Method for controlling voltage-dependent ion channels via Type I taste receptors (T1Rs)

Abstract

The present invention pertains to a method for controlling a membrane potential-dependent ion channel (VGSC or the like) through a type I taste receptor present in a nerve cell or the like.　In the present invention, it has been found that an Aβ peptide, or a sweet amino acid or an umami substance specifically binds to a type I taste receptor on the surface of a nerve cell to exert an agonist-like or antagonist-like action, thereby amplifying or suppressing a VGSC active current.　Moreover, with the binding of an Aβ peptide or the like to a type I taste receptor, the amplification of a VGSC active current occurs, the overactivity of nerve cells causing epileptiform attack occurs, and a large number of substances, which can effectively suppress the amplification of the VGSC active current, among ligand substances that specifically bind to the type I taste receptor, can be found.　The present invention provides: a type I taste receptor-specific ligand substance that can control the amplification or suppression of a VGSC active current; and a pharmaceutical composition for preventing or treating various neurodegenerative diseases, such as Alzheimer's disease (AD), due to the amplification of a VGSC active current caused by the binding of an Aβ peptide or the like to a type I taste receptor. Moreover, a method for using, as a target receptor, a type I taste receptor present in a nerve cell or the like to screen a ligand substance for controlling a VGSC or the like in the cell is also provided.　Furthermore, the present invention provides a method in which a solution that reproduces the type and concentration of an Aβ peptide contained in the cerebrospinal fluid and serum of a patient with Alzheimer-type dementia is used, and the Aβ peptide contained in the CFS and serum of the patient assesses pathology. Specifically, provided is: a method for assessing the form and dynamic state of cells and the activity of a neurite by using an optical microscope; or a method for assessing the impedance change in a vascular epithelial cell.

Description

[Technical Field]

[0001] This invention is based on the first discovery that Type I taste receptors, which are umami and sweetness receptors, are involved not only in taste perception but also in the polymerization / depolymerization of the cytoskeleton and the regulation of ion channels such as voltage-gated sodium channels (VGSCs) and voltage-gated potassium channels (VGKCs). The present invention relates to a method for controlling VGSC and VGKC via umami and sweet GPCR receptors (Type I taste receptors, "T1Rs" gene family) present in nerve cells and cardiomyocytes. Furthermore, the invention relates to a ligand substance that specifically binds to Type I taste receptors (such as T1R3) and can control the VGSC activation current amplified by the binding of sweet amino acids or umami substances to Type I taste receptors (such as T1R3) on the surface of nerve cells, and to a method for screening such a substance. Furthermore, the invention relates to a pharmaceutical composition for the prevention and / or treatment of neurological diseases caused by the amplification of VGSC activation current induced by sweet amino acids or umami substances via Type I taste receptors on the surface of nerve cells, and to a method for screening such a composition, comprising the ligand substance as an active ingredient.

[0002] Furthermore, this invention is based on the discovery that Type I taste receptors are activated not only by amino acids and sweet substances, but also by amyloid-beta peptides, and are involved in the control of the polymerization state of the cytoskeleton and the control of VGSC and VGKC. This strongly suggests that nerve dysfunction caused by amyloid-beta peptides, which has been reported in large numbers in neurodegenerative diseases such as Alzheimer's disease, may be due to Type I taste receptors (T1R1, T1R2, T1R3) acting as receptors for amyloid-beta peptides and influencing nerve function and morphology. That is, the present invention relates to an invention regarding a method for controlling voltage-gated sodium channels (VGSCs) via Type I taste receptors (the "T1Rs" gene family) that exist particularly in nerve cells and the like. In particular, it relates to a method for suppressing voltage-dependent sodium channels (VGSCs) by using a binding inhibitor of amyloid-β peptide, which is a T1R3 binding inhibitor that acts on the taste GPCR receptor "T1R3" in nerve cells. Further, it relates to a therapeutic agent for Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS) using these binding inhibitors of amyloid-β peptide to T1R3 and a screening method therefor.

[0003] Furthermore, in cancer treatment, regarding neuropathic pain that occurs when an anticancer agent (paclitaxel) is used, etc., it relates to a method for suppressing voltage-dependent sodium channels (VGSCs) via the T1R2 / T1R3 sweet taste GPCR receptor and a pain treatment preparation using a binding inhibitor of Aβ peptide such as an agonist or antagonist of the sweet taste GPCR receptor (T1R2 / T1R3) and a screening method therefor.

Background Art

[0004] In pain and epileptic seizures, action potentials that occur frequently are observed in nerve cells. This phenomenon is due to the hyperactivity of nerve cells. Therefore, suppressing frequently occurring action potentials is the main utility of analgesics and antiepileptic drugs. It has also been reported that in brain regions such as the hippocampus of patients with neurodegenerative diseases, particularly Alzheimer's disease (AD), seizure-like symptoms due to the hyperactivity of nerve cells occur, and it is thought that after this hyperactive period, nerve cell death occurs. Therefore, the development of drugs that suppress nerve hyperactivity is also effective as a therapeutic agent for improving the symptoms of AD patients.

[0005] Furthermore, it is known that changes in amyloid-beta peptides in cerebrospinal fluid (CSF) occur in Alzheimer's disease (AD), Lewy body dementia (DLB), and other neurodegenerative dementias (Bibl et al., (2007)), and amyloid-beta peptides in CSF are used as biomarkers for AD and other conditions. It has been reported that overexpression of amyloid precursor protein (APP) increases sodium channel activity. Specifically, experiments using iPS-derived nerve cells have reproduced the phenomenon where amyloid-beta peptide causes hyperactivity in nerve cells and amplifies sodium channels. It has also been reported that this hyperactivity can be suppressed by cleaving APP and inhibiting BACE1, which produces amyloid-beta peptide (Non-Patent Literature 1). However, the mechanism by which amyloid-beta peptide enhances sodium channels remained unclear.

[0006] Also, voltage-gated Na + Channels (VGSCs) are many types of Na with similar structures. + The channel exists and is widely distributed not only in brain regions. In particular, it plays an important role in supporting cardiac contraction and nerve function, so blocking it unnecessarily can be life-threatening. Therefore, Na + In drug discovery targeting the channel molecule itself, the target Na + It is difficult to selectively suppress channels.

[0007] On the other hand, voltage-gated sodium channels (VGSCs) are known to have their activity (kinetics) regulated by the activity of G proteins. When G proteins are activated, the magnitude of the VGSC current and the activation threshold are affected, and as a result, the ease (threshold) of action potential expression is controlled. Many GPCR receptors on the cell membrane are known to activate serine / threonine kinases (PKA / PKC) through G proteins, thereby modifying VGSC activity. Activation of pertussis toxin-sensitive Gαi and Gαo shifts the SS curve of VGSC in the hyperpolar direction, and activation of Gαi and Gαo by thrombin further enhances this hyperpolarization (Ma et al., 1994).

[0008] Furthermore, in umami and sweet GPCR receptors (Type I taste receptors, "T1Rs" gene family) present in nerve cells, Gαi binds to T1R1 / T1R3 (Husted et al., 2017) and T1R2 / T1R3 (Yang et al., 2020), suggesting that the action of T1R GPCRs on VGSCs is also mediated by Gαi. In addition to the effects of the G protein α subunit on VGSC, the effects of Gβγ have also been reported. Ma et al. (1997) reported that when Gβ2γ3, Gβ1γ3, or Gβ5γ3 bind to the carboxyl end of VGSC, the inactivation of VGSC is delayed. However, this delayed inactivation effect is dependent on the Gβγ subtype, and no such effect is observed with Gβ1γ1 (Ma et al., 1997). This effect is thought to be a direct effect of Gβ2γ3 on VGSC, as the action of Gβ2γ3 can be reversed by adding adenylyl cyclase 2 and the Gβγ-binding motif, Gln-XX-Glu-Arg, which is present at the carboxyl end of VGSC. As an example of Gβγ directly acting on and regulating the activity of ion channels, the activation of a GPCR coupled with Gi / Go resulted in the activation of GIRK (G-protein activated Inwardly Rectifying K₂). + It is known to act on and regulate the activity of (Oliveira et al., 2019).

[0009] Mantegazza et al. (2005) investigated the delay in VGSC current inactivation by Gβγ for different VGSCs (Nav1.1, Nav1.2, Nav1.4, Nav1.5) and reported that the delay in inactivation by Gβγ occurred only in Nav1.1 and Nav1.2, but not in Nav1.4 and Nav1.5. Therefore, the delay in VGSC inactivation by Aβ1-42 is thought to be due to the action of a different Gβγ type or a different mechanism.

[0010] As a supplement, it has been reported that T1R3 GPCRs couple with Gαs, causing microtubule depolymerization, which in turn activates the Rho / ROCK pathway via Gα12 / 13 (Masubuchi et al., 2017), and that T1R1 / T1R3 (Husted et al., 2017) and T1R2 / T1R3 (Yang et al., 2020) both bind to Gαi.

[0011] On the other hand, in the central nervous system, numerous cases have been reported where VGSC activity is actually suppressed by phosphorylation of VGSC through activation of PKA / PKC. For example, it has been reported that VGSC is suppressed by PKA activation in muscarinic acetylcholine receptors in hippocampal pyramidal neurons (Cantrell et al. (1996)), 5-HT2a / c receptors in cortical pyramidal neurons (Carr et al. (2002)), and D1 class dopamine receptors in hippocampal pyramidal neurons and striatal medium spiny neurons (Cantrell et al. (1997); Surmeier et al. (1992)). Furthermore, it has been reported that PKC activation by activation of D2 dopamine receptors in striatal medium spiny neurons and cholinergic interneurons (Carr et al. (2003)) suppresses VGSC.

[0012] Furthermore, because VGSCs play a major role in action potential generation in both the peripheral and central nervous systems, changes in the VGSC activity threshold significantly affect nerve excitability and are deeply involved in the pathogenesis of neuropathic pain. As a typical example of neuropathic pain, it has been reported that when the anticancer drug (paclitaxel) is used as a cancer treatment, chemotherapy (paclitaxel)-induced neuropathic pain occurs because paclitaxel increases the expression of VGSCs in the nerve cell bodies of the spinal dorsal root ganglia (DRG), which are suppressed by ProTx II, a VGSC blocker specific to Nav1.7. As a result, the frequency of spontaneous firing of sensory neuronal action potentials in peripheral nerves increases, leading to neuropathic pain (Non-Patent Literature 3). Neuropathic pain is estimated to affect 6% to 17% of the general population and is associated with a reduced quality of life (QOL) and functional impairment (North, RY, et al, (2018)). In 2016, an estimated 20.4% of adults in the United States had chronic pain, and 8.0% of adults in the United States reported having chronic pain severe enough to affect their daily lives (Dahlhamer J., et al. (2016)). Statistical results in Japan showed a prevalence of 16.6% for chronic pain, and a prevalence of 3.2% for chronic pain accompanied by a reduced quality of life (Inoue, S., et al. (2017)).

[0013] Diseases and conditions such as failback surgery syndrome (FBSS), complex regional pain syndrome (CRPS), and diabetic polyneuropathy are known to cause chronic neuropathic pain syndrome (Huygen, Frank, et al. (2019)). Typical causes of neuropathic pain include peripheral neuropathy such as diabetic neuropathy and postherpetic neuralgia. Neuropathic pain in the peripheral nerves (primary sensory neurons) increases the excitability of primary sensory neurons, leading to chronic pain. This increased excitability of primary sensory neurons also affects central nervous system excitability, making the pain difficult to treat. Chronic pain and neuropathy during and after chemotherapy significantly impact patients' quality of life (QOL) (Xiao et al. (2008), Authier et al. (2009), Quintao, et al. (2019)). Chemotherapy-induced peripheral neuropathy is one of the most serious side effects of anticancer drugs, including platinum and taxane-derived drugs (oxaliplatin, cisplatin, carboplatin, paclitaxel). Due to its severity, chemotherapy-induced peripheral neuropathy can even be a factor in treatment discontinuation, and has been reported to potentially increase the risk of death (Quintao, et al. (2019)). Electrophysiological studies suggest that chronic pain and neuropathy are caused by spontaneous action potential activity observed in sensory afferent nerves, and that painful peripheral neuropathy caused by chemotherapy is due to abnormal activity of spontaneous afferent nerves.

[0014] In recent years, there has been a growing emphasis on patients' quality of life (QOL) in the medical field, and there is a general trend towards actively trying to control pain. Voltage-dependent Na in terminal nerves + Channels (VGSCs) are located in the cell membrane of sensory neurons and have the function of opening and closing in a membrane potential-dependent manner to allow ions to pass through. And VGSCs are Na + Because it is a channel with high ion selectivity and plays an important role in the generation of action potentials, it has long been known as an important drug target in the treatment of pain. In the peripheral and central nervous systems, VGSCs greatly influence nerve excitability, not only in the generation of action potentials but also in the suppression of excitability by controlling the VGSC activation threshold, and are deeply involved in the pathogenesis of neuropathic pain. Nerve cells transmit pain signals to the central nervous system using electrical pulses called action potentials, so inhibiting VGSCs can block pain. Therefore, Na + Channel (VGSC) blockers are important candidates for analgesics.

[0015] However, since VGSCs support functions such as heart contraction and nerve function, random blocking can pose a life risk. Also, there are many types of Na + channels, and because their structures are similar, it is difficult to selectively inhibit the target Na + channel in drug development targeting the Na + channel molecule itself. In particular, it is very difficult to develop a drug that can make a subtle adjustment such as restoring a slightly hypersensitive reaction to normal instead of completely inhibiting the Na + channel. That is, a system that can continuously provide a substance that specifically acts on VGSCs on peripheral nerve cells and appropriately regulates the degree of their susceptibility to activation is needed.

[0016] Also, as described above, considering that amyloid-β peptide expression increases in the brain regions affected by AD and that overexpression of amyloid precursor protein (APP) increases VGSC activity, elucidating how amyloid-β peptide enhances VGSC activity through which receptors expressed in the brain regions and by what mechanism, and finding a substance that inhibits the binding of amyloid-β peptide to the receptors that cause the enhancement of VGSC activity are expected to lead to the development of pharmaceuticals for treating or preventing the onset or exacerbation of AD.

[0017] However, according to Shakoor et al. (2017), "While we have been able to treat the symptoms of over 500 types of Alzheimer's dementia mice created through genetic engineering, the results from these mouse models have not led to treatments for human dementia." This indicates that the unknown mechanisms by which amyloid-beta peptide affects / inhibits the function of human (nerve) cells, and what its receptors are, make Alzheimer's dementia research and drug discovery difficult. For this reason, in order to effectively treat and prevent AD, it has been urgent to identify the GPCR receptor present on the surface of nerve cells in human cerebrospinal fluid (CSF) that amyloid-beta peptide acts as a ligand to activate VGSC currents and cause central nervous system hyperactivity. [Prior art documents] [Non-patent literature]

[0018] [Non-Patent Document 1] Ghatak, et al., ELife 8 (November 29, 2019). https: / / doi.org / 10.7554 / eLife.50333. [Non-Patent Document 2] Li, et al. (2002) Proceedings of the National Academy of Sciences 99, no. 7: 4692-96. https: / / doi.org / 10.1073 / pnas.072090199. [Non-Patent Document 3] Li, et al. (2018) The Journal of Neuroscience 38, no. 5: 1124-36. https: / / doi.org / 10.1523 / JNEUROSCI.0899-17.2017. [Non-Patent Document 4] Maillet, et al. (2015) Chemical Senses 40, no. 8: 577-86. https: / / doi.org / 10.1093 / chemse / bjv045. [Non-Patent Document 5] Martin, et al. (2017) Journal of Biological Chemistry 292, no. 27: 11508-30. https: / / doi.org / 10.1074 / jbc.M116.773820. [Non-Patent Document 6] Martinez, et al. (1993) Journal of Neural Transmission - Parkinson's Disease and Dementia Section 6, no. 1: 1-9. https: / / doi.org / 10.1007 / BF02252617. [Non-Patent Document 7] Mattheisen, et al. (2018) Cell Reports 23, no. 9 (May 2018): 2770-81. https: / / doi.org / 10.1016 / j.celrep.2018.04.109. [Non-Patent Document 8] Vetter, et al. (2012) Biochemical Pharmacology 83, no. 11: 1562-71. https: / / doi.org / 10.1016 / j.bcp.2012.02.022. [Non-Patent Document 9] Xu, et al. (2015) Journal of Biological Chemistry 290, no. 27 (July 3, 2015): 16619-32. https: / / doi.org / 10.1074 / jbc.M115.638932. [Overview of the Initiative] [Problems that the invention aims to solve]

[0019] This invention provides an effective method for controlling VGSC activity in nerve cells by indirectly enhancing or suppressing VGSC activity in nerve cells via T1Rs receptors, which are GPCR-type taste receptors such as sweet taste receptors (T1R2 / T1R3). Furthermore, this invention aims to prevent nerve cell death and improve epilepsy-like symptoms caused by nerve hyperactivity, thereby treating or preventing AD and ALS, by indirectly and efficiently suppressing central nervous system hyperactivity caused by enhanced voltage-gated sodium channel (VGSC) activity due to overexpression of amyloid-beta peptide (Aβ) and / or APP (Amyloid precursor protein) in the brain regions of patients who are considered one of the causes of Alzheimer's disease (AD) and ALS. In addition, this invention aims to treat or prevent neuropathic pain caused by enhanced VGSC activity in peripheral nerves by indirectly suppressing VGSC activity via T1Rs receptors and restoring the excessively active state of nerves to normal. To achieve this, the challenge is to identify GPCR-type receptors that use amyloid-beta peptide as a ligand, and that enhance VGSC currents in nerve cells via these receptors, thereby causing neuronal hyperactivity. [Means for solving the problem]

[0020] Type I taste receptors, consisting of T1R1, T1R2, and T1R3 GPCRs that sense umami and sweetness, are expressed not only in the oral cavity where taste is perceived, but also in many tissues, including nerves. In addition to T1R2 / T1R3, which sense sweetness, T1R1 / T1R3 are known to function as amino acid sensors for umami and other tastes. The inventors have discovered for the first time a novel biological phenomenon in which Type I taste receptors are activated by amino acids including umami and sweet substances, and that they regulate not taste perception, but the polymerization / depolymerization of the cytoskeleton, and voltage-gated sodium channels (VGSC) and voltage-gated potassium channels (VGKC). Furthermore, they discovered that Type I taste receptors are activated not only by amino acids and sweet substances, but also by amyloid-beta peptides, and that these are involved in the control of the polymerization state of the cytoskeleton and the regulation of VGSC and VGKC. This strongly suggests that the nerve dysfunction caused by amyloid-beta peptides, which has been reported in numerous cases of neurodegenerative diseases such as Alzheimer's disease, is likely due to Type I taste receptors (T1R1, T1R2, T1R3) acting as receptors for amyloid-beta peptides, and thus affecting nerve function and morphology. In other words, the present invention is based on these discoveries and is specifically described below.

[0021] <Alzheimer's disease and taste receptors> Perceptual abnormalities such as taste and smell have been reported in neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, as well as in neuropsychiatric disorders including ADHD and schizophrenia (Field et al., (2015)). In patients with mild cognitive impairment, olfactory discrimination disorder, in particular, where there is a lack of recognition of olfactory dysfunction, may have clinical utility as an early diagnostic marker for Alzheimer's disease. Olfactory dysfunction is observed in the early stages of Alzheimer's disease and amyotrophic lateral sclerosis (ALS), and olfactory and gustatory dysfunction is observed in the early stages of Parkinson's disease, and these have been reported to be useful as early biomarkers (Field et al. (2015)). Li et al. (2002) discovered and reported that T1R2 / T1R3 recognizes sweetness, including artificial sweeteners, and T1R1 / T1R3 recognizes umami (monosodium glutamate; MSG), and that the T1R1 / T1R3 response to MSG is enhanced by other umami substances, the 5'-ribonucleotides IMP and GMP (Non-Patent Literature 2). Meanwhile, Martin et al. analyzed the functional role of T1R3 using T1R3-KO mice and confirmed that T1R3 subunits are widely expressed throughout the brain and are abundant in the hypothalamus, hippocampus, and cortex (Non-Patent Literature 5). This suggests that the sweet taste receptor system plays an important role in central nervous tissue other than the tongue, supporting synaptic function, memory acquisition, and social behavior.

[0022] Furthermore, the inventors focused on the association between Alzheimer's disease (AD) and taste receptors, particularly sweet or umami receptors containing the T1R3 subunit, which are widely expressed in the brain and nervous system, including the hippocampus and cortex. Using human neuroblastoma SH-SY5Y (functionally expressing Nav1.2, Nav1.3, and Nav1.7; Non-Patent Literature 8) and HEK cells that stably express Nav1.5 (endogenously expressing T1R1, T1R2, and T1R3), they observed the sodium channel activation effect of taste stimulants. By reducing the expression of T1R1 and T1R3 genes through the expression of a silence vector, they confirmed that the effect of taste stimulants on sodium channel currents actually acts via taste receptor GPCRs. In SH-SY5Y cells, Nav1.7 is the most abundant mRNA expression, followed by Nav1.3 >> Nav1.2 > Nav1.4 > Nav1.5. However, the functionally expressed genes that generate voltage-gated sodium channel (VGSC) currents are Nav1.7, Nav1.2, and Nav1.3, which are TTX-sensitive (Non-Patent Literature 8).

[0023] Next, to elucidate the mechanism by which amyloid-beta (Aβ) peptide, which is overexpressed in the hippocampus of patients with Alzheimer's disease (AD) and ALS, enhances voltage-gated sodium channels (VGSCs), detailed experiments were first conducted using the Aβ25-35 peptide (ANASPEC). Experiments involving the aforementioned SH-SY5Y cells and HEK cells demonstrated that the Aβ25-35 peptide indirectly enhances sodium channel (VGSC) activity by binding to T1R3. Specifically, administering the Aβ25-35 peptide to the SH-SY5Y cells enhances the activity of VGSC Nav1.3 and Nav1.7. On the other hand, HEK Nav1.5 We confirmed that expressing a T1R3-KO vector in the cells and reducing the T1R3 expression level in the cells eliminated the Nav1.5 activity-enhancing effect of the amyloid β25-35 peptide. This indicates that the binding of Aβ25-35 peptide to the T1R3 receptor expressed in nerve cells in cerebrospinal fluid (CSF) is the cause of VGCS activation. Therefore, it strongly suggests that T1R3 binding inhibitors, such as T1R3 antagonists that competitively bind to T1R3 with Aβ25-35 peptide, can suppress the enhancement of VGCS by Aβ25-35 peptide.

[0024] Furthermore, in this invention, we have observed that other Aβ peptides besides Aβ25-35 peptides, such as Aβ1-38 (Peptide Research Institute), Aβ1-40 (Wako Pure Chemical Industries, Ltd.), and Aβ1-42 (Wako Pure Chemical Industries, Ltd.), also regulate the activity of sodium channels (VGSC) via sweet taste receptors (T1R2 / T1R3, T1R3 / T1R3) or umami taste receptors (T1R1 / T1R1, T1R1 / T1R3). Furthermore, the inventors recently demonstrated that the effects of these Aβ peptides on VGSCs cease in cells in which T1R3 expression is silenced (Data not shown). This suggests that, not only in the case of Aβ25-35, whose binding to T1R3 has been confirmed, but also in the case of other Aβ peptides, the regulation of VGSC activity is due to binding to T1R3, or at least the presence of T1R3 is essential for the regulation of VGSC activity by Aβ peptides. For example, even if the effect of Aβ peptides (such as Aβ1-42) on umami receptors (T1R1 / T1R1, T1R1 / T1R3) is due to binding to T1R1, it can be inferred that the loss of the effect of T1R3 on umami receptors prevents Aβ peptides from binding to T1R1, or that the transmission of information from bound T1R1 to VGSC is impaired, resulting in the inhibition of VGSC activity regulation by Aβ peptides. Since all four Aβ peptides used in the above experiment contain the Aβ25-35 sequence (Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met: Sequence ID No. 1), it can be said that the Aβ25-35 sequence in Aβ peptides is the T1R3 binding activity region, and that Aβ peptides containing this sequence are highly likely to have T1R3 binding activity. In that case, understanding the physical relationship between this amino acid sequence structure and T1R3 will elucidate the mechanism of action of Aβ peptides on T1R3, which is expected to provide important information for the search for compounds and peptides that further inhibit the action of Aβ peptides on T1R3. In particular, the latter half of the Aβ25-35 sequence (Ala-Ile-Ile-Gly-Leu-Met) has hydrophobic side chains except for Gly, and therefore does not participate in hydrogen bonding that binds ligand to receptor (O'Hara et al. (1993), Hampson et al. (1999)). Therefore, the first half of the Aβ25-35 sequence, Gly-Ser-Asn-Lys-Gly, especially Ser-Asn-Lys, is considered to be important for the binding of Aβ25-35 to T1R3.

[0025] As described above, the present invention is the first to discover that "Type I taste receptors (T1R1, T1R2, T1R3) can act as receptors for amyloid-beta peptides," which makes it possible to develop drugs targeting amyloid-beta peptide receptors. One major reason why mouse models of Alzheimer's dementia have not been useful in developing drugs for human dementia is thought to be species differences in the sensitivity of Type I taste receptors to umami and sweet ligands. In fact, mice have significantly different umami and sweet sensitivities than humans, while only great apes such as chimpanzees, gorillas, orangutans, cercomas, baboons, and rhesus monkeys have similar umami and sweet sensitivities to humans (Li et al. (2011), Toda et al. (2021)). Therefore, it can be said that it is impossible to perform in vivo drug discovery in animals other than the great apes mentioned above without replacing the Type I taste receptors with human-type receptors for drug screening. Therefore, the cells used in in vitro experiments in this invention are limited to human-derived cells, animal-derived cells that endogenously express human-type Type I taste receptors, or cells that express Type I taste receptors having a human-specific amino acid sequence through gene transfer or other means.

[0026] <Sweet taste receptor (T1R2 / T1R3, or T1R3 / T1R3) binding substance> As candidate inhibitors of Aβ25-35 peptide binding to T1R3, we investigated the effects of lactisole, a known T1R3 antagonist that binds to T1R3 and has a sweetness-inhibiting effect, and its structural analogues, chlorofibrate and dichlorprop ((R)-2-(2,4-dichlorophenoxy)propanoic acid:DichlorProp), on VGSC using the aforementioned T1R3-expressing cells. We confirmed that all of them suppressed VGSC activity. Furthermore, we confirmed that dichlorprop was the most effective in suppressing VGSC compared to lactisole and clofibrate. IC of these compounds along with experiments on the inhibitory effect of sweetening 50The results showed that lactizol > clofibrate > dicloprop, with dicloprop inhibiting sodium channels at the lowest concentration. These compounds exhibit VGSC inhibitory effects through T1R3 antagonist-like action. Similar results were obtained in an experimental system (Non-Patent Literature 4) that compared the degree of sucralose-induced response inhibition using an overexpression system of T1R2 / T1R3 and G16-gust44, as shown in the experimental system, using intracellular calcium assay. Jiang et al. reported that lactitol binds to TMD3, TMD5, and TMD6 of TR3 (Jiang et al. (2005)). This suggests that, like sweeteners, it may limit the transformation of T1R3 to its activated structure by amyloid beta, an agonist of T1R3.

[0027] Next, we investigated the effects of the T1R2 / T1R3 receptors, which sense sweetness, on enhancing or inhibiting VGSC activity. We discovered that T1R2 / T1R3 receptors, which sense sweetness, have enhanced VGSC activity when exposed to sweet-tasting amino acids found in CSF, such as L-glutamine (L-Gln) and L-serine (L-Ser). Martinez et al. have reported that there are changes in the concentrations of many amino acids in cerebrospinal fluid in Alzheimer's disease and vascular dementia (Non-Patent Literature 6). In particular, Smith et al. measured the amount of amino acids in the cerebrospinal fluid of patients with epileptic seizures complicated by dementia (12 people) and patients with epileptic seizures only (10 people) compared to healthy individuals (23 people), and found that the L-glutamine concentration was (497) in healthy individuals. + In contrast to patients with only epileptic seizures (715 + 145 nmol / ml), patients with epilepsy complicated by dementia (733 nmol / ml) + A statistically significant increase was reported at 165 nmol / ml (Smith et al. (1985)). This report suggests that changes in amino acid concentration in cerebrospinal fluid (CSF) may be influencing neuronal hyperactivity in conditions such as epilepsy and dementia. From the above, it can be concluded that, in the case of sweet amino acids such as glutamine and serine, contrary to the T1R3 antagonists such as lactisol mentioned above, they are a group of substances that exhibit a VGSC-enhancing effect via the T1R2 / T1R3 receptor, similar to Aβ25-35 peptides, suggesting the possibility that they are T1R2 / T1R3 agonists, but at the same time, it is also suggested that they may be T1R1 / T1R3 agonists.

[0028] Substances that enhance or inhibit VGSCs via "T1Rs" receptors such as sweet taste receptors (T1R2 / T1R3, T1R3 / T1R3) In this invention, substances that enhance VGSC (sodium channel) current by acting on sweet taste receptors (T1R2 / T1R3) are referred to as "Group 1," which includes substances such as glutamine and serine, which are sweet amino acids. The substances belonging to "Group 1" are as follows: "Group 1": In addition to sweet amino acids such as serine and glutamine, agonist-like activity that acts on sweet taste receptors (T1R2 / T1R3) and amplifies VGSC current is also observed with sodium lactate. According to Shirahata et al. (2005), the strength of the binding relationship between Ankyrin G and VGSC may be a key player in controlling the left and right shifts of the IV curve. Therefore, it is possible that glutamine, serine, etc., promote the polymerization of microfilaments via T1R2 / T1R3, strengthening their binding to Ankyrin G and resulting in the enhancement of VGSC.

[0029] "Group 2": This group consists of peptides that act on sweet taste receptors (T1R2 / T1R3), including amyloid-beta (Aβ) 25-35 peptides, which are considered one of the substances that cause dementia. Similar to "Group 1," these peptides have agonist-like activity that amplifies VGSC currents.

[0030] "Group 3": This group consists of artificial sweeteners that act on sweet taste receptors (T1R2 / T1R3) and exhibit antagonist-like activity that suppresses VGSC current. Among artificial sweeteners, saccharin and aspartame act only on T1R2, while sucralose, which has a similar structure to sucrose, has been reported to act on both T1R2 and T1R3 (Neiers et al. (2016)). We have confirmed that overexpression of T1R2 has an effect on VGSC similar to that caused by artificial sweetener stimulation. In summary, in the T1R2 / T1R3 heterodimer, stimulating T1R3 leads to VGSC enhancement, while stimulating T1R2 leads to inhibition. In other words, it is highly probable that the substances belonging to "Group 1" and "Group 2" stimulate T1R3 in the T1R2 / T1R3 group, while the substances belonging to "Group 3" stimulate T1R2.

[0031] "Group 4": Substances that act on umami receptors (T1R1 / T1R3) include nucleotides (NMP: nucleoside monophosphate) such as glutamic acid and monosodium glutamate (MSG), known as amino acid seasonings, and IMP (inosine phosphate) and GMP (guanosine phosphate), known as nucleic acid seasonings. Of these, MSG delays the inactivation of VGSC current, while IMP transiently enhances VGSC current, and subsequently, the IV curve shifts by approximately 10 mV towards a negative potential over time. Similar changes to VGSC are also observed in MSG / IMP mixtures. GMP enhances VGSC on its own but does not affect the inactivation process or the properties of the IV curve. Note that IMP and GMP, used as nucleic acid-based umami seasonings, have a structure in which the 5' position is phosphorylated. Therefore, to avoid confusion, they are referred to as inosinic acid and guanylic acid, respectively, in this specification.

[0032] Unlike the Aβ25-35 peptide, which acts as a T1R3 agonist, amyloid-beta (Aβ) 1-42 peptide (12.5 nM-1 μM) acts on the T1R1 / T1R3 umami receptor, similar to the effects of MSG and IMP, and belongs to "Group 4". It delays the inactivation of VGSC and shifts the activation threshold in the hyperpolarization direction by 5-10 mV. In experiments using Nav1.5, the effect of Aβ1-42 in delaying the inactivation process was weaker than that of MSG and very similar to that of IMP. It is equivalent to the effect of MSG in SH-SY5Y.

[0033] When we observed the effects of saccharin and aspartame, artificial sweeteners that act on sweet T1R2 / T1R3 receptors, on VGSCs, we found that the degree of VGSC reduction differed depending on the artificial sweetener, and there was considerable variability between cells. The mechanism of action of artificial sweeteners in suppressing VGSCs differs from that of lidocaine and other substances that directly inhibit VGSC channels. Instead, it involves shifting the steady-state (SS) inactivation curve to the left (shifting the potential towards the negative direction), thereby reducing VGSC availability (the number of VGSCs that can be activated by potential stimulation) and decreasing the number of activated VGSCs. Since intercellular variability was observed in the experimental results of VGSC suppression with artificial sweeteners, we considered the possibility that there was intercellular variability in the expression levels of T1R2 / T1R3 receptors and attempted to overexpress T1R2 / T1R3 receptors, but no improvement in intercellular variability was observed. Therefore, when we overexpressed only T1R2, we confirmed the suppression of VGSC current based on the shift of the steady-state inactivation curve (SS inactivation curve) towards the negative voltage direction, similar to the experimental results described above. This confirms that in cell types that express a large amount of T1R2, T1R2 can be a drug target for VGSC suppression. These T1R2 binding inhibitors belong to "Group 3".

[0034] Unlike sweet substances, umami substances, including monosodium glutamate (MSG), are detected by umami receptors (T1R1 / T1R3), which are amino acid sensors. In addition to enhancing the umami effect of monosodium glutamate (MSG), inosine phosphate (IMP) and guanosine phosphate (GMP), which are also known as umami substances, are known to stimulate umami receptors (T1R1 / T1R3) on their own. In this invention, in order to investigate the possibility that umami receptors (T1R1 / T1R3) function as amino acid sensors in CSF and regulate VGSC activity, the effect of umami receptors (T1R1 / T1R3) endogenously expressed in HEK cells or SH-SY5Y cells activated by monosodium glutamate (MSG), IMP (inosine phosphate), and GMP (guanosine phosphate) on VGSC activity was confirmed using the patch-clamp method. As a result, we discovered that umami substances, depending on their type, have the effect of enhancing VGSC current, shifting the threshold potential for VGSC activation in the direction of hyperpolarization (approximately 10 mV), and slowing the deactivation rate of VGSC current via T1R1. This demonstrates for the first time that VGSC, like taste perception, is a downstream effector, as umami substances control VGSC activity (Example 4, Figure 35, Figures 36A, B, Figure 36C). Furthermore, it was confirmed that the effect of amyloid-β(Aβ)1-42 peptides on VGSC differs from the effect of amyloid-β(Aβ)25-35 peptides in that they also act on umami receptors (T1R1 / T1R3), and that this effect is similar to that of MSG and IMP (Figure 37). Furthermore, when we investigated the effects of lysine and aspartic acid, which are found to be at elevated concentrations in CSF of Alzheimer's disease patients compared to healthy individuals, on VGSCs, we confirmed that they, like MSG, delay the inactivation of VGSCs (Data not shown).

[0035] We observed that when umami receptor (T1R1 / T1R3) expressing cells were pre-treated with artificial sweeteners such as aspartame to suppress VGSC, and then treated with monosodium glutamate (MSG) or MSG2.5 (MSG, IMP, GMP mixed composition: Ajinomoto®), the suppression of VGSC current by aspartame, etc., was partially reversed (the VGSC IV curve was shifted to the right (+ direction)). Furthermore, we found that this effect of monosodium glutamate on T1R1 occurs independently of the effect of "Group 3" substances such as aspartame that inactivate VGSC via T1R2, and therefore functions antagonistically to the inactivation effect of VGSC via T1R2. In other words, the VGSC-enhancing effect of umami substances such as monosodium glutamate, mediated through T1R1, is considered to be an independent effect on VGSCs, separate from the effects mediated through T1R2 and T1R3. This is supported by the observation that the effect of MSG2.5 on T1R1 was observed in experiments using HEK-Nav1.5 cells in which T1R3 was silenced, similar to that observed in control cells. Substances that target these umami receptors (T1R1 / T1R3) are called "Group 4".

[0036] As will be discussed later, at high concentrations, Aβ25-35 peptide inhibits VGSC current. In other words, the VGSC current enhancement effect that occurs at low concentrations of Aβ25-35 peptide disappears at high concentrations and returns to the control level. This is the same phenomenon observed when T1R2 is activated by stimulation with artificial sweeteners. While it is necessary to suppress the enhancement of VGSC current (neuronal hyperactivity) caused by low concentrations of Aβ25-35 peptide, the suppression of VGSC current by high concentrations of Aβ25-35 peptide corresponds to a point where nerves are unable to transmit information. In that case, the effect of monosodium glutamate, etc., on VGSC via T1R1 (the effect of shifting the VGSC activation threshold in the negative direction) shifts the membrane potential threshold required for VGSC activation in the hyperpolarization direction compared to before T1R1 activation, bringing it closer to the resting membrane potential. As a result, VGSC can be activated by a small shift in membrane potential depolarization from the resting membrane potential. As a result, action potentials are more easily generated, which may play an important role in partially restoring function.

[0037] In terms of the effects of Aβ peptides, Aβ25-35 and Aβ1-42 exhibit completely different behaviors. Furthermore, even focusing solely on Aβ25-35, high concentrations can cause desensitization, indicating that consistent VGSC-enhancing activity is not always observed. In other words, identifying the type and concentration of Aβ peptide is essential. Aβ1-42 has been reported as a major peptide in the nervous system (Agarwal et al., 2011, Lue et al., 1999, Mo et al., 2015), suggesting that it is a major cause of disease symptoms such as Alzheimer's disease (AD). Similar to MSG, Aβ1-42 delays VGSC inactivation, and the degree of this delay increases significantly in a concentration-dependent manner, resulting in VGSCs remaining continuously open. The ratio of Aβ1-40 to Aβ1-42 has been reported as a diagnostic indicator for Alzheimer's disease. According to these reports, Aβ1-42 levels in CSF are lower in Alzheimer's patients than in healthy individuals (Agarwal et al., 2011, Lue et al., 1999, Mo et al., 2015). In this case, Aβ1-40 is thought to shift the VS curve of VGSC in the hyperpolarization direction, thereby reducing neuronal activity. Furthermore, it is clinically known that as AD progresses, the Aβ1-40 / Aβ1-42 ratio in CSF changes, with an increase in Aβ1-40 (Lehmann et al. (2020)). Lue et al. (1999) measured soluble and insoluble Aβ1-40 and Aβ1-42 concentrations in the entorhinal cortex, a component of the medial temporal lobe memory system where lesions of Alzheimer's disease are observed from the early stages, and the superior frontal gyrus, which occupies approximately one-third of the lateral surface of the brain's frontal lobe and is responsible for higher-order cognitive functions. They compared these concentrations in healthy individuals and those with Alzheimer's disease. The results showed that while Aβ1-42 levels decreased in cerebrospinal fluid in Alzheimer's disease patients, Aβ1-42 concentrations, like Aβ1-40, increased in the entorhinal cortex and superior frontal gyrus, both soluble and insoluble. Particularly noteworthy is the detection of soluble Aβ1-42 in the entorhinal cortex and superior frontal gyrus, which were not detected in healthy individuals. The following is a summary (Table 1) of the section from Lue et al. (1999) comparing the concentrations of soluble and insoluble Aβ1-40 and Aβ1-42 measured in the entorhinal cortex and superior frontal gyrus of healthy individuals and patients with Alzheimer's disease.

[0038] [Table 1]

[0039] In addition to the above, Aβ1-42 also tends to suppress outward potassium currents, as seen in the series of compounds in "Group 4," so it is highly likely that its activity will be suppressed not only in nerve cells but also in cardiomyocytes. In that case, in cardiomyocytes as well, voltage-gated calcium channels will be activated, and at the same time, sodium / calcium (Na) will be activated to eliminate excess intracellular sodium. + / Ca 2+ The reversal of the ion exchange mechanism (NCX) leads to calcium-dependent cell death by transporting calcium into the cell. Aβ1-42 administration causes depolymerization of microtubules (Pianu et al. (2014)) (Figure 33), and Aβ25-35 causes depolymerization of microfibrils (Figure 34). The loss of the cytoskeleton is also thought to contribute to the progression of cell death.

[0040] Since the VGSC effect of MSG or a solution of MSG and IMP or GMP could not be confirmed in experiments using cells with silenced T1R1 receptors, it can be confirmed that the effect is mediated by umami receptors (T1R1 / T1R3) and not by the glutamate GPCR receptor (metabotropic glutamate receptor). The effect of MSG in slowing the inactivation rate of VGSCs via umami receptors is thought to have a physiologically important role. Specifically, this property of inducing delayed inactivation is important for the expression of high-frequency action potentials and burst action potentials, and for the integration and accumulation of inputs from multiple synapses. Therefore, when treating neuronal activity and synaptic integration, T1R1 / T1R3 or T1R1 homodimers are effective targets for therapeutic drug development. Vanderstichele et al., (2016) reported a measured concentration of Aβ1-40 in the cerebrospinal fluid of Alzheimer's disease (dementia) patients of 1.9 μM, and Lehmann et al., (2020) also reported a concentration of Aβ1-40 in the cerebrospinal fluid of 2.85 μM.

[0041] Furthermore, according to Mattheisen et al. (2018), cinacalcet, an agonist of the calcium-sensitive receptor (CaSR), and NPS-2143, an antagonist of CaSR, have an inhibitory effect on VGSCs, and this inhibitory effect was induced via an unknown GPCR different from "CaSR". The present inventors hypothesized that the VGSC inhibitory effect of cinacalcet might also be mediated through sweet taste receptors including "T1R3". Using HEK-Nav1.5 expressing cells in which T1R3 was silenced, the effects of cinacalcet and NPS-2143 on VGSCs were observed, but no VGSC inhibitory effect was observed. This strongly suggests that cinacalcet and NPS-2143 induce an inhibitory effect on VGSCs via sweet taste receptors, including T1R3. Furthermore, when we applied cinacalcet and NPS-2143 to SH-SY5Y cells, we confirmed that they suppressed sodium channel currents in a similar manner to the experiments with HEK-Nav1.5 expressing cells. Based on the above, calcium-sensitive receptor (CaSR) agonists and antagonists such as cinacalcet and NPS-2143 can be considered a type of sweet taste receptor binding inhibitor, including T1R3, and therefore belong to "Group 3" as described above.

[0042] As mentioned above, excessive brain nerve activity, such as epileptic seizures, has been observed in AD patients (especially in the early stages) on electroencephalograms, and it has been reported that one of the main causes of this is VGSC. However, in this invention, for the first time, VGSC (Na), which is widely expressed in nerve cells, has been shown to suppress epileptic-like neuronal hyperactivity. + It was revealed that the inhibition of the channel is mediated by the sweet GPCR receptor, particularly "T1R3," and that this inhibition plays a crucial role. Furthermore, the inhibition of Na by sweetness-inhibiting substances (lactisol, clofibrate, dicloprop) is also important. + The inhibitory effect on channels (suppression of neuronal hyperactivity) was overwhelmingly stronger with dicloprop (dicloprop >> clofibrate > lactizol).

[0043] Furthermore, we observed the effect of an anti-T1R3 (229-258) antibody (anti-N-terminal T1R3 Venus Flytrap Domain (VFD, 229-258 aa) polyclonal antibody (AP16368a, Abcepta, Inc.)), which can act as a potent T1R3 binding inhibitor, on the enhancement of VGSC activity by Aβ peptides. As a result, we observed that in neurons pre-treated with the anti-T1R3 antibody, the enhancement of VGSC activity by Aβ25-35 peptides was suppressed, and the binding of Aβ peptides to neurons was also prevented. We also confirmed that the VGSC current enhanced by Aβ25-35 peptides was restored to control levels by the above-mentioned T1R3 antibody (Data not shown).

[0044] <Various T1R3 antibodies and Aβ peptide binding sites on the T1R3 protein> The three antibodies used in this invention—anti-T1R3(229-258) antibody, anti-T1R3(303-396) antibody, and anti-T1R3(400-570) antibody—are polyclonal antibodies produced using the 229-258 aa, 303-396 aa, and 400-570 aa regions of the T1R3 protein as antigens, respectively. Experimental results show that the anti-T1R3(229-258) antibody inhibits the binding of the Aβ25-35 peptide, the anti-T1R3(303-396) antibody inhibits the binding of the Aβ1-40 peptide, and the anti-T1R3(400-570) antibody inhibits the binding of the Aβ1-42 peptide to T1R3. As a result, we found that each Aβ peptide suppresses the enhancement of VGSC current via T1R3 and improves changes in cell morphology and dynamics. This indicates that each of these T1R3 antibodies, or anti-T1R3 antibodies that use as an epitope a peptide containing at least eight consecutive amino acids from the amino acid sequence of each peptide used as an epitope for these antibodies, are inhibitors of the binding of each Aβ peptide to nerve cells and other tissues, and also act as suppressors of the pathological physiological effects caused by each Aβ peptide in nerve cells and other tissues, making them promising as treatments for Alzheimer's disease, among others.

[0045] <Mechanism of Inhibition of VGSC by the T1R3 Binding Inhibitor Group (Group 3)> The action and inhibitory mechanism of DichlorProp on VGSC are very similar to those of the casein kinase inhibitor TBB (4,5,6,7-tetrabromobenzotriazole), which inhibits casein kinase 2 (CK2). Therefore, the inhibitory effect of DichlorProp via T1R3 is considered to have a similar inhibitory mechanism to that of casein kinase 2. Inhibition of CK2 by TBB inhibits the phosphorylation of VGSC, which is essential for the binding of VGSC to ankyrin G, and has the effect of inhibiting the binding to ankyrin G. Protein phosphatase also dephosphorylates VGSC in the same way as TBB. There is a report that the KCNQ2 channel, which binds to ankyrin G like VGSC, anchors both CK2 and protein phosphatase 1 to the channel itself (Kang et al., 2014). Furthermore, Pradhan et al. (2017) reported that Gβ1 binds to the catalytic subunit of protein phosphatase 1 in platelets. From this, it is considered possible that the delayed inactivation effect of VGSC by the umami receptor T1R (T1R GPCR) occurs through a mechanism that regulates the degree of phosphorylation at the ankyrin G binding site of VGSC via Gβ.

[0046] As described above, when MSG (sodium glutamate), IMP (inosine phosphate), GMP (guanosine phosphate), and MSG2.5 (mixing ratio contained in "Ajinomoto (registered trademark)": 97.5% MSG + 1.25% IMP, GMP mixture), which are well-known umami substances for the umami receptor (T1R1 / T1R3), act on the umami receptor, the sodium channel current of VGSC is activated, contrary to the sweet substances that act on the sweet receptor ((T1R2 / T1R3)). However, when sodium glutamate (MSG) is further administered to VGSC in a state where VGSC is suppressed by more than 80% by the action of DichlorProp, it was observed that a part of the inactivation curve in which the magnitude of VGSC shifted in the negative direction by the action of DichlorProp was restored. From this, it can be understood that MSG acting through T1R1 suppresses VGSC using a different pathway from DichlorProp acting through T1R3.

[0047] <Regarding the control mechanism of the VGSC activity by the T1R3 binding inhibitor> Paclitaxel (Taxol), which has an effect of stabilizing the cytoskeleton (microtubules: Microtubules), is used as an anticancer agent for the treatment of breast cancer, but it is known that neuropathic pain occurs as a side effect. Since it is strongly suggested that this pain is also caused by overactivity of the nerves, the presence or absence of the VGSC enhancing effect by Paclitaxel (Taxol) was investigated. As a result, it was also found that the enhancement of VGSC occurs by acting Paclitaxel (Taxol) on VGSC-expressing cells. This indicates that neuropathic pain, which develops as a side effect of the anticancer agent Paclitaxel (Taxol), is also considered to be caused by the enhancement of VGSC, similar to the overactivity of cranial nerves. Therefore, it is expected that the VGSC inhibitor via T1R3 has an analgesic effect on neuropathic pain. That is, it suggests that T1R3 binding inhibitors such as DichlorProp are effective for the treatment of neuropathic pain.

[0048] <Consideration of the control mechanism of T1R3 binding inhibitor on VGSC activity> Based on the above, the control mechanism of the T1R3 binding inhibitor on VGSC activity can be considered as follows. However, the control mechanism of the T1R3 binding inhibitor on VGSC activity in the present invention is not limited to only the restriction mechanism considered below. When the sweet receptor (T1R2 / T1R3) is bound and activated by a ligand, it modifies the cytoskeleton structure, and the structural change modifies the activity of VGSC. VGSC is known to bind to the cytoskeleton via ankyrin G / spectrin. In particular, the binding of ankyrin G to VGSC is known to require phosphorylation of the ankyrin binding site, which is conserved in many voltage-gated sodium channels in the intracellular loop between domains II and III of VGSC, by CK2 (Casein kinase2) (Brechet et al. (2008), Non-Patent Document 9). Specifically, the cytoskeleton binds to VGSC via ankyrin G / spectrin, and the binding of ankyrin G to VGSC is necessary for the localization of VGSC to the cell membrane surface. This binding of ankyrin G to VGSC plays an important role in the dense expression of VGSC at the axon initial segment and Node of Ranvier of neurons. At that time, it has been reported that when ankyrin G is knocked out, the magnitude of the VGSC current decreases (Lowe et al. (2008)), and the reason has been considered to be a decrease in the expression density of VGSC. However, in this invention, we found that activation of T1R2 / T1R3 does not reduce the number of VGSCs themselves, but rather inactivates VGSCs. That is, while reports on knockout of ankyrin G have concluded that the decrease in VGSC current is due to a decrease in VGSC expression density, we discovered that the regulatory mechanism by the T1R2 / T1R3 receptor controls VGSC current not only by the actual number of VGSCs on the cell membrane surface, but also by reducing the number of activating VGSCs through inactivation. In this invention, we have discovered that dephosphorylation of VGSCs by a CK2 inhibitor (such as TBB) not only affects the degree of VGSC expression on the cell membrane, but also acts on the properties of VGSCs functioning on the cell membrane, thereby inhibiting their function.

[0049] The binding site for ankyrin G in VGSCs has been identified, and it is known that the presence / degree of phosphorylation of this site by casein kinase 2 (CK2) controls the degree of VGSC expression in the cell membrane. Experiments in which TBB, a CK2 inhibitor, was administered revealed that TBB-induced inhibition of CK2 suppresses VGSC. Thus, it was discovered that CK2 inhibitors also suppress VGSC, and therefore have an analgesic effect. Considering the VGSC-enhancing effect of paclitaxel (Taxol), a microtubule stabilizer that constitutes the cytoskeleton, and the inhibition of CK2, which controls the binding strength between the cytoskeleton and ankyrin G, it was found that taste receptors regulate VGSC through the cytoskeleton (mainly microtubule-ankyrin G). From the above, we were able to confirm the mechanism by which T1R3 binding inhibitors enhance and suppress VGSCs.

[0050] As described above, it was confirmed that all compounds that bind to sweet and umami GPCR receptors (T1R3, T1R2, and T1R1), including T1R3 binding inhibitors, are substances that are involved in the regulation (inhibition and enhancement) of VGSC activity via sweet and umami GPCR receptors. Table 2 shows these compounds categorized by their agonist-like and antagonist-like effects on the target T1Rs and VGSC currents.

[0051] [Table 2]

[0052] Among these compounds (Table 2), in particular, dicloprop, a binding inhibitor to the T1R3 sweet taste receptor, and anti-T1R3 antibodies were found to have therapeutic and preventive effects on AD by preventing the binding of Aβ peptides to nerve cells in the brains of AD patients and preventing hyperexcitability of nerves mediated by VGSCs. Furthermore, it was confirmed that these compounds also have potential effects as therapeutic agents for neuropathic pain, thus completing the present invention. Furthermore, artificial sweeteners such as aspartame, which bind to T1R2 or T1R2 / T1R3 and suppress VGSC current, are also expected to have a similar therapeutic effect because they can suppress nerve hyperexcitability. Furthermore, although the primary binding site of the Aβ1-42 peptide is T1R3, it also partially acts on T1R1. Therefore, we confirmed the potential for glutamic acid, sodium glutamate, and nucleic acid seasonings that bind to T1R1 to be used as therapeutic agents or for screening purposes by utilizing the inhibitory activity of Aβ1-42 on VGSC activity via T1R1.

[0053] <Effects of artificial sweetener, aspartame, on changes in cell morphology> The effect of the artificial sweetener aspartame, a T1R2 / T1R3 agonist, on cell morphology was investigated (Figure 53-1). As shown in the example of MSG stimulation in Figure 33B, not only Aβ peptides but also taste substances control the polymerization state of cytoskeletal fibers. In this experiment, undifferentiated SH-SY5Y cells were treated with 120 μM aspartame, and changes in cell morphology were observed. As a result, it was observed that T1R2 / T1R3 stimulation by the artificial sweetener affected cell morphology and caused contraction. Considering this in parallel with the results of patch-clamp experiments, it is thought that umami and sweet substances control cell morphology through T1R receptors.

[0054] <Effect of the concentration ratio of Aβ42 / Aβ40 on cell morphological changes> Both Aβ1-42 and Aβ1-40 are present in cerebrospinal fluid or blood, and it is known that the concentration ratio of Aβ42 / Aβ40 varies depending on the presence or absence of cognitive impairment in Alzheimer's dementia. A correlation is observed between the ratio of Aβ1-42 being 10% or less and the presence of cognitive impairment. In the Aβ42 / Aβ40 ratio with cognitive impairment in Alzheimer's dementia (AD-D), cells shrink, the motility of neurites is impaired, and they retract. On the other hand, when the Aβ42 / Aβ40 ratio is high and the concentration of Aβ1-42 is high, except for the physiological morphological changes of the cells, no shrinkage is observed, and Ruffling, a wavy movement, is observed at the periphery of the cells (Figure 55). Similarly, a change similar to Ruffling of neurites (Lamellipodia) was also observed by β-alanine (Figure 53-2). That is, at the Aβ42 / Aβ40 ratio of healthy individuals, cells are activated and do not suffer from harmful effects such as cell shrinkage. Therefore, cell-cell junctions (capillaries, blood-brain barrier, glomerular membrane of the kidney) are maintained, and the movement of cell processes is activated, so the plasticity of nerve function is retained or improved. On the contrary, at the Aβ42 / Aβ40 ratio with cognitive impairment in Alzheimer's dementia, cells shrink, and as a result, cell-cell junctions become weak, causing basic changes in many diseases including chronic nephritis. Similarly, in the nerves, in addition to the shrinkage of cell bodies and neurites, the plasticity of nerves decreases due to the decrease in the motility of neurites.

[0055] That is, the present invention includes the following aspects. 〔1〕One aspect of the present invention relates to the use as a target receptor for controlling the active current of a voltage-dependent ion channel (for example, voltage-gated sodium channel (VGSC), voltage-gated potassium channel (VGKC), or voltage-gated calcium channel (VGCC)) of a TypeI taste receptor present on the cell surface (particularly, on the surface of nerve cells, cardiomyocytes, or skeletal muscle cells), and is described as an invention of a method as follows. In this context, the target cells are human-derived cells or cells expressing human-type Type I taste receptors (such as primate-derived cells or cells derived from humanized experimental animals) that require control of the active current of voltage-gated ion channels on the surface of their cell membrane. Furthermore, while all cells that fire VGSC-dependent action potentials are included in this study, skeletal muscle cells, in particular, show more T1R3 RNA messages than nerves and cardiomyocytes (normalized transcripts per million (nTPM) averaged across samples for RNA expression is 1.1 for skeletal muscle cells, 0.9 for cardiomyocytes, 0.9 for the cerebellum, 0.7 for the cerebral cortex, 0.4 for the hippocampus, and 0.4 for the kidneys). In addition, the presence of Aβ peptides in skeletal muscle cells has been confirmed (Kuo et al. 2000), and muscle weakness and disuse atrophy have been reported in diabetes (Perry et al. 2016). Therefore, it is thought that Type I taste receptors exist in skeletal muscle-type VGSCs (Nav 1.4) in the same way as in nerves and cardiomyocytes. For the reasons stated above, skeletal muscle cells are listed on the same level as nerve cells and cardiomyocytes. Other cells that fire VGSC-dependent action potentials include neurosecretory cells and β-cells of the islets of Langerhans, which are considered to be potential targets for application of the present invention.

[0056] The present invention also includes newly added measurement methods (assays using cell morphology / motility, impedance assays, and assays using changes in intracellular calcium concentration as indicators). Furthermore, since species differences are important in research on Alzheimer's disease, chronic nephritis, and many other diseases, and therefore it is important to use humanized mice (experimental animals), this also includes the commercial use of mice with humanized T1R genes, as well as the commercial use of the Aβ peptide binding region and the extracellular region of T1R2 (targeted drug discovery). Specifically, since the T1R2 / T1R3 GPCR protein is an Aβ peptide-binding protein, drugs that can inhibit the binding of Aβ peptides to the T1R2 / T1R3 GPCR protein are highly effective in treating Aβ peptide-related diseases (such as Alzheimer's disease and chronic nephritis). Therefore, in drug discovery aimed at treating the above diseases, the T1R2 / T1R3 GPCR protein, which contains the Aβ peptide binding site, is an important drug target. Furthermore, the 229-258 (LB2) region of the VFD domain where Aβ25-35 binds, the 303-396 (LB1) region where Aβ1-40 binds, and the 400-570 region which inhibits the action of VGSC currents of Aβ1-42 are also important drug targets. Thus, in addition to the T1R2 / T1R3 GPCR protein, the T1R3 site indicated by the above amino acid sequences is also included as a drug target. Since Aβ40 mixed peptide exhibits significantly different effects in human and mouse cells regarding the enhancement of VGSC current and its impact on cell morphology, future research will require that the proteins derived from the T1R1, T1R2, and T1R3 genes be of human origin. Otherwise, experiments using mice will not yield research results applicable to human clinical practice, nor will it be possible to develop therapeutic drugs applicable to clinical practice. Therefore, it is necessary to replace the T1R GPCR genes from the host mouse with human-derived genes. This includes the development of mice with humanized T1R genes for use in drug discovery research and development by commercial companies. When performing drug discovery screening at the cellular level, we have shown that methods using changes in intracellular calcium concentration as an indicator, and methods observing changes in cell sheet impedance, are effective for primary screening. Therefore, the invention includes aspects of screening methods using the above-mentioned methods. The type of Aβ peptide, particularly the Aβ42 / Aβ40 ratio, significantly alters cell morphology and dynamics, especially the morphology and motility of neurites when observed using nerve cells. This phenomenon can be easily measured under a microscope, making it useful for screening drugs like β-alanine that restore the activity of neurites inactivated by the Aβ42 / Aβ40 ratio in Alzheimer's patients, or for functional diagnosis using the patient's CSF and serum. Therefore, aspects of this measurement method are included.

[0057] [1-1] A method for controlling the activation current of voltage-gated ion channels in nerve cells, cardiomyocytes, or skeletal muscle cells, comprising acting on the Type I taste receptors present on the cell surface with a ligand that specifically binds to a particular Type I taste receptor and exerts an antagonist-like or agonist effect. [1-2] The method according to [1-1], wherein the voltage-gated ion channel is a voltage-gated sodium channel (VGSC), a voltage-gated potassium channel (VGKC), or a voltage-gated calcium channel (VGCC). [1-3] The method according to [1-1] or [1-2], wherein the Type I taste receptor is a sweet taste receptor or an umami taste receptor, and is T1R1, T1R2, or T1R3, or a homodimer or heterodimer thereof. [1-4] The method according to [1-3], characterized in that the ligand specifically binds to T1R2 and / or T1R3, thereby suppressing the activity current of VGSC. [1-5] The method according to [1-4], wherein the ligand is a T1R3 ligand that specifically binds to T1R3 and exerts an antagonist-like effect, thereby suppressing the active current of VGSC or suppressing the active current of VGCC, and is characterized in that it is selected from an anti-human T1R3 antibody, lactisol, clofibrate and dicloprop. Here, there are typically three types of antibodies that act as anti-human T1R3 antibodies. (1) A monoclonal or polyclonal antibody that specifically binds to an epitope comprising at least a continuous 3-5 amino acid sequence, preferably a 5-8 amino acid sequence, more preferably an 8-10 amino acid sequence, even more preferably an 11-13 amino acid sequence, and most preferably a 13-15 amino acid sequence, in the 229-258 aa region of the human T1R3 protein, characterized in that it inhibits the binding of Aβ25-35 peptide to human T1R3 and suppresses the active current of VGSC amplified by Aβ25-35 peptide. (2) A monoclonal or polyclonal antibody that specifically binds to an epitope comprising at least a continuous 3-5 amino acid sequence, preferably a 5-8 amino acid sequence, more preferably an 8-10 amino acid sequence, even more preferably an 11-13 amino acid sequence, and most preferably a 13-15 amino acid sequence, in the 303-396 aa region of the human T1R3 protein, characterized in that it inhibits the binding of Aβ1-40 peptide to human T1R3 and suppresses the active current of VGSC amplified by Aβ1-40 peptide. (3) A monoclonal or polyclonal antibody that specifically binds to an epitope comprising at least a continuous 3-5 amino acid sequence, preferably a 5-8 amino acid sequence, more preferably an 8-10 amino acid sequence, even more preferably an 11-13 amino acid sequence, and most preferably a 13-15 amino acid sequence, in the 400-570 aa region of the human T1R3 protein, characterized in that it inhibits the binding of Aβ1-42 peptide to human T1R3 and suppresses the active current of VGSC amplified by Aβ1-42 peptide. [1-6] The method according to [1-4], wherein the ligand is a T1R2 or T1R2 / T1R3 ligand that specifically binds to T1R2 or T1R2 / T1R3 and exerts an antagonist-like effect, thereby suppressing the active current of VGSC or suppressing the active current of VGCC, and is characterized in that the ligand is selected from artificial sweeteners such as saccharin, aspartame, and sucralose, and structural analogs such as Gymnema tea extract. [1-7] The method according to [1-3], characterized in that the ligand specifically binds to T1R3, T1R2 / T1R3, or T1R1 / T1R3 and exerts an agonist-like effect, thereby amplifying the activity current of VGSC. [1-8] The method according to [1-7], wherein the ligand is a sugar or a sweet amino acid. [1-9] The method according to [1-8], wherein the ligand is selected from glucose, sucrose, fructose, glutamine, serine, and mixtures thereof. [1-10] The method according to [1-3] for controlling the active current of intracellular VGSC and / or VGKC by having the ligand specifically bind to T1R1 and exert an agonist-like or antagonist-like effect. [1-11] The method according to [1-10], wherein the ligand is selected from glutamic acid (L-Glu), sodium glutamate (MSG), inosine phosphate, guanylate, and mixtures thereof.

[0058] [2] Another aspect of the present invention is an invention relating to the ligand (substance) itself used in the invention of the "method" described above. The invention relating to the ligand substance is described as follows.

[0059] [2-1] A ligand substance for controlling the activation current of voltage-gated ion channels in nerve cells, cardiomyocytes, or skeletal muscle cells, characterized in that it is a Type I taste receptor ligand that specifically binds to Type I taste receptors present on the cell surface and exerts agonist-like or antagonist-like activity. [2-2] The ligand substance according to [2-1], wherein the voltage-gated ion channel is a voltage-gated sodium channel (VGSC), a voltage-gated potassium channel (VGKC), or a voltage-gated calcium channel (VGCC), and the Type I taste receptor is a sweet taste receptor or an umami taste receptor, which is T1R1, T1R2, or T1R3, or a homodimer or heterodimer thereof. [2-3] The ligand substance according to [2-2], characterized in that the ligand specifically binds to T1R2 and / or T1R3, thereby suppressing the activity current of VGSC. [2-4] The ligand substance according to [2-3], characterized in that the ligand specifically binds to T1R3 and exerts an antagonist-like effect, thereby suppressing the active current of VGSC, and is a T1R3 ligand selected from an anti-human T1R3 antibody, lactisol, clofibrate, and dicloprop. [2-5] The ligand substance according to [2-3], characterized in that the ligand specifically binds to T1R2 or T1R2 / T1R3 and exerts an antagonist-like effect, thereby suppressing the active current of VGSC, wherein the ligand is a T1R2 ligand or T1R2 / T1R3 ligand selected from saccharin, aspartame, sucralose, and Gymnema tea extract and their structural analogs. [2-6] The ligand substance according to [2-2], characterized in that the ligand specifically binds to T1R3, T1R2 / T1R3, or T1R1 / T1R3 and exerts an agonist-like effect, thereby amplifying the active current of VGSC or VGCC. [2-7] The ligand substance according to [2-6], wherein the ligand is a sugar or a sweet amino acid. [2-8] The ligand substance according to [2-7], wherein the ligand is selected from glucose, sucrose, fructose, glutamine, serine, and mixtures thereof. [2-9] The ligand substance according to [2-2], wherein the ligand is a T1R1 ligand that specifically binds to T1R1 or T1R1 / T1R3 and exerts an agonist-like effect, thereby shifting the VGSC activity current to the left of the IV curve, delaying inactivation, or suppressing the VGKC activity current. [2-10] The ligand substance according to [2-9], wherein the T1R1 or T1R1 / T1R3 ligand is selected from glutamic acid (L-Glu), sodium glutamate (MSG), inosine phosphate (IMP), guanylate (GMP), and mixtures thereof.

[0060] [3] A further aspect of the present invention relates to a pharmaceutical composition for the prevention or treatment of various diseases caused by the amplification of VGSC activity current and / or the suppression of VGKC activity current caused by the binding of sweet amino acids, which are ligands (agonists) of T1R2 / T1R3, or umami substances, which are ligands (agonists) of T1R1 / T1R3, to T1R2 / T1R3 or T1R1 / T1R3 present on the cell surface (particularly the surface of nerve cells, cardiomyocytes, or skeletal muscle cells). In particular, it addresses neurological diseases associated with hyperactivity of nerve cells caused by the amplification of VGSC activity current due to the binding of sweet amino acids glutamine and serine, which have been confirmed to be present in CSF, and umami substances glutamic acid and its sodium salt (MSG), as well as nucleic acid seasonings IMP and GMP to T1R2 / T1R3 or T1R1 / T1R3.

[0061] [3-1] A pharmaceutical composition for preventing and / or treating diseases caused by amplification of VGSC current and / or suppression of VGKC current resulting from the binding of sweet amino acids or umami substances to T1R2 / T1R3 or T1R1 / T1R3 on the surface of nerve cells, cardiomyocytes, or skeletal muscle cells, wherein the pharmaceutical composition comprises a ligand as an active ingredient that specifically binds to T1R2 or T1R3, or their homodimer or heterodimer, and exerts an antagonist-like effect. [3-2] The pharmaceutical composition according to [3-1], wherein the disease is a neurological disease and / or neuropathic pain disorder in which an epileptic hyperactivity state is observed. [3-3] The pharmaceutical composition according to [3-2], wherein the neurological disease in which the epileptic-like neuronal hyperactivity state is observed is a neurological disease selected from the group consisting of Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), Lewy body dementia, focal cranial nerve symptoms of cerebrovascular disease, epilepsy, Parkinson's disease, progressive supranuclear palsy, multiple system atrophy, focal cranial nerve symptoms in an area damaged by trauma, Huntington's disease, spinocerebellar degeneration, and multiple sclerosis. [3-4] The pharmaceutical composition according to [3-2], wherein the neuropathic pain disorder is neuropathic pain associated with nerve hyperactivity due to amplification of VGSC current, and is selected from the group consisting of peripheral neuropathy caused by anticancer drug administration, failback surgery syndrome (FBSS), complex regional pain syndrome (CRPS), and diabetic polyneuropathy. [3-5] The pharmaceutical composition according to [3-1] or [3-2], wherein the ligand is a ligand that specifically binds to T1R3 and exerts an antagonist-like effect, and is selected from anti-human T1R3 antibody, cinacalcet, NPS-2143, lactizol, clofibrate, dicloprop, and structural analogs thereof. Here, the following three types of antibodies are typical candidates for anti-human T1R3 antibodies in pharmaceutical compositions. (1) A monoclonal or polyclonal antibody that specifically binds to an epitope comprising at least a continuous 3-5 amino acid sequence, preferably a 5-8 amino acid sequence, more preferably an 8-10 amino acid sequence, even more preferably an 11-13 amino acid sequence, and most preferably a 13-15 amino acid sequence, in the 229-258 aa region of the human T1R3 protein, characterized in that it inhibits the binding of sweet amino acids or umami substances to human T1R3 and suppresses the active current of VGSC amplified by sweet amino acids or umami substances. (2) A monoclonal or polyclonal antibody that specifically binds to an epitope comprising at least a continuous 3-5 amino acid sequence, preferably a 5-8 amino acid sequence, more preferably an 8-10 amino acid sequence, even more preferably an 11-13 amino acid sequence, and most preferably a 13-15 amino acid sequence, in the 303-396 aa region of the human T1R3 protein, characterized in that it inhibits the binding of sweet amino acids or umami substances to human T1R3 and suppresses the active current of VGSC amplified by sweet amino acids or umami substances. (3) A monoclonal or polyclonal antibody that specifically binds to an epitope comprising at least a continuous 3-5 amino acid sequence, preferably a 5-8 amino acid sequence, more preferably an 8-10 amino acid sequence, even more preferably an 11-13 amino acid sequence, and most preferably a 13-15 amino acid sequence, in the 400-570 aa region of the human T1R3 protein, characterized in that it inhibits the binding of sweet amino acids or umami substances to human T1R3 and suppresses the active current of VGSC amplified by sweet amino acids or umami substances. [3-6] The pharmaceutical composition according to [3-1] or [3-2], wherein the ligand is a ligand that specifically binds to T1R2 or T1R2 / T1R3 and exerts an antagonist-like effect, and is selected from saccharin, aspartame, sucralose, and Gymnema tea extract and structural analogs thereof.

[0062] [4] A further aspect of the present invention relates to a pharmaceutical composition for the prevention or treatment of various diseases caused by the amplification of the active current of VGSC and / or the suppression of the active current of VGKC, which occurs when an Aβ peptide (the shortest Aβ peptide is the Aβ25-35 peptide (SEQ ID NO: 1), and others such as Aβ1-38 (SEQ ID NO: 2), Aβ1-40 (SEQ ID NO: 3), and the typical Aβ1-42 (SEQ ID NO: 4), which are ligands (agonists) of Type I taste receptors, binds to Type I taste receptors (particularly T1R2 / T1R3 or T1R3) present on the cell surface (particularly the surface of nerve cells, cardiomyocytes or skeletal muscle cells). In particular, this invention relates to a pharmaceutical composition for the prevention or treatment of neurological diseases in which neuronal hyperactivity is observed, caused by the amplification of the active current of VGSC resulting from the binding of the Aβ25-35 peptide, a ligand (agonist) of T1R2 / T1R3, to T1R2 / T1R3 present on the surface of nerve cells.

[0063] [4-1] A pharmaceutical composition for the treatment and / or prevention of a disease caused by hyperexcitability of nerve cells resulting from the binding of an Aβ peptide containing the amino acid sequence shown in "Sequence ID 1" to Type I taste receptors on the surface of nerve cells, wherein the active ingredient is a ligand that specifically binds to T1R1, T1R2, or T1R3, or their homodimer or heterodimer, and exerts an antagonist-like effect. [4-2] A pharmaceutical composition for the treatment and / or prevention of a disease caused by hyperexcitability of nerve cells via T1R2 / T1R3 by any Aβ peptide selected from Aβ25-35 peptide, Aβ1-38 peptide, and Aβ1-40 peptide, comprising as an active ingredient a ligand that specifically binds to T1R2 or T1R3, or its homodimer or heterodimer, and exerts an antagonist-like effect. [4-3] The pharmaceutical composition according to [4-1] or [4-2], wherein the disease is a neurological disease and / or neuropathic pain disorder in which an epileptic hyperactivity state is observed. [4-4] The pharmaceutical composition according to [4-3], wherein the neurological disease in which the epileptic-like hyperactivity state is observed is a neurological disease selected from the group consisting of Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), Lewy body dementia, focal cranial nerve symptoms of cerebrovascular disease, epilepsy, Parkinson's disease, progressive supranuclear palsy, multiple system atrophy, focal cranial nerve symptoms in an area damaged by trauma, Huntington's disease, spinocerebellar degeneration, and multiple sclerosis. [4-5] The pharmaceutical composition according to [4-3], wherein the neuropathic pain disorder is neuropathic pain associated with nerve hyperactivity due to amplification of VGSC current, and is a neuropathic pain disorder selected from the group consisting of peripheral neuropathy caused by anticancer drug administration, failback surgery syndrome (FBSS), complex regional pain syndrome (CRPS), and diabetic polyneuropathy. [4-6] The pharmaceutical composition according to [4-1] or [4-2], wherein the ligand is a ligand that specifically binds to T1R3 and exerts an antagonist-like effect, and is selected from anti-human T1R3 antibody, cinacalcet, NPS-2143, lactisol, clofibrate, dicloprop, and structural analogs thereof. Here, the following three types of antibodies are typical candidates for anti-human T1R3 antibodies in pharmaceutical compositions. (1) A monoclonal or polyclonal antibody that specifically binds to an epitope comprising at least a continuous 3-5 amino acid sequence, preferably a 5-8 amino acid sequence, more preferably an 8-10 amino acid sequence, even more preferably an 11-13 amino acid sequence, and most preferably a 13-15 amino acid sequence, in the 229-258 aa region of the human T1R3 protein, characterized in that it inhibits the binding of Aβ25-35 peptide to human T1R3 and suppresses the active current of VGSC amplified by Aβ25-35 peptide. (2) A monoclonal or polyclonal antibody that specifically binds to an epitope comprising at least a continuous 3-5 amino acid sequence, preferably a 5-8 amino acid sequence, more preferably an 8-10 amino acid sequence, even more preferably an 11-13 amino acid sequence, and most preferably a 13-15 amino acid sequence, in the 303-396 aa region of the human T1R3 protein, characterized in that it inhibits the binding of Aβ1-40 peptide to human T1R3 and suppresses the active current of VGSC amplified by Aβ1-40 peptide. (3) A monoclonal or polyclonal antibody that specifically binds to an epitope comprising at least a continuous 3-5 amino acid sequence, preferably a 5-8 amino acid sequence, more preferably an 8-10 amino acid sequence, even more preferably an 11-13 amino acid sequence, and most preferably a 13-15 amino acid sequence, in the 400-570 aa region of the human T1R3 protein, characterized in that it inhibits the binding of Aβ1-42 peptide to human T1R3 and suppresses the active current of VGSC amplified by Aβ1-42 peptide. [4-7] The pharmaceutical composition according to [4-1] or [4-2], wherein the ligand is a ligand that specifically binds to T1R2 or T1R2 / T1R3 and exerts an antagonist-like effect, and is selected from saccharin, aspartame, sucralose, and Gymnema tea extract and structural analogs thereof.

[0064] [5] A further aspect of the present invention relates to a method for screening substances that can control the amplification or suppression of the activation current of voltage-gated ion channels induced via Type I taste receptors on the surface of human nerve cells, cardiomyocytes, or skeletal muscle cells. Here, nerve cells, cardiomyocytes, or skeletal muscle cells are cells that express voltage-gated ion channels along with Type I taste receptors on their cell surface. Typical voltage-gated ion channels include voltage-gated sodium channels (VGSC), voltage-gated potassium channels (VGKC), or membrane voltage-gated calcium channels (VGCC).

[0065] [5-1] A method for screening substances capable of controlling the amplification or suppression of the activation current of voltage-gated ion channels induced via Type I taste receptors on the surface of nerve cells, cardiomyocytes, or skeletal muscle cells, comprising the following steps (1) to (3); (1) A step of culturing cultured human-derived cells that express voltage-gated ion channels on their cell surface along with Type I taste receptors by administering a fluorescently labeled known Type I taste receptor ligand to the culture medium. (2) A step in which the test substance is administered to the culture medium, or not administered, and the cultures are performed simultaneously with, or before or after, step (1). (3) If it is observed or measured that the fluorescence intensity on the surface of cultured cells is significantly amplified or decreased when the test substance is administered compared to when it is not administered, the test substance is selected as a candidate substance capable of controlling the voltage-gated ion channel current on the surface of nerve cells, cardiomyocytes, or skeletal muscle cells. [5-2] The method according to [5-1], characterized in that the control of amplification or inhibition of the activation current of the voltage-gated ion channel is amplification of VGSC current and / or inhibition of VGKC current, the Type I taste receptor is T1R1, T1R2, or T1R3, or a homodimer or heterodimer thereof, and the cultured human-derived cells used in step (1) are cultured human nerve cells, cultured human cardiomyocytes, or human-derived cultured cells that forcibly express VGSC and / or VGKC. Here, "human-derived cultured cells that forcibly express VGSC and / or VGKC" refers to cells possessing endogenous Type I taste receptors, such as human HEK cells. The same applies hereafter. [5-3] The method according to [5-1], wherein the Type I taste receptor is T1R2 / T1R3, the cultured human-derived cells used in step (1) are cultured human nerve cells or human-derived cultured cells that forcibly express VGSC, and the known Type I taste receptor ligand administered to the culture medium in step (1) is Aβ25-35 peptide. [5-4] A method for screening substances that can control the amplification of VGSC current and / or the suppression of VGKC current, which are generated when Aβ peptides, sweet amino acids, or umami substances bind to Type I taste receptors on the surface of human nerve cells, cardiomyocytes, or skeletal muscle cells, comprising the following steps (1) to (4); (1) Using the patch-clamp method (membrane potential fixation method), measure the VGSC and / or VGKC currents of cultured human nerve cells expressing Type I taste receptors on the cell surface, cultured human cardiomyocytes, or human-derived cultured cells that forcibly express VGSC and / or VGKC, and establish control values. (2) A step of administering the Aβ peptide or sweet amino acid or umami substance to the cell culture medium, measuring the VGSC and / or VGKC currents of the cells using the same method as in step (1), and measuring the amount of amplification of the VGSC current and / or suppression of the VGKC current caused by the binding of the Aβ peptide or sweet amino acid or umami substance to the Type I taste receptors. (3) Next, administer the test substance to the cell culture medium and measure the VGSC and / or VGKC current of the cells using the same method as in step (1). (4) If the measured values ​​of VGSC and / or VGKC current measured in step (3) show a value that significantly inhibits either or both of the amplification amount of VGSC current and / or the inhibition amount of VGKC current measured in step (2), the test substance is evaluated as a substance capable of controlling the amplification of VGSC current and / or the inhibition of VGKC current via Type I taste receptors of the Aβ peptide or sweet amino acid or umami substance in human nerve cells or cardiomyocytes. [5-5] A method for screening substances that can control changes in the properties of action potentials (magnitude, width, and firing frequency of action potentials) resulting from the amplification of VGSC current and / or suppression of VGKC current, which occur when Aβ peptides, sweet amino acids, or umami substances bind to Type I taste receptors in human nerve cells, cardiomyocytes, or skeletal muscle cells, the method comprising the following steps (1) to (4); (1) Using the patch clamp method (current clamp mode), NanoTouch method, or MEA method, measure the spontaneous action potential of cultured human nerve cells, cultured human cardiomyocytes, or human-derived cultured cells that forcibly express VGSC and / or VGKC, which are cells that spontaneously or in response to external stimuli and express Type I taste receptors on their cell surface, and / or record the waveform thereof, and establish a control value. (2) A step of administering the Aβ peptide, sweet amino acid, or umami substance to the cell culture medium, and measuring and / or recording the waveform of the action potential induced by the binding of the Aβ peptide, sweet amino acid, or umami substance to Type I taste receptors using the same method as in step (1). (3) Next, administer the test substance to the culture medium of step (2), and measure and / or record the waveform of the action potential induced by the administration of the test substance using the same method as in step (1). (4) If the measured value of the frequency of action potentials or the waveform shape measured in step (3) shows a significant change compared to the frequency of action potentials or the waveform shape measured in step (2), the test substance is evaluated as a candidate substance capable of controlling the amplification of VGSC current and / or the suppression of VGKC current via Type I taste receptors for the Aβ peptide or sweet amino acid or umami substance in human nerve cells or cardiomyocytes. [5-6] A method for screening substances that can control the modification of the cytoskeleton caused by the binding of Aβ peptides, sweet amino acids, or umami substances to Type I taste receptors in human nerve cells, comprising the following steps (1) to (4) (1) A step of culturing cultured human nerve cells or human-derived cultured cells expressing Type I taste receptors on the cell surface in a culture medium containing a fluorescent reagent, measuring the intracellular calcium concentration using the fluorescent reagent by an intracellular calcium assay method, and establishing a control value. (2) A step of administering the Aβ peptide, sweet amino acid, or umami substance to the cell culture medium and measuring the intracellular calcium concentration which has increased due to the binding of the substance to Type I taste receptors, using the same method as in step (1). (3) Next, the test substance is administered to the culture medium of step (2), and the intracellular calcium concentration is measured in the same manner as in step (1). (4) If the intracellular calcium concentration measured in step (3) is significantly lower than the calcium concentration measured in step (2), the test substance is evaluated as a substance that activates Type I taste receptors in nerve cells and causes a change in intracellular calcium concentration ([Ca2+]i), and is selected as a candidate substance capable of controlling the modification of the cytoskeleton via Type I taste receptors by the Aβ peptide, sweet amino acids, or umami substances in human nerve cells. [5-7] A method for screening substances capable of controlling the amplification of VGSC current and / or the suppression of VGKC current, which are generated when Aβ peptides, sweet amino acids, or umami substances bind to Type I taste receptors in human nerve cells or cardiomyocytes, comprising the following steps (1) to (4); (1) A step of forming a cell sheet from cultured human nerve cells, cultured human cardiomyocytes, or human-derived cultured cells that forcibly express VGSC and / or VGKC, which are cells that express Type I taste receptors on their cell surface, and measuring the changes in the cytoskeleton and intercellular adhesion of the cultured cells as the resistance on the cell sheet by impedance method, and establishing a control value. (2) A step in which the Aβ peptide, sweet amino acid, or umami substance is administered to the cell culture medium, and the changes in the cytoskeleton and intercellular adhesion caused by the binding of the substance to Type I taste receptors are measured as resistance on the cell sheet in the same manner as in step (1); (3) Next, a test substance is administered to the cell culture medium, and the resistance on the cell sheet is measured in the same manner as in step (1); (4) If it is confirmed that the change in cell sheet resistance is significantly greater when the test substance is administered than when it is not administered, the test substance is selected as a candidate substance capable of controlling the amplification of VGSC current and / or the suppression of VGKC current caused by structural changes in the cytoskeleton (microfibrils) and / or changes in intracellular calcium that occur via the Aβ peptide or sweet amino acid or umami substance's Type I taste receptor. Other screening methods include the following: [5-8] A method for screening compounds that modify / inhibit the effects of Aβ peptides, sweet amino acids, or umami substances on VGSC and VGKC activity via T1Rs in CSF, (1) Human epithelial cells are cultured in a sheet form after being treated with Aβ peptide, sweet amino acids, or umami substances on Type I taste receptors, and the impedance (resistance) values ​​at both ends of the cell sheet are measured. (2) Next, measure the impedance value when the test substance is applied to the cells. (3) If a decrease in impedance value is observed due to the action of the test substance, the barrier function in the cell sheet is evaluated as having deteriorated, and the test substance is selected as a candidate compound capable of controlling the amplification of VGSC current and / or the suppression of VGKC current via Type I taste receptors of the Aβ peptide, sweet amino acid, or umami substance. [5-9] A method for evaluating cell barrier function (degree of cell adhesion) mediated by T1Rs based on the difference in the ratio of Aβ42 / 40 contained in CSF or serum, (1) Measure the impedance (resistance) values ​​at both ends of a cell sheet cultured with human vascular epithelial cells. (2) Next, the Type I taste receptors of the cells are treated with a test substance such as Aβ peptide, sweet amino acids, or umami substances, and the impedance value of the cell sheet is measured. (3) A step to evaluate whether or not a decrease in impedance value occurred due to the action of the test substance, and whether or not there is a pathological effect on the barrier function in the cell sheet.

[0066] [6] A further aspect of the present invention relates to a method for screening candidate compounds for active ingredients of a pharmaceutical composition for preventing and / or treating diseases caused by amplification or inhibition of the activation current of voltage-gated ion channels mediated via Type I taste receptors on the surface of human nerve cells, cardiomyocytes, or skeletal muscle cells.

[0067] [6-1] A method for screening candidate compounds for active ingredients of a pharmaceutical composition for preventing and / or treating diseases caused by the amplification of VGSC current and / or inhibition of VGKC current resulting from the binding of Aβ peptide, sweet amino acids, or umami substances to Type I taste receptors in human nerve cells or cardiomyocytes, comprising the following steps (1) to (4); (1) Using the patch-clamp method (membrane potential fixation method), measure the VGSC and / or VGKC currents of cultured human nerve cells expressing Type I taste receptors on the cell surface, cultured human cardiomyocytes, or human-derived cultured cells that forcibly express VGSC and / or VGKC, and establish control values. (2) A step of administering Aβ peptide, sweet amino acids, or umami substances to the culture medium; (1) A step of measuring the VGSC and / or VGKC currents of the cells using the same method as in (1), and measuring the amount of amplification of the VGSC current and / or the amount of suppression of the VGKC current from the control value; (3) Next, administer the test substance to the cell culture medium and measure the VGSC and / or VGKC current of the cells using the same method as in step (1). (4) If the measured values ​​of VGSC and / or VGKC current measured in step (3) show a value that significantly inhibits either or both of the amplification amount of VGSC current and / or the inhibition amount of VGKC current measured in step (2), the test substance is evaluated as a substance capable of controlling the amplification of VGSC current and / or the inhibition of VGKC current via Type I taste receptors of Aβ peptides, sweet amino acids, or umami substances in human nerve cells or cardiomyocytes, and is selected as a candidate compound for the active ingredient of the pharmaceutical composition. [6-2] A method for screening candidate compounds for active ingredients of a pharmaceutical composition for preventing and / or treating diseases caused by the amplification of VGSC current and / or the suppression of VGKC current resulting from the binding of Aβ peptide, sweet amino acids, or umami substances to Type I taste receptors in human nerve cells or cardiomyocytes, comprising the following steps (1) to (4); (1) Using the patch-clamp method (current-clamp mode), NanoTouch method, or MEA method, measure the action potentials of cultured human nerve cells, cultured human cardiomyocytes, or cultured human cells that forcibly express VGSC and / or VGKC, which are cells that express Type I taste receptors on their cell surface and can fire action potentials spontaneously or in response to external stimuli, record the magnitude, width, and firing frequency of the waveform, and establish control values. (2) A step of administering an Aβ peptide, a sweet amino acid, or an umami substance to the cell culture medium, and measuring and / or recording the waveform width of the action potential induced by the binding of the Aβ peptide, sweet amino acid, or umami substance to a Type I taste receptor using the same method as in step (1). (3) Next, administer the test substance to the culture medium of step (2), and measure and / or record the waveform width of the action potential induced by the administration of the test substance using the same method as in step (1). (4) If the measured value of the frequency of action potentials or the shape of the waveform width measured in step (3) shows a significant change compared to the frequency of action potentials or the shape of the waveform width measured in step (2), the test substance is evaluated as a substance capable of controlling the amplification of VGSC current and / or the suppression of VGKC current via Type I taste receptors for the Aβ peptide or sweet amino acid or umami substance in human nerve cells or cardiomyocytes, and is selected as a candidate compound for the active ingredient of the pharmaceutical composition. [6-3] A method for screening candidate compounds for active ingredients of a pharmaceutical composition for preventing and / or treating diseases caused by cytoskeletal modification resulting from the binding of Aβ peptides, sweet amino acids, or umami substances to Type I taste receptors in human nerve cells, comprising the following steps (1) to (4); (1) A step of culturing cultured human nerve cells or human-derived cultured cells expressing Type I taste receptors on the cell surface (e.g., HEK cells) in a culture medium containing a fluorescent reagent, measuring the intracellular calcium concentration using the fluorescent reagent by an intracellular calcium assay method, and establishing a control value. (2) A step in which the Aβ peptide, sweet amino acid, or umami substance is administered to the cell culture medium, and the intracellular calcium concentration, which has increased due to the binding of the substance to Type I taste receptors, is measured using a fluorescent reagent in the same manner as in step (1). (3) Next, the test substance is administered to the culture medium of step (2), and the intracellular calcium concentration is measured using a fluorescent reagent in the same manner as in step (1). (4) If the intracellular calcium concentration measured in step (3) is significantly lower than the calcium concentration measured in step (2), the test substance is evaluated as a substance capable of activating Type I taste receptors in nerve cells to cause a change in intracellular calcium concentration ([Ca2+]i), and capable of controlling the modification of the cytoskeleton via Type I taste receptors by the Aβ peptide, sweet amino acids, or umami substances in human nerve cells, and is selected as a candidate compound for the active ingredient of the pharmaceutical composition. Furthermore, in experiments measuring intracellular calcium changes or staining the cytoskeleton, since these are reactions that occur prior to VGSC regulation, human-derived cultured cells (HEK cells) that express Type I taste receptors that do not express VGSC on their cell surface can be used. [6-4] A method for screening candidate compounds for active ingredients of a pharmaceutical composition for preventing and / or treating diseases caused by the amplification of VGSC current and / or inhibition of VGKC current resulting from the binding of Aβ peptides consisting of Aβ25-35, Aβ1-38, Aβ1-40, or Aβ1-42 to Type I taste receptors in human nerve cells or cardiomyocytes, comprising the following steps (1) to (3); (1) A step of confirming that fluorescently labeled actin or tubulin is expressed in cultured human nerve cells, cultured human cardiomyocytes, or human-derived cultured cells that express Type I taste receptors on their cell surface, or that VGSC and / or VGKC are forcibly expressed in the cell culture medium, and that the Aβ peptide selected from Aβ25-35, Aβ1-38, Aβ1-40, and Aβ1-42 binds to the Type I taste receptors, causing the cytoskeleton of the cells to polymerize and emit fluorescence in a fibrous manner. (2) In parallel with step (1), the Aβ peptide is administered to the cell culture medium that has been previously administered with the test substance, and the degree of depolymerization of the fluorescently labeled fibrous cytoskeleton by the Aβ peptide is observed. (3) If the degree of depolymerization of the cytoskeleton observed in step (2) significantly suppresses the degree of depolymerization of the cytoskeleton compared to the control value in step (1), the test substance is evaluated as a candidate compound of a substance with high inhibitory activity against the amplification of VGSC current and / or suppression of VGKC current by the Aβ peptide, and selected as a candidate compound of the active ingredient of the pharmaceutical composition. Alternatively, instead of expressing fluorescently labeled actin or tubulin, methods such as staining actin with rhodamine phalloidin staining or using an anti-tubulin antibody as the primary antibody and a fluorescently labeled secondary antibody can also be used. [6-5] A method for screening substances that can control the amplification of VGSC current and / or the suppression of VGKC current, which are generated when Aβ peptides, sweet amino acids, or umami substances bind to Type I taste receptors in human nerve cells or cardiomyocytes, comprising the following steps (1) to (4); (1) A step of forming a cell sheet from cultured human nerve cells, cultured human cardiomyocytes, or human-derived cultured cells that forcibly express VGSC and / or VGKC, which are cells that express Type I taste receptors on their cell surface, and measuring the changes in the cytoskeleton and intercellular adhesion of the cultured cells as resistance values ​​on the cell sheet by impedance method, and establishing control values. (2) The process involves administering the Aβ peptide, sweet amino acid, or umami substance to the cell culture medium and measuring the changes in the cytoskeleton and intercellular adhesion caused by the binding of the substance to Type I taste receptors as a resistance value on the cell sheet, in the same manner as in step (1); (3) The process then involves administering the test substance to the cell culture medium and measuring the resistance on the cell sheet in the same manner as in step (1); (4) If it is confirmed that the change in cell sheet resistance is significantly greater when the test substance is administered than when it is not administered, the test substance is selected as a candidate substance capable of controlling the amplification of VGSC current and / or the suppression of VGKC current caused by structural changes in the cytoskeleton (microfibrils) and / or changes in intracellular calcium that occur via the Aβ peptide or sweet amino acid or umami substance's Type I taste receptor. Here, as with the nanoTouch method, the impedance method completely covers the electrode surface in a sheet-like manner, so the impedance of the cell sheet can be measured by the change when a current is applied between the ground placed in the extracellular fluid and the electrode covered by the cells (a conductive material such as conductive glass). When a constant current (I) is applied, from V=IR (Ohm's law), if the impedance (resistance R) increases, the voltage (V) increases, and if R decreases, since I is constant, the output voltage V decreases. [6-6] A method for screening candidate compounds for active ingredients of a pharmaceutical composition for preventing and / or treating diseases caused by the amplification of VGSC current and / or the suppression of VGKC current resulting from the binding of Aβ peptide, sweet amino acids, or umami substances to Type I taste receptors in human nerve cells or cardiomyocytes, comprising the following steps (1) to (4); (1) Using the patch-clamp method (membrane potential fixation method), measure the VGSC and / or VGKC currents of cultured human nerve cells expressing Type I taste receptors on the cell surface, cultured human cardiomyocytes, or human-derived cultured cells that forcibly express VGSC and / or VGKC, and establish control values. (2) A step of administering Aβ peptide, sweet amino acids, or umami substances to the culture medium, measuring the VGSC and / or VGKC currents of the cells using the same method as in step (1), and measuring the amount of amplification of the VGSC current and / or suppression of the VGKC current from the control value. (3) Next, administer the test substance to the cell culture medium and measure the VGSC and / or VGKC current of the cells using the same method as in step (1). (4) If the measured values ​​of VGSC and / or VGKC current measured in step (3) show a value that significantly inhibits either or both of the amplification amount of VGSC current and / or the inhibition amount of VGKC current measured in step (2), the test substance is evaluated as a substance capable of controlling the amplification of VGSC current and / or the inhibition of VGKC current via Type I taste receptors of Aβ peptides, sweet amino acids, or umami substances in human nerve cells or cardiomyocytes, and is selected as a candidate compound for the active ingredient of the pharmaceutical composition. [6-7] A method for screening candidate compounds for active ingredients of a pharmaceutical composition for preventing and / or treating diseases caused by the amplification of VGSC current and / or the suppression of VGKC current resulting from the binding of Aβ peptide, sweet amino acids, or umami substances to Type I taste receptors in human nerve cells or cardiomyocytes, comprising the following steps (1) to (4); (1) A step of measuring the action potential and / or recording the waveform width of cultured human nerve cells, cultured human cardiomyocytes, or cultured human cells that forcibly express VGSC and / or VGKC, which are cells that express Type I taste receptors on their cell surface and can fire action potentials spontaneously or in response to external stimuli, and establishing a control value, using the patch clamp method (current-clamp mode), NanoTouch method, or MEA method, or a method using a fluorescent membrane potential-sensitive reagent or a fluorescent intracellular calcium-sensitive reagent. (2) A step of administering an Aβ peptide, a sweet amino acid, or an umami substance to the cell culture medium, and measuring and / or recording the waveform width of the action potential induced by the binding of the Aβ peptide, sweet amino acid, or umami substance to a Type I taste receptor using the same method as in step (1). (3) Next, administer the test substance to the culture medium of step (2), and measure and / or record the waveform width of the action potential induced by the administration of the test substance using the same method as in step (1). (4) If the measured value of the frequency of action potentials or the shape of the waveform width measured in step (3) shows a significant change compared to the frequency of action potentials or the shape of the waveform width measured in step (2), the test substance is evaluated as a substance capable of controlling the amplification of VGSC current and / or the suppression of VGKC current via Type I taste receptors for the Aβ peptide or sweet amino acid or umami substance in human nerve cells or cardiomyocytes, and is selected as a candidate compound for the active ingredient of the pharmaceutical composition. [6-8] A method for screening candidate compounds for active ingredients of a pharmaceutical composition for preventing and / or treating diseases caused by the amplification of VGSC current or modification of the cytoskeleton resulting from the binding of Aβ peptides, sweet amino acids, or umami substances to Type I taste receptors in human nerve cells, comprising the following steps (1) to (4); (1) A step of culturing cultured human nerve cells expressing Type I taste receptors on the cell surface, or human-derived cultured cells that have been forced to express VGSC, in a culture medium containing a fluorescent reagent, measuring the intracellular calcium concentration using the fluorescent reagent by an intracellular calcium assay method, and establishing a control value. (2) A step in which the Aβ peptide, sweet amino acid, or umami substance is administered to the cell culture medium, and the intracellular calcium concentration, which has increased due to the binding of the substance to Type I taste receptors, is measured using a fluorescent reagent in the same manner as in step (1). (3) Next, the test substance is administered to the culture medium of step (2), and the intracellular calcium concentration is measured using a fluorescent reagent in the same manner as in step (1). (4) If the intracellular calcium concentration measured in step (3) is significantly lower than the calcium concentration measured in step (2), the test substance is evaluated as a substance capable of activating Type I taste receptors in nerve cells to cause a change in intracellular calcium concentration ([Ca2+]i), and capable of controlling the modification of the cytoskeleton via Type I taste receptors or the amplification of VGSC current by the Aβ peptide or sweet amino acid or umami substance in human nerve cells, and is selected as a candidate compound for the active ingredient of the pharmaceutical composition. Here, when measuring intracellular calcium concentration, high-throughput screening using a plate reader can also be performed. This allows for procedures such as pre-treating the test substance with a control well that is treated only with Aβ peptide, sweet amino acids, or umami substances, or simultaneously administering a solution mixed with Aβ peptide, sweet amino acids, or umami substances. [6-9] A method for screening candidate compounds for active ingredients of a pharmaceutical composition for preventing and / or treating diseases caused by the amplification of VGSC current and / or inhibition of VGKC current resulting from the binding of Aβ peptides consisting of Aβ25-35, Aβ1-38, Aβ1-40, or Aβ1-42 to Type I taste receptors in human nerve cells or cardiomyocytes, comprising the following steps (1) to (3); (1) A step of confirming that fluorescently labeled actin or tubulin is expressed in cultured human nerve cells, cultured human cardiomyocytes, or human-derived cultured cells that express Type I taste receptors on their cell surface, or that VGSC and / or VGKC are forcibly expressed in the cell culture medium, and that the Aβ peptide selected from Aβ25-35, Aβ1-38, Aβ1-40, and Aβ1-42 binds to the Type I taste receptors, causing the cytoskeleton of the cells to polymerize and emit fluorescence in a fibrous manner. (2) In parallel with step (1), the Aβ peptide is administered to the cell culture medium that has been previously administered with the test substance, and the degree of depolymerization of the fluorescently labeled fibrous cytoskeleton by the Aβ peptide is observed. (3) If the degree of depolymerization of the cytoskeleton observed in step (2) significantly suppresses the degree of depolymerization of the cytoskeleton compared to the control value in step (1), the test substance is evaluated as a candidate compound of a substance with high inhibitory activity against the amplification of VGSC current and / or suppression of VGKC current by the Aβ peptide, and selected as a candidate compound of the active ingredient of the pharmaceutical composition. [6-10] A method for screening candidate compounds for active ingredients of a pharmaceutical composition for preventing and / or treating diseases caused by the amplification of VGSC current and / or inhibition of VGKC current resulting from the binding of Aβ peptide, sweet amino acids, or umami substances to Type I taste receptors in human nerve cells or cardiomyocytes, comprising the following steps (1) to (4); A method for screening substances whose properties can be controlled, comprising the following steps (1) to (4); (1) A step of forming a cell sheet from cultured human nerve cells, cultured human cardiomyocytes, or human-derived cultured cells that forcibly express VGSC and / or VGKC, which are cells that express Type I taste receptors on their cell surface, and measuring the changes in the cytoskeleton and intercellular adhesion of the cultured cells as the resistance on the cell sheet by impedance method, and establishing a control value. (2) A step in which the Aβ peptide, sweet amino acid, or umami substance is administered to the cell culture medium, and the changes in the cytoskeleton and intercellular adhesion caused by the binding of the substance to Type I taste receptors are measured as resistance on the cell sheet in the same manner as in step (1); (3) Next, a test substance is administered to the cell culture medium, and the resistance on the cell sheet is measured in the same manner as in step (1); (4) If it is confirmed that the change in cell sheet resistance is significantly greater when the test substance is administered than when it is not administered, the test substance is evaluated as a candidate substance capable of controlling the amplification of VGSC current and / or the suppression of VGKC current caused by structural changes in the cytoskeleton (microfibrils) and / or changes in intracellular calcium that occur via the Aβ peptide or sweet amino acid or umami substance's Type I taste receptor, and selected as a candidate compound for the active ingredient of the pharmaceutical composition. [6-11] A method for screening candidate compounds for active ingredients of a pharmaceutical composition for preventing and / or treating diseases caused by the amplification of VGSC current resulting from the binding of Aβ peptide to Type I taste receptors derived from human nerve cells or humanized experimental animals, the method comprising the step of observing changes in the area or volume of cultured human nerve cells or humanized mouse nerve cells. [6-12] A method for screening active ingredients of a pharmaceutical composition, wherein when the VGSC current, which is enhanced by applying the Aβ42 / 40 ratio (AD-D) observed in the cerebrospinal fluid of AD patients to Type I taste receptors of human nerve cells or humanized experimental animal-derived cells, is subsequently reduced by the application of the test substance, the test substance is evaluated as a candidate compound for an active ingredient of a pharmaceutical composition for preventing and / or treating diseases caused by the amplification of VGSC current due to the binding of Aβ peptides. [Effects of the Invention]

[0068] In this invention, we have for the first time discovered that Type I taste receptors (T1R1, T1R2, T1R3) control the properties and magnitude of voltage-gated sodium channel currents (VGSC) and voltage-gated potassium channel currents (VGKC) through the cytoskeleton. While it was previously believed that glutamate receptors (AMPA and NMDA types) play a crucial role in the memory formation mechanism in the hippocampus, this discovery suggests that voltage-gated ion channels, in addition to glutamate receptor dynamics, may also play an important role in neuroplasticity and memory formation. This indicates that Type I taste receptors are effective drug targets for the treatment of memory disorders such as dementia. First, T1R1, which functions as a receptor for umami substances such as glutamate (and partially acts on Aβ1-42), can be used to identify potential therapeutic agents by searching for its agonists and positive allosteric modulators (PAMs). Next, T1R2 acts as a receptor for artificial sweeteners and suppresses voltage-gated sodium channel currents via microfilaments, making it a promising drug target for analgesics and anticonvulsants. Furthermore, T1R3 was identified as a receptor for amyloid-beta peptides (Aβ25-35), which are of paramount importance in the clinical manifestations of Alzheimer's disease. Since Aβ25-35 amplifies VGSC currents and causes hyperactivity in nerves or myocardium, T1R3 antagonists and other T1R3 binding inhibitors of Aβ25-35 are promising candidates for the treatment of Alzheimer's disease. As described above, in this invention, we have found that Type I taste receptors (T1Rs) are receptors for amyloid-beta peptides (Aβ25-35 receptors are T1R3, and Aβ1-42 receptors are T1R1 and T1R3), and that Aβ peptides regulate VGSC and VGKC currents via T1Rs, at least in nerve cells. Therefore, we have shown that these can be drug targets to prevent or suppress the binding / deposition of amyloid-beta peptides, which cause Alzheimer's disease, ALS, and functional decline of other tissues. Furthermore, since the therapeutic agent for neuropathic pain provided by the present invention acts on VGSCs via sweet taste receptors, it is effective even under inflammatory acidic conditions, and is therefore expected to be particularly effective as a local anesthetic.

[0069] In addition, this invention investigated, using various anti-T1R3 antibodies, which region of T1R3 or T1R2 / T1R3 enhances VGSC currents for Aβ25-35, Aβ1-42, and Aβ1-40, which are among the most toxic amyloid-beta peptides. Furthermore, the effects on VGSC currents were compared using a mixture of Aβ1-42 / Aβ1-40 (Aβ42 / 40) in cerebrospinal fluid adjusted to the concentration ratio of Alzheimer's disease (AD) patients (AD-D) and healthy individuals (NDC). As a result, no significant change occurred with the action of NDC, but VGSC currents were significantly enhanced with AD-D. This suggests that the epileptic-like symptoms observed in patients with Alzheimer's disease may be caused by neuronal hyperactivity resulting from the enhancement of VGSC currents by Aβ42 / 40 (AD-D). This enhancement was restored by β-alanine. Furthermore, we found that when AD-D was applied to mouse / rat-derived neuronal-like cells, the effect of AD-D decreased, the opposite of what was observed in human cells. This suggests a fundamental reason why research findings from mice, an experimental model, are not always applicable to human clinical practice in drug discovery and research for Alzheimer's disease. Similarly, we observed morphological changes in human neuronal-like cells and confirmed that morphological changes caused by AD-D concentration are significantly involved in the onset of AD. We then found that these morphological changes could be reversed by β-alanine. In other words, observing the morphological / dynamic changes that AD patients' cerebrospinal fluid or serum affects cultured human neuronal-like cells makes it possible to determine the progression of the disease, and this strongly suggests that β-alanine or its structural analogs could be promising therapeutic agents for AD. Furthermore, electrophysiological methods were used to demonstrate that there are species differences in the effects of Aβ peptides on T1Rs. [Brief explanation of the drawing]

[0070] [Figure 1]Western blot showing endogenous T1R3 silencing in HEK293 cells: The silencing effect of T1R3 protein expression was confirmed using a T1R3 antibody. Left: Lane HEK293 Control, Right: HEK293 cells with Lane Silencing vector introduced. [Figure 2] The expression levels of T1R1, T1R2, and T1R3, which are endogenously expressed in HEK cells and SH-SY5Y cells, were confirmed by quantitative PCR. [Figure 3] Glutamine-induced VGSC enhancement: The effect of 500 μM glutamine on VGSC current in SH-SY5Y was measured using the membrane potential fixation method. The left figure shows the membrane potential fixation recordings before and after the action of 500 μM glutamine; a transient increase in downward VGSC current was confirmed. The right figure shows the results of the analysis of the left figure using an IV plot. [Figure 4] Effect of Aβ25-35 on SH-SY5Y VGSC current. The effect of 2 μM Aβ25-35 peptide on SH-SY5Y VGSC current was measured using the membrane potential fixation method. (A) Membrane potential fixation recordings before and after the action of 2 μM Aβ25-35 peptide; transient downward VGSC enhancement was confirmed after the action. (B) Results of the analysis of the results in the left figure are shown in the IV plot. [Figure 5] The Aβ25-35 peptide, like glutamine, increased the excitability of SH-SY5Y cells. Current-clamp recordings were performed using SH-SY5Y cells to confirm the effect of the Aβ25-35 peptide on action potentials. Cells were stimulated using the following current patterns: short-term stimulation (25 ms), followed by a long-term stimulation (250 ms) after a 200 ms interval. The current intensity was increased in the order of 6, 8, 10, and 12 pA. The cell membrane voltage was maintained at -80 mV by applying current as needed. (A) shows the response to each stimulus at 125 mV intervals. (B) shows four traces superimposed. [Figure 6]The effect of Aβ1-42 on HEK Nav1.5VGSC current was measured using the membrane potential fixation method. (A) Membrane potential fixation recordings before and after the action of the 74nM Aβ1-42 peptide. Transient downward VGSC did not show an enhancement effect like Aβ25-35, but showed a decreasing trend (n=4). It also showed a tendency to shift the peak of the IV plot in the direction of hyperpolarization by 5-10mV. It delayed the inactivation of VGSC (Figure 37). (B) shows the results of the IV plot analysis of the results from (A). [Figure 7] Comparison of the effect of Aβ25-35 peptide on VGSCs in control HEK Nav1.5 cells and HEK Nav1.5 cells with silenced T1R3 (A)(B) shows a comparison of VGSCs recorded by voltage clamp from control HEK Nav1.5 cells before and after Aβ25-35 administration. VGSC enhancement was confirmed with 1 μM Aβ25-35. (C)(D) shows recordings of VGSCs from HEK Nav1.5 cells with silenced T1R3. No change was observed in VGSCs even when treated with 2 μM Aβ25-35 peptide, double the control dose. (E) shows that lactisole, which specifically binds to the T1R3 TM (TransMembrane) domain, suppresses the VGSC enhancement effect of Aβ25-35. The figure above shows VGSC current under membrane potential fixation. From left to right, it shows control and VGSC enhancement by Aβ25-35. The subsequent administration of 1 mM lactisole suppressed the VGSC current enhancement effect of Aβ25-35. The bar graph below shows the changes induced by Aβ25-35 and lactisole across three experiments, with the control peak current set at 100%. The symbols represent each experiment, and the bar graph shows the relative average value of the changes. [Figure 8]HEK Nav1.5 cells cultured on coverslips were kept at room temperature for 10 to 30 minutes in a culture medium containing a 1000-fold dilution of anti-T1R3 antibody (Abcepta, Inc.). These coverslips were then transferred to HBPS solution, and the membrane potential-dependent activation process of VGSC(Nav1.5) current, specifically the steady-state inactivation property, was measured using the membrane potential fixation method and used as a control. Subsequently, Aβ25-35 peptide was added to the recording solution, and the change in VGSC current was measured using the same experimental method as above, observing the changes before and after Aβ25-35 peptide administration. In cells not pre-treated with T1R3 antibody, Aβ25-35 peptide enhanced the maximum VGSC current, but no enhancement was observed in cells pre-treated with T1R3 antibody. The change in the VGSC IV curve caused by Aβ25-35 peptide was similar to that observed when T1R2 was stimulated with artificial sweeteners, representing a shift in the direction of hyperpolarization of the steady-state inactivation curve. This suggests that the enhancement of VGSC current by the Aβ25-35 peptide occurs through T1R3. This experimental result suggests that when the Aβ25-35 peptide cannot act on T1R3 due to the T1R3 antibody, it may act on T1R2. This indicates that T1R2 may secondarily act as a low affinity binding site for the Aβ25-35 peptide. At present, there is a possibility that the Aβ25-35 peptide acts only on T1R3, or that it acts on both T1R2 and T1R3, like sucrose and sucralose. [Figure 9] The concentration-dependent amplification effect of the Aβ25-35 peptide on VGSC current was observed, and the EC50 value was 86.8 nM. [Figure 10]The perforated patch-clamp method was used to record the concentration-dependent VGSC current and membrane capacity amplification effects of the Aβ25-35 peptide via T1R3, as well as the desensitization process. The upper half of the figure shows the change in cell membrane capacity, and the lower half shows the magnitude of the VGSC current. At 120 nM Aβ25-35, both cell membrane capacity and VGSC current increased, but when the Aβ25-35 concentration was increased to 240 nM, both decreased and returned to the control level (before Aβ25-35 action). [Figure 11] Similar to (Figure 10), the concentration-dependent amplification of VGSC current and membrane capacity by Aβ25-35 peptide via T1R3 was reproduced using the perforation patch-clamp recording method. (A) Shows the increase in cell membrane capacity and VGSC current (HEKNav1.5) after the control and 12.5 nM Aβ action. With the membrane potential fixed at the top of the figure, the membrane capacity is determined by integrating the change in current waveform caused by depolarizing by 10 mV from the holding voltage. The dot pattern represents the control charge capacity, and the white area above it represents the increase after Aβ action. The VGSC current also increases along with the increase in capacity. (B) The increase in membrane capacity due to Aβ action is plotted on the time axis. The waveform on the plot represents the membrane capacity in the state where VGSC is not activated. (Left: control, Right: after Aβ action) (C) Simultaneously with the capacity measurement in (B), the change in the magnitude (increase) of VGSC current after Aβ action was measured and plotted on the time axis. [Figure 12]Comparison of VGSC current density due to the action of Aβ25-35 peptide (including experimental results in Figures 10 and 11). Bar graph: Current density (pA / pF) obtained by dividing the peak current value of VGSC by the cell membrane volume measured in the control, with the denominator being the cell membrane volume. From left to right, the bars show control, low concentration Aβ25-35, and high concentration Aβ25-35. Following this, Inserted and Retrieved show the current density of intracellular vesicles obtained by dividing the increase in VGSC current by the difference between the membrane volume measured under each experimental condition and the membrane volume obtained in the control. In other words, this indicates the increased area of ​​the cell membrane due to the fusion of the membrane component of intracellular vesicles with VGSC on their surface to the cell membrane. The current densities of VGSC derived from vesicles inserted into the cell membrane by low concentration Aβ25-35 and VGSC taken up after receptor desensitization occurred by high concentration Aβ25-35 and endocytosis from the cell membrane were almost the same. This suggests that VGSCs derived from inserted vesicles may remain within the vesicle-derived membrane rather than diffusing into the surrounding cell membrane during short-term experiments like this one. The table on the right shows the measurements from the individual experiments used to create the bar graph on the left. [Figure 13] Effect of DichlorProp on VGSC current: DichlorProp reduced the amplitude of the VGSC current. Voltage-dependent VGSC activity was recorded by applying depolarization stimuli at 10mV intervals from -80mV to 50mV, following a pre-pulse of -120mV. The inward current peak decreased in a DichlorProp concentration-dependent manner. The curve on the left of the figure shows steady-state inactivation, plotting the magnitude of the VGSC current activated by a -20mV test pulse applied after all pulses from -160mV to -120mV to 0mV against the membrane potential. The curve gradually shifts to the left with DichlorProp. [Figure 14]The slope of the steady-state inactivation curve of DichlorProp and its effect on Vh were investigated. Vh and the slope of the slope were determined using the Boltzman equation (1 / (1+exp((V50-x) / slope)). The analysis software used was CurveExpert Professional (Hyams Development) (n=4). (A) Comparison of the slopes of the curves for Control, 300, and 500 μM DichlorProp. There was no significant difference in the t-test among these three conditions. (B) Comparison of the effect on Vh. A statistically significant difference was confirmed from the measured value of 500 μM DichlorProp. It can be seen that the Vh value shifts to a more negative potential value with higher concentrations of DichlorProp. [Figure 15] Effect of TBB on VGSC current (A) TBB reduced the amplitude of the VGSC current. Voltage-dependent VGSC activity was recorded by applying depolarization stimulation at 10mV intervals from -80mV to 50mV following a pre-pulse of -120mV. The peak current of the inward current decreased in a TBB concentration-dependent manner. The left side of the figure shows the steady-state inactivation curve, plotting the magnitude of the VGSC current activated by a -20mV test pulse applied after all pulses from -170mV to -120mV to 0mV against the membrane potential. It is gradually shifted to the left by TBB. (B) Prepulse potentials are shown from top to bottom as -80mV (top), -100mV (middle), and -120mV (bottom). The magnitude of the VGSC current generated by the -20mV test stimulation pulse from the prepulse potential was recorded. The TBB concentration was increased from right to left. From left to right, the changes with Control and 10μM TBB are shown, the middle row shows 20μM TBB, and the right row shows 30μM TBB. In the top row, at -80mV, the VGSC current was almost completely suppressed by the 10μM TBB, but in the middle row, at -100mV, it was about half unaffected, and in the bottom row, at -120mV, it was more than 80% unaffected. With the 10μM TBB, the current was almost completely suppressed except in the bottom row at -120mV. [Figure 16](A) IC50 of the concentration-dependent inhibitory effect of TBB on VGSC current and Prepulse: VGSC activated by stepping from -80mV to -20mV was almost completely suppressed at 10μM, and stimulation from -120mV suppressed it by only about 50% at the TBB concentration used. Therefore, using measurement results with -100mV as the Prepulse, the TBB IC50 (15.8μM) was calculated using a Hill plot. The INa activated when stepping from -100mV to -20mV was plotted against the TBB concentration. The vertical axis represents the average amplitude of INa after TBB action, with the control INa set to 1. (B) The Stayday-state inactivation curve was analyzed using the Boltzman equation to calculate Vh before (control) and after TBB administration, and the difference was plotted against the TBB concentration. This plot was further analyzed using the Hill equation to calculate the IC50 (12.9μM). Although the inhibitory effect on TBB was analyzed using different methods (A) and (B), nearly identical IC50 values ​​were obtained. [Figure 17] VGSC current recordings were observed using the Pulse & Ramp Protocol. With the membrane potential fixed at -120mV, a -20mV square wave was initially applied to induce a transiently activated VGSC current component (A), and then a ramp waveform stimulation from -120mV to +60mV was applied to induce a slowly deactivating VGSC current component (B). Due to the CK2 inhibitor TBB effect, the amplitudes of both the transient component (A) and the slowly deactivating component (B) were suppressed (reduced) upon TBB administration, and the effect of 10mM MSG was observed. The VGSC current components suppressed by TBB, along with the VGSC currents induced by the square wave (A) and the VGSC currents induced by the ramp (B), were partially recovered. (C) The process of VGSC current enhancement by MSG is plotted on a time axis. The enhancement of VGSC current by MSG administration may have a mechanism of action independent of the inhibitory mechanism by TBB. ● indicates the VGSC current caused by rectangular wave stimulation, and ▲ indicates the VGSC current caused by a ramp. [Figure 18]Delayed effect of MSG on VGSC inactivation in the presence of TBB: The SS curve in the left figure shows the VGSC stimulation by changing the VGSC membrane potential in a step-like manner from different steady-state holding voltages to -20mV. When CK2 was suppressed by TBB, the SS curve shifted significantly in the hyperpolarization direction (leftward arrow). Subsequently, when MSG was applied, the leftward shift caused by TBB was partially recovered and a rightward shift occurred (rightward arrow). The right figure shows the trace of the actual VGSC current for which the SS curve was measured. It can be confirmed that the inactivation process is delayed by the action of MSG compared to the VGSC current treated with TBB. Comparing the current waveform after MSG action with the control waveform, it can be interpreted that the slow inactivation seen in the control waveform is enhanced. [Figure 19] Paclitaxel (Taxol), like glutamine and Aβ25-35 peptide, enhances VGSCs in HEK Nav1.5 cells. (A) Shows VGSC current under membrane potential fixation before (left control) and after (right) application of 2.4 μM Taxol. (B) IV plot of the VGSC peak current shown in (A). (C) The same experiment was performed using 1 μM Taxol. In the IV curve, two cases were created: when the stimulation pulse was applied from a Prepulse of -120 mV (●) and when the stimulation pulse was applied from -80 mV (〇). In both cases, the magnitude of the peak current was amplified by Taxol. However, although the measured values ​​for -70 mV and -80 mV Prepulse increased slightly with Taxol, the overall effect on the SS curve was limited. A detailed examination of the effect confirmed that Taxol (which stabilizes the microtubule) enhances VGSCs in HEK Nav1.5 cells, similar to glutamine and Aβ25-35 peptide. [Figure 20] Inhibitory effect of 5μM NPS-2143 on HEK Nav1.5 VGSC. (A) shows the process by which the actual VGSC current is inhibited by NPS-2143. (B) is an IT plot showing the decrease in current amplitude associated with the action of NPS-2143 along the time axis. Stimulation pulses of -120mV to -20mV were applied every 8 seconds. [Figure 21]Inhibitory effect of 5 μM cinacalcet on VGSC inward current and potassium outward current in SH-SY5Y cells. (A) shows the waveform before cinacalcet administration and the waveform after the suppression by cinacalcet is complete. (B) is an IT plot showing the decrease in current amplitude associated with the action of cinacalcet along the time axis. Stimulation pulses of -120 mV to -20 mV were applied every 8 seconds. Both VGSC inward current and potassium outward current were suppressed by cinacalcet. [Figure 22] The VGSC inhibitory effect of cinacalcet occurs via T1R3. In HEK Nav1.5 cells with silenced T1R3 expression, the inhibitory effect of cinacalcet was ineffective. (A) shows VGSC current recordings before and after cinacalcet administration. In cells with suppressed T1R3 expression, no change in VGSC current was observed due to the effect of cinacalcet. Pre-pulse -120mV, -20mV test pulse. The graph in (B) is an IT plot showing the amplitude of VGSC current and its change over time. 20μM cinacalcet was applied twice at the points indicated by the arrows. The amplitude of VGSC with a -20mV test pulse was measured when the pre-pulse was -80mV and when it was -120mV, and the change is shown. [Figure 23] The inhibitory effect of the CaSR agonist cinacalcet on VGSCs via T1R3 is inhibited depending on the polymerization state of the cytoskeleton. (A) When actin polymerization is inhibited with cytochalasin D, the inhibitory effect of cinacalcet on VGSCs is lost. The IV curve and steady-state inactivation curve of VGSC current in the presence of control, cytochalasin D, and high concentrations of cinacalcet are shown. (B) When tubulin polymerization is inhibited with colchicine, unlike the actin polymerization inhibitor cytochalasin D, the inhibitory effect of cinacalcet on VGSCs via T1R3 was observed, and no effect of colchicine was seen. From the above, it was concluded that cinacalcet inhibits VGSCs by acting on actin via T1R3. [Figure 24]Inhibitory effect of CaSR antagonist NPS-2143 on VGSC: (A) shows that when 5 μM NPS-2143 is applied, the VGSC current is slowly suppressed, similar to cinacalcet. The vertical axis shows the suppression process with the control current magnitude set to 1. The horizontal axis shows time (SEC). (B) shows the suppression process of VGSC current by NPS-2143. ● indicates the VGSC current peak, and ○ indicates the baseline level. The time course changes from left to right, similar to (A). When the suppression of VGSC current by NPS-2143 is almost complete, the baseline (leakage) current increases, but when the CaR agonist cinacalcet is applied, the baseline-leakage current decreases (B upper panel). (B lower panel) shows the peak current and baseline measured values ​​plotted from actual experimental values. [Figure 25] Inhibitory effect of cinacalcet on VGSCs under acidic conditions. (A) Cinacalcet inhibited VGSC-dependent action potentials in SH-SY5Y cells under acidic conditions of pH 6 (current-clamp). (B) The inhibitory effect on HEK Nav1.5 VGSC current using membrane potential fixation was exactly the same as under normal pH 7.4 conditions. Voltage clamp traces of the Na+ current are shown in (B above). The Na+ current was activated by repeating voltage steps from -120mV to -20mV every 8 seconds. Upon application of 5 μM cinacalcet, the Na+ current was gradually inhibited. The results are shown in the IT relationship in (C). The initial low trace amplitude corresponds to the size of the Na+ current in the raw recorded trace on the left. [Figure 26]The effect of saccharin on VGSC current. (A) Saccharin reduced the amplitude of the VGSC current in a concentration-dependent manner. Voltage-dependent VGSC activity was recorded by applying depolarization stimuli at 10mV intervals from -80mV to 50mV following a pre-pulse of -120mV. Plot (B) shows that the inward current peak current decreases in a saccharin concentration-dependent manner. The curve on the left right of the figure shows the steady-state inactivation curve, plotting the magnitude of the VGSC current activated by a -20mV test pulse applied after all pulses from -120mV to 0mV against the membrane potential. [Figure 27] The effects of T1R2 overexpression in HEK Nav1.5 cells on VGSC current were investigated by examining the IV curve to study its activation properties. When the prepulse was set to the normal -120mV stimulation pulse, the peak current of VGSC was small. However, when the membrane potential range for measuring the Stead-state inactivation curve was expanded to a negative -200mV range compared to normal recording, the maximum current amplitude of the peak current of VGSC increased by approximately 5 times. Therefore, when the prepulse was set to -140mV and -180mV and the same stimulation pulse as in the -120mV experiment was applied, the maximum peak current of VGSC increased, similar to that obtained in the Stead-state inactivation curve. From these results, it is considered that, similar to stimulation with artificial sweeteners acting on T1R2, T1R2 overexpression has a function that affects VGSC inactivation. [Figure 28]Effect of Gymnema sylvestre (GTE) on VGSC current: (A) GTE reduced the amplitude of the VGSC current. Voltage-dependent VGSC activity was recorded by applying depolarization stimulation at 10mV intervals from -80mV to 50mV, following a pre-pulse of -120mV. The peak current of the IV curve decreased by 40-50% before and after GTE action. The curve on the left of the figure shows Stead-state inactivation, plotting the magnitude of the VGSC current activated by a -20mV Test pulse applied after all pulses from -120mV to 0mV against the membrane potential. It is shifted to the left by GTE. (B) VGSC current was recorded from SH-SY5Y, Nav1.5-HEK, and NG108-15, and the Stead-state inactivation curve was plotted. Vh (the membrane potential at which the magnitude of the VGSC current is halved) was calculated from the data using the Botzman eq. described above. Vh was determined between the control and after treatment with 10 ul / ml GTE, and the difference in Vh values ​​(Vhdiff) and the difference in Slope(K) (Kdiff) were summarized in Table (B). In all cells tested, Vh shifted towards hyperpolarization, and it was confirmed that the peak current of VGSC was reduced (suppressed) to around 60%. [Figure 29] Changes in sodium current enhancement in SH-SY5Y cells pretreated with anti-T1R3 antibody: SH-SY5Y cells differentiated with retinoic acid were pretreated with anti-T1R3 antibody, similar to the HEK Nav1.5 cell experiment (Figure 8). Stimulation pulses were applied from a holding voltage of -120mV to activate voltage-dependent VGSCs, and an IV curve was created to be used as a control. Subsequently, when Aβ25-35 peptide was applied, a slight leftward shift of the IV curve was observed, similar to (Figure 8), but no increase in VGSC current occurred, as recorded from SH-SY5Y cells that had not been treated with anti-T1R3 antibody. This suggests that the involvement of T1R3 GPCR is essential for the enhancement of VGSC current by Aβ25-35 peptide. [Figure 30]Binding of fluorescently labeled Aβ25-35 peptide (FL-Aβ25-35) to SH-SY5Y cells. The left image shows the results of the Positive Control when SH-SY5Y cells were treated with the Aβ25-35 peptide. Under a fluorescence microscope, fluorescence derived from the Aβ25-35 peptide was confirmed to be bound to the cells. The right image shows the case when cells that had been pre-treated with anti-T1R3 antibody were used. Although FL-Aβ25-35 was applied for the same duration and at the same concentration as the Control, almost no fluorescence derived from FL-Aβ25-35 was observed on the cell surface. [Figure 31] The effect of Aβ25-35 on enhancing VGSC current in the presence of ratrtrunclin A, which inhibits actin polymerization. (A) Effect of ratrtrunclin A on VGSC current. The magnitude of VGSC current before and after the action of 12.6 μM ratrtrunclin A was plotted in correspondence with the stimulation pulse potential (● before ratrtrunclin A action, □ after action). (B) The effect of ratrtrunclin A was measured by applying stimulation pulses from holding voltages of -80, -100, and -120 mV to -20 mV. The inhibition by ratrtrunclin A was membrane voltage dependent, and its inhibitory effect was significantly greater at -80 mV compared to at -120 mV. (C) To investigate whether the actin polymerization state is involved in the mechanism of action of Aβ25-35, the action of 12.5 nM Aβ25-35 was observed under conditions where actin polymerization was inhibited by ratrunklin A, and the magnitude of the VGSC current was plotted against the membrane potential (□ before Aβ25-35 action, ◇ after action). Amplification of VGSC by Aβ25-35 was confirmed. The degree of amplification was more pronounced in the presence of ratrunklin A than in the absence of ratrunklin A. (D) The process of amplification of the VGSC current by Aβ25-35 in the presence of ratrunklin A is shown in time series. [Figure 32]In the presence of colchicine, no enhancement of VGSC current by Aβ25-35 was observed. (A) The VGSC current was stimulated by changing the potential of the pre-pulse of the stimulation pulse, as in the SS inactivation protocol, with a -20mV stimulation pulse. The control trace is shown on the left with the current after colchicine action, and on the right with Aβ25-35 applied in the presence of colchicine. (B) The magnitude of the VGSC current against the stimulation pulse voltage is plotted for the control, after colchicine action, and after Aβ25-35 action. In the presence of colchicine, the enhancement effect by Aβ25-35 was suppressed. (C) In the presence of colchicine, there was no change in the waveform of the VGSC current due to Aβ25-35. (D) The peak current during measurement shown in (C) is plotted against the time axis, showing that the effect of Aβ25-35 is suppressed. (E) is an example of the effect of colchicine on increasing VGSC current in cells different from those used in (A) to (D) above. [Figure 33A] When the Aβ25-35 peptide was applied, the fibrous structure of microtubules observed before application disappeared (by GFP-Tublin labeling). [Figure 33B] Applying MSG, an agonist of the umami receptor, also caused the disappearance of the microtubule fibrous structure, similar to the effect of Aβ25-35 peptide (by GFP-Tublin labeling). [Figure 34] Changes / disappearance of the fibrous structure of microfibrils due to the action of Aβ1-42 peptide and cinecalset (by GFP-Actin labeling). [Figure 35] Delay inactivation process of VGSC current by glutamic acid (MSG), an umami substance and T1R1 agonist (HEK NaV1.5 cells): When MSG is applied, the transient VGSC current inactivation process is significantly delayed. As a result, the transient VGSC current component is reduced, but at the same time, the amplitude of the small, persistent VGSC current observed following the transient VGSC is increased. The transiently activated current was investigated using rectangular wave stimulation (upper left figure), and the persistent current was investigated using a ramp waveform (upper right figure). The measured values ​​of the experiment using rectangular wave stimulation (upper left figure) are plotted in time series (current amplitude vs. time) in the figure below. [Figure 36]Effects of the umami substance glutamate (MSG), a T1R1 agonist, and Aβ1-42 on voltage-gated potassium channels. (A) Experimental results using the membrane potential fixation method with SH-SY5Y cells showed that after MSG action, in addition to a delay in the inactivation process of VGSC current, a decrease in voltage-gated potassium channel current was confirmed. The upper left figure shows the experimental results using the membrane potential fixation method before MSG administration, and the upper right figure shows the results after administration. The lower right figure shows the upward outward potassium current and the IV curve below the current trace. In the lower right figure of (A), it was confirmed that the potential was maintained at 40mV following ramp wave stimulation, and the magnitude of the potassium current was slowly suppressed / decreased. (B) Using HEK cells expressing Kv1.2 and the above ramp waveform, the effect of Aβ1-42 on potassium channels, which causes a delay in VGSC inactivation similar to MSG, was confirmed. Aβ1-42 reduced Kv1.2 potassium, similar to the effect of MSG in the recording of SH-SY5Y cells. The upper part of (B) shows the actual experimental trace, and the lower part plots the inhibition process of potassium channel current by Aβ1-42 over time. [Figure 37] The inhibitory effect of IMP and Aβ1-42 on VGSC current and the delaying effect on the inactivation process of VGSC current; (A) The upper panel shows the decrease in VGSC current observed after the action of control and 1 mM IMP. (B) The Steady-state inactivation curve and IV plot of the experimental results (A) are shown. IMP reduces VGSC current, but shows a transient enhancing effect from immediately after action to about 5 minutes later. (C) The effect of IMP on VGSC current was evaluated by a membrane potential fixation experiment using a ramp waveform (an increase in VGSC current occurs as indicated by the downward arrow, followed by a decrease in VGSC current as indicated by the upward arrow). (D) and (E) show the analysis of the effect of Aβ1-42 by a membrane potential fixation experiment similar to (A) and (B). It was confirmed that increasing the active concentration to 12.5 nM and 25 nM caused a concentration-dependent decrease in VGSC current, similar to MSG and IMP. In addition, a delay in VGSC inactivation was confirmed, similar to the case of IMP. [Figure 38]The effects of GMP on inward VGSC current and outward potassium current were investigated by whipping SH-SY5Y cells. (A) Total currents observed by membrane potential fixation are shown before (left), after (center) administration of 2 mM GMP, and after further administration of 5 mM MSG. (B) An IV curve of the inward current due to VGSC was created, and an enhancement of the VGSC current was confirmed by GMP administration. Here, not only the VGSC current but also the voltage-dependent potassium current was plotted. In addition, the Steady-state inactivation curve was also included in the same plot. Black symbols indicate the control, and white symbols indicate the change after GMP action. The VGSC current showed a slight increase and a left shift of the IV curve (downward (inward) arrow at -20mV), and the peak current of the upward (outward) potassium current showed a decrease (downward arrow at +50mV). No change was observed in the Steady-state inactivation curve. The enhancement of VGSC current by GMP alone was smaller than that by IMP, and there was variability depending on the cells recorded. The difference between the effect of GMP on VGSC current and that of IMP is that the effect of GMP is sustained. Also, the difference from MSG is that it has little effect on the inactivation process. As shown in (A), no changes in the inactivation process like those caused by MSG were observed before and after GMP administration. Therefore, we administered MSG following GMP and investigated the changes in VGSC current by creating an IV plot. It was confirmed that in the presence of GMP, MSG caused the IV curve to shift approximately 10 mV to the left (leftward arrow) compared to the control (C). [Figure 39]Effect of MSG on the VGSC current inactivation rate in the presence of artificial sweetener (saccharin): The transient activation of HEK Nav1.5 (T1R2 activated by saccharin) was investigated using rectangular wave stimulation (left), and the sustained current was investigated using a ramp waveform (right) with membrane potential fixation. The effect of 5 mM glutamate (MSG) on the kinetics of VGSC current was examined. The top row shows the current waveform before intervention (control), the middle row (30 seconds after administration), and the bottom row (10 minutes after administration) after MSG administration. The inactivation process of the transient VGSC current activated by rectangular wave stimulation was significantly delayed after MSG intervention. While the degree of delay weakened over time, the effect persisted even after 10 minutes (bottom row). The bottom row of the figure shows the potential waveform used for stimulation. [Figure 40] The effect of MSG2.5 on VGSC current in SH-SY5Y cells and HEK Nav1.5 cells. The effect of MSG2.5 on VGSC in (A) SH-SY5Y cells and (B) Nav1.5 HEK cells was investigated using the membrane potential fixation method. MSG2.5 amplified the peak VGSC current, but unlike MSG alone, its effect on the inactivation process was limited. In the upper panels of (A) and (B), the left side shows the VGSC current measured by the membrane potential fixation method before and after the effect of MSG2.5. The IV relationship of the magnitude of the peak potential relative to the membrane potential is shown on the right side of the panel. In the lower panel of (C), instead of the transient peak current of VGSC, the sustained VGSC current component 540 ms after the start of stimulation was analyzed using Nav1.5 HEK cells to see how it changes with MSG2.5. The IV relationship is shown in the center of (C). The experiment using a ramp waveform instead of the rectangular wave stimulation shown on the left of (C) is shown on the right of (C). Compared to before the action of MSG2.5, the peak temporarily shifted in the hyperpolarization direction after the action (1 minute 12 seconds later), but returned to almost the same waveform as before the action after 4 minutes. [Figure 41]The effect of MSG2.5 on VGSC current in HEK Nav1.5 cells with silenced T1R1 expression. VGSC current was recorded using membrane potential fixation. (A) In cells with silenced T1R1 expression, the change in VGSC current induced by 5 mM MSG2.5 was suppressed. (C) In the same cells as in (A), the sustained effect of MSG2.5 on VGSC current was observed and analyzed by stimulating with a ramp waveform every 8 seconds before and after MSG2.5 action. Similar to the results obtained with rectangular wave stimulation, no effect of MSG2.5 was confirmed. (B) On the other hand, in cells with silenced T1R3 expression, the effect of MSG2.5 on VGSC current was observed to be similar to the changes recorded from cells without silencing. From this, it was confirmed that T1R1 expression is essential for the effect of MSG2.5 on VGSC current, and that the presence or absence of T1R3 does not affect the effect of MSG2.5 on VGSC current. [Figure 42] Mechanisms of pain and epileptic-like symptoms due to nerve hyperactivity: In addition to the mechanisms of pain and epileptic-like symptoms caused by sweet amino acids such as amyloid-beta peptide and glutamine (L-Gln) mentioned above, this section describes the mechanism of neuropathic pain caused by chemotherapy. Chemotherapy drugs such as paclitaxel increase VGSC current by polymerizing microtubules, causing nerve hyperactivity. In addition to paclitaxel, which stabilizes microtubules, colchicine, which inhibits their polymerization, was also observed to similarly increase VGSC current. In this state, the VGSC current per nerve cell is enhanced, the action potential threshold is lowered, and action potentials are more easily generated. Furthermore, stimuli that induce a single action potential generate burst-like action potentials consisting of multiple segments. [Figure 43]Measurement of VGSC enhancement by Aβ25-35 peptide via T1R3 using the nano Charge method: Aβ25-35 peptide was added to differentiated SH-SY5Y cell culture medium, and changes in intracellular potential were measured from before addition. In the upper control group, the rate of action potential generation was low, but in the lower group, an increase in the frequency of spontaneous action potentials was observed after the action of the Aβ25-35 peptide. [Figure 44] Changes in VGSC current when Aβ1-38 and Aβ1-40 were applied to HEK Nav1.5 cells, which are cardiomyocyte-type cells: (A) Case for Aβ1-38 (B) Case for Aβ1-40. In both (A) and (B), the actual Steady-state inactivation protocol current traces using membrane potential fixation are shown in the lower panel. Control (left), after 12.5 nM Aβ peptide application (right). The upper panel shows the analyzed IV curve and Steady-state inactivation protocol curve. Here, ● indicates control, and ○ indicates after Aβ peptide application. The IV curves are measurement results when the Prepulse is -80 mV and -120 mV. Aβ1-38 shifted the Steady-state inactivation curve to the right, increasing the current from the small Prepulse of -80 mV. On the other hand, the current from -120 mV decreased. In the case of Aβ1-40, the Steady-state inactivation curve was shifted to the left, and as a result, the VGSC current decreased in both prepulses. [Figure 45]Effects of Aβ1-40 and Aβ1-42 on intracellular calcium. Changes in intracellular calcium concentration in HEK cells expressing endogenous T1R GPCRs were measured using a fluorescence microscope after administration of Aβ1-40 and Aβ1-42 peptides using a fluorescent dye (Cal-520, AAT Bioquest). (A) shows the dynamics of intracellular calcium in response to Aβ1-40, and (B) shows the dynamics in response to Aβ1-42. Aβ1-40 increased intracellular calcium. On the other hand, Aβ1-42 responded to intracellular calcium in a biphasic manner, initially decreasing slightly before increasing. Previously, when studying the taste response to T1R GPCRs using changes in intracellular calcium as an indicator, the common method involved overexpressing the T1R GPCR gene and the calcium-responsive G16 or Gustducin / G16. However, this discovery demonstrates that it is possible to measure and screen for intracellular calcium responses using only the T1R GPCR and the G protein coupled to its receptor, which are endogenously expressed in HEK cells. [Figure 46] (A) shows the ligand action of the Aβ1-40 peptide using HEK cells that stably express Nav1.5, and (B) shows the inhibitory effect of the anti-T1R3 (303-396) antibody on the Aβ1-40 peptide against T1R3 ligand binding site 2 (303-396aa), which was identified from patch-clamp experiments and inhibits the action of the Aβ1-40 peptide. (C) shows the inhibitory effect of the antibody simultaneously on the same scale to compare it with the effect of the Aβ1-40 peptide alone. The vertical axis represents impedance (resistance, kilohms), and the horizontal axis represents time (seconds). [Figure 47]Cav1.2 activity was measured by measuring changes in intracellular calcium concentration. (A) shows the actual experimental results. The vertical axis RLU (Relative Light Unit) is averaged with the strongest response set to 1. The horizontal axis is time (in seconds). The cell membrane was depolarized with 77.5 mM KCl, activating the Cav1.2 channel. At the same time, the extracellular calcium concentration was increased by 5 mM to increase the amount of calcium flowing into the cell through the Cav1.2 channel, making it easier to measure the increase in intracellular calcium. The Cav1.2 channel was activated by increasing the extracellular KCl concentration, followed by the application of Aβ1-42. (B) The hyperbolic decline equation was fitted to the response in the region unaffected by the action of Aβ1-42, showing the activation of the Cav1.2 channel specifically induced by Aβ1-42. [Figure 48] Whole-cell currents from Jurkat cells are shown in (A) and (B). (A) shows the control, and (B) shows the response after Aβ1-42 action. In (A-1) and (B-1), the downward signals indicated by arrows are VGSC currents. In (A-2) and (B-2), the time axis is enlarged to easily observe the changes caused by Aβ1-42. Here, the change in membrane potential used by the membrane potential fixation method is shown above the current response. The membrane potential was set to a holding voltage of -120mV and varied from -50mV to +50mV in 10mV steps. In (C), the inward (downward) VGSC current and the outward (upward) current measured during the last 10ms of the voltage pulse are measured and summarized in an XY plot. The horizontal axis represents the stimulation voltage, and the vertical axis represents the magnitude of the current. [Figure 49] The concentration-dependent enhancement effect of Aβ25-35 on Nav1.5 current. By changing the solvent for Aβ25-35 from ddH2O to 10 mM NaOH to maintain more Aβ25-35 in the monomer state, the EC50 value was calculated to be approximately 10 times lower (7.9 nM). [Figure 50]The upper panel of the figure shows a bright field image, and the lower panel shows a fluorescence image of Aβ1-40 bound to cells. In (A), the binding of the fluorescently labeled Aβ1-40 peptide to the cell surface can be confirmed as a fluorescence image. In (B), when cells treated with fluorescent Aβ1-40 were treated with anti-T1R3(303-396) antibody, the binding of fluorescent Aβ1-40 almost disappeared. To avoid the effect of fluorescence bleaching, areas that had not been previously irradiated with fluorescence were observed. [Figure 51] (A) Nav1.6 VGSC current was recorded using the patch clamp method in membrane potential fixation mode. ● represents the control, △ represents the VGSC current after Aβ1-42 action, and ■ represents the VGSC current after T1R3 antibody action. The VGSC current was amplified at each step. (B) In a similar experiment, thaumatin was applied last instead of the antibody. Unlike with the T1R3 antibody, no further amplification of the VGSC current was observed after Aβ1-42 action. (C) On the other hand, thaumatin amplified the VGSC current similarly to Aβ1-42. No further amplification of the VGSC current was observed even when the concentration was increased. [Figure 52] The Nav1.5 VGSC current was measured using the membrane potential fixation method, and an IV plot was created. (A) ● indicates the control before Aβ1-42 action, △ indicates the VGSC current enhanced by 12.5 nM Aβ1-42, and ■ indicates the VGSC current attenuated by anti-T1R3 (400-570) antibody (Epitope 400-570). (B) When T1R2 expression was suppressed by expressing a miRNA vector, the effect of Aβ1-40 on VGSC current disappeared. ● indicates before Aβ1-42 action, and △ indicates after action. [Figure 53](53-1) To investigate the effect of the artificial sweetener aspartame, a T1R2 / T1R3 agonist, on cell morphology, undifferentiated SH-SY5Y cells were treated with 120 μM aspartame and changes in cell morphology were observed. As a result, it was confirmed that T1R2 / T1R3 stimulation by the artificial sweetener affected cell morphology and caused contraction. (53-2) The effect of β-alanine on cell morphology was investigated using SH-SY5Y cells differentiated to resemble nerve cells. SH-SY5Y cells were differentiated in the presence of 1% FBS and retinoid acid. SH-SY5Y cultured cells observed under a light microscope are shown (A). Here, the morphological changes of the lamellipodia in the area circled in white were observed over time. (B) shows the changes observed after β-alanine treatment at 5-minute intervals. The branched structure in the upper left circle changed to a thin, sheet-like morphology over time. Furthermore, the Neuroite terminal structure within the circle on the right was initially fan-shaped, but then contracted and then expanded back into a fan shape. [Figure 54] The effects of Aβ1-40 on the cell morphology of HEK cells (A1) and HUEhT-1 cells (A2) were observed. The left column shows the control, and the right column shows the cell morphology after Aβ1-40 treatment, compared with the control in the same field of view. In both (A1) and (A2), individual cells can be seen to have contracted. In (B1) and (B2), the same cells as in (A) were treated with a mixture of Aβ1-42 and Aβ1-40, where the Aβ42 / 40 ratio is the ratio observed in AD dementia (AD-D). Cell contraction was observed as in (A), but the effect was weaker compared to when Aβ1-40 was treated alone in HUEhT-1 cells. [Figure 55] This figure shows the changes in cell morphology observed when Aβ42 / 40 was applied using the ratio of Aβ42 / 40 to healthy individuals (Aβ ND). Compared to patients exhibiting cognitive symptoms, the effect of this mixture did not significantly result in cell body contraction, but ruffling was observed around the cell periphery. [Figure 56] This figure shows the effects of AβAD and ND on cell morphology using a mouse-derived brain microvascular endothelial cell line (βEnd.3). Unlike human cells, no morphological changes were observed in most cells, with the exception of a few. [Figure 57] When the terminal Ile-Ala (Isoleucyl-alanine) is cleaved from Aβ1-42, it becomes Aβ1-40. Isoleucyl-alanine has a structural formula similar to that of β-alanine. (A) Diagram showing that Aβ1-42 is formed by adding Ile-Ala (Isoleucyl-alanine) to the end of Aβ1-40. (B) Shows structural analogs containing isoleucyl-alanine and β-alanine. Shows the structural formulas of alanine with an amino group attached to the α-carbon, β-alanine with an amino group attached to the β-carbon, GABA with an amino group attached to the γ-carbon, and carnosine, which is composed of histidine and β-alanine. [Figure 58]VGSC currents were recorded using the whole-cell patch-clamp method from human-derived cells expressing human Nav1.5 (HEK, SH-SY5Y) or mouse / rat hybridoma cells NG108-15 differentiated to resemble neurons by treatment with forskolin and low-concentration FBS, and the effects of Aβ peptides (a mixed solution of Aβ40 and Aβ42) were observed. Two types of mixed solutions were used: one with the Aβ42 / 40 ratio observed in the cerebrospinal fluid of healthy individuals (NDC), and another with the Aβ42 / 40 ratio observed in the cerebrospinal fluid of AD patients exhibiting cognitive impairment (AD-D). (A-1) When Aβ42 / 40 in the AD-D ratio was applied, the VGSC current was increased compared to before application (n=6). This increase was greater than when other Aβ peptides were applied individually. This is qualitatively similar to the experiment shown in (Figure 31) where Aβ25-35 was applied after the action of the actin depolymerizer latruncrin A. In the figure, ● indicates the control, and ▲ indicates the cell after treatment with the Aβ peptide mixture. VGSC current was recorded using the membrane potential fixation method, with the membrane potential fixed at -120mV and a stimulation pulse of 80ms in length applied. The pulse was increased in 10mV increments from -80mV to 50mV. The effect of Aβ peptide was compared and analyzed across multiple cells by averaging based on the maximum VGSC current recorded in the control. In the IV plot, the mean value is shown with a symbol, and the error bar uses the standard error. (A-2) When cell morphology was observed, β-alanine activated lamellipodia, causing ruffling, suggesting the possibility of increased cell motility and enhanced synaptic plasticity. When β-alanine was applied to the VGSC current enhanced by the Aβ42 / 40 AD-D ratio, the increase in VGSC current decreased and recovered to almost the control level (n=2). Here, the bar graph shows the control as 1, the increase and degree of recovery are shown as averages, and the horizontal line shows the actual experimental measurements. (B) A peptide mixture with the Aβ41 / 40 ratio observed in healthy individuals was applied. Unlike AD-D, almost no change in VGSC current was observed (n=5).(C) When AD-D's Aβ42 / 40, which significantly enhanced the VGSC current in human cells (human T1R2 / T1R3), was applied to the VGSC current expressed in mouse / rat hybridoma cells NG108-15, the VGSC current did not increase in mouse / rat T1R2 / T1R3 cells, unlike in human cells expressing human-type T1R2 / T1R3, but rather decreased (n=3). [Figure 59] The positions of the peptides used to create the anti-T1R3 antibody are shown on the crystal structure (Nuemket et al. (2017)). (A) shows the crystal structure of the T1R2 / T1R3 heterodimer (PDB, 5X2M, Crystal structure of the medaka fish taste receptor T1r2a-T1r3 ligand binding domains in complex with L-glutamine). (B) shows the crystal structure of T1R3 only, with the positions of the peptide sequences used as each antigen indicated by white bars. It can be confirmed that 229-258 is LB2, 303-396 is LB1, and 400-570 is an amino acid sequence that includes the range between LB1 and LB2, including the CRD. [Modes for carrying out the invention]

[0071] 1. Definition of Terms (1) In this invention, we discovered for the first time that sweet G protein-coupled receptors (GPCRs) (T1R2 / T1R3) regulate VGSC. In addition, since T1R2 / T1R3 is expressed in many tissues, including nerve tissue, it is thought to play a regulatory role in VGSC in excitable cells in general, such as nerves and muscles. In this invention, the term "sodium channel" or "Na" is used instead of simply "sodium channel". + When we say "channel," we are referring to "voltage-gated sodium channels (VGSCs)."

[0072] It is known that G proteins regulate voltage-gated sodium channels (VGSCs). The action of G proteins also affects the action potential threshold and influences the ease with which action potentials are generated. In addition, especially in neurons, they affect the ease with which repeated action potentials occur and how long they persist. This is recognized as a major regulator that determines how inputs to many synapses are integrated, how action potentials are generated as a result, and the degree of dendrite membrane potential depolarization that affects the degree of neurotransmitter release. (Gonzalez-Burgos and Barrionuevo, (2001); Schwindt and Crill, (1995); Williams, (1999)).

[0073] Many GPCRs have been reported to phosphorylate and modify the activity of VGSCs through the action of serine / threonine kinases (PKA, PKC). The suppression of VGSCs by PKA activation has been reported in many studies, including muscarinic acetylcholine receptors in hippocampal pyramidal neurons (Cantrell et al., 1996), 5-HT2a / c receptors in cortical pyramidal neurons (Carr et al., 2002), and D1 class dopamine receptors in hippocampal pyramidal neurons and striatal medium spiny neurons (Cantrell et al., 1997; Surmeier et al., 1992). Examples of VGSC suppression by PKC activation include the activation of D2 dopamine receptors in striatal medium spiny neurons and cholinergic interneurons (Carr et al., 2003).

[0074] (2) Substances that act on T1Rs (T1R1, T1R2, and T1R3) and control of the VGSC current of those substances Table 3 summarizes the effects of compounds acting on T1R3 (T1R3 / T1R3), Table 4 summarizes the effects of compounds acting on T1R2 (T1R2 / T1R2 or T1R2 / T1R3), and Table 5 summarizes the effects of compounds acting on T1R1 (T1R1 / T1R1 or T1R1 / T1R3). Here, the sweet amino acids such as glutamine and serine in Table 3 are also present in CSF and, like Aβ25-35, have a VGSC-enhancing effect, causing hyperexcitability of nerve cells and are highly likely to be amino acids that cause epileptic-like neurological disorders and / or neuropathic pain. Furthermore, considering that both glutamic acid (MSG) and inosinic acid (IMP), which act as umami substances on T1R1 (Table 5) and delay the inactivation of VGSCs, are compounds present in CSF (Barnett, et al., (2020)), it is possible that both, individually or in combination, enhance VGSCs via T1R1, potentially contributing to epileptic-like neurological disorders and / or neuropathic pain. (Hereafter, amino acids that act as umami seasonings, such as glutamic acid (MSG), and nucleic acids such as inosinic acid (IMP), may also be referred to as "umami substances.")

[0075] [Table 3]

[0076] [Table 4]

[0077] [Table 5]

[0078] 2. Control of VGSC current via the disease / T1Rs targeted for treatment in the present invention (1) Central nervous system disorders (1-1) Pain generation and epileptic hyperactivity symptoms due to excessive VGSC activity One of the main target diseases of this invention is a disease in which the clinical symptoms are thought to be caused by the generation of pain due to excessive activity of VGSC, and ultimately by neuronal cell death resulting from epileptic-like hyperactivity. When sodium channels are activated, a large amount of sodium cations transiently flow into the cell, playing a crucial role in the generation of action potentials. At this time, the inactivation of sodium channels within a few milliseconds after activation, and the subsequent activation of potassium channels leading to the outflow of potassium cations, alleviate the accumulation of cations in the cell caused by the sodium (cation) influx, restoring the intracellular potential to the resting membrane potential. This interplay between sodium and potassium channels is important for completing spike-like action potentials quickly, and is essential for the efficient and safe transmission of electrical signals from the periphery to the central nervous system, or from the central nervous system to the periphery. Delayed inactivation of sodium channels (VGSCs) slows the recovery of the membrane potential from the depolarized state caused by the action potential, leading to neuronal hyperactivity, arrhythmias, or, in nerves, epileptic seizures, and ultimately, neuronal cell death.

[0079] (1-2) Consideration of the mechanism of action of Aβ peptide (Aβ25-35) involved in the onset of Alzheimer's disease (AD) and AD treatment drugs While we do not intend to limit the mechanisms of neuronal hyperactivity (epileptic seizures) observed in Alzheimer's disease to only those described below, the following explanation can be used to describe the results of the embodiments of this invention. Specifically, amyloid-beta peptides (Aβ25-35) bind to T1R3 and recruit VGSCs derived from intracellular endosomes onto the cell membrane, thereby increasing neuronal activity. This is thought to be the mechanism behind the epileptic hyperactivity reported in Alzheimer's disease. Since the above VGSC enhancement does not occur in cells where T1R3 is silenced, drugs that inhibit the binding of amyloid-beta peptides (Aβ25-35) to T1R3, or antibodies against T1R3, are drug targets for treating neuronal hyperactivity. Furthermore, in hyperactive neurons, VGSC activity can be suppressed via T1R2 / T1R3 by using substances (drugs) that depolymerize the cytoskeleton (microfilaments), such as dicloprop or artificial sweeteners (aspartame). These T1R2 / T1R3 antagonists (or T1R2 agonists) belonging to "Group 3," such as dicloprop and artificial sweeteners (aspartame), inhibit the binding of amyloid-beta peptides (Aβ25-35) to T1R3. Therefore, it is strongly suggested that they are effective as treatments for Alzheimer's disease (AD), in which amyloid-beta peptides (Aβ25-35) are considered to be the cause of the disease. Furthermore, when anti-T1R3 antibodies are made to compete with fluorescent Aβ25-35 peptides and their binding to T1R3 on the cell surface is observed, the binding of Aβ25-35 peptides to T1R3 is completely blocked. Therefore, anti-T1R3 antibodies could be the most potent treatment for Alzheimer's disease. Furthermore, as a symptomatic treatment to suppress VGSC overactivity, the suppression of VGSCs using aspartame, Gymnema tea extract (Gymnetic acid), calcium-sensitive receptor (CaSR) agonists (such as cinacalcet), and CaSR antagonists (such as NPS-2143) is effective.

[0080] (1-3) Neurological disorders in which an epileptic-like hyperactivity state similar to that of Alzheimer's disease is observed. Furthermore, there are numerous neurological diseases in which epileptic-like neuronal hyperactivity is frequently observed, similar to Alzheimer's disease, and it is assumed that these diseases all develop through the same mechanisms as Alzheimer's disease described above. Diseases that may develop through such mechanisms include, for example, the following: Alzheimer's disease, memory impairment Lewy body dementia: Dementia, hallucinations • Cerebrovascular disorders: Localized cranial nerve symptoms appear in the area affected by cerebral infarction (hemorrhage). • Epilepsy: Partial seizures, complex seizures. However, symptoms vary depending on the type of seizure. Parkinson's disease requires bradykinesia (slowness of movement). In addition, muscle rigidity or resting tremor is present. Other symptoms include postural instability, and non-motor symptoms such as constipation, REM sleep disturbances, and decreased sense of smell. • Progressive supranuclear palsy: Parkinsonism (bradykinesia, muscle rigidity, gait disturbance), dysphagia, unsteadiness while walking, oculomotor dysfunction (impaired vertical eye movement). Multiple system atrophy: Parkinsonism (bradykinesia, muscle rigidity, gait disturbance), dysphagia, unsteadiness while walking (cerebellar symptoms), falls, orthostatic hypotension, bladder and bowel dysfunction (autonomic neuropathy) • Amyotrophic lateral sclerosis (ALS): Progressive muscle weakness and atrophy of the limbs, dysphagia, and tongue atrophy. • Traumatic brain injury: The symptoms vary depending on the area of ​​the brain damaged by the trauma. Focal cranial nerve symptoms appear in the area damaged by the trauma. Huntington's disease: Involuntary movements, primarily chorea, and psychiatric symptoms, as well as dementia. Spinocerebellar degeneration: symptoms include unsteadiness, difficulty walking, difficulty using hands, and impaired movement. • Multiple sclerosis: Characterized by demyelination, with neurological symptoms such as motor paralysis, visual impairment, and sensory impairment that repeatedly relapse and remission.

[0081] (1-4) Pathological conditions induced by delayed VGSC inactivation The delay in VGSC inactivation by MSG induces the pathological conditions shown above (Table 4). While MSG-induced neuronal hyperactivity was previously thought to be solely due to glutamate receptors, the delayed inactivation of VGSC current via T1R1 and T1R1 / T1R3 induces neuronal hyperexcitability. Therefore, inhibitors of T1R1 and T1R1 / T1R3 (preventing MSG from binding to the T1R1 receptor) are highly effective drug targets for the treatment of the aforementioned diseases. When enhancement of VGSC current via T1R2 / T1R3 by sweet amino acids such as glutamine and serine, and amyloid-beta peptides (Aβ25-35), and the delay in VGSC inactivation by MSG occur simultaneously, it is easy to imagine that the nerves will become hyperexcitable and lead to neuronal cell death. This phenomenon is thought to occur particularly when nerve cells, astrocytes, etc., undergo necrotic cell death due to mechanical trauma, such as in stroke or spinal cord injury, and when large amounts of amino acids are released from inside the cell to the outside.

[0082] (1-5) Physiological roles of Aβ25-35 and Aβ1-42 Aβ peptides are also found in CSF (Lehmann et al. (2020)) and are known to be released from synapses (Groemer et al. (2011)). This paper examines the physiological function of Aβ peptides in long-term potentiation (LTP) in synaptic cells. Applying high-frequency, repetitive stimulation (tetanic stimulation) to nerve cells induces synaptic plasticity, which in turn leads to long-term potentiation (LTP). In this process, it is thought that the presynaptic terminal releases glutamate, a neurotransmitter, into the synaptic cleft, while Aβ peptide is also released simultaneously. The prevailing theory was that the massive release of glutamate activated NMDA-type glutamate receptors, and the influx of calcium into the postsynaptic terminal inserted AMPA-type glutamate receptors, which are inherent in intracellular endosomes, into the cell surface (synaptic cleft). This increased the number of glutamate receptors, thus sustainably improving the efficiency of synaptic signal transmission. In this invention, we discovered that, similar to the mechanism by which AMPA-type glutamate (Park et al. (2004)) is inserted from intracellular endosomes into the cell membrane (postsynaptic membrane) in LTP, VGSC is inserted into the cell membrane via the T1R3 receptor by Aβ25-35, thereby increasing its current density. In addition, we discovered that glutamate itself, via the T1R1 receptor, delays the inactivation of VGSCs, increases the sustained inward current of VGSCs, and facilitates the generation of burst activity in cells. Furthermore, we found that glutamate suppresses voltage-dependent potassium channel currents such as Kv1.2 via the T1R1 receptor. This means that glutamate, via T1R1, increases the sustained inward current of VGSCs and simultaneously decreases the outward potassium channel current, creating a situation where cells are easily overexcited. We also found that Aβ1-42, at nanomolar concentrations, has a similar effect on VGSCs and VGKCs via the T1R1 / T1R3 receptors to glutamate. This discovery demonstrates that Aβ peptides are responsible for cell death and disease symptoms not only in Alzheimer's disease but also in other diseases involving Aβ peptides, such as amyloidosis, and that they play an important role in physiological functions such as neuronal plasticity. Therefore, exploring positive allosteric modulators (PAMs) that amplify T1R1 and regulate it allosterically is considered an important drug target for improving symptoms such as memory impairment.

[0083] (2) Neuropathic pain (2-1) Mechanism of action of neuropathic pain and the scope of the therapeutic agent of the present invention as inferred from said mechanism of action The present invention also addresses the effective treatment of neuropathic pain associated with nerve hyperactivity based on the amplification of VGSC current. A typical mechanism of action for such neuropathic pain is shown in Figure 42. The neuropathic pain targeted by this invention typically includes neuropathic pain that occurs as a complication of anticancer drug administration (such as Taxol), but also chronic neuropathic pain syndromes caused by diseases such as failback surgery syndrome (FBSS), complex regional pain syndrome (CRPS), and diabetic polyneuropathy. It is also believed that other neuropathic disorders can be caused by similar mechanisms, as described below. • Cancer neuropathy (paraneoplastic peripheral neuropathy): Effects on nerves other than those directly compressed by cancer. • Peripheral neuropathy caused by anticancer drug treatment: Numbness, pain, and numbness in the hands and feet caused by drugs such as paclitaxel. Furthermore, in neurological diseases such as Alzheimer's disease (AD) as exemplified in (1) above, VGSC action potentials rapidly increase in all nerve cells, including not only the central nervous system (CNS) but also the peripheral nervous system (PNS), leading to a prolonged state of excitation and simultaneously causing neuropathic pain. In other words, it is also effective as a preventive and therapeutic agent for neuropathic pain caused in the diseases exemplified in (1).

[0084] One possible mechanism for the development of "neuropathic pain" is the enhancement of VGSC currents on the cell membrane and the resulting hyperactivity of nerves, leading to pain. Potential causative substances include chemotherapeutic agents used in chemotherapy, as well as amyloid-beta peptides, sweet amino acids such as glutamine (L-Gln), and umami substances such as glutamic acid and inosinic acid. Figure 42 illustrates one possible mechanism of action for such neuropathic pain. While there is no intention to limit the explanation to this mechanism alone, it allows for the explanation of the results of the embodiments of the present invention. Specifically, chemotherapy drugs such as paclitaxel increase VGSC currents by polymerizing microtubules, thereby hyperactivating nerves. In addition to paclitaxel, which stabilizes microtubules, colchicine, which inhibits their polymerization, was also observed to similarly increase VGSC currents. In this state, the VGSC current per nerve cell is enhanced, the action potential threshold is lowered, and action potentials are more easily generated. Furthermore, stimuli that would normally induce a single action potential generate multiple, continuous burst-like action potentials. As shown in Figure 42, the mechanism by which sweet amino acids such as amyloid-beta peptide (Aβ25-35) and glutamine (L-Gln) enhance VGSC currents within CSF and cause nerve hyperactivity is thought to have a high degree of commonality with the mechanism of neuropathic pain development caused by chemotherapeutic agents.

[0085] (3) Other diseases involving amyloid-beta peptide (3-1) Cardiac diseases involving abnormalities in VGSC (or VGKC) currents mediated by T1Rs due to amyloid-beta peptide Heart failure, like Alzheimer's disease, is a serious modern disease that not only affects the quality of life but also endangers life. Troncone et al. (2016) reported that when they examined cardiac function using echocardiography, in addition to memory impairment, patients with Alzheimer's disease showed impaired cardiac diastolic function, and the expression levels of Aβ1-40 and Aβ1-42 in the myocardium were elevated in Alzheimer's disease patients compared to healthy individuals. Since Type I taste receptors (T1R1, T1R2, and especially T1R3) are receptors for amyloid-beta peptides, they are thought to play an important role in the pathogenesis of various diseases in nerve cells. In nerve cells, amyloid-beta peptides insert ion channels such as VGSC and VGKC from the intracellular pool onto the cell membrane, inducing hyperactivity. This leads to epileptic-like hyperactivity in nerves, and ultimately to nerve cell death due to hyperactivity (Alzheimer's disease, ALS). In this process, amyloid-beta 25-35 peptides act on T1R3, increasing ion channels such as VGSC on the cell membrane and causing hyperactivity, while amyloid-beta 1-42 peptides act on T1R1 and T1R1 / T1R3 umami receptors, delaying VGSC inactivation and inducing persistent VGSC components, thus contributing to the expression of action potentials such as bursts. However, it is thought that elevated amyloid-β1-42 peptide levels, like MSG (glutamate), reduce VGSCs and inactivate nerve activity, and the same is likely true for cardiomyocytes that express a large amount of T1R3. Kim et al. (2011) reported that the Cav1.2 current, which corresponds to VGCC (voltage-gated calcium channel) expressed in cardiomyocytes, similar to VGSC in the present invention, is amplified by the action of amyloid-β25-35 peptide. This phenomenon can also be explained by considering that T1R3, identified in the present invention, functions as a receptor for amyloid-β25-35 peptide.

[0086] Troncone et al. (2016) reported that amyloid-β1-40 peptide is present in the myocardium of patients with Alzheimer's disease who suffer from cardiac diastolic dysfunction. Furthermore, Shibata et al. (2006) reported that NaV1.5, CaV1.2a, and KV1.5 coexist in caveolae within the recycling endosome (cardiomyocyte cellular reservoirs-caveolae) of cardiomyocytes, and Palygin et al. (2008) reported that the α subunit of G proteins (Gs alpha) translocates to the cell membrane by binding to caveolin 3, a membrane protein localized in recycling endosomes. Considering these findings together, it is possible that amyloid-β1-40 peptide induces myocardial hyperactivity and may be a cause of tachyarrhythmias. Since the action of Aβ1-40 peptide, or its deposition, has been observed in many diseases, suggesting a correlation with these diseases, further research into how Aβ1-40 peptide acts as a ligand for T1R GPCRs and controls the polymerization state of the cytoskeleton, and how this is involved in the downstream pathogenesis of diseases, is expected to lead to the establishment of treatments for many diseases and even valuable discoveries as drug targets.

[0087] (3-2) Other heart diseases in which amyloid-beta peptide may be involved Troncone et al. (2016) pointed out that Alzheimer's disease may be a systemic disease or metastatic disease that causes multiple organ failure, including heart disease, because amyloid-beta peptide deposition in the myocardium and cardiac dysfunction are observed simultaneously in patients with Alzheimer's disease. Furthermore, there have been many studies on the correlation between amyloid-beta peptides and heart disease. Since amyloid-beta peptides localized in brain nerves (Aβ1-42) and platelet-derived amyloid-beta peptides in the blood localized in non-nerve tissues such as the heart (Aβ1-40) are peptides of different sizes, it has been suggested that the development of heart disease due to amyloid-beta peptides may not be limited to patients with Alzheimer's disease (Troncone et al., 2016). If this is the case, then the deposition of amyloid-beta peptide in the myocardium and cardiac dysfunction are thought to be due to platelet-derived Aβ1-40, not Aβ1-42 found in cranial nerves. In fact, the effect of Aβ1-40 on VGSCs differs from that of Aβ1-42; in Nav1.5 expressed in the myocardium, it suppresses VGSCs by shifting the SS curve in the hyperpolarization direction. This effect differs from experimental results showing that Aβ1-42 acts on T1R1 / T1R3 umami receptors, and is similar to the effect of the artificial sweetener dichloroprop, which acts on T1R2 / T1R3 sweet taste receptors. Since cardiac contraction is functionally caused by the syncytium of the myocardium, if the generation of action potentials in some myocardial cells is suppressed by Aβ1-40, the periodic transmission of action potentials is hindered, which can induce arrhythmias, etc.

[0088] Inyushin et al (2020) pointed out that platelet-derived amyloid-beta peptides are involved in Aβ deposition in the brain, cancer, glaucoma, and skin, as well as in pre-eclampsia, Alzheimer's disease, and late-stage Parkinson's disease. In particular, in glaucoma, it has been confirmed that amyloid-beta peptides deposit in the retinal ganglion cell layer where cell death occurs, and also cause changes in the surrounding microvessels, and the therapeutic effect of using anti-amyloid-beta antibodies has been confirmed for glaucoma. Given that T1R and T3R are common GPCP-type receptors expressed in all tissues (The Human Protein Atlas (https: / / ww.proteinatlas.org / )), it is possible that the deposition of amyloid-beta peptides on T1R or T3R is involved in retinal ganglion cell death. According to Stamatelopoulos et al. (2017), amyloid-beta 1-40 (Aβ40) primarily accumulates in cerebral blood vessels (not nerve cells), inducing cerebral amyloid angiopathy. In addition, Aβ40 is present in the myocardial tissue of patients with Alzheimer's disease and heart failure, promoting atherosclerosis by forming plaques and thus having detrimental effects on the cardiovascular system. The amount of Aβ1-40 circulating in the blood is associated with the presence and degree of atherosclerosis and is linked to cardiovascular mortality in patients with coronary artery disease. Plasma-derived circulating Aβ1-40 levels were increased in heart failure patients compared to control participants and were positively correlated with risk factors for heart failure such as renal impairment, diabetes, and coronary artery disease, suggesting that it is an important predictor of mortality due to heart failure. Similarly, Bayes-Genis et al. (2017) reported that circulating Aβ1-40 levels were elevated in all outpatients with heart failure and that it is a predictor of heart failure. A noteworthy point in this study is that, unlike Aβ1-42, serum Aβ1-40 concentration is not associated with cognitive decline. These findings suggest that Aβ1-40 binding to T1Rs in the cardiovascular system may trigger heart failure.

[0089] A history of atherosclerosis has been reported to promote electrophysiological remodeling and arrhythmia development after acute myocardial infarction, similar to hypertension (Guvenc et al. (2010), De Jesus et al. (2015)). Therefore, it is highly probable that Type I taste receptors (T1Rs), which are thought to be receptors for Aβ peptides, act on the polymerization state of myocardial cytoskeletal fibers and influence the function of ion channels, thereby contributing to the promotion of arrhythmia development. In this invention, taxol, which causes VGSC enhancement, has been reported to not only stabilize microtubules but also increase the proportion of detyrosylated microtubules along with cytoskeletal density, resulting in increased cytoskeletal rigidity (Kerr et al., 2015). Furthermore, the relationship between myocardial function and the cytoskeleton has been clarified in clinical studies. The increase in cytoskeletal rigidity due to detyrosinization of microtubules occurs similarly in myocardium, and it has been reported that when many microtubules (polymerized tubulin) are detyrosinized, the mechanical resistance (rigidity) of the muscle increases, making it more difficult for muscles, including myocardium, to contract (Chen et al., 2018). In addition, Chen et al. stated that, based on the results of proteomics analysis, the thickened microtubule network (Microtubule Network), which is characteristically observed in heart failure, is consistently highly detyrosinized in the left ventricle of people with heart failure, and that this correlates with increased rigidity and impaired contractility of myocardial cells. Considering these factors, it is quite possible that amyloid-beta peptides act on the cytoskeleton of cardiomyocytes via Type I taste receptors (T1Rs), triggering cardiac dysfunction.

[0090] (3-3) Other diseases that may be associated with amyloid-beta 1-40 peptide Regarding the Aβ1-40 peptide, given the positive correlation between blood Aβ1-40 concentration and renal dysfunction and diabetes, it is possible that Type I taste receptors, similar to those in the heart, act as receptors for the Aβ1-40 peptide, and that this may be one of the factors causing cellular dysfunction in the kidneys and pancreas. For example, regarding kidney disorders such as nephrotic syndrome and renal failure, just as amyloid-beta 25-35 and amyloid-beta 1-42 have the effect of depolymerizing microtubules and microfilements in nerve cells (Pianu et al. (2014)), it is possible that Aβ1-40 also depolymerizes microtubules and microfilements in some kidney cells. The kidneys are vital organs that filter waste products from the blood and excrete them as urine. When kidney function declines, waste products accumulate in the blood, impairing the function of various organs in the body. Considering that nephrotic syndrome, characterized by symptoms such as proteinuria, develops when the cytoskeletal structure of glomerular epithelial cells (podocytes), which play a crucial role in kidney function, is inhibited, it is possible that chronic stimulation of Type I taste receptors on glomerular epithelial cells (podocytes) by amyloid-β1-40 peptide could lead to a decline in kidney function (Schell et al. (2014)) due to depolymerization of the podocyte cytoskeleton (especially the microfilament), thus causing kidney dysfunction. This suggests that polymerization / depolymerization of the cytoskeleton may affect cell morphology.

[0091] In diseases potentially caused by Aβ1-40, Type I taste receptors act as amyloid-beta peptide receptors, potentially leading to dysfunction in various organs and the development of various diseases due to their effects on ion channel (especially VGSC and VGKC) function or on cytoskeletal structures (promoting or inhibiting polymerization and depolymerization). In particular, when structural effects on microtubules and microfilaments are considered to be the cause of disease development, measuring the polymerization state of microtubules and microfilaments using markers such as GFP is an effective diagnostic and drug discovery screening method.

[0092] (4) Method and dosage of the therapeutic pharmaceutical composition according to the present invention The pharmaceutical composition provided by the present invention, which contains a substance acting on type I taste receptors (T1Rs), particularly a substance stimulating T1R2 / T1R3, as an active ingredient, can suppress epileptic-like hyperactivity in CSF, such as in Alzheimer's disease (AD), and excessive activity of VGSCs that cause inflammatory pain in nerve cells. Therefore, it can be used as a pharmaceutical composition for the prevention or treatment of Alzheimer's disease (AD) and the like. In this case, it may be used in combination with existing therapeutic agents for Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), and the like. Furthermore, the T1R2 / T1R3 stimulating substance of the present invention can be used as an active ingredient in pharmaceutical compositions for the prevention or treatment of neuropathic pain, and can be used in combination with existing analgesics such as opioid preparations, or with other drugs such as vasoconstrictors like epinephrine, which are used to keep the drug local and suppress side effects on other organs. The pharmaceutical composition of the present invention can be administered orally, nasally, rectally, or parenterally. For oral administration, the dosage forms can include powders, granules, capsules, pills, tablets, elixirs, suspensions, emulsions, and syrups, from which appropriate formulations can be selected. Furthermore, these formulations can be modified to include sustained release, stabilization, easy disintegration, poor disintegration, enteric coating, and improved absorption. Furthermore, for intravenous, intramuscular, or subcutaneous administration, the dosage form can be an injectable preparation or an intravenous drip preparation (including a dried product prepared immediately before use), and can be selected as appropriate. The pharmaceutical composition of the present invention may contain pharmaceutically acceptable carriers and additives depending on its dosage form (oral administration or various parenteral administration dosage forms). Examples of pharmaceutically acceptable carriers and additives include solvents, excipients, coating agents, bases, binders, lubricants, disintegrants, solubilizers, suspending agents, viscosity modifiers, emulsifiers, stabilizers, buffers, isotonic agents, analgesics, preservatives, flavoring agents, fragrances, and colorants.

[0093] When the pharmaceutical composition of the present invention is used as a preventive or therapeutic agent for diseases caused by VGSC activation due to binding of Aβ peptides (such as Aβ25-35 peptides) or sweet amino acids, umami substances, etc., in CSF to T1Rs (for example, as a preventive or therapeutic agent for Alzheimer's disease, epilepsy, ALS, and neuropathic pain), the oral dose is preferably in the range of 0.03 to 300 mg / kg body weight, more preferably 0.1 to 50 mg / kg body weight, converted to the amount of the compound of the present invention. When administered intravenously, a dose can be given such that the effective blood concentration of the compound of the present invention is in the range of 0.2 to 50 μg / mL, more preferably 0.5 to 20 μg / mL. The same applies when used as a pharmaceutical composition for the prevention or treatment of neuropathic pain. These dosages may vary depending on age, sex, weight, the nature and severity of the situation, and the route of administration.

[0094] Among the compounds belonging to "Group 3" that can stimulate typical T1R2 / T1R3 receptors and suppress VGSC activity, the artificial sweetener aspartame, dicloprop, and cinacalcet are described in detail below, along with examples of dosage and usage. However, the compounds that constitute the active ingredients of the pharmaceutical composition of the present invention are not limited to these compounds and their specific numerical ranges.

[0095] For example, in the case of the artificial sweetener aspartame, IC 50 At a concentration of 15-17 μM, VGSC is suppressed. Since aspartame is completely digested in the small intestine, parenteral administration, such as intravenous injection, is preferred over oral administration. The purpose of administration is not to completely suppress VGSC, but to reduce its firing frequency and prevent bursts; therefore, a concentration of 4.5-10 mg / kg is preferred when administered intravenously. Regarding Nav1.5 expressed in the myocardium, more than twice the concentration is required at -100mV, near the resting membrane potential of the myocardium, suggesting that aspartame has little effect on myocardial activity. Since the effects of aspartame tend to be weakened in the presence of Aβ peptide, it is considered more effective as a pharmaceutical composition for preventing and treating pain caused by inflammation, injury, etc., than as a pharmaceutical composition for treating Alzheimer's disease.

[0096] In the case of dichlor-prop, it acts at lower concentrations than aspartame on VGSC (Nav1.7, Nav1.6) expressed in nerve cells (SH-SY5Y), and increases the membrane potential before action potential expression to the extent of its effect. In particular, in nerve activity exhibiting burst-like activity (pain, epileptic seizures, AD, etc.), the membrane potential shifts towards depolarization, so even lower concentrations (IC) are effective. 50 It is effective at concentrations between 9 μM and 17.3 μM. A dosage of 2-10 mg / kg is considered effective whether administered orally or parenterally. Further higher concentrations may be possible as needed, but the upper limit of the dosage concentration should be avoided by not inhibiting Nav1.5, a myocardial VGSC, at the resting membrane potential of the myocardium.

[0097] In the case of cinacalcet, since it completely inhibits VGSC activity at a concentration of 5 μM, significant side effects are expected when administered systemically. Therefore, its role as a local anesthetic is recommended. The method of administration is local injection as a local anesthetic. A preferred administration concentration is 1-2 mg / kg (containing 1:100,000 epinephrine). When the area requiring pain relief is inflamed or otherwise acidic (e.g., terminal cancer pain, acute periodontitis (dental treatment)), conventional local anesthetics (such as lidocaine) are pH-dependent and therefore ineffective for pain relief. In such cases, by incorporating epinephrine, similar to conventional local anesthetics, and constricting local blood vessels, systemic side effects can be suppressed, ensuring safety during administration and allowing the anesthetic to function as an effective pain reliever.

[0098] 3. Screening Methods (1) Type I taste receptors (T1Rs) as drug targets for diseases associated with increased levels of sweet amino acids (serine, glutamine, etc.) and umami substances (glutamic acid, inosinic acid, etc.) in Aβ peptides and CSF. In this invention, we confirmed that among the Aβ peptides, Aβ25-35 peptides enhance VGCS via T1R3, while nanomolar concentrations of Aβ1-42 peptides delay VGSC inactivation via T1R1 in addition to T1R3, suppress voltage-gated potassium channels (such as Kv1 and Kv2), and enhance nerve (myocardial) hyperactivity. On the other hand, glutamate, known as an "excitatory neurotransmitter" as a glutamate receptor agonist, was confirmed to delay VGSC inactivation via T1R1, suppress voltage-gated potassium channels (such as Kv1 and Kv2), and enhance nerve (myocardial) hyperactivity (data not shown). Furthermore, considering that the enhancement of VGSCs causes neuronal cell death and is one of the causes of AD, as described below, Aβ25-35 peptides or Aβ1-42 peptides that exhibit agonist-like activity against T1R3 or T1R1, as well as sweet amino acids such as glutamine and serine present in cerebrospinal fluid (CSF), and umami substances such as glutamic acid, aspartic acid, and inosinic acid also found in CSF, could be potential drug targets. In particular, substances that inhibit the binding of glutamine (L-Gln), a sweet amino acid whose levels are elevated in CSF in Alzheimer's disease, epilepsy, and ALS, or substances that inhibit the function of its conjugates, have a high potential as therapeutic agents for AD and neuropathic pain. Here, substances that inhibit the function of T1Rs conjugates refer to substances that inhibit the binding of Aβ peptides to T1Rs themselves, and substances that do not inhibit the binding of the target substance to T1Rs itself, but inhibit the signal transduction emitted after the target substance binds to T1Rs. This invention provides a method for efficiently screening such "T1Rs" inhibitors.

[0099] (2) In vitro screening method for substances that inhibit the binding of Aβ peptides or sweet amino acids or umami substances in CSF to T1Rs, or substances that inhibit the function of their conjugates: It is hypothesized that Aβ peptides or sweet amino acids or umami substances within CSF bind to T1Rs on the surface of target cells, thereby increasing the VGCS or VGKS currents of the target cells and ultimately causing cell death. This paper describes an effective screening method for identifying substances that inhibit the binding of such Aβ peptides or sweet amino acids or umami substances within CSF to T1Rs, or substances that inhibit the function of such binding products. The effect on VGSC currents resulting from the binding of Aβ peptides or sweet amino acids or umami substances within CSF to Type I taste receptors (T1Rs), including T1R3, is thought to occur through multiple steps. i) First, the process by which Aβ peptides or sweet amino acids or umami substances within CSF bind to Type I taste receptors (T1R1 / T1R3, T1R2 / T1R3 GPCRs); ii) the activation of G proteins resulting from the activation of Type I taste receptors by the binding of Aβ peptides or sweet amino acids or umami substances within CSF; iii) Polymerization or depolymerization of microfilaments (microfibers) or microtubules (microtubules) that result from this process. iv) At the end of this cascade is a control action that enhances or suppresses the VGSC or VGKC current. Furthermore, the activation of Adenylate cyclase and Phospholipase C via the G protein β and γ subunits, which occurs upon activation of T1RsGPCR, activates IP3 channels and Ryanodine receptor channels, leading to the release of calcium from the ER and an increase in intracellular calcium concentration. Based on the above, it is thought that the following sites of action could be drug targets to suppress or inhibit the effect of Aβ peptides or sweet amino acids or umami substances within CSF on the cellular VGSC or VGKC current, or to increase or change the dynamics of intracellular calcium as a result of calcium leakage from intracellular calcium stores such as the ER, rather than calcium flowing in from outside the cell through calcium channels. (1) Methods to prevent Aβ peptides or sweet amino acids or umami substances within CSF from binding to Type I taste receptors such as T1R3: For example, this includes the search for anti-T1R3 antibodies or compounds with equivalent activity. (2) Search for compounds that suppress the activation of Type I taste receptors resulting from the binding of Aβ peptides or sweet amino acids or umami substances within CSF to Type I taste receptors: For example, in the case of T1R3, the search for compounds that specifically bind to T1R3, such as lactisole and dichlor-prop, and prevent conformational changes in T1R3 caused by the binding of Aβ peptides or sweet amino acids or umami substances within CSF, thereby inhibiting the activity of G proteins downstream of T1R3 signal transduction. (3) In the case of sweet taste receptors (T1R2 / T1R3), for example, one could search for compounds that have an inhibitory effect on VGSC current by activating T1R2 / T1R3, such as artificial sweeteners (saccharin, aspartame, Gymnema tea extract, sucralose, etc.) which are agonists of T1R2 that form a heterodimer with T1R3, and which suppress the amplification of VGSC current induced by the binding of Aβ peptides (especially Aβ25-35) or sweet amino acids in CSF (such as glutamine) to T1R3 by their ability to suppress VGSC current. The approaches described in (2) and (3) above can also be applied to the search for substances using changes in intracellular calcium concentration as the output.

[0100] The membrane potential fixation method (patch clamp method) shown in (2-3) below is the most accurate and useful method for measuring the effects of Aβ peptides or sweet amino acids or umami substances in CSF. However, since the membrane potential fixation method requires a complex operation that requires skill and concentration under a high-magnification microscope, the fluorescent labeling observation methods shown in (2-1) and (2-2) below are effective for simplicity and improved screening efficiency. For example, by applying the drug discovery screening method from the perspective of (1) above in (2-1), the changes in the cytoskeleton caused by the binding of fluorescently labeled Aβ peptides or sweet amino acids or umami substances in CSF to T1R3 can be observed with a fluorescence microscope. By confirming the presence or absence of such changes, it is possible to search for substances that prevent the changes caused by the binding of Aβ peptides or sweet amino acids or umami substances in CSF. Furthermore, when performing screening from the perspectives of (2) and (3) above, by measuring the polymerization state of microfilaments and microtubules visualized with fluorescently labeled actin and tubulin using the method of (2-2), it is possible to observe the polymerization state of the cytoskeleton caused by the binding of Aβ peptides or sweet amino acids or umami substances in CSF to Type I taste receptors including T1R3, and to screen for substances that can suppress the change in VGSC or VGKC current caused by the binding of Aβ peptides to Type I taste receptors. Similarly, the same screening can be performed by monitoring intracellular calcium concentration.

[0101] (2-1) Method for observing fluorescent labeling on the surface of target cells As a typical example, a screening method for inhibitors of Aβ peptide (Aβ25-35) binding to T1R3 is described below. It goes without saying that the target combinations of Aβ peptide, T1Rs, and cells are not limited to these combinations. As mentioned above, anti-T1R3 antibodies are cultured together with fluorescently labeled Aβ peptides (Aβ25-35) in cultured nerve cells (SH-SY5Y cells differentiated in RA), or in Nav1.5 and other Na +When the fluorescent Aβ peptide is added to HEK cells that forcibly express the channel, its binding to T1R3 on the cell surface can be completely blocked. By observing the fluorescent labeling on the cell surface, the degree of competitive inhibition of binding to T1R3 can be visually evaluated. In other words, following this method, cells that highly express T1R3, such as cultured nerve cells (for example, SH-SY5Y cells differentiated for RA), are used. By administering the test substance together with a fluorescent Aβ peptide into the culture medium of these cells, it is possible to determine whether or not the binding of the fluorescent Aβ peptide to the cell surface is inhibited by observing the fluorescence on the cell surface. By applying this method, it is possible to screen for drugs that are effective as treatments for Alzheimer's disease from the test substance. If an anti-T1R3 antibody is targeted as the test substance, it is possible to screen for neutralizing antibodies that have a higher efficacy among anti-T1R3 antibodies. Specifically, cultured nerve cells expressing a large amount of T1R3 (or T1R3) or human-derived cultured cells in which sodium channels such as Nav1.5 are forcibly expressed are administered with a pre-fluorescently labeled Aβ peptide (Aβ25-35 or Aβ1-42). The fluorescence of the cell surface is confirmed by imaging, and then the amount of fluorescence on the cell surface is checked to see if it decreases after administration of the test substance. This allows for the determination of the presence and strength of the T1R3 binding inhibitory activity of the Aβ peptide of the test substance.

[0102] (2-2) Other methods for observing fluorescent labels; In this invention, we have shown that T1R GPCRs expressed on the cell membrane alter the function of effectors such as VGSCs through changes in the polymerization state of the cytoskeleton, such as actin and tubulin, depending on the agonist applied. This property can be utilized. By expressing fluorescently labeled actin or tubulin in cells that endogenously express T1R GPCRs, such as HEK cells, and observing the changes in the cytoskeleton with a fluorescence microscope, it is possible to screen for inhibitors of the action of Aβ peptides or sweet amino acids or umami substances in CSF. Similarly, drug discovery screening targeting T1R GPCRs with pharmacological effects such as analgesic and anticonvulsant effects can be performed. Furthermore, this method is just as effective as the impedance-based method described in (2-6) below, even when the final effector of the Aβ peptide or sweet amino acid or umami substance in the CSF is not an ion channel. Control experiments to distinguish this from direct action on the cytoskeleton or action by other GPCRs can be performed using a GFP-labeled T1R GPCR silencing vector and RFP-labeled actin or tubulin.

[0103] (2-3) Patch clamp method: The patch-clamp method is a technique for recording intracellular potentials, and it can also stimulate cells by injecting an electric current while simultaneously recording these potentials. Patch clamp recording modes include Voltage-clamp (membrane potential fixation) and Current-clamp (current clamp). Voltage-clamp mode records ion channel currents such as VGSCs. Current-clamp mode records changes in intracellular potential. Specifically, it can record action potentials, which are changes in cell membrane potential, and its width, magnitude, and frequency. Furthermore, in patch-clamp experiments, control data and the response after reagent action are recorded from the same cell. The results after reagent action and the control data before action are then compared to determine whether or not the reagent had an effect. Single-cell electrophysiological observation (patch-clamp method) is unique compared to other experimental methods in that it allows for the observation of changes over time from the same cell, both for control and after reagent action. The experimental results obtained by patch-clamp allow for the direct measurement of reagent changes over time, making it the most accurate experimental method. The delayed inactivation phenomenon of VGSCs mediated by monosodium glutamate (MSG) via T1R1 has potential applications in antiarrhythmic drug screening. The increase in sustained sodium current resulting from delayed inactivation plays an important pathophysiological role in cardiac diseases, including rhythm disorders (Horvath et al., (2020)). As described in this invention, Nav1.5, the main VGSC in myocardium, generates a sustained sodium current due to the delayed inactivation by MSG.

[0104] Incidentally, in conventional drug screening, a sustained sodium current was pharmacologically induced using Nav1.5 mutants, veratridine, or sea anemone toxin (ATX-II) to confirm drug efficacy. However, in the present invention, it was found that umami stimulation delays the inactivation of Nav1.5. Therefore, it is now possible to conduct safety pharmacology tests on Nav1.5 without inactivation, using only umami stimulation, without using such special mutants or expensive drugs. In other words, by administering MSG to the culture medium of cultured cardiomyocytes, the inactivation of VGSC current is delayed via the umami receptor (T1R1), generating a sustained sodium current (VGSC) in the cultured cardiomyocytes, and drug screening can be performed using the change in VGSC current as an indicator.

[0105] The effect of Aβ peptides or umami substances on sustained potassium current (VGKC) in cardiomyocytes can be investigated using human cultured cells expressing VGKC such as Kv1.2, with SH-SY5Y cells expressing VGKC (Kv1.2, Kv3.2, Kv3.3, etc.) or HEK cells endogenously expressing T1R GPCRs as host cells. In fact, as shown in the experimental example in Figure 36B, when Aβ peptides were applied to VGKC-expressing human cultured cells, the VGKC current was recorded using the membrane potential fixation method to obtain a lamp-like voltage waveform or a square wave voltage, and the change (inhibition) of the VGKC current by Aβ peptides could be measured. The inhibitory effect on VGKC can be similarly measured when using the umami substance L-sodium glutamate (MSG) (Figure 36A).

[0106] The method used to demonstrate that the regulatory effect of the aforementioned Aβ peptides (Aβ25-35, Aβ1-38, Aβ1-40, Aβ1-42) on VGSC current is lost by silencing T1R3 was this membrane potential fixation method (patch clamp method).

[0107] (2-4) Method for measuring membrane potential changes of target cells using the "NanoTouch method" Another method involves applying the "NanoTouch method" (Saito, 2019) (International Publication Number: WO2018 / 199334), previously developed by the inventors, to measure changes in the membrane potential of target cells. The "NanoTouch" method involves using conductive nanoparticles to penetrate the cell membrane and utilize them as intracellular electrodes. Specifically, a method combining this with a capacitive potential measurement device (nanoCharge) is used to record spontaneously firing action potentials in cultured nerves or myocardium. Specifically, the cells are cultured on conductive glass, polyethyleneimine and conductive magnetic nanoparticles (gold magnetic nanoparticles) are mixed, and this mixture is applied to cells washed with PBS(-), and left in a 37°C incubator for 15 minutes. Subsequently, this mixture is replaced with physiological saline or culture medium, the conductive glass containing the cultured cells is connected to the positive electrode, and a thin metal film (such as aluminum foil) is placed on the underside of the conductive glass without cultured cells and connected to the negative electrode. This conductive glass is then placed on a 180-200 millitesla magnet. This magnetic force attracts the conductive magnetic nanoparticles already introduced into the cells towards the cell membrane in contact with the glass surface, and by penetrating the cell membrane, the intracellular potential is conducted to the conductive glass, allowing the intracellular potential to be measured. In this method, the principle of a capacitive potential measurement device is utilized, forming a capacitor between conductors placed on the upper and lower surfaces of the conductive glass. This allows changes in intracellular potential to be captured as electric charge, which can then be detected by the conductor on the opposite side of the conductive glass. Since the relationship between charge (Q) and electrocautery (CV) is Q=CV, the potential (V) can be measured in proportion to the capacitance (C) of the electrode glass. By using this method, intracellular potential can be recorded without using techniques such as membrane potential fixation (patch clamp method), and the activity and inhibitory effects of Aβ peptides or sweet amino acids or umami substances in CSF on nerves or myocardium can be measured.

[0108] (2-5) Method for observing the frequency of action potentials in cultured nerve cells using extracellular recording techniques In the case of multielectrode arrays (such as MEAs), the frequency of cellular action potentials can be observed, but the waveform of the action potential or slow changes in synaptic potentials cannot be observed, as in the "nanoTouch" method described above. When using an MEA, each cell is seeded and cultured on an MEA probe embedded with recording electrodes, and extracellular recording is performed using an MEA device. The effects of Aβ peptide on nerve activity and the effects of inhibitors can be used in the same way as the above experiments using a fluorescent membrane potential-sensitive reagent or a fluorescent intracellular calcium-sensitive reagent. The cells that spontaneously generate action potentials (note that "spontaneous firing" refers to the generation of action potentials spontaneously, rather than in response to external stimuli. Typical "spontaneously firing cells" are nerve cells and cardiomyocytes, and nerve cells in particular have a high natural firing activity.) are seeded and cultured. In the case of a calcium-sensitive reagent, after administering the reagent mixture, they are cultured in an incubator at 37°C for 60 - 90 minutes, and then measured using the same measuring instrument as in the case of the fluorescent membrane potential reagent. Since the fluorescence wavelength varies depending on the reagent used, follow its specifications.

[0109] (2 - 6) A method for screening a compound that modifies / suppresses the changes caused by Aβ peptide or sweet amino acids or umami substances in CSF: A method for measuring the impedance (resistance) of cells The measured value of the impedance of the cell sheet reflects the polymerization state of ATP and actin, and the value fluctuates. This change occurs in linkage with cell morphological changes. Since the actin cytoskeleton is an important downstream effector of GPCR, the activation state of GPCR can be highly sensitively measured as an increase or decrease in impedance due to the change in the adhesion state of the cells seeded on the electrode substrate to the substrate and the spread of the adhesion area, resulting in cell morphological changes. In the GPCR pathway that activates Gαi of G protein, an increase in impedance is observed when activated. In the pathway that activates Gαq, first, a transient impedance phenomenon is observed, followed by a large increase. And in the pathway that activates Gαs, a decrease in impedance has been reported (Scott and Matthew., 2010).

[0110] When measuring the activity of GPCR as a change in cell morphology caused by a change in the cytoskeleton as a change in the impedance value of the cell sheet, it is usually performed using measuring instruments such as the ×CELLigence System (ACEA Biosciences) and CellKey (Molecular Devices). Specifically, cells are cultured in a sheet-like structure on an electrode plate such as gold foil or conductive glass. By applying an electric current between the conductive glass, which is covered by the cells, and the extracellular solution, the change in the impedance value of the cell sheet can be measured. By culturing the cells in a sheet, the electrode surface is completely covered by the cells and isolated from the extracellular solution. Therefore, by passing an electric current (I) between the extracellular solution and the electrode plate and measuring the resulting potential difference (V), the resistance (R) of the entire cell sheet can be calculated using Ohm's law (V=IR>R=V / I). Since impedance is the reciprocal of resistance, 1 / R is the measured impedance. Here, GPCR activity causes changes in the cytoskeleton, leading to changes in cell morphology and changes in gap junctions connecting cells. When the adhesion between cells weakens, current flows more easily, resulting in a decrease in resistance, i.e., an increase in impedance. First, by applying an Aβ peptide (or a sweet amino acid or umami substance within CSF) to a ligand for a type I taste receptor agonist, such as a sweet or umami receptor (T1Rs), we measure how the impedance changes as described above. Then, we identify which G protein (Gαi, Gαq, or Gαs) is being stimulated. We will analyze the impedance changes that occur when T1Rs GPCRs are treated with various Aβ peptides (or sweet amino acids or umami substances in CSF). Using this experimental system, we will screen for compounds that modify or suppress the changes caused by Aβ peptides (or sweet amino acids or umami substances in CSF). In other words, we will search for compounds that suppress or modify the changes in the cytoskeleton caused by Aβ peptides (or sweet amino acids or umami substances in CSF) upon administration of the test substance. This invention demonstrates that T1R GPCRs expressed on the cell membrane alter the function of effectors such as VGSCs through changes in the polymerization state of the cytoskeleton, including actin and tubulin, depending on the agonist applied. In addition to the impedance method, another effective method for preliminary screening is to monitor the intracellular calcium concentration using cells that endogenously express T1R GPCRs such as HEK cells. In this discovery, it was found that Aβ1-40 and Aβ1-42 have different patterns of intracellular calcium changes. By further accumulating the response patterns of other lengths of Aβ peptides, umami, and sweet substances, associating the response patterns with the T1R GPCR subunits involved, and combining the results with the screening method shown below, accurate drug discovery can be carried out. It is also effective to use this method as one of the preliminary screenings and then perform the aforementioned screenings (effects on the polymerization states of fluorescently labeled actin and tubulin, "nanoTouch" method, effects on cell excitability using MEA, etc., and detailed analysis of the effects by the membrane potential clamping method). It is known that the measurable impedance changes depending on the activity of GPCRs expressed on the cell surface.

[0111] (3) In vivo screening method: (Method using experimental animals with humanized T1Rs genes) For in vivo screening, it is necessary to use experimental animals such as mice with humanized T1Rs genes. This is because compounds represented by lactisole, which inhibits the conformational change of T1R3 by ligands, show different responses in mice and rats compared to human genes. Lactisole suppresses the perception of umami and sweetness only in humans, and its effect is not confirmed in mice and rats (Jiang et al. (2005)). And Toda et al. (2018) reported the allosteric modulation of T1R1 / T1R3 by methional, but the effect on T1R1 / T1R3 acts as a PAM (positive allosteric modulator) in humans and as a NAM (negative allosteric modulator) in mice. Additionally, the sweet protein thaumatin is detectable in humans but not in mouse T1R3. Gymnemic acid is also effective in human T1R2 but not in mouse T1R2. The above thaumatin was confirmed to enhance VGSC current, similar to Aβ peptide, in experiments using Nav1.6-expressing HEK cells with membrane potential fixation (patch clamp method) (Data not shown). Based on the above, in addition to in vivo drug screening using mice, mouse experiments to analyze the function of taste receptors require the use of humanized mice, such as those created by knockout (KO) of relevant mouse-derived genes or insertion of the human T1Rs gene. [Examples]

[0112] Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these. Other terms and concepts in this invention are based on the meanings of terms conventionally used in the art, and the various techniques used to carry out this invention, except for those techniques whose sources are specifically indicated, can be easily and reliably performed by those skilled in the art based on known literature, etc. Furthermore, various analyses were performed by applying methods mutatis mutandis to those described in the instruction manuals, catalogs, etc., of the analytical instruments or reagents used, or the kits. Furthermore, the contents of the technical documents, patent publications, and patent application specifications cited herein shall be referred to as the contents of the present invention. Furthermore, in this invention, when we simply refer to "cells," we mean human-derived cells. However, cells derived from any animal that endogenously expresses human-type Type I taste receptors, or cells that express Type I taste receptors with a human-specific amino acid sequence through gene transfer or other means, can also be used. Typical animals that have cells that endogenously express human-type Type I taste receptors include great apes such as chimpanzees, gorillas, orangutans, cercomas, baboons, and rhesus monkeys. However, the invention is not limited to these, as long as the cells are derived from animals with similar properties.

[0113] <Experimental method used in this embodiment> 1. Electrophysiological experiments The electrophysiological experiments in this embodiment were performed using the "patch clamp method (membrane potential fixation method, current clamp)". Patch clamp experiments were conducted using an Axopatch 200A amplifier (Axon Instruments Inc., Union City, CA), and the data was digitized using a Data Digidata 1332a (Axon Instruments Inc.). Voltage control and data acquisition were performed using Clampex Software (pClamp9.0, Axon Instruments Inc.). The data digitized by Clampex was analyzed using Clampfit (Clampfit10.6.2.2. (Molecular Devices, San Jose, CA)). Statistical analysis was performed using Microsoft Excel. The Boltzmann equation and Hill plot analysis were performed using Curve Expert Professional 2.6.0 (Hyams Development). Specifically, a patch electrode containing intracellular fluid, connected to an Axopatch amplifier, is attached to the target cell under a microscope. The cell membrane at the opening of the patch electrode tip is disrupted, and the intracellular potential is recorded through the patch electrode, which has become integrated with the inside of the cell. During this process, stimulation is repeated at intervals of 8 to 10 seconds until the recording state stabilizes. The IV curve and SS curve are then measured and used as the control (baseline). At this point, the test substance is applied while stimulating again at intervals of 8 to 10 seconds. Once the effect of the test substance is stably observed, or after 5 minutes or more have passed, the IV curve and SS curve are measured again, and the results are compared with the control to determine whether or not the test substance has an effect. If the opening of the patch electrode in the cell membrane narrows during the experiment and a deterioration in measurement accuracy is confirmed, the result is removed.

[0114] The patch clamp experiment solution was prepared as follows: Extracellular fluid: (in mM) 126 NaCl, 4 KCl, 1.8 CaCl2, 1 MgCl2, 24 HEPES, 10 glucose (pH 7.4 adjusted with NaOH). Intracellular solution (electrode solution): (in mM) 130 KCl, 5 MgCl2, 5 EGTA, 4 Tris-ATP, 10 HEPES (pH 7.2 adjusted with KOH). The chemicals used were purchased from Nakalai Tescue, Sigma-Aldrich, and Tokyo Chemical Industry Co., Ltd.

[0115] 2. Cell lines used in the present invention and their cell culture conditions: (1) Creation of a HEK cell line that stably expresses Nav1.5 We created a hygromycin-resistant cell line by introducing Nav1.5-pcDNA3.1 (hygromycin, Invitrogen, USA) into HEK293 cells (JCRB bank) using a Superfect transfection reagent (Qiagen, DE). The HEK293 cell culture medium used in this study was "DMEM (Sigma-Aldrich, St. Louis, MO, USA), 10% FBS (BioWest, FR)". Hereafter, when "HEK293 cells" are referred to in this specification, it refers to this "Nav1.5 stably expressing HEK293 cell line."

[0116] (2) Culture of the SH-SY5Y cell line and differentiation into neuronal-like cells For the SH-SY5Y cell culture medium, DMEM (Sigma-Aldrich) and F-12 (Sigma-Aldrich) were mixed in a 1:1 ratio, and "5% FBS (BioWest), 5% 40mM L-Glutamine (Fujifilm), and 1% MEM Non-essential Amino Acids (Wako Pure Chemical Industries)" were added. To differentiate SH-SY5Y cells into neuronal-like cells, the above solution was diluted to 1% FBS, 10 μM / ml retinoic acid (Fujifilm) was added, and the cells were cultured for at least 5 days to induce differentiation. Hereafter, when "SH-SY5Y cells" are referred to in this specification, it refers to this "SH-SY5Y cell line differentiated into neuronal-like cells."

[0117] 3. Electrophysiological measurement protocol: (1) Activation protocol for voltage-gated sodium channels (VGCS): The membrane potential was set to -80mV, followed by a pre-pulse (-120mV, 500ms), and finally a 200ms depolarization pulse was applied at 10mV intervals from -80mV to 40mV. Double pulse protocol: Similar to the activation protocol above, but after the initial multiple pulses, another set of pulses is applied with a Prepulse of -120mV.

[0118] (2) Protocol to investigate the deactivation of VGCS in a steady state: The membrane potential was set to -80 mV. First, a pulse of -20 mV (200 ms) was applied, followed by a change from 0 mV to -120 mV (1 s) in 10 mV increments to create an inactivated state. Finally, a test pulse (-20 mV, 200 ms) was applied to examine how the magnitude of the VGSC activated by the test pulse was affected by the membrane potential before the test pulse. For Nav1.5 expressed in the HEK cell line and Nav1.7 and Nav1.3 expressed in SH-SY5Y, when different test pulses were used in experiments with SH-SY5Y, it should be specified each time.

[0119] (3) Pulse and lamp protocol: A depolarization step of 20 ms and -20 mV was performed from the prepulse (-120 mV, 320 ms) to restore the membrane potential to 2 nd As a prepulse, it was held at -120 mV for 360 ms, and a ramp waveform was applied from -120 mV to 60 mV over 20 ms. Subsequently, the membrane potential was held at 60 mV for 10 ms and then at -40 mV for 100 ms.

[0120] (Experimental Example 1) Confirmation of the effectiveness of the T1R3 silencing vector. : Proteins were extracted from control HEK cells and HEK cells expressing the T1R3 knockout vector to examine the effect of silencing of endogenously expressed T1R3 on the protein level of T1R3 using Western blot as follows (Figure 1). The antibodies used in Western blot were the T1R3 antibody LifeSpan Biosciences, Inc. (LSP) LS-C49686,8-100 and the anti-T1R3 / AS1R3 Human (Rabbit) antibody. Specifically, the cells to be used were centrifuged at 6000 rpm for 3 minutes at 4°C to form a cell pellet. This pellet was suspended in a solution containing 100 μL of lysis buffer (20 mM / L Tri-HCl, 150 mM / L NaCl, 2 mM / EDTA, 1% Nonidet P-40), a protease inhibitor cocktail (Complete Mini; Roche Applied Science), and a phosphatase inhibitor cocktail (PhosSTOP; Roche Applied Science), and incubated on ice for 20 minutes. Subsequently, the lysis buffer containing the cells was centrifuged at 15,000 rpm at 4°C for 20 minutes, the supernatant concentration was measured, and 10 μg of this supernatant was subjected to electrophoresis (7.5% Tris-HCl polyacrylamide gel) and used for Western blotting. The electrophoretic proteins were transferred to a polyvinylidene difluoride membrane (Immobilon P; Millipore, Burlington, MA, USA). The primary antibody used was Tas1R3 antibody (LifeSpan Biosciences, Inc. (LSP) LS-C496868), and the secondary antibody was sheep anti-mouse HRP-conjugated secondary antibody (Amersham Pharmacia Biotech, Buckinghamshire, UK). Visualization was performed using enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech).

[0121] (Experimental Example 2) Creation of expression vectors for hT1R1, hT1R2, and hT1R3, and Measurement of T1R1, T1R2, and T1R3 expression levels endogenously expressed in HEK cells and SH-SY5Y cells by quantitative PCR. cDNA used and Accession numbers: hTAS1R1 (BC136515), hTAS1R2 (BC141437), hTAS1R3 (BC152912) )(Kabushiki Kaisha DNAFORM). The purchased cDNA was inserted into the expression vector using the following method. cDNA cloning was performed using Thermo's cloning kit (Gateway® cloning technology). First, the cDNA was ligated to the BamH1 / Xho1 site of the pENTR1.0A (Entry Clone) vector, and then the LR reaction (Gateway TM LR Clonase TM II Enzyme mix (Catalog number 11791100) uses Vivid Color. TM pcDN TM I cloned it into the 6.2 / EmGFP-Bsd / V5-DEST Vector (Catalog number: V36620). Using quantitative PCR (qPCR), we examined the expression levels of T1R1, T1R2, and T1R3, which are endogenously expressed in HEK cells and SH-SY5Y cells, and confirmed that they were expressed in sufficient quantities. (Figure 2) shows the results of quantitative PCR from HEK cells. It can be seen that T1R3 is expressed in significantly greater abundance than T1R1 and T1R2.

[0122] (Experimental Example 3) Knockdown vector creation The miRNA Oligo (Thermofisher) used for silencing was annealed to double-stranded DNA, which was then ligated to the miR RNAi Expression Vector using T4 DNA ligase to create a knockdown vector. Specifically, the miR RNAi Expression Vector Kit manual (BLOCK-i TM I followed the instructions for the Pol II miR RNAi Expression Vector Kit with EmGFP (Catalog number: K493600).

[0123] (Example 1) Activation of VGSC via T1R2 / T1R3, a sweet taste GPCP receptor for glutamine (Gln) Glutamine is a sweet amino acid and is recognized as a taste through T1R2 / T1R3 receptors. Since it is known to be significantly elevated in the cerebrospinal fluid of AD patients compared to healthy controls, we hypothesized the potential effect of glutamine on VGSCs and investigated whether or not this effect exists. Administration of 0.5-1 mM glutamine resulted in an amplification of VGSC current (10-20%). No further enhancement was observed with glutamine concentrations higher than 1 mM. The VGSC enhancement effect of glutamine was observed in both SH-SY5Y (n=8) and HEK-Nav1.5 (n=4). Figure 3 shows the records from SH-SY5Y. Amplification of the inward current of VGSC was confirmed before and after administration of 500 μM glutamine. Figure 3 (left) shows the actual current waveform from the experiment, and Figure 3 (right) shows the IV plot (a graph plotting the change in current magnitude in relation to the stimulation voltage).

[0124] (Example 2) Activation of VGSC via T1R2 / T1R3 by amyloid-beta peptide (Aβ25-35) (2-1) Enhancement effect of Aβ25-35 on VGSC inward current Amyloid-beta peptide is used as a biomarker for diseases such as Alzheimer's disease (AD), dementias including Lewy body dementia (Bibl et al. (2007)), and amyotrophic lateral sclerosis (ALS, Lanznaster et al. (2020)), as immunohistochemical staining using Aβ antibodies has been reported to enhance amyloid-beta peptide levels in the brain. Clinically, the relationship between amyloid-beta peptide and neuronal hyperexcitability has also been studied in ALS patients (Palop et al. (2009), Sperling et al. (2009)). It has been suggested that neuronal hyperexcitability caused by amyloid-beta peptide induces burst activity (seizure) in AD or ALS patients, which may be a cause of neurodegeneration (Hartley et al. (1999), Brunet et al.). (2020). Furthermore, it has been suggested that overexpression of amyloid-beta peptide enhances VGSCs such as Nav1.6, and that this is the cause of hyperexcitability of brain neurons (Li et al. (2016), Ciccone et al., (2019), Non-Patent Literature 1). Furthermore, Non-Patent Literature 1 demonstrates, using electrophysiological methods (membrane potential fixation), that cultured cortical neurons differentiated from iPS cells derived from AD patients exhibit increased excitability compared to controls derived from healthy individuals, resulting in burst-like neuronal hyperactivity and simultaneous amplification of VGSCs, which supports this neuronal hyperactivity. Moreover, since this neuronal hyperactivity and VGSC amplification disappear upon application of a BACE1 inhibitor, it suggests a causal relationship between Aβ peptide and neuronal hyperactivity and the onset of epileptic seizures in AD patients.

[0125] Amyloid-beta peptides, specifically amyloid-beta 1-42 (Aβ1-42) peptides, are formed from amyloid precursors. Among these amyloid-beta peptides, Aβ25-35, formed by proteases in the brain, has been reported to be the most toxic. Immunohistochemical analysis has shown that amyloid-beta peptides 25-35 are not found in healthy individuals but are found in senile plaques and in degenerated neurons of the hippocampal CA1 region in AD patients (Kubo et al. (2002)).

[0126] (2-2) Amplification of concentration-dependent VGSC current by Aβ25-35 peptide For the reasons stated above, in this example, we first treated VGSC with the Aβ25-35 peptide and investigated its effects. Specifically, experiments similar to those in Example 1, where glutamine (Gln) was administered, were performed using 1-6 μM Aβ25-35 peptide. As shown in Figure 4, an enhancement of the VGSC inward current was observed in SH-SY5Y cells (n=5), similar to the case when glutamine was applied. A similar enhancing effect was also observed in HEK-Nav1.5 cells (n=4). The concentration-dependent effect of various concentrations of Aβ25-35 peptide on the VGSC current of SH-SY5Y cells was investigated. At the highest concentration of 6 μM, the magnitude of the VGSC current was amplified 1.65 times. Then, using the membrane potential fixation method (perforated patch-clamp recording method) described in detail below (2-4), the change with 6 μM Aβ25-35 peptide was assumed to be the maximum change (100%), and the EC (Emission Control Value) was calculated. 50 The Hill slope was calculated. As a result, EC 50 The values ​​obtained were 86.8 nM for the saturation and 1.33 for the Hill Slope (Figure 9).

[0127] Next, using the current-clamp mode, electrical stimulation was applied, and action potentials from differentiated SH-SY5Y cells were recorded (Figure 5; a rectangular wave electrical stimulation was applied from the bottom to the top of the figure. The current strengths from bottom to top were 6, 8, 10, and 12 pA). Before the action of the Aβ25-35 peptide, no action potentials were induced at stimulation currents of 6 pA and 8 pA, but action potentials were recorded at stronger stimulation currents (10 pA and 12 pA). From this, it can be concluded that the threshold for generating action potentials was lowered by the Aβ25-35 peptide. In addition, the amplitude of the action potentials also increased, indicating that, similar to how glutamine's VGSC-enhancing effect lowers the excitability threshold of neurons, the Aβ25-35 peptide enhances VGSCs, making neurons more prone to hyperexcitability.

[0128] Since the Aβ25-35 peptide is said to be the most toxic, we used HEK-Nav1.5 to investigate whether the Aβ1-42 peptide has a similar effect on VGSC current using the voltage clamp method (Figure 6). Unlike Aβ25-35, no enhancing effect was observed with Aβ1-42 (n=4). Rather, unlike Aβ25-35 peptide, the sodium channel current in VGSC was suppressed with the Aβ1-42 peptide. Vitvitsky et al. showed that when Aβ25-35 peptide was applied to astrocytes, the amount of Na in the cells increased. + and K + While an increase in [protein level] occurs, Vitvitsky et al. (2012) reported that such an effect was not observed with the Aβ1-42 peptide, which is consistent with the results of this example using VGSC. Vitvitsky et al. also described that similar effects were observed with Aβ25-35 and Aβ1-40, but not with Aβ1-42, suggesting that the presence or absence of amino acid positions 40-42 may bring about some important structural change.

[0129] (2-3) The Aβ25-35 peptide enhances VGSC activity via T1R2 / T1R3. (i) Observation of T1R3 by silencing or treatment with anti-T1R3 antibody: The experimental results in the above-mentioned Example 1 showed that the sweet amino acid glutamine acts through sweet taste receptors (T1R2 / T1R3), and it is understood that the VGSC-enhancing effect of glutamine lowers the excitability threshold, making it easier to generate action potentials, which leads to an increase in the amplitude of the action potentials. Since the Aβ25-35 peptide produced effects very similar to glutamine (VGSC-enhancing effect, lowering the excitability threshold), it was considered possible that the Aβ25-35 peptide also acts on the T1R2 / T1R3 receptors in the same way as glutamine. Therefore, in this example, HEK Nav1.5 cells with silenced T1R3 cells were used to compare the effect of Aβ25-35 peptide on VGSCs with that of control HEK Nav1.5 cells.

[0130] Figures 7A and 7B show a comparison of VGSCs before and after Aβ25-35 administration, recorded using voltage clamping from control HEK Nav1.5 cells. A 1 μM Aβ25-35 effect was confirmed to enhance the VGSC current. (B) shows the actual experimental current waveforms (left: control, right: after 1 μM Aβ25-35 treatment). (A) shows the IV plot measured from experiment B. On the other hand, in HEK Nav1.5 cells with silenced T1R3 cells, no change was observed in VGSCs even when treated with 2 μM Aβ25-35 peptide, double the concentration used for control recording (n=4). (A) IV plot, (B) VGSC current traces before and after Aβ25-35 peptide treatment. In addition to the observations above, VGSCs enhanced by the Aβ25-35 peptide were restored to the size recorded in the control before enhancement by 1-1.5 mM lactisole (n=2) (Figure 7E). Given that lactisole specifically binds to T1R3, this observation also indicates that the Aβ25-35 peptide acts on T1R3. The suppression of the enhancing effect by lactisole, which is located at the carboxylate terminus rather than the extracellular ligand-binding site (Venus flytrap Domain) and binds to the TM domain near the G protein-binding site, and is thought to inhibit T1R3 activation, strongly suggests that the action of Aβ25-35 is mediated through T1R3.

[0131] (ii) Changes in sodium current enhancement in HEK Nav1.5 cells treated with anti-T1R3VFD antibody: Changes in sodium current enhancement in HEK Nav1.5 cells pretreated with anti-T1R3VFD antibody were observed (Figure 8). HEK Nav1.5 cells were incubated with a 1000-fold dilution of anti-T1R3 antibody for 10-20 minutes, and patch-clamp experiments were performed to confirm whether the effect of Aβ25-35 peptide on VGSCs could be suppressed. The IV and SS curves (n=2) of VGSC current recorded from HEK Nav1.5 cells treated with a T1R3 N-terminal recognition antibody are shown before and after the action of the Aβ25-35 peptide. In experiments where T1R3 was silenced, the effect of the Aβ25-35 peptide disappeared. In the control without anti-T1R3 antibody pretreatment, a reproducible 10-20% amplification of the VGSC current amplitude was confirmed, but no enhancement was observed in cells pretreated with the anti-T1R3 antibody. This antibody was created targeting the amino acid sequence region 229-258 of the human T1R3 VFD region and reacts specifically with human cells. The fact that this antibody suppresses the action of the Aβ25-35 peptide suggests the possibility of species differences (differences from mice) in the action of the Aβ25-35 peptide.

[0132] (iii) Potential effects on both T1R2 and T1R3: On the other hand, when T1R3 was treated with an antibody that recognizes the N-terminal side of T1R3, the enhancement of VGSC current by the Aβ25-35 peptide was suppressed, but a left shift (negative potential direction) of the IV curve and SS curve was observed (Figures 8, 29). Since the phenomenon of a left shift in the IV curve and SS curve is mainly observed when reagents / compounds that act on T1R2 are applied, it is thought that T1R2 and T1R3 are functionally closely related. This suggests that when the Aβ25-35 peptide cannot act on T1R3 due to the T1R3 antibody, T1R2 may function as a low-affinity binding site. Therefore, it is suggested that the Aβ25-35 peptide, like sucrose and sucralose, may act on both T1R2 and T1R3.

[0133] From the above, it was confirmed that T1R2 / T1R3 heterodimers or T1R3 / T1R3 homodimers act as receptors for Aβ25-35 peptides, enhancing VGSCs through a mechanism similar to that of glutamine, and potentially causing neuronal hyperactivity.

[0134] (2-4) Enhancement effect of Aβ25-35 peptide on VGSC current via T1R3 Re-examination using the perforated patch-clamp method. The VGSC current-enhancing effect of the Aβ25-35 peptide via T1R3 acts on the effector (VGSC) via intracellular second messengers mediated by G proteins. In the experiment described in Example (2-2) above, the opening in the inner diameter of the whole-cell membrane-fixed patch electrode disrupts the cell membrane, creating a hole that integrates the inside of the cell with the patch electrode. This allows for the recording of intracellular potentials, control of the cell's membrane potential, and measurement of ion currents using the membrane potential fixation method. During this process, the solution inside the patch electrode and the intracellular solution mix, and because the volume of solution inside the patch electrode is vastly greater than the volume of intracellular solution, the intracellular solution and intracellular substances are replaced by the solution inside the patch electrode. For intracellular second messengers to function properly, it is essential to maintain the composition of the intracellular solution and prevent the outflow of intracellular substances such as ATP to the patch electrode. Therefore, we re-examined the effects of the Aβ25-35 peptide using the perforated patch-clamp method, which is a membrane potential fixation method. The perforated patch-clamp method is a method in which antibiotics such as Amphotericin B and Gramicidin are mixed into the patch electrode solution to form a passage for monovalent cations to pass through lipid membranes containing cholesterol, such as cell membranes, without making holes in the cells that can be accessed by the patch electrode. The VGSC current is then recorded using the membrane potential fixation method without making holes in the cell membrane within the patch electrode (without causing changes in the intracellular environment). When the Aβ25-35 peptide was applied, the VGSC current was amplified, similar to what was recorded using the standard membrane potential fixation method (patch-clamp method). Subsequently, when the concentration of the Aβ25-35 peptide was further increased, the amplification of the VGSC current caused by the Aβ25-35 peptide disappeared, and the magnitude of the VGSC current recovered to almost the level before the Aβ25-35 peptide was applied (Figure 10). This phenomenon is thought to be due to desensitization of the VGSC receptor. What is particularly noteworthy here is that, along with the amplification of the VGSC current, an amplification of the cell membrane area was observed simultaneously. When a square wave voltage is applied during membrane potential fixed recording, the amplifier charges the capacitance generated by the cell membrane before recording the ionic current. This charge is proportional to the size of the cell (size of the cell membrane), and this charge (amount of charge) is represented by Q, which can be measured experimentally. This value Q is expressed by the equation Q = CV. The capacitance (C) of the cell membrane can be calculated by applying the voltage pulse value (V=10mV) used in this experiment and the experimentally measured charge amount (Q) to the formula C=Q / V.

[0135] As shown in (2-2) above (Figure 9), the cell membrane charge and the magnitude of the VGSC current were measured after the action of the Aβ25-35 peptide using the membrane potential fixation method. It was observed that the cell membrane charge capacitance was amplified simultaneously with the VGSC current. To determine how the increase in cell membrane capacitance relates to the increase in VGSC current, the current density of the VGSC current was calculated and analyzed. The current density (pA / pF) is obtained by dividing the VGSC current (pA) by the cell membrane capacitance (pF). (Figure 11B) After Aβ peptide action, it was found that not only did the VGSC current increase, but the VGSC current density (pA / pF) also rose. An increase in cell membrane volume indicates that the surface area of ​​the cell membrane increased due to Aβ peptide action. In other words, it means that vesicles, similar to intracellular endosomes, were attached to the cell membrane. Furthermore, the increase in VGSC current density after Aβ peptide action indicates that the loaded intracellular vesicles contain more VGSC than the VGSC already present in the cell membrane. When the Aβ peptide concentration was further increased, both the VGSC current and cell membrane volume decreased, returning to almost control levels (Figure 11A).

[0136] Since this experiment using the perforated patch-clamp method is transient, it is unclear whether VGSCs originating from intracellular vesicles with high VGSC current density, generated by Aβ peptide, diffuse to cell membranes not originating from intracellular vesicles. However, since the differential current density returns to normal when the cell volume returns to control levels, it is thought that in experiments lasting just over 10 minutes like this one, membranes originating from intracellular vesicles with high VGSC current density that were inserted into the membrane are endocytose into the vesicles while maintaining their VGSC current density. The increase or decrease in VGSC current density due to exocytosis or endocytosis was confirmed not only in HEK Nav1.5 (n=3) but also in SH-SY5Y neuroblasts differentiated in RA (n=1). Stimulation was applied every 8 seconds. The X-axis in (Figure 11C) indicates the number of stimulations. Therefore, for example, stimulation 10 means 80 seconds after the start of the experiment.

[0137] (2-5) Comparison of changes in VGSC current density due to increases and decreases in Aβ25-35 peptide concentration. In this experiment, given the striking similarity between the enhancement of AMPA-type glutamate receptors on the cell membrane in LTP and the enhancement of VGSCs on the cell membrane by Aβ25-35, we applied the method used in LTP research and adopted a method to evaluate the insertion of VGSC-containing endosomes into the cell membrane using membrane potential fixation (patch-clamp method) (Figure 12). The measurement results of the current and current density of the VGSC via T1R3 using the perforated patch-clamp, obtained in (2-4) above, are also shown in (Figure 10) and (Figure 11), and are illustrated in (Figure 12).

[0138] As shown in Figure 12, the current of VGSC increased due to the action of Aβ25-35 peptide. When the concentration of Aβ25-35 peptide was further increased, the current of VGSC decreased to the value before the action of Aβ25-35 peptide. It was observed that the increase and decrease of VGSC current proceeded simultaneously with the increase and decrease of cell membrane volume. Therefore, the current density (pA / pF) was calculated by dividing the VGSC current (pA) by the cell volume (pF). It was confirmed that the current density of VGSC increased due to the action of Aβ25-35 peptide, and that the current density decreased with increasing the amount of Aβ25-35 peptide. This suggests that intracellular endosomes expressing VGSC fuse with the cell membrane and migrate to the extracellular surface (increase in current density) due to the action of Aβ25-35 peptide, and are then taken back into the cell (decrease in current density) due to an increase in the amount of Aβ25-35 peptide. Furthermore, when calculating the current density using only the current and cell volume changes measured after the Aβ25-35 peptide action, compared to the current density before the action, it was confirmed that the change in current density caused by the Aβ25-35 peptide was greater than the current density before the action. The decrease in current density caused by the increased amount of Aβ25-35 peptide was also larger than the increase observed when the current density was increased, compared to the current density before and after the action. In other words, the amount of VGSC per unit area of ​​the membrane derived from endosomes newly added to the cell membrane by the Aβ25-35 peptide was greater than the amount of VGSC per unit area that existed on the cell membrane before the action of the Aβ25-35 peptide, and similarly, the amount of VGSC per unit area of ​​the membrane absorbed from the cell membrane by T1R3 desensitization was also greater than the amount of VGSC per unit area that existed on the cell membrane before the action of the Aβ25-35 peptide. From the above, it was concluded that the current density of VGSCs on endosomes inserted by the Aβ25-35 peptide is larger than the size (current density) per unit area of ​​VGSCs present on the cell membrane surface.

[0139] The enhancement of synaptic currents observed in LTP (Long-term potentiation), which is considered important in explaining the formation process of early memories, has been reported to occur when AMPA-type glutamate receptors expressed in intracellular vesicles fuse to the cell membrane (Park et al. (2004) Science 24:305(5692):1972-5). This is very similar to the process by which VGSCs localized in vesicles fuse and are inserted into the cell membrane in this discovery. It is thought that Rab11a and Rab5, which are involved in the transport (trafficking) of recycling endosomes, are involved in the fusion and repair (retrieval) of vesicles expressing AMPA-type glutamate receptors as part of the LTP expression mechanism (Park et al. (2004)). Therefore, as shown in (2-4) above, it is possible to measure the current density of VGSCs in cellular vesicles, or to evaluate the degree / process of vesicular fusion of labeled VGSC-expressing vesicles to the cell membrane by incorporating FITC-dextran, FM1-43 fluorescent reagent, etc., into cellular vesicles that have previously expressed VGSCs. Using the above method, it is possible to evaluate the process / degree of VGSC enhancement that occurs when VGSCs are inserted into the cell membrane by Aβ peptides. This method is effective for screening inhibitors that suppress neuronal hyperactivity caused by Aβ peptides, etc. Furthermore, it is a technique that can be applied to studying the effects of many amino acids contained in CSF (cerebrospinal fluid), particularly glutamine (L-Gln) and lysine, which are known to increase in neurodegenerative diseases such as epilepsy and Alzheimer's disease, on neuronal activity.

[0140] (Example 3) VGSC inhibitory effect targeting sweet taste receptors (T1R2 / T1R3 heterodimer or T1R3 homodimer) and its concentration dependence. (3-1) VGSC inhibitory effect targeting T1R2 / T1R3 or T1R3 / T1R3 Mailliet et al. reported that they used G16-gust44 (Ueda et al., (2003)), which has the function of converting the responses of these receptors into an intracellular calcium enhancement reaction, along with T1R2 and T1R3, in HEK cells as an experimental system in which the response to sweet stimuli can be observed as an increase in intracellular calcium, and observed the effect of T1R3 inhibitors on the response to sweet stimuli (Mailliet et al. (2009)). In that study, they used the artificial sweetener sucralose as a stimulant and used the resulting response of sweet receptors (T1R2 / T1R3) as a control, and reported the inhibitory effects of lactisole, its structural analogues fibrates, and phenoxy herbicides on this sweet stimuli response. According to the report, each IC 50 The concentration was 70 μM for lactisole, 28 μM for clofibric acid (a fibrate used to reduce cholesterol), and 5.3 μM for dichlorprop (2,4DP), a phenoxy-herbicides. Dichlorprop has been reported to bind to the transmembrane region of T1R3, similar to lactisole (Nagakita et al. (2019)).

[0141] Therefore, in this experiment, we applied substances similar to sweet taste receptors (T1R2 / T1R3), such as clofibrate and dichlorprop, which were shown to inhibit the binding of sweet taste receptors (T1R2 / T1R3) in the experimental system described above by Mailiet et al., to VGSCs and observed their IC (integration). 50 The value obtained from the above experimental results by Mailiet et al. is the IC 50We conducted experiments to determine whether the values ​​showed a similar trend. In addition, we observed whether the effects on VGSCs of artificial sweeteners saccharin and sucralose (Non-Patent Literature 2), which exhibit agonist-like activity on sweet taste receptors (T1R2 / T1R3), and Gymnema tea extract (GTE, Sanematsu et al. (2014) J. of Biol. Chem. 289(37): 25711-20.), which exhibit antagonist activity, were activating or inhibitory.

[0142] The concentration-dependent effect of dichlorprop was observed based on the membrane potential-dependent activation curve and steady-state inactivation curve of VGSC (Figure 13). Unlike saccharin, sucralose, and Gymnema tea extract (GTE), dichlorprop causes a concentration-dependent decrease in VGSC current by shifting the steady-state inactivation curve to the left (towards hyperpolarization). Therefore, when depolarization stimulation is performed using a holding voltage of -120mV, which is frequently used in this experiment to activate Nav1.5, the steady-state inactivation curve shifts further to the left (towards hyperpolarization), and since there are no VGSCs that can be activated by depolarization stimulation, it acts as an inhibitor. However, saccharin, sucralose, and Gymnema tea do not affect the steady-state inactivation curve and purely inhibit VGSC. Therefore, dichlorprop differs significantly from saccharin, sucralose, and Gymnema tea extract in that, similar to T1R2 overexpression, T1R2 silencing, and TBB (Casein kinase 2 inhibitor), it shifts the steady-state inactivation curve parallel to the negative direction of membrane potential. This confirms that while dichlorprop, saccharin, and Gymnema tea extract all have the same effect of reducing VGSC activity in a concentration-dependent manner by acting on sweet taste receptors (T1R2 / T1R3), their mechanisms of action are different. Specifically, saccharin, sucralose, and Gymnema tea are suggested to be agonists and antagonists that share a common target of action, increasing or decreasing VGSC current without affecting kinetics such as glutamine (L-Gln) and Aβ peptide (inactivation kinetics).

[0143] The steady-state inactivation curve was analyzed using Boltzmann's equation, and the slope factor and the intermediate potential (Vh) of the pre-pulse membrane potential required to halve the current were calculated. No significant change was observed in the slope (Figure 14A), but the intermediate potential (Vh) shifted in the hyperpolarization direction with increasing dicloprop concentration (Figure 14B). The analysis results, along with the results for clothifibrate, are shown below (Tables 6 and 7).

[0144] (3-2) Concentration-dependent effect of dichlorprop on VGSC amplitude Next, we analyzed the concentration-dependent effect of dicloprop on VGSC amplitude. Experimental results of steady-state inactivation recorded at different dicloprop concentrations allow for the analysis of the degree of inhibition dependent on the VGSC current amplitude and dicloprop concentration in response to a test pulse stimulus (-20mV) at a preconditioning pulse potential. Therefore, the concentration-dependent inhibitory effect of dicloprop at preconditioning pulse potentials of -80mV, -100mV, and -120mV was analyzed (Tables 6 and 7). The more hyperpolarized the preconditioning pulse potential, the greater the IC50. 50 It was found that the value increases significantly. In other words, it is highly effective at the resting potential of normal cells (around -80mV), and when used in treatment aimed at correcting the hyperexcitatory state of nerves caused by VGSC enhancement by Aβ25-35 peptide as shown in Example (2-2) (Figures 3, 4, 7), a sufficient therapeutic effect can be expected at concentrations of 100μM or less. Similar therapeutic effects can be expected not only for epilepsy and ALS, but also for neuropathic pain that occurs concurrently with anticancer drug administration (the anticancer drug Taxol, like Aβ25-35 peptide, causes a similar degree of VGSC enhancement; see Figure 8).

[0145] ICs showing the degree of VGSC suppression using dicloprop and clofibrate 50 The results calculated under different pre-pulse conditions are shown in (Tables 6 and 7). These results also show that DichlorProp is significantly more effective than chlorofibrate. 50 The value is the value measured by Mailiet et al. (2009) using calcium imaging (dicloprop IC5). 50 = 5.3 μM, Crofibrate IC 50 It is more than an order of magnitude larger than (=28μM). However, in this experiment, the degree of hyperpolarization of the holding potential (Prepulse) that activates VGSC is large and IC 50 It can be seen that it is having an effect.

[0146] [Table 6]

[0147] The Nav1.5 used in this experiment has a more hyperpolarized steady-state inactivation compared to Nav1.6 expressed in SH-SY5Y cells. Therefore, as described above, the IC of dicloprop and clofibrate 50 If the value increases as the holding voltage is hyperpolarized, it can be reasonably inferred that it will differ from the calcium imaging measurement results. IC obtained by calcium imaging 50 To further explore the differences from the values, we used SH-SY5Y cells expressing Nav1.6 to perform dicloprop IC. 50 The result was obtained when the holding voltage is -60mV. 50 The IC value was 9 μM, which is almost the same as the IC value of 5.3 μM obtained from calcium imaging. 50 A value was obtained. This suggests that dicloprop has a high safety profile for cardiac function when considered for use as an analgesic or anticonvulsant. However, with the same holding voltage (-80mV), the dicloprop IC 50 Since the values ​​differ between HEK Nav1.5 and SH-SY5Y (Nav1.6), the difference in VGSC genes (Nav1.5, Nav1.6) also plays a role in IC compared to the action of the T1R gene. 50 This is likely influencing the difference in values.

[0148] [Table 7]

[0149] As described above, dicloprop, which is known to bind to T1R3 similarly to clofibrate and lactisole, was found to have the effect of shifting the membrane dependence of the steady-state inactivation curve of VGSCs toward hyperpolarization. This phenomenon is very similar to the phenomenon observed in brain trauma, where shearing or stretching forces act on nerves, disrupting the connection between the cytoskeleton and VGSCs, and causing a shift in the membrane dependence of the steady-state inactivation curve of VGSCs toward hyperpolarization (Wang et al. (2009), Shcherbatko et al. (1999)). This suggests that GPCR sweet taste receptors (T1R2 / T1R3) may also influence the relationship between the cytoskeleton and VGSCs, and may regulate VGSC function. As described above, the cytoskeleton binds to VGSCs via Ankyrin G / Spectric, and the presence and degree of phosphorylation by casein kinase 2 at the Ankyrin G binding site on VGSCs plays a crucial role in the binding of VGSCs to Ankyrin G.

[0150] Next, in the experimental results shown in (Table 6) and (Table 7), the prepulse potential was IC 50 We considered the factors that significantly influenced the value. According to Nin et al., "hyperpolarization of the membrane potential stabilizes the actin cytoskeleton, strengthening adhesion junctions (adhesion zones) in epithelial cells" (Nin et al. (2009)). From this, it is thought that the more hyperpolarized the prepulse potential, the more stable the cytoskeleton becomes, and the weaker the effect of T1R3 blockers (sweetness-inhibiting compounds). In other words, when nerves are hyperactive, the membrane potential is depolarized compared to the prepulse potentials used in (Table 6) and (Table 7), so it is thought that the above compounds exert their VGSC inhibitory effect at even lower concentrations. This has the advantage in treatment that the effect is weak on normally functioning nerves, and selectively acts only on hyperactive nerve cells.

[0151] Based on the above, it is thought that inhibiting casein kinase 2 (CK2) would dephosphorylate the ankyrin G binding site of VGSCs, thereby weakening the relationship between VGSCs and the cytoskeleton. This would likely cause changes such as a shift in the membrane dependence of the steady-state inactivation curve toward hyperpolarization.

[0152] (Example 4) Effect of VGSC on dephosphorylation of the Ankyrin G binding site of VGSC using a casein kinase 2 inhibitor (TBB). (4-1) Inhibitory effect of casein kinase 2 inhibitor (TBB) on concentration-dependent VGSC current We discovered that TBB, known as a casein kinase 2 inhibitor, reduces the amplitude of VGSC current. Specifically, we utilized the property that the activity of voltage-dependent VGSC is affected by the potential of the pre-pulse, and that the amplitude of VGSC current due to depolarization stimulation decreases when the pre-pulse is depolarized in 10mV increments from -80mV to -50mV compared to when the pre-pulse is depolarized from -120mV to -20mV. We administered TBB to Nav1.5-expressing HEK cells and observed the change in the magnitude of the VGSC current. We observed that the inward current peak current decreased in a TBB concentration-dependent manner. This inhibitory effect by TBB was highly dependent on the membrane potential of the pre-pulse, and when the pre-pulse was -80mV, it was almost completely suppressed at the minimum concentration used (Figure 15B), which is 30μM on the left side (Figure 15B). The IV curve in Figure 15A shows the experimental results when the pre-pulse was -120mV. In the steady-state inactivation (SS) curve on the left side of the graph in Figure 15A, the SS curve for 10 μM TBB (white circle) is shifted to the hyperpolarization direction (left) compared to the control (black circle), and the VGSC current is almost completely suppressed with a -80 mV pre-pulse. However, the degree of suppression is weaker with a -100 mV pre-pulse, and almost no suppression is observed with -120 mV. In the steady-state inactivation curve experiment, the magnitude of the VGSC current activated by a -20 mV test pulse applied after pre-pulses of -170 mV to -120 mV to 0 mV is plotted against the membrane potential. As the TBB concentration increases, it gradually shifts to the left (towards hyperpolarization). (Figure 15A) Figure 15B shows the actual experimental data waveform used for plotting 15A. Next, a similar experiment was conducted using SH-SY5Y cells, yielding comparable results (data not shown).

[0153] The experimental results in Figure 15 show that the inhibitory effect of TBB on VGSCs is significantly dependent on membrane potential. This phenomenon can be attributed to a shift in the steady-state inactivation curve towards hyperpolarization (to the left), as shown above (Figures 13 and 14).

[0154] Figure 16 shows the effect of TBB on suppressing concentration-dependent VGSC current in IC. 50 The values ​​(Figure 16A) and the shift in the hyperpolarization direction of the steady-state inactivation curve of the concentration-dependent VGSC current due to TBB (Figure 16B) are shown as the difference between Vh before and after TBB action (control).

[0155] Next, a similar experiment was conducted using SH-SY5Y cells, yielding comparable results (data not shown).

[0156] The phenomenon of VGSC suppression due to a leftward shift in VGSC SS is remarkably similar to the suppression of VGSC by aspartame, which exhibits T1R2 overexpression and T1R2 agonist-like activity, as described later. This suggests that the inhibitory effect of TBB on VGSC may also be due to suppression of VGSC activity via T1R2 or other T1Rs. IC of dicloprop and aspartame with VGSC partially suppressed by TBB 50 If it can be confirmed that this has an effect, it would mean that the suppression of VGSC via T1R2 or T1R3 is related to the degree of suppression of VGSC phosphorylation by CK2 via TBB, which strongly suggests that the relationship between VGSC and the cytoskeleton with Ankyrin G and other elements is related to the mechanism of VGSC suppression via T1Rs.

[0157] VGSCs colocalize with the membrane skeletal protein ankyrin G in the initial axonal segment (AIS), nodes of Ranvier, and synaptic clefts at the neuromuscular junction in mammals. Zhou et al. reported that ankyrin G is essential for clustering VGSCs in the initial axonal segment and is therefore essential for VGSC activity (Zhou, et al., (1998)). Similarly, Brechet et al. reported that VGSCs possess an ankyrin-binding motif, and that the serine contained within this motif is phosphorylated by CK2, which is essential for the binding of VGSCs to ankyrin G. They also reported that inhibition of CK2 activity reduces sodium channel accumulation in the neuronal axonal origin (AIS) (Brechet et al., (2008)). In other words, this indicates that phosphorylation of VGSCs by CK2 regulates the degree of binding between ankyrin G and VGSCs. Xu et al. (2015) also reported that phosphorylation of VGSC by CK2 is important for binding to ankyrin G (Non-Patent Document 9).

[0158] When TBB is applied during patch-clamp recording of VGSC current, the steady-state inactivation curve (hereinafter abbreviated as SS) of VGSC shifts significantly in the hyperpolarization direction (Figure 15). In other words, if the holding potential (the membrane potential set before applying the stimulation pulse) is not set to a significantly hyperpolarized state (<-140mV) when activating the VGSC current, the magnitude of the VGSC current will be significantly reduced (suppressed).

[0159] (4-2) Delay in VGSC inactivation process by MSG Even in this suppressed state, the magnitude of the VGSC current showed a tendency to recover when MSG was applied (Figure 17), confirming that it works in the direction of correcting the hyperpolarization shift of SS (Figure 18). In addition to this, since the effect of MSG also occurs in T1R3 knockout (KO), it is suggested that while T1R1 umami receptors and sweet receptors have a cooperative relationship, there are pathways that act on VGSC through different mechanisms.

[0160] The results in Figure 18 show that the effect of the casein kinase 2 (CK2) inhibitor TBB on the SS curve of VGSC (shift towards hyperpolarization), and that subsequent application of MSG partially restores the effect of TBB on the SS curve, suggesting that the effect of MSG on VGSC via the T1R1 umami receptor may be independent of the effect caused by CK2. In Figure 18 (left), the SS curve was stimulated by changing the VGSC membrane potential in a step-like manner from different steady-state holding voltages to -20mV. When CK2 was suppressed by TBB, the SS curve shifted significantly in the hyperpolarization direction (leftward arrow). Subsequently, the application of MSG partially recovered the leftward shift caused by TBB and a rightward shift occurred (rightward arrow). The current-membrane potential curve (IV curve) on the right side of the SS curve was performed with the holding potential set to -120mV, so the maximum current was influenced by the magnitude of the VGSC current that could be activated at -120mV due to the action of the reagent. Figure 18 (right) shows the traces of the actual VGSC currents measured by the SS curves. Since the vertical axis scale is the same, the magnitude of the maximum VGSC current for each reagent can be clearly seen. It can also be confirmed that the VGSC inactivation process is delayed by the action of MSG.

[0161] The experimental results in (Figure 18) confirmed that TBB-mediated inhibition of casein kinase 2 (CK2) suppresses VGSCs. This was observed by patch clamp, where the CK2 inhibitor shifted the membrane potential conditions necessary for VGSC activity from the physiologically tolerable range of the cell to a negative potential, thereby promoting inactivation and reducing the number of activatable VGSCs within the membrane potential range in which cells are active. This invention is the first report to describe the above. Considering all the findings, including the VGSC-enhancing effect of paclitaxel (Taxol), a microtubule stabilizer constituting the cytoskeleton, the VGSC-enhancing effect of colchicine, a microtubule depolymerizer, and the inhibition of VGSC current by latentrin A, a microfibrillation inhibitor, we concluded that, regarding the relationship between the cytoskeleton and VGSCs, stimulation from taste receptors controls the activity of VGSC currents through the cytoskeleton. In other words, given that the relationship between VGSCs and the cytoskeleton strongly influences the increase or decrease of VGSC currents, TBB, a CK2 inhibitor, is also suggested to have efficacy as a treatment for pain relief and epileptic seizures.

[0162] (Example 5) Effects on neuropathic pain complicated by paclitaxel (Taxol) We hypothesized that pain caused by nerve hyperactivity, other than neurological disorders, such as neuropathic pain associated with paclitaxel (Taxol), an anticancer drug used in breast cancer treatment, is also a result of amplified VGSC currents, and investigated the effect of Taxol on VGSC currents. Specifically, the effect of Taxol on VGSC current was investigated using HEK Nav1.5 (Figure 19). VGSC current was recorded from HEK Nav1.5, and Taxol concentrations ranging from 0.4 μM to 2.4 μM were applied. The results were compared before and after administration. As a result, enhancement of VGSC was confirmed in all experiments (Figure 19, n=4). This experiment observed that Taxol, like glutamine and Aβ25-35 peptide, enhances VGSC current. This suggests that the enhancement of VGSC current may lower the nerve action potential generation threshold, making it easier for burst-like activity of action potentials, which cause pain, to occur. At the same time, it suggests that the degree of microtubule polymerization may be involved in VGSC activity. This suggests that VGSC can be enhanced by acting directly on the cytoskeleton (microtubules) without involving taste receptors, similar to sweet amino acids such as L-glutamine and Aβ25-35 peptide. Furthermore, we observed that dicloprop shifted the VGSC current SS curve towards hyperpolarization in the inactivation direction even in the presence of Taxol, resulting in the suppression / reduction of the VGSC current. This suggests that compounds with VGSC inhibitory effects mediated by T1R3 inhibitors may also be applicable to the treatment of neuropathic pain complicated by Taxol.

[0163] (Example 6) Substances that target T1R3 and have VGSC inhibitory activity (6-1) The VGSC inhibitory effect of cinacalcet is mediated through T1R3. Mattheisen et al. reported that cinacalcet, calindol, calhex, and NPS2143, allosteric modifiers of the calcium-sensing receptor (CaSR), completely suppress VGSCs in the majority of neocortical neurons in mouse cultures. In their report, they stated that the suppression of VGSCs by these reagents acts via GPCRs, that PKA and PKC are not involved, and furthermore, that the suppressive effect is not affected by CaSR knockout mice (Non-Patent Literature 7). However, the GPCRs involved were not identified in this report. In this example, since sweet amino acids act on VGSC function through T1R3 or T1R2 / T1R3 sweet taste (GPCR) receptors, we hypothesized that the effect of CaSR allosteric modifiers on VGSCs used in Mattheisen et al.'s experiment was mediated through sweet taste receptors, and conducted the following experiment. Specifically, using the CaSR agonist cinacalcet and the antagonist NPS-2143 from the above reagents, we acted them on VGSCs expressed in human-derived HEK Nav1.5, SH-SY5Y cells to verify whether similar effects were observed not only in mouse neurons but also in human-derived cells (GPCRs). As a result, both cinacalcet and NPS-2143 suppressed human VGSCs (NPS-2143 (n=10), cinacalcet (n=11)). The fact that the concentration required for VGSC suppression was 5 μM, and that it took a relatively long time of 3-5 minutes for the suppression to be completed, was consistent with the report by Mattheisen et al. (Non-Patent Literature 7). An experimental example showing the inhibitory effect of 5 μM NPS-2143 on HEK Nav1.5 VGSCs is shown in (Figure 20), and an experimental example showing the effect of 5 μM cinacalcet on SH-SY5Y is shown in (Figure 21). Figure 21A shows the SH-SY5Y endogenous VGSC current, followed by the outward potassium channel current. After the action of cinacalcet, both the VGSC current and the potassium channel current were suppressed. Figure 21B shows the changes in the VGSC current and potassium channel current after the action of cinacalcet in a time series. Cinacalcet completely suppressed not only the VGSC current but also the potassium channel current over almost the same time period.

[0164] (6-2) Analgesic effect due to VGSC inhibition via T1R3 To test the hypothesis that the above reagents act via sweet taste receptors, we pre-treated the samples with lactisole (Nakagita et al. (2019)), a sweet taste inhibitor that specifically binds to T1R3, at a concentration of 1-1.5 mM, before applying cinacalcet. This confirmed that it suppressed the cinacalcet-induced action of HEK Nav1.5 VGSC (n=3, data not shown). This experimental result strongly suggests that the inhibitory effect of cinacalcet may be mediated through T1R3 as a receptor.

[0165] To further strengthen these experimental results, a similar experiment was conducted using a HEK Nav1.5 with T1R3 silenced. (Figure 22) VGSC current was measured in HEK Nav1.5 cells in which T1R3 expression was silenced. Stimulation was repeated every 8 seconds. In control HEK Nav1.5 cells, no effect was observed even when cinacalcet, which completely inhibits expression at 5 μM, was applied twice at a concentration of 20 μM. This result confirms that suppressing T1R3 expression renders the inhibitory effect of cinacalcet ineffective (n=7).

[0166] (6-3) Observation of the inhibitory effect of cinacalcet on VGSCs in cells with altered cytoskeletal polymerization states. Based on the results in (6-2) above, it can be inferred from the observations above that the polymerization state of the cytoskeleton influences the suppression of VGSC current via T1R3 by cinacalset. Therefore, the inhibitory effect of cinacalset on VGSC was observed in cells treated with cytochalasin D, a compound that acts on the polymerization state of microfilaments, or colchicine, which acts on the polymerization state of microtubules (Figure 23). The lower panel of Figure 23A shows the VGSC currents from left to right: control, after cytochalasin D action, and after cinacalcet action. The plots above Figure 23A show the IV plots and SS curves for each recording condition. The action of cinacalcet is suppressed by cytochalasin D action. This indicates that the action of cinacalcet is carried out by controlling the polymerization state of microfibrils. Figure 23B shows cells in which microtubules were depolymerized by cortisin and then treated with cinacalcet. Cinacalcet suppressed the VGSC current as well as cells without cortisin treatment. This suggests that microtubules are not involved in the suppression of VGSC current by cinacalcet. Based on the above results, we were able to conclude that the suppression of VGSC by synacalcet is mediated at least through T1R3 in HEK cells and SH-SY5Y neuroblasts.

[0167] (6-4) Inhibitory effect of the CaSR antagonist NPS-2143 on VGSC On the other hand, NPS-2143, a CaSR antagonist, also suppressed VGSC current, similar to synacalset. However, although NPS-2143 inhibited VGSCs similarly to cinacalcet, one important difference was observed between the two reagents. After administration, once VGSC inhibition was nearly complete, NPS-2143 produced non-selective leakage currents in both outward and inward directions in many cases. This phenomenon was not observed with cinacalcet. Conversely, cinacalcet was confirmed to reduce leakage currents (Figure 24). Figure 24A shows the suppression of VGSC current by NPS-2143 over time. The upper part of Figure 24B shows the actual trace of VGSC current. Black symbols represent the peak VGSC current, and white symbols represent the baseline value. The lower plot of Figure 24B shows the VGSC current and baseline values ​​over time for NPS-2143 and after the application of synacalset. It was observed each time that the baseline leakage current increased at the timing when the VGSC current was almost completely suppressed. Therefore, since this does not happen with synacalset, we investigated whether this leakage current could be reversed by applying synacalset after NPS-2143. As expected, the leakage current decreased with synacalset.

[0168] This suggests that while NPS-2143 and cinacalcet exhibit similar effects in suppressing VGSCs, they have diametrically opposed effects in maintaining cytoskeleton and cell membrane integrity. In other words, this suggests that cinacalcet may promote the polymerization of microfilaments, while NPS-2143 may conversely inhibit polymerization.

[0169] (Example 7) Application of cinacalcet as a local anesthetic and analgesic for neuropathic pain It is known that only uncharged (unionized) local anesthetics effectively diffuse through cell (lipid) membranes. However, since the pKa value of almost all local anesthetics is greater than pH 7.6 at 37°C, under inflammatory acidic conditions, most local anesthetics exist in a charged (ionized) state, and only a portion of them can maintain membrane permeability and membrane activity. Therefore, the effectiveness of local anesthetics is significantly reduced under inflammatory acidic conditions (Tsuchiya, (2008)). Therefore, we hypothesized that compounds like cinacalcet, which target extracellular sweet taste receptor GPCRs rather than local anesthetics that directly act on VGSCs, would effectively suppress VGSCs and exert analgesic effects even under inflammatory acidic conditions. Accordingly, we measured the VGSC inhibitory effect of cinacalcet using extracellular fluid adjusted to pH 6 under acidic conditions. (Figure 25) As a result, it was confirmed that cinacalcet exhibits VGSC inhibitory effects even under acidic conditions due to inflammation (pH 6) and at a concentration of 5 μM, demonstrating its effectiveness as a local anesthetic. Pain in terminal cancer metastasis is also caused by H2C released from invasive cancer cells. + This is thought to be due to the acidic environment created by the substance. Therefore, its use as an analgesic in terminal cancer patients is being considered.

[0170] The plasma concentration of cinacalcet used to treat hypercalcemia is said to be 50 nM (Padhi, Harris (2009)). The concentration of 5 μM used in this experiment is almost 100 times higher. However, local anesthesia is usually injected locally along with vasoconstrictors such as epinephrine, so its effect is limited to the injection site and unlikely to widely disperse and diffuse into the surrounding area. For these reasons, cinacalcet is a promising candidate for local anesthesia.

[0171] (Example 8) Pain relief effect of artificial sweeteners In this example, the effects of the artificial sweeteners saccharin, aspartame, and sucralose on VGSC were investigated. As a result, all artificial sweeteners used shifted steady-state inactivation to the left (negative) in a concentration-dependent manner. (Figure 26) shows the experimental results when saccharin was administered. Compared to the control, administration of 2 mM saccharin and 4 mM saccharin resulted in a decrease in amplitude without a significant change in the VGSC waveform. From this, we concluded that using the appropriate concentration has the effect of suppressing hyperactivity and correcting nerve activity to a normal state.

[0172] This is because VGSCs are inactivated shortly after activation, preventing current from passing through (transient current generation). Therefore, if the membrane potential is not kept at a negative value (e.g., -120mV) to allow sufficient recovery from inactivation, the number of VGSCs that can be activated by depolarization stimulation decreases, resulting in a smaller VGSC current that can be activated by polarizing stimulation. It is thought that saccharin reduces the VGSC current and shifts the steady-state inactivation curve to the left, resulting in a state where VGSCs cannot recover sufficiently at -120mV. Similar changes were observed with aspartame, but the concentration required to cause a similar shift was about 1 / 5, confirming that aspartame is more effective than saccharin.

[0173] [Table 8]

[0174] The effective concentration of aspartame was calculated using the inhibition of VGSC as an indicator. The IC of aspartame based on the analysis results. 50 The value was <17 μM (49.7 μM when the membrane potential was set to -100 mV and hyperpolarized in Nav1.5). 50 The values ​​are effective concentrations determined using a T1R2 / T1R3 calcium assay performed by Masuda et al. (2012) by overexpressing G16-gust44 (here, the degree of intracellular calcium increase by aspartame is used as an indicator, so IC) 50 Instead, EC 50 ) is EC 50 The value of 756 μM was more than 10 times higher than the value obtained using VGSC as an indicator. The mechanism by which VGSC inhibits this is thought to be different from the pathway by which artificial sweeteners are recognized as sweet.

[0175] We attempted to enhance stimulation to artificial sweeteners by simultaneously introducing the T1R2 and T1R3 genes, but no significant changes were observed in the response of endogenous sweet taste receptors. Therefore, when only T1R2 was expressed, we obtained reproducible results showing a shift in the steady-state inactivation curve toward hyperpolarization. (Figure 27) shows the recording from cells expressing T1R2 in HEK Nav1.5. While the VGSC current was very small in these cells, overexpression of T1R2 resulted in a large leftward shift of the steady-state inactivation curve, similar to that observed when an artificial sweetener, an antagonist of T1R2, was applied. Specifically, when a stimulation pulse was applied with a holding voltage set to -100mV, VGSC was completely suppressed. However, when the pre-pulse holding voltage was set to -120mV, -140mV, and -180mV in the direction of hyperpolarization, the amplitude of the VGSC current in response to the stimulus increased significantly and recovered (n=3). Since the inhibitory effect disappeared when the holding voltage was set in the direction of hyperpolarization, it is thought that the principle of VGSC inhibition by artificial sweeteners via the T1R2 sweet receptor is due to promoting the inactivity of VGSC in the steady state.

[0176] (Example 9) Gymnema tea extract's effect of suppressing hyperactivity Next, as a traditional medicine, it has been used in India for over 2,000 years to treat diabetes and obesity. Similar experiments were conducted using Gymnema Tea Extract (GTE) as with saccharin. Gymnema tea is obtained from Gymnema sylvestre, a tropical plant with anti-sweet properties, and its active ingredient is gymnemic acids. Gymnema sylvestre is currently gaining recognition in Western countries as an alternative therapy for treating obesity, as the anti-sweetening substances in plants like Gymnema can lower high blood sugar levels (Mailiet et al. (2009)). Experiments using Gymnema sylvestre extract (GTE) showed a leftward shift in the steady-state inactivation curve, similar to saccharin. This experiment was conducted not only with HEK Nav1.5 cells but also with SH-SY5Y cells and NG108-15 cells. Before and after GTE administration, the negative shift in the midpoint of the membrane potential between the peak of the steady-state inactivation curve (expressed as Vh) and zero response current, and the decrease in the peak current peak were analyzed. These values ​​are summarized in the table in Figure 28 (n=2). These results suggest an effect on AD treatment similar to that of diclopropen.

[0177] (Example 10) Suppression of VGSC overactivity caused by Aβ25-35 peptide using anti-T1R3 antibody (10-1) Suppression of the effect of anti-T1R3 antibody on enhancing the inward current of Aβ peptides in VGSCs. HEKNav1.5 cells seeded on coverslips were pre-cultured for at least 10 minutes in a culture medium containing 1,000-fold diluted anti-T1R3 antibody. The coverslips containing HEK Nav1.5 cells were then transferred to a recording solution (HBPS), and the VGSC current was recorded using a patch clamp. After the VGSC current stabilized, Aβ peptide was administered, and its effects were measured. In cells fully treated with anti-T1R3 antibody, the VGSC current was not amplified at the Aβ peptide concentration that amplifies the VGSC current; instead, a shift to the left (hyperpolarized) direction occurred in the threshold for VGSC activation. The cells were cultured in a medium containing Aβ25-35 peptide, and the VGSC inward current was measured in the same manner as described above (Example 2-1, Figures 4, 6, 7). Similar experiments using SH-SY5Y cells showed that the enhancement effect of VGSC current by Aβ25-35 was suppressed when anti-T1R3 antibodies were applied (Figure 29). In contrast, we confirmed that in both SH-SY5Y cells and HEK Nav1.5 cells, when incubation times with anti-T1R3 antibody were 5 minutes and 7 minutes, the effect of anti-T1R3 antibody was insufficient, and the enhancing effect of Aβ25-35 peptide on VGSCs did occur.

[0178] (10-2) Inhibition of Aβ peptide binding to nerve cells by anti-T1R3 antibodies We confirmed that SH-SY5Y cells become fluorescently labeled when exposed to a fluorescently labeled Aβ25-35 peptide (FL-Aβ25-35) for 20-30 minutes. Next, we investigated whether the binding affinity of the fluorescently labeled Aβ25-35 peptide (FL-Aβ25-35) to SH-SY5Y cells changed when treated with the T1R3 antibody. Fluorescence from the FL-Aβ25-35 peptide was almost absent in cells pre-treated with the T1R3 antibody. (Figure 30 left) shows the control, and (Figure 30 right) shows that the binding of FL-Aβ25-35 to cells pre-treated with anti-T1R3 antibody was suppressed compared to the control. The anti-T1R3 antibody not only suppressed the amplification effect of the Aβ25-35 peptide on VGSC current, but also tended to suppress the binding of the FL-Aβ25-35 peptide to SH-SY5Y cells. These findings suggest that anti-T1R3 antibodies could be potential therapeutic agents that prevent Aβ peptide binding to nerve cells and thus prevent VGSC-mediated neuronal hyperexcitability.

[0179] (10-3) Enhancement of VGSC current by colchicine, which has microtubule resolution capabilities, and reduction of VGSC current by ratrunculin A, which has microfiber resolution capabilities. Colchicine, which disassembles microtubules, enhances VGSCs, while latenculin A, which disassembles microfibrils, decreases VGSCs. We confirmed that VGSC enhancement by Aβ25-35 occurs even in the presence of latrunculin A. Similar to TBB, artificial sweeteners, and dichlorprop, 12.6 μM latrunculin A suppressed the VGSC current by shifting the SS curve in the hyperpolarization direction. In addition, a suppression of the magnitude of the VGSC current itself was observed. In this state, when 12.5 μM Aβ25-35 was applied, the magnitude of the VGSC current, which had been reduced by latrunculin A, doubled (Figure 31). This suggests that the polymerization state of microfilaments does not play a significant role in the mechanism by which Aβ25-35 enhances VGSC current. In particular, it is thought that the enhancing effect of Aβ25-35 suppresses (regulates) the enhancing effect of Aβ25-35 via microtubules in cells where the polymerization state of microfilaments is not artificially affected (normal state).

[0180] Next, to investigate how the polymerization state of microtubules affects the kinetics of VGSCs, we checked whether VGSC current was enhanced by Aβ25-35 in the presence of colchicine, a microtubule (tubulin) polymerization inhibitor. 2.25 μM colchicine was applied. In these cells, colchicine slightly shifted the IV curve towards hyperpolarization. At -20 mV, the change in VGSC current was limited, but at -40 mV, the magnitude of the VGSC current nearly doubled. When 12.5 nM Aβ25-35 was applied, no further enhancement of the VGSC current was observed (Figure 32). In these cells, the VGSC current-enhancing effect of corticin was relatively small, but the enhancement effect by Aβ25-35 was completely inhibited. From these results, it can be concluded that the enhancement of VGSC current by Aβ25-35 depends on the polymerization state of microtubules. An example of the effect of cortisin on increasing VGSC current is shown in Figure 32E, although it is from an experiment using different cells.

[0181] (Example 11) A screening method for inhibitors of the effects of Aβ peptides on cellular proteins, including VGSCs, using changes in the fibrous structure of microtubules induced by GFP-Tubulin as an indicator. Patch-clamp experiments revealed that the effect of Aβ peptides (Aβ25-35 peptides) on VGSC current was observed even after the action of latrunculin A, which degrades microfibrils. This suggests that the polymerization state of cytoskeletal fibers (including microtubules), such as microfibrils, may be affected by Aβ peptides. Therefore, GFP-Tubulin was expressed in HEK Nav1.5, and the polymerization state of microtubules mediated by Aβ peptides was observed. The fibrous structure of microtubules, which was clearly visible in the control state, disappeared upon application of Aβ peptides. From this, it was concluded that observing the fibrous structure of microtubules in GFP-Tubulin-labeled cells is a useful screening method for inhibitors of the effects of Aβ peptides on cellular proteins, including VGSCs (Figure 33A). To confirm that the structural changes in the microfibril structure are mediated not by the direct action of Aβ peptide on the microfibrils but via Type I taste receptors, we applied MSG, a ligand for the umami receptor, and demonstrated that this induced structural changes in the microfibril structure (Figure 33B). After application of 5 mM MSG, the fibrous structure disappeared due to the depolymerization of microtubules (tubulin).

[0182] Similar to TBB, artificial sweeteners, and dichlorprop, latenchrin A significantly inhibits VGSC current by shifting the SS curve toward hyperpolarization. Furthermore, the inhibitory effect of cinacalcet on VGSC current is rendered ineffective by cytochalasin D, a compound that acts on the polymerization state of microfilaments. Therefore, the effects of cinacalcet and Aβ1-42 peptide on the fibrous structure of microfilaments were observed in cells in which the fibrous structure of microfilaments was labeled with GFP-Actin (Figure 34). In this experiment, a clear fibrous structure was not observed with GFP-Actin compared to GFP-Tubulin. However, condensation of GFP-Actin was observed in cells treated with both Cinacalcet and Aβ1-42 peptide. These findings suggest that the kinetics of VGSC current are influenced by the polymerization state of microfibrils and microtubules. The above experimental method, by labeling microtubules and microfibrils with GFP-Tubulin, GFP-Actin, etc., can be used as a screening method for compounds that control the activity of proteins that regulate ion channels and neuronal activity, including VGSC inhibition (analgesia, epilepsy, and inhibition of neuronal hyperactivity). Furthermore, tubulin and actin can be simultaneously labeled by labeling with different fluorescent proteins. In this case, after fixing the test cells, tubulin and actin fibers can be visualized using fluorescently labeled antibodies or fluorescent phaloidin.

[0183] This invention demonstrates that changes in the fibrous structure of the cytoskeleton enhance or inhibit ion channels. Therefore, a screening method using cells labeled with GFP-Tublin, GFP-Actin, etc., is effective in the drug discovery process for excluding substances that directly or indirectly affect the cytoskeleton structure in order to ensure safety. In particular, it is considered to be an effective screening method in cardiac safety tests where changes in VGSC function have a significant impact.

[0184] (Example 12) Screening methods for developing safe artificial sweeteners —Regarding artificial sweeteners and migraines— A link between migraines and Nav1.1 VGSC mutants has been reported. Nav1.1 migraine mutants include types that induce seizure-like hyperactivity and types that reduce Nav1.1 expression. Since aspartame inhibits VGSC, it is speculated that excessive in...

Claims

1. A composition for controlling the activation current of voltage-gated sodium channels (VGSC), voltage-gated potassium channels (VGKC), or voltage-gated calcium channels (VGCC) of nerve cells or cardiomyocytes, characterized in that it contains as an active ingredient a Type I taste receptor ligand as described in any of (1) to (5) below, which specifically binds to a Type I taste receptor selected from T1R1, T1R2, or T1R3, or their homodimers or heterodimers, on the cell surface and exerts an agonist or antagonist effect; (1) The Type I taste receptor ligand is a T1R3 ligand selected from lactisol, clofibrate, dicloprop, and a structural analog of lactisol, fibrate, phenoxy herbicide, and cinacalcet, which specifically binds to the T1R3 and exerts an antagonist effect, and is characterized by controlling the activation current of VGSC or VGKC. (2) The Type I taste receptor ligand is a T1R3 ligand or a T1R2 / T1R3 ligand selected from anti-human T1R3 antibodies, characterized in that the Type I taste receptor ligand controls the activation current of the VGSC by specifically binding to the T1R3 or T1R2 / T1R3. (3) The Type I taste receptor ligand is characterized in that it specifically binds to T1R2 or T1R2 / T1R3 and exerts an antagonist effect, thereby controlling the activation current of the VGSC, and is a T1R2 ligand or T1R2 / T1R3 ligand selected from saccharin, aspartame, sucralose, and Gymnema tea extract. (4) A T1R1 ligand selected from inosine phosphate (IMP) and guanosine phosphate (GMP), characterized in that the Type I taste receptor ligand specifically binds to T1R1 or T1R1 / T1R3 and exerts an agonist effect to control the VGSC activation current or the VGKC activation current. or (5) The Type I taste receptor ligand is a human T1R3 receptor ligand selected from thaumatin and blazein, which are sweet-tasting proteins, characterized in that it specifically binds to an epitope containing at least eight consecutive amino acid sequences in the 400-570aaCRD region (SEQ ID NO: 7) of the human T1R3 protein, thereby controlling the VGSC activation current of the nerve cells.

2. The anti-human T1R3 antibody described in claim 1(2) above is (1) An anti-human T1R3 antibody that specifically binds to an epitope containing at least eight consecutive amino acid sequences in the 229-258aaVFD region of the human T1R3 protein (SEQ ID NO: 5), thereby inhibiting the binding of Aβ25-35 peptide (SEQ ID NO: 1) to the said region and suppressing activated VGSC current. (2) An anti-human T1R3 antibody that specifically binds to an epitope containing at least eight consecutive amino acid sequences in the 303-396aaLBI region of the human T1R3 protein (SEQ ID NO: 6), thereby inhibiting the binding of Aβ1-40 peptide (SEQ ID NO: 3) to that region and acting on human T1R2 to suppress activated VGSC current, or The composition according to claim 1, characterized in that (3) an anti-human T1R3 antibody that specifically binds to an epitope containing at least eight consecutive amino acid sequences in the 400-570aaCRD region of human T1R3 protein (SEQ ID NO: 7), thereby inhibiting the binding of Aβ1-42 peptide (SEQ ID NO: 4) to the said region and suppressing activated VGSC current.

Images