the evaluation. The electrophysiological method also includes the so-called patch clamp method.

Pharmaceutical Composition The pharmaceutical composition comprises a compound represented by the above formula (I) or (II) or a pharma- ceutical acceptable salt thereof. The pharmaceutical composition is used for treating epilepsy, particularly for treating Dravet syndrome. The pharmaceutical composition may also be used for treating a disease (hereinafter also referred to as a specific disease) resulting from a mutation in a gene encoding a specific subtype of sodium channel. Here, the treatment of a disease may be any treatment performed for a disease, such as treatment, improvement, inhibition of progression (prevention of aggravation), prevention, and alleviation of symptoms caused by a disease.

The pharmaceutical composition can be prepared according to conventional methods using at least one compound represented by formula (I) or (II) or a pharma- ceutically acceptable salt thereof and a pharma- ceutically acceptable carrier.

Pharmaceutical compositions can be administered parenterally or orally depending on their dosage form. Dosage forms for parenteral administration include, for example, injections, drops, eye drops, nasal preparations, and pulmonary preparations. Dosage forms for oral administration include, for example, solid preparations such as tablets, capsules, granules, powders, lozenges, syrups, emulsions, and suspensions, as well as liquid and semi-liquid preparations.

The pharmaceutical composition may contain the compound represented by formula (I) or (II) itself as an active ingredient, or may contain a pharma- ceutically acceptable salt thereof as an active ingredient. Specific examples of pharma- ceutically acceptable salts include acid addition salts, metal salts, ammonium salts, and organic amine addition salts. Examples of acid addition salts include inorganic acid salts such as hydrochloride, sulfate, nitrate, and phosphate; and organic acid salts such as acetate, maleate, fumarate, citrate, malate, lactate, α-ketoglutarate, gluconate, and caprylate. Examples of metal salts include alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as magnesium salt and calcium salt; aluminum salt, zinc salt, and the like. Examples of ammonium salts include salts of ammonium and tetramethylammonium. Examples of organic amine addition salts include salts of morpholine and piperidine.

As a pharma- ceutically acceptable carrier, any carrier commonly used in pharmaceutical preparations can be used. Examples of carriers include excipients, disintegrants, binders, flow agents, lubricants, etc. in solid preparations, and solvents, solubilizers, suspending agents, isotonicity agents, buffers or pH adjusters, soothing agents, etc. in liquid preparations. If necessary, the preparations may further contain additives such as preservatives, antioxidants, colorants, sweeteners, cooling agents or flavoring agents, antifoaming agents, thickening agents, etc.

Examples of excipients include lactose, sucrose, D-mannitol, D-sorbitol, corn starch, dextrin, microcrystalline cellulose, crystalline cellulose, carmellose, carmellose calcium, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, and gum arabic. Examples of disintegrants include carmellose, carmellose calcium, carmellose sodium, sodium carboxymethyl starch, croscarmellose sodium, crospovidone, low-substituted hydroxypropyl cellulose, hydroxypropyl methylcellulose, and crystalline cellulose. Examples of binders include hydroxypropyl cellulose, hydroxypropyl methylcellulose, povidone, crystalline cellulose, sucrose, dextrin, starch, gelatin, carmellose sodium, and gum arabic. Examples of flow agents include light anhydrous silicic acid and magnesium stearate. Examples of lubricants include magnesium stearate, calcium stearate, and talc.

Examples of solvents in liquid preparations include purified water, ethanol, propylene glycol, macrogol, sesame oil, corn oil, and olive oil. Examples of solubilizing agents include propylene glycol, D-mannitol, benzyl benzoate, ethanol, triethanolamine, sodium carbonate, and sodium citrate. Examples of suspending agents include benzalkonium chloride, carmellose, hydroxypropyl cellulose, propylene glycol, povidone, methylcellulose, and glycerin monostearate. Examples of isotonicity agents include glucose, D-sorbitol, sodium chloride, and D-mannitol. Examples of buffers or pH adjusters include sodium hydrogen phosphate, sodium acetate, sodium carbonate, and sodium citrate. Examples of soothing agents include benzyl alcohol.

Examples of preservatives include methyl parahydroxybenzoate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate, chlorobutanol, benzyl alcohol, sodium dehydroacetate, and sorbic acid. Examples of antioxidants include sodium sulfite and ascorbic acid. Examples of colorants include food dyes (e.g., Food Red No. 2 or No. 3, Food Yellow No. 4 or No. 5, etc.), and β-carotene. Examples of sweeteners include sodium saccharin, dipotassium glycyrrhizinate, and aspartame. Examples of refreshing agents or flavoring agents include l-menthol and peppermint water. Examples of defoamers include dimethylpolysiloxane and silicone defoamers. Examples of thickening agents include xanthan gum, tragacanth, methylcellulose, and dextrin.

The pharmaceutical composition may contain other antiepileptic drugs as necessary, and may be used in combination with other antiepileptic drugs. Examples of other antiepileptic drugs include stiripentol (STP), topiramate (TPM), bromide (Br), levetiracetam (LEV), phenobarbital (PB), zonisamide (ZNS), valproic acid (VPA), clonazepam (CZP), etc.

The content of the active ingredient in the pharmaceutical composition varies depending on the dosage form, dosage amount, etc., but is, for example, 0.1% to 20% by weight, or 0.1% to 10% by weight of the entire composition. The dosage of the pharmaceutical composition is appropriately selected depending on the subject, disease, symptoms, dosage form, administration route, etc. For example, when orally administered to an adult cancer patient, the dosage is usually about 0.1 mg to 500 mg, or about 0.5 mg to 100 mg, of the active ingredient per day, and can be administered once or in divided doses.

The method for treating a disease comprises administering an effective amount of the pharmaceutical composition to a subject, and is a method for treating epilepsy, specific diseases, and the like. Details of the pharmaceutical composition and the method of administration are as described above. The subject of the treatment is, for example, a mammal, and mammals include humans. The subject of the treatment may also be a non-human animal.

In other aspects, the present invention also includes the use of a compound represented by formula (I) or (II) in the manufacture of a pharmaceutical composition for use in the treatment of epilepsy, specific diseases, etc., the use of a compound represented by formula (I) or (II) in the treatment of epilepsy, specific diseases, etc., and a compound represented by formula (I) or (II) used in the treatment of epilepsy, specific diseases, etc.

The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

As the candidate compound group, low molecular weight compounds registered in the Chembl database, low molecular weight compounds registered in the compound database provided by Kishida Chemical Co., Ltd., and low molecular weight compounds registered in the compound database provided by Namiki Shoji Co., Ltd. were selected. The number of compounds included in the candidate compound group, including duplicates, was approximately 13 million. For the candidate compounds included in the candidate compound group, 1,449 types of compounds were selected based on molecular weight, topological polar surface area (TPSA), and predicted BBB activity value. The topological polar surface area and predicted BBB activity value were calculated using MOE (Molecular Operating Environment; Fujitsu Kyushu Systems and MOLSIS Co., Ltd.), an integrated computational chemistry platform.

Next, the binding ability value of each candidate compound for the sodium channel Na _v 1.1 was calculated by docking simulation using MOE, and 834 candidate compounds were selected as compounds with high binding ability. For the three-dimensional structures of the candidate compounds, stable conformations calculated using MOE were adopted. For the three-dimensional structures of Na _v 1.1, those registered in the PDB (Protein Data Bank) of the RCSB (Research Consortium for Structural Bioinformatics) were adopted. The docking simulation was performed by selecting 100 binding sites contained in the binding site of δ-teraphotoxin for Na _v 1.1 using Triangle Matcher, a ligand placement method provided by MOE, and calculating the binding ability value (S value) at each binding site.

Next, for the candidate compounds selected by the docking simulation, the binding ability values for Na _v 1.2 were calculated in the same manner as described above, and candidate compounds having S values for Na _v 1.1 larger than the S value for Na _v 1.2 were selected. For the selected candidate compounds, the binding ability values for each subtype were calculated in the order of Na _v 1.3 to Na _v 1.9, and candidate compounds having high binding ability values for Nav1.1 were successively narrowed down to finally select 56 candidate compounds.

Of the selected candidate compounds, the following four compounds were available and their efficacy was evaluated in vitro as follows:

Evaluation The efficacy of the candidate compounds was evaluated using the Maestro system (Axion Biosystems), which is an MEA (The microelectrode array). With reference to Molecular Brain 2013, 6:19, an iPS cell line derived from a patient with Dravet syndrome was established from skin cells collected from a patient with Dravet syndrome. Also, with reference to Stem Cell Research, 28 (4); 100-104 (2018), an isogenic iPS cell line derived from a patient with Dravet syndrome was established.

GABAergic inhibitory neurons were produced using selective differentiation induction method from iPS cells derived from healthy subjects, iPS cells derived from Dravet syndrome patients, and isogenic iPS cells derived from Dravet syndrome patients. Specifically, GABAergic inhibitory neurons were produced as follows, with reference to Plasmid construction and Neuronal differentiation by transcription factor overexpression in Material and method in eNeur, 6(5);0403-18(2019).

The feeder-free cultured iPS cells established above (B7: control, D1: derived from a Dravet patient, D1T: isogenic control derived from a Dravet patient) were seeded onto 48-well Maestro plates (Axion Biosystems) coated with iMatrix-511 silk and poly-L-lysine, and differentiation was induced. For neural differentiation, the cells were cultured for 6 days using differentiation induction medium (BrainPhys Neuronal Medium and N2-A/SM1, STEMCELL Technologies; containing 10 μM DAPT and 1 μg/mL doxycycline), and then cultured for more than 6 weeks in neural maturation medium (BrainPhys Neuronal Medium and N2-A/SM1, STEMCELL Technologies; containing 20 ng/ml BDNF, 20 ng/ml GDNF, 200 nM L-ascorbic acid, 1 mM dibutyryl c-AMP).

The expression of neuronal markers in GABAergic inhibitory neurons in neurons cultured for more than 6 weeks was confirmed by qRT-PCR and immunostaining. Next, the function of GABAergic inhibitory neurons was evaluated by measuring spontaneous firing using Maestro. A sampling rate of 12.5 kHz was used to acquire data, and a Butterworth bandpass filter of 200 Hz to 3000 Hz was used. The detection threshold was set to +6.0 x SD of baseline electrode noise. Spontaneous neural activity of neurons was recorded while culturing at 37°C for 5 minutes.

Considering the large variation in spontaneous spike counts between Maestro plates and wells, we focused on spontaneous burst firing, which is an indicator of neuronal maturity. A firing frequency of >5 spikes/100 ms was used as the detection threshold for burst firing. Axion Integrated Studio program (Axion Biosystems) and Neural Metric Tool (Axion Biosystems) were used to analyze data on neuronal firing measurements. The results are shown in Figure 1. A decrease in the number of spikes during burst firing was confirmed in Dravet patient-derived GABAergic inhibitory neurons (D1) compared with healthy subjects (B7) and Dravet patient-derived isogenic neurons (D1T). This decrease in firing frequency (functional attenuation) was used as an indicator for subsequent drug efficacy evaluation.

Test compounds were added at various concentrations to the culture medium of GABAergic inhibitory neurons. While culturing at 37°C for 5 minutes, the action potential of the neurons was recorded, and the change in firing frequency at each concentration of the test compound was measured as the number of spikes during spontaneous burst firing. The total number of experiments (number of wells recorded) was n = 3 to 6. Statistical analysis was performed using Dunn's multiple comparisons test. A probability value (p value) of less than 0.05 was considered statistically significant.

Figure 2 shows the results when the spider venom component Hm1a was used as the test compound. As shown in Figure 2, by adding Hm1a to the culture medium, a concentration-dependent increase in firing frequency (functional recovery) was confirmed in GABAergic inhibitory neurons derived from Dravet patients. In the figure, * indicates p<0.05.

Figure 3 shows the results for compound 2-1, Figure 4 shows the results for compound 1-2, Figure 5 shows the results for compound 3-1, and Figure 6 shows the results for compound 3-2. In the figures, * means p<0.05, and ** means p<0.01. As shown in Figures 3 and 4, compounds 1-2 and 2-1, like Hma1, showed functional recovery of GABAergic inhibitory neurons derived from Dravet patients. On the other hand, compounds 3-1 and 3-2 did not show functional recovery of GABAergic inhibitory neurons derived from Dravet patients.

Claims (4)

A sodium channel binding agent represented by the following chemical formula 1-2 or 2-1 , which selectively binds to the sodium channel Nav1.1.

A pharmaceutical composition comprising a compound represented by the following chemical formula 1-2 or 2-1, or a pharma- ceutical acceptable salt thereof:

3. The pharmaceutical composition according to claim 2 for use in the treatment of epilepsy. The pharmaceutical composition according to claim 2, which is used for the treatment of Dravet syndrome.

Images