Method for screening for medicine, and agent

Abstract

A main purpose of the present technique is to provide a technique for identifying a novel indicator capable of affecting sleep and screening for a compound on the basis of the indicator, thereby newly identifying a compound capable of favorably affecting sleep.　The present inventors have made intensive studies and found that it becomes possible to identify a compound capable of affecting synaptic strength, i.e., synaptic connection strength between nerve cells constituting the brain, by using the synaptic strength as an indicator, and to favorably screen the identified compound as a candidate for a medicine capable of affecting sleep.

Description

Drug screening method and drug

[0001] The present technology relates to a drug screening method and agent, and more particularly to a technology for identifying a compound that can affect sleep, and an agent that affects sleep and contains the identified compound as an active ingredient.

[0002] Conventionally, research has been conducted on the mechanism of sleep and compounds that can affect sleep.

[0003] For example, Non-Patent Document 1 below focuses on the process of activating the GABA system in the ventrolateral preoptic area of ​​the hypothalamus to suppress the wakefulness system, and discloses a compound that has the effect of inducing this process.

[0004] The role and mechanism of sleep / Nihon University Journal of Medicine 79 (6): 327-331 (2020)

[0005] The main purpose of this technology is to provide a technology for identifying novel compounds that can favorably affect sleep by defining novel indicators that can affect sleep and screening compounds based on these indicators.

[0006] As a result of extensive research, the inventors have identified compounds that affect synaptic strength, which is the strength of synaptic connections between nerve cells that make up the brain, using this synaptic strength as an indicator, and have found that these identified compounds can be suitably screened as candidates for drugs that affect sleep.

[0007] That is, the present technology provides a drug screening method that uses synaptic strength, which is the strength of synaptic connections between neurons constituting the brain, as an index to identify compounds that affect the synaptic strength, and identifies the identified compounds as candidates for drugs that affect sleep. In the drug screening method of the present technology, the brain is preferably the cerebral cortex, the cerebral cortex is preferably the frontal lobe, and the frontal lobe is preferably the prefrontal cortex or the motor cortex. In the drug screening method of the present technology, the strengthening of the synaptic strength may be achieved by activating AMPA receptors or NMDA receptors. Furthermore, in the drug screening method of the present technology, the synaptic strength may be determined by the strength of electricity flowing between the neurons, the volume of the dendritic spines of the neurons that form the synapse, the extracellular potential recording of neurons constituting the brain, or an increase in calcium concentration at the synapse.

[0008] Next, the present technology provides an agent for influencing sleep, comprising as an active ingredient a compound characterized by affecting synaptic strength, which is the strength of synaptic connections between neurons constituting the brain, using synaptic strength as an index. In the agent of the present technology, the compound may be a compound involved in activating or inhibiting receptors on the cell membrane of neurons adjacent to the synapse, or a compound involved in signal transmission to receptors in neurons adjacent to the synapse. The compound may be any of ligands for amino acid neurotransmitter receptors, ligands for monoamine neurotransmitter receptors, compounds that act directly on neurons, compounds that regulate calcium-induced signal transduction at synapses, inhibitors or activators of molecules involved in intracellular signal transduction pathways, and compounds that act on hormone receptors. The ligand for the amino acid neurotransmitter receptor may be any of an AMPA receptor agonist and an AMPA receptor antagonist.

[0009] 1 is a graph showing changes in the current flowing between cultured brain neurons when a compound that affects synapse strength is administered to the neurons. This graph compares the current flowing between the neurons when a compound that affects synapse strength is administered to the cultured brain neurons with or without administration. This graph compares extracellular potentials recorded on the scalp by electroencephalography with or without administration of a compound that affects synapse strength. This graph compares the dendritic spine volume of the neurons when a compound that affects synapse strength is administered to the cultured brain neurons with or without administration. This graph is an illustration of measuring the extracellular potential of iPS cells using electrodes laid out under the cells. This graph shows the results of measuring the extracellular potential of iPS cells using electrodes laid out under the cells with and without administration of a compound that affects synapse strength. This graph compares the extracellular potential of iPS cells using electrodes laid out under the cells with and without administration of a compound that affects synapse strength. This example shows how the screening method of the present technology was used to screen for compounds that affect synaptic strength, which is the strength of synaptic connections between neurons that make up the brain. This graph shows the effect on sleep induced when synaptic strength in the motor cortex was enhanced by administering a compound that affects synaptic strength.

[0010] Preferred embodiments of the present technology will be described below. However, the embodiments shown below are examples of typical embodiments of the present technology, and the present technology is not limited to only the preferred embodiments below and can be freely modified within the scope of the present technology.

[0011] [Screening Method] In the drug screening method of the present technology, compounds that affect synaptic strength, which is the strength of synaptic connections between nerve cells that make up the brain, are identified using the synaptic strength as an index, and the identified compounds can be newly identified as drug candidates that affect sleep.

[0012] In the present technology, the term "agent" refers to a substance in general that is used for the purpose of treatment or prevention, and refers to the entirety of what a patient takes, including not only the medicinal ingredients involved in the treatment but also the auxiliary ingredients used in the formulation. Examples of such auxiliary ingredients include the capsules of capsule medicines and the solvents of liquid medicines. In contrast, the term "drug" refers to the medicinal ingredients contained in the "agent." Examples of medicinal ingredients include a single compound with medicinal effects and a mixture of two or more compounds with medicinal effects.

[0013] In the present technology, "sleep" refers to a periodically recurring state in an organism in which the organism's brain is reduced in its ability to respond to external stimuli. During sleep, the organism's brain's electroencephalograms exhibit waveforms with specific patterns of electrical activity defined by non-REM (NREM) sleep and REM sleep.

[0014] In the present technology, "influencing sleep" refers to inducing an organism into the aforementioned sleep state or maintaining the sleep state. For example, by inducing the brain waves of the organism to waveforms of specific electrical activity patterns defined by non-REM (NREM) sleep and REM sleep or by maintaining the brain waves in a state showing the waveforms, the sleep state can be induced or the sleep state can be maintained.

[0015] In the present technology, the term "drug that affects sleep" refers to a medicinal ingredient that can induce a state of sleep or maintain a state of sleep.

[0016] The drug screening method of the present technology can suitably identify components that may affect sleep as "candidate drugs that affect sleep."

[0017] In the drug screening method of the present technology, synaptic strength, which is the strength of synaptic connections between neurons that constitute the brain, is used as an index for screening compounds. In particular, the neurons that are the target of the present technology are preferably neurons that constitute the brain, and among the brain, neurons in the cerebral cortex are more preferred, among the cerebral cortex, neurons in the frontal lobe are even more preferred, and among the frontal lobe, neurons in the prefrontal cortex or motor cortex are particularly preferred.

[0018] "Affecting synaptic strength" refers to strengthening synaptic strength or maintaining a strengthened state, and "a compound that affects synaptic strength" refers to a compound that can strengthen synaptic strength or maintain a strengthened state.

[0019] In the drug screening method of the present technology, strengthening of synaptic strength can be achieved, for example, by activating or inhibiting receptors on the cell membrane of nerve cells adjacent to the synapse.

[0020] For example, when synaptic strength is enhanced by activating a receptor such as an AMPA receptor, activation of the AMPA receptor can be regulated by an agonist, which binds to the receptor and promotes its activity, or an antagonist, which binds to the receptor and inhibits its activity.

[0021] In the present technology, synaptic strength can be determined by any method.For example, it can be determined by the strength of the electricity flowing between neurons, or by the volume of the dendritic spine of the neurons that form synapses, or by the waveform of electroencephalograms, or by the increase in calcium concentration in synapses.In the present technology, synaptic strength can be determined by these methods alone, or can be determined by combining these methods.

[0022] The methods listed above for determining synaptic strength are described in more detail below. As mentioned above, the method for determining synaptic strength is not limited to the methods described below, and any method can be used.

[0023] <When synaptic strength is determined by the strength of electricity flowing between neurons> In the present technology, when synaptic strength is determined by the strength of electricity flowing between neurons, the strength of electricity refers to the strength of the current or the magnitude of the voltage based on the potential difference. In this case, the method for measuring the strength of electricity flowing between neurons is not particularly limited and can be measured by any method. For example, there are methods such as directly measuring the strength of electricity flowing between neurons using the patch clamp method, or expressing a protein or the like that emits fluorescence in response to the strength of the potential or current in the target neurons and measuring the intensity of the fluorescence by any means, such as imaging using a microscope.

[0024] In the drug screening method of the present technology, when a compound is administered to a living organism or a culture of cells that make up a living organism, if the strength of the electricity flowing between nerve cells is increased compared to before administration of the compound, the compound can be identified as a compound that affects synaptic strength, and the identified compound can be newly identified as a candidate drug that affects sleep.

[0025] The method of administering a compound to a living organism or a culture of cells constituting the living organism is not particularly limited, and can be measured by any method. When a compound is administered to a living organism, it can be preferably administered by, for example, oral administration, injection, rectal administration, inhalation, etc. When a compound is administered to a cell culture, it can be preferably administered by, for example, adding the compound to the culture medium of the cells.

[0026] <When synaptic strength is determined by changes in the volume of dendritic spines of neurons that form synapses> In the present technology, when synaptic strength is determined by the volume of dendritic spines of neurons that form synapses, the method for measuring the dendritic spine volume is not particularly limited and can be any method. Examples include a method of visualizing the shape of a target dendritic spine using an electron microscope and measuring the volume of the visualized dendritic spine, a method of imaging the fluorescence of a fluorescent protein locally expressed in a dendritic spine using a fluorescence microscope or the like and measuring the volume, and a method of staining a protein localized in a dendritic spine with an antibody or RNA labeled with a fluorescent substance, imaging the fluorescence of the fluorescent substance using a fluorescence microscope or the like and measuring the volume.

[0027] Even in this case, when a compound is administered to a living organism or a culture of cells that constitute a living organism using the above-mentioned method, if the volume of the dendritic spines of the nerve cells that form synapses increases compared to before administration of the compound, the compound can be identified as a compound that affects synapse strength, and the identified compound can be newly identified as a candidate drug that affects sleep.

[0028] <When synaptic strength is determined by recording extracellular potentials of neurons constituting the brain> In the present technology, when synaptic strength is determined by recording extracellular potentials of neurons constituting the brain, the method for measuring the extracellular potentials of neurons constituting the brain is not particularly limited and can be measured by any method. For example, suitable methods include measuring extracellular potentials from the scalp of the brain using an electroencephalograph, attaching electrodes to a subdural position of an experimental animal and measuring cortical electroencephalograms, attaching electrodes to the cerebral cortex of an experimental animal and measuring local field potentials, and measuring extracellular potentials of cultured cells of neurons constituting the brain.

[0029] As a method for measuring extracellular potential recordings of cultured cells that constitute the brain, for example, when the cultured cells are tissue fragments derived from brain tissue, cell clusters (cells), or induced pluripotent stem cells (iPS cells) derived therefrom, extracellular potential recordings can be measured using electrodes laid under these cells. Also, when the cultured cells are dispersed in a culture medium, extracellular potential recordings can be measured by inserting electrodes into the culture medium of the cells.

[0030] In this case, when a compound is administered to a living body using the above-mentioned method, if the extracellular potential recordings of neurons after administration are compared with those before administration within a specified time period and show waveforms with specific electrical activity patterns defined by non-REM (NREM) sleep and REM sleep, the compound can be identified as a compound that affects synaptic strength, and the identified compound can be newly identified as a candidate drug that affects sleep.

[0031] <When synaptic strength is determined by calcium concentration in synapses> In the present technology, when synaptic strength is determined by an increase in calcium concentration in synapses, the method for measuring the calcium concentration in synapses is not particularly limited and can be any method. Examples include a method in which calcium present in synapses is stained with a dye that specifically binds to calcium and emits fluorescence, and the intensity of the fluorescence emitted by the dye is measured by any means, such as imaging using a microscope, a method in which calcium concentration is measured by introducing a calcium-sensing protein or peptide and detecting changes in the structure of the protein or peptide, and a method in which calcium concentration is estimated based on the potential between neurons measured by patch clamping.

[0032] Even in this case, when a compound is administered to a living organism or a culture of cells that make up a living organism using the above-mentioned method, if the calcium concentration in the synapse increases compared to before administration of the compound, the compound can be identified as a compound that affects synaptic strength, and the identified compound can be newly identified as a candidate drug that affects sleep.

[0033] [Agents Affecting Sleep] The agents affecting sleep provided by the present technology contain, as an active ingredient, a compound identified by the drug screening method of the present technology described above. That is, the agents affecting sleep provided by the present technology contain, as an active ingredient, a compound that affects synaptic strength, which is the strength of synaptic connections between neurons that constitute the brain, using synaptic strength as an indicator.

[0034] Here, as defined above, the term "agent" in this specification refers to any substance used for the purpose of treatment or prevention, etc., and the term "agent" refers to the entirety of what is taken by a patient, including not only the medicinal ingredients involved in the treatment but also the auxiliary ingredients used in the formulation. Furthermore, the term "active ingredient" refers to the medicinal ingredient contained in the "agent," and is a component contained in the "agent" to an extent that ensures that the medicinal ingredient effectively achieves the purpose of treatment or prevention, etc.

[0035] In addition, in the agent that affects sleep provided by the present technology, the compound contained as the active ingredient may be a single compound or a combination of two or more compounds.

[0036] The sleep-affecting agent provided by the present technology can induce or maintain a state of sleep by containing, as an active ingredient, a compound identified by the drug screening method of the present technology.

[0037] The compound contained as an active ingredient in the agent according to the present technology is not particularly limited as long as it is a compound that can be identified by the drug screening method of the present technology. That is, using synaptic strength, which is the strength of synaptic connections between nerve cells constituting the brain, as an index, a compound that affects the synaptic strength can be suitably used as an active ingredient in the agent according to the present technology that affects sleep.

[0038] Examples of compounds that affect synaptic strength include compounds that are involved in activating or inhibiting receptors on the cell membrane of neurons adjacent to the synapse, and compounds that are involved in signal transmission to receptors in neurons adjacent to the synapse.

[0039] Examples of compounds involved in activating or inhibiting receptors on the cell membrane of neurons adjacent to synapses include ligands for receptors of amino acid neurotransmitters such as AMPA and NMDA, and ligands for receptors of monoamine neurotransmitters.

[0040] Here, examples of the ligand include agonists, antagonists, positive allosteric modulators, etc. Furthermore, examples of the monoamine neurotransmitters include dopamine, 5-HT (serotonin), noradrenaline, etc.

[0041] Ligands for receptors of amino acid neurotransmitters such as AMPA and NMDA are specifically agonists, antagonists, or positive allosteric modulators of AMPA receptors, and agonists or antagonists of NMDA receptors. These agonists, antagonists, or positive allosteric modulators are compounds that can act as agonists, antagonists, or positive allosteric modulators of the target AMPA receptor or NMDA receptor.

[0042] Ligands for receptors of monoamine neurotransmitters are compounds that can act as ligands for receptors such as those listed above for dopamine, 5-HT (serotonin), and noradrenaline.

[0043] Examples of compounds involved in signal transmission to receptors in neurons adjacent to synapses include compounds that act directly on neurons, compounds that regulate calcium-induced signal transmission in synapses, inhibitors or activators of molecules involved in intracellular signal transduction pathways, and compounds that act on hormone receptors.

[0044] Compounds that act directly on nerve cells include, for example, TrkB agonists and cAMP activators.

[0045] Examples of compounds that regulate calcium-induced signal transduction in synapses include CaMKII (calcium / calmodulin-dependent kinase II inhibitors), etc. Examples of CaMKII include KCN1, KN-93, Autocamtide-2-Related Inhibitory Peptide (AIP), KN-62, and STO-609.

[0046] Examples of molecules involved in intracellular signaling pathways include small G proteins, which interact with specific G proteins in intracellular signaling pathways. Examples of small G proteins include Rac1, Cdc42, RhoA, PAK, ROCK, LIMK1, and PKC.

[0047] The inhibitors or activators of molecules involved in intracellular signaling pathways are inhibitors or activators of the above-mentioned small G protein, etc. Specifically, they include inhibitors or activators of Rac1, inhibitors or activators of Cdc42, inhibitors of RhoA, inhibitors of PAK, inhibitors of ROCK, inhibitors of LIMK1, inhibitors or activators of PKC, etc.

[0048] Examples of Rac1 inhibitors include Rac1 inhibitor W56, NSC23766, EHT1864, GYS32661, MBQ-167, CAS 1177865-17-6, etc. Examples of Rac1 activators include EHT 1864, steffimycin, etc.

[0049] Examples of Cdc42 inhibitors include ML141, CID-2950007, CASIN (Cdc42 activity-specific inhibitor), Secramine A, ZCL278, MBQ-167, NSC23766, Simvastatin, Lovastatin, etc. Examples of Cdc42 activators include Bradykinin, etc.

[0050] Examples of RhoA inhibitors include C3 Exoenzyme, Rhosin, CCG-1423, Y-27632, and statins.

[0051] Examples of PAK inhibitors include FRAX597, PF-3758309, FRAX486, PIR3.5, G-5555, KD-1, and ZM 449829.

[0052] Examples of ROCK inhibitors include Y-27632, Fasudil (HA-1077), H-1152, GSK269962A, RKI-1447, SAR407899, Thiazovivin, Atorvastatin, and the like.

[0053] Examples of LIMK1 inhibitors include BMS-3, LIMKi 3, Pyridone 6, T56-LIMKi, and Dihydroindirubin-3'-oxime (DIO).

[0054] Examples of PKC inhibitors include ruboxistaurin, cheleythrine, miyabenol C, myricitrin, gossypol, verbascoside, BIM-1, Ro31-8220, bryostatin 1, tamoxifen, etc. Examples of PKC activators include ingenol mebutate, bryostatin 1, 12-O-tetradecanoylphorbol-13-acetate (PMA or TPA), etc.

[0055] Examples of compounds that act on hormone receptors include estrogen modulators that act on estrogen receptors and regulate signal transduction at synapses in nerve cells.

[0056] Furthermore, the drug screening method of the present technology can also be used to screen for drugs that have not previously been suggested to be involved in synaptic potentiation, and such drugs can also be suitably used as active ingredients in the agents that affect sleep according to the present technology.

[0057] The drug screening method of the present technology is expected to be able to screen a variety of drugs, such as drugs involved in DNA repair pathways and metabolic pathways, and drugs involved in cell stress responses.

[0058] In particular, among the compounds listed above, compounds that are already being used as drugs for purposes other than the purpose of the present technology of inducing or maintaining a sleep state are expected to be suitable for use as active ingredients in the agents that affect sleep of the present technology from the standpoint of safety to living organisms.

[0059] The agent according to the present technology may contain, in addition to the active ingredient described above, auxiliary ingredients used in the formulation described above. Furthermore, the agent according to the present technology may contain other ingredients as needed, as long as the desired physical properties are not significantly impaired.

[0060] The present technology may be configured as follows: [1] A drug screening method, using synaptic strength, which is the strength of synaptic connections between neurons constituting the brain, as an index, to identify a compound that affects the synaptic strength, and designate the identified compound as a candidate drug that affects sleep. [2] The drug screening method according to claim 1, wherein the brain is the cerebral cortex. [3] The drug screening method according to [2], wherein the cerebral cortex is the frontal lobe. [4] The drug screening method according to [3], wherein the frontal lobe is the prefrontal cortex. [5] The drug screening method according to [3], wherein the frontal lobe is the motor cortex. [6] The drug screening method according to any one of [1] to [5], wherein the enhancement of synaptic strength is achieved by activation of AMPA receptors or NMDA receptors. [7] The drug screening method according to any one of [1] to [6], wherein the synaptic strength is determined by the strength of electricity flowing between the neurons. [8] The drug screening method according to any one of [1] to [7], wherein the synaptic strength is determined by the volume of the dendritic spine of the neuron that forms the synapse. [9] The drug screening method according to any one of [1] to [8], wherein the synaptic strength is determined by the waveform of an electroencephalogram.

[10] The drug screening method according to any one of [1] to [9], wherein the synaptic strength is determined by an increase in calcium concentration at the synapse.

[11] An agent that affects sleep, using synaptic strength, which is the strength of synaptic connections between neurons that constitute the brain, as an index, and comprising as an active ingredient a compound that affects the synaptic strength.

[12] The agent according to

[11] , wherein the compound is a compound involved in activating or inhibiting receptors on the cell membrane of a neuron adjacent to the synapse, or a compound involved in signal transmission to receptors in a neuron adjacent to the synapse.

[13] The agent according to

[11] or

[12] , wherein the compound is any one of a ligand for a receptor of an amino acid neurotransmitter, a ligand for a receptor of a monoamine neurotransmitter, a compound that acts directly on neurons, a compound that regulates calcium-induced signal transduction in synapses, an inhibitor or activator of a molecule involved in an intracellular signal transduction pathway, and a compound that acts on a hormone receptor.

[14] The agent according to

[13] , wherein the ligand for a receptor of an amino acid neurotransmitter is any one of an agonist of an AMPA receptor and an antagonist of an AMPA receptor.

[0061] The present technology will be described in detail below based on specific examples, but the present technology is not limited to the contents of the examples shown below.

[0062] <When synaptic strength is determined by the strength of electricity flowing between neurons> Figure 1 is a graph showing the change in current flowing between neurons when 80 μM of S18986 (Cayman, Canada), an AMPA agonist compound to be screened, was administered to cultured neurons isolated from the brain of a fetal mouse (Jcl:ICR). The current flowing between the neurons in this case was measured by the patch clamp method using a microelectrode amplifier (product name MultiClamp 700B, manufactured by Axon Instruments).

[0063] The vertical axis of the graph in Figure 1 represents the change in the amount of current when the amount of current at the time of administration of the compound to be screened is set to 100%, and the horizontal axis represents time. Measurements were carried out for a sample group of cultured cells administered with an AMPA agonist and a sample group of cultured cells not administered with an AMPA agonist, with the number of samples (n = 4) for each group.

[0064] As shown in the graph in Figure 1, in the cultured cells administered with an AMPA agonist, it can be confirmed that the amount of current flowing between neurons increases after administration of the AMPA agonist (after 0 minutes on the horizontal axis of the graph). On the other hand, in the cultured cells not administered with an AMPA agonist, it can be confirmed that there is no significant change in the amount of current flowing between neurons over the above time period.

[0065] Figure 2 is a graph showing the current flowing between neurons after administration of the AMPA agonist (after 0 minutes on the horizontal axis of the graph) as a relative value of the average value measured for cultured cells administered with an AMPA agonist over the same period, relative to the average value measured for cultured cells not administered with an AMPA agonist, based on the measurement data of Figure 1. This data also confirms that administration of an AMPA agonist significantly increases the current flowing between neurons.

[0066] Based on these results, we identified AMPA agonists as compounds that affect synaptic strength, and measured extracellular potentials from the scalp of mice intraperitoneally administered with an AMPA agonist using an electroencephalograph. The results are shown in Figure 3.

[0067] The upper row <3A> of FIG. 3 shows the measurement results of mice administered with an AMPA agonist, and the lower row <3B> of FIG. 3 shows the measurement results of subjects not administered with an AMPA agonist.

[0068] The upper graph <3A> in Figure 3 shows the waveforms of the characteristic electrical activity patterns defined by non-REM (NREM) sleep and REM sleep, while the lower graph <3B> in Figure 3 does not show the waveforms of the characteristic electrical activity patterns described above, indicating a wakeful state.

[0069] The above results confirm that the AMPA agonists identified by the screening method of the present technology can be candidates for drugs that affect sleep, i.e., AMPA agonists can be used as active ingredients in drugs that affect sleep.

[0070] Next, when 10 μM of (±)-trans-ACPD (Tocris Bioscience), an NMDA agonist compound to be screened, was administered to cultured neurons extracted from the brains of fetal mice (Jcl:ICR), and synaptic strength was measured by the same method to measure the electrical current flowing between neurons. It was confirmed that administration significantly increased the current flowing between neurons. These results also identify (±)-trans-ACPD as a compound that affects synaptic strength, and this compound may be a candidate for a drug that affects sleep.

[0071] <When synaptic strength is determined by changes in the volume of dendritic spines of neurons that form synapses> Figure 4 shows images of cultured neurons extracted from the brain of a fetal mouse (Jcl:ICR) that were administered 80 μM of S18986 (Cayman, Canada), an AMPA agonist compound to be screened, and the fluorescence emitted by a fluorescently stained antibody against PSD-95 (postsynaptic density protein 95), a protein that is locally present in dendritic spines, imaged using a confocal microscope (product name A1R, manufactured by Nikon Corporation).

[0072] In Figure 4, the left panel shows data before administration of the AMPA agonist, and the right panel shows data after administration of the AMPA agonist. Comparing the two panels, it can be seen that administration of the AMPA agonist increased the volume of dendritic spines.

[0073] Figure 5 is a graph showing the relative values ​​of the dendritic spine volume calculated after administration of the AMPA agonist to the value calculated before administration of the AMPA agonist, based on the imaging data in Figure 4. This data also confirms that administration of the AMPA agonist significantly increases the dendritic spine volume.

[0074] These results also identify the AMPA agonist S18986 as a compound that affects synaptic strength. That is, this compound is a potential candidate for a drug that affects sleep, and can be used as an agent that affects sleep, containing this compound as an active ingredient.

[0075] Next, we administered 10 μM of (±)-trans-ACPD (Tocris Bioscience), an NMDA agonist compound to be screened, to cultured neurons isolated from the brains of fetal mice (Jcl:ICR). Using the same method, we examined changes in dendritic spine volume in neurons that form synapses, confirming that administration significantly increased dendritic spine volume. These results also identify (±)-trans-ACPD as a compound that affects synapse strength, making it a potential candidate for drugs that affect sleep.

[0076] <Determining synaptic strength by recording extracellular potentials of neurons constituting the brain> iPS cells were generated from tissue slices of the brain of a fetal mouse (Jcl:ICR), and extracellular potentials were recorded using electrodes placed under the resulting iPS cells. The extracellular potentials were measured using a high-density microelectrode array (product name: MaxOne MEA System / MaxWell Biosystems). Figure 6 is a conceptual diagram of the extracellular potential recording using multiple electrodes 2 placed under the iPS cells 1.

[0077] The iPS cell culture medium was administered 80 μM of S18986 (Cayman, Canada), an AMPA agonist compound to be screened. The left graph in Figure 7 is a graph showing the time course of extracellular potential recordings before administration of the AMPA agonist, and the right graph is a graph showing the time course of extracellular potential recordings after administration of the AMPA agonist. The waveform of the graph before administration of the AMPA agonist confirms that each cell fires independently, and that the cells do not fire synchronously. On the other hand, the waveform of the graph after administration of the AMPA agonist confirms that the cells fire synchronously.

[0078] Figure 8 is a graph comparing the delta wave band (0.5 to 4.0 Hz) extracted from the measurement data in Figure 7 by Fourier transforming it into frequency components before and after administration of the AMPA agonist. This data also confirms that administration of the AMPA agonist increases the intensity of the extracellular potential recording.

[0079] Based on the above results, AMPA agonists can be identified as compounds that affect synaptic strength, i.e., these compounds are potential candidates for drugs that affect sleep, and drugs containing these compounds as active ingredients that affect sleep can be used.

[0080] Next, similar measurements were performed using iPS cells generated from brain tissue slices of fetal mice (Jcl:ICR), and 10 μM of the NMDA agonist (±)-trans-ACPD (Tocris Bioscience), the compound being screened, was administered. These results also confirmed that administration increased the intensity of extracellular potential recordings. These results also identify (±)-trans-ACPD as a compound that affects synaptic strength, and this compound may be a candidate for drugs that affect sleep.

[0081] <Screening of Compounds Using Synaptic Strength as an Indicator> As in the experimental system shown in Figure 4, cultured cells of neurons extracted from the brains of fetal mice (Jcl:ICR) were used, and 80 µM of each compound to be screened was administered. The dendritic spine volume calculated before administration of the compound was compared with the dendritic spine volume calculated after administration of the compound, and compounds that induce an increase in dendritic spine size were screened.

[0082] Figure 9 shows data on compounds that increased dendritic spine volume when administered to cultured neuronal cells. In this experiment, 4-nitroquinoline-1-oxide (4NQO), 3-(4-morpholinyl)sydnonimine (SIN), neocarzinostatin (NCS), 4-hydroxynonenal (4HNE), tunicamycin, IL1-β, MG132, TNF-α, and KL-11743 were screened. Based on these results, these compounds are considered to be potential drug candidates that affect sleep.

[0083] Furthermore, because the above compounds are drugs that have not previously been proposed to be involved in synaptic potentiation, the screening method of this technology may enable the identification of compounds that could not be predicted based on previous knowledge as candidate drugs that affect sleep.

[0084] <Confirmation of the effect on sleep of administering compounds that affect synaptic strength> We confirmed the effect on sleep when synaptic strength was increased by drugs that affect sleep, which were identified using the screening method of this technology.

[0085] Based on the results, we identified an AMPA agonist, S18986 (Cayman, Canada), as a potential drug candidate for influencing synaptic strength and sleep. We administered 80 μM of S18986 to the motor cortex of mice. Figure 10 is a graph showing the sleep time before and after administration of the drug.

[0086] The results confirmed that administering drugs that affect synaptic strength to the motor cortex can significantly increase sleep time. Furthermore, they demonstrated that sleep induction through synaptic strengthening can be achieved even when using areas of the brain other than the prefrontal cortex of the frontal lobe of the cerebral cortex as an indicator.

[0087] 1 iPS cells 2 electrodes

Claims

1. A drug screening method that uses synaptic strength, which is the strength of synaptic connections between nerve cells that make up the brain, as an indicator to identify compounds that affect said synaptic strength, and identifies said identified compounds as candidates for drugs that affect sleep.

2. The drug screening method according to claim 1, wherein the brain is the cerebral cortex.

3. The drug screening method according to claim 2, wherein the cerebral cortex is the frontal lobe.

4. The drug screening method according to claim 3, wherein the frontal lobe is the prefrontal cortex.

5. The drug screening method according to claim 3, wherein the frontal lobe is the motor cortex.

6. The drug screening method according to claim 1, wherein the enhancement of synaptic strength is achieved by activating AMPA receptors or NMDA receptors.

7. The drug screening method according to claim 1, wherein the synaptic strength is determined by the strength of the electricity flowing between the nerve cells.

8. The drug screening method according to claim 1, wherein the synaptic strength is determined by the volume of the dendritic spine of the nerve cell that forms the synapse.

9. The drug screening method according to claim 1, wherein the synaptic strength is determined by recording extracellular potentials of neurons that constitute the brain.

10. The drug screening method according to claim 1, wherein the synaptic strength is determined by an increase in calcium concentration in the synapse.

11. An agent that affects sleep, which contains as an active ingredient a compound that affects synaptic strength, which is the strength of synaptic connections between nerve cells that make up the brain, using synaptic strength as an indicator.

12. The agent according to claim 11, wherein the compound is a compound involved in activating or inhibiting receptors on the cell membrane of neurons adjacent to synapses, or a compound involved in signal transmission to receptors in neurons adjacent to synapses.

13. The agent according to claim 11, wherein the compound is any one of a ligand for a receptor of an amino acid neurotransmitter, a ligand for a receptor of a monoamine neurotransmitter, a compound that acts directly on nerve cells, a compound that modulates calcium-induced signal transduction in synapses, an inhibitor or activator of a molecule involved in an intracellular signal transduction pathway, and a compound that acts on a hormone receptor.

14. The agent according to claim 13, wherein the ligand for the receptor of the amino acid neurotransmitter is either an agonist or an antagonist of the AMPA receptor.

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