JP2005336081A - Reexpression inhibitor against nr2b-nmda receptor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a technique for controlling expression of an NR2B-NMDA receptor and to provide a prophylactic and therapeutic agent for pain, an inhibitor against fixation of memories related to psychic traumata and an inhibitor against neurocyte death. <P>SOLUTION: The reexpression inhibitor against the NR2B-NMDA receptor, the prophylactic and therapeutic agent for pain, the inhibitor against fixation of memories related to psychic trauma and the inhibitor against neurocyte death comprise an NF-κB decoy as an effective ingredient. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a re-expression inhibitor of NR2B-NMDA receptor, and more particularly to a preventive or therapeutic agent for pain, an inhibitor of memory colonization related to trauma, and an inhibitor of neuronal cell death.

  NMDA-type glutamate receptors are known to play an important role in the plasticity of neural networks, and play a very important role in the development of the nervous system, memory and learning. NR2B type NMDA receptor (NR2B-NMDAR) is a receptor molecule that is highly expressed in early childhood, and is known to be important for the formation of neural circuits in early childhood. On the other hand, NR2B-NMDAR is less expressed in mature individuals.

  The present inventor has shown that re-expression of NR2B-NMDAR is induced by suppressing neural activity even in the mature period (Non-patent Document 1). In addition, it has been clarified that re-expression of the receptor molecule greatly changes the way the neural circuit is connected, and one cell synapses with more cells (Non-patent Document 1). The mechanism of reconnection of the neural circuit by re-expression of this receptor is an important function of the brain, and if this control becomes possible, it is expected to greatly contribute to brain function disorders and diseases.

  On the other hand, excessive neurotransmission is expected to occur due to re-expression of NR2B-NMDAR. This is thought to be the cause of “pain” due to spinal cord injury and excessive excitement of peripheral nerves. It is also considered to be the causative mechanism of the phantom limb phenomenon. If this expression control of NR2B-NMDA becomes possible, it will greatly benefit humanity.

  For example, with regard to the recognition of `` pain '' at the center, mice that overexpress NR2B in the forebrain anterior cingulate cortex insular cortex have a response to inflammatory pain stimuli. It is known to rise (Non-Patent Document 1).

  Regarding the transmission of “pain” in the periphery, it is known that NMDAR is involved in the reception of transmission from the sensory nerve to the spinal cord (Non-patent Document 3).

There is no known drug that fundamentally treats such neuropathic pain.
Nakayama K et al ,, Society for Neuroscience 2003.11.8 Wei F et al., Nature neuroscience, vol.4, p164-, 2001) Ryotaro Kuroda et al., Pharmaceutical Journal, 123 (7), 533-546, 2003

An object of the present invention is to provide a technique for controlling the expression of the NR2B-NMDA receptor.
Furthermore, an object of the present invention is to provide a preventive or therapeutic agent for pain, an inhibitor of memory fixation related to trauma, and an inhibitor of neuronal cell death.

The present invention relates to the following inventions.
1. A re-expression inhibitor of NR2B-NMDA receptor, comprising NF-κB decoy as an active ingredient.
2. A preventive or therapeutic agent for pain comprising NF-κB decoy as an active ingredient.
3. Item 3. The preventive or therapeutic agent according to Item 2, wherein the pain is neuropathic pain.
4). An inhibitor of fixing of memory related to trauma, comprising NF-κB decoy as an active ingredient.
5). A neuronal cell death inhibitor comprising NF-κB decoy as an active ingredient.
6). Item 6. The inhibitor according to Item 5, wherein the neuronal cell death is a continuous neuronal cell death accompanying the damage of another neuronal cell.
7). Item 7. The inhibitor or preventive or therapeutic agent according to any one of Items 1 to 6, wherein the NF-κB decoy is a cyclic decoy.

Hereinafter, the present invention will be described in more detail.
In the following, the NR2B-NMDA receptor re-expression inhibitor, pain preventive or therapeutic agent, memory colonization inhibitor related to trauma, and neuronal cell death inhibitor are collectively referred to as “the present invention”. May be described as “drugs”.
Expression control of NR2B type NMDA receptor (NR2B-NMDAR) using NFkB decoy The present inventor found for the first time that re-expression of NR2B-NMDAR can be suppressed by using NFkB decoy.

  The NMDA glutamate receptor (NMDAR) plays an important role in neural network plasticity and plays a very important role in the development of the nervous system, memory and learning. In particular, the NR2B type NMDA receptor (NR2B-NMDAR) is a receptor molecule that is highly expressed in early childhood, and is known to be important for the formation of neural circuits in early childhood. On the other hand, expression is low in mature individuals.

  The present inventor has revealed that reexpression of NR2B-NMDAR is induced even in the mature period, for example, by suppressing neural activity with tetrodotoxin (TTX).

  Re-expression of NR2B-NMDAR changes the way the neural circuit is connected, resulting in one cell synapsing with more cells. This increase in synaptic connections is involved in the establishment of memory that causes trauma, such as traumatic experiences that involve reduced neuronal activity, resulting in serious life threatening events. . Therefore, when such an event is encountered, it is possible to reduce or treat trauma such as PTSD by temporarily attenuating memory that is traumatic early after the encounter.

  NR2B-NMDAR can also cause pain due to excessive neurotransmission. For example, when NR2B-NMDAR is increased at the synapse of peripheral nerve cells due to spinal cord injury or peripheral nerve damage, the threshold for pain is lowered and it becomes easy to feel pain. When NFkB decoy is used in such a situation, overexpression of NR2B-NMDAR at the synapse of nerve cells is suppressed, and pain sensitivity is reduced to a normal level. Since administration of NFkB decoy can suppress pain, particularly compensatory increase in pain that occurs as a result of nerve damage, the present invention can be used as a preventive or therapeutic agent for pain, such as an analgesic. Increased pain in peripheral nerves associated with nerve damage such as the central nerve, spinal cord, and peripheral nerve is temporary, and is suppressed as time elapses from nerve damage. Accordingly, the drug of the present invention can be reduced immediately after nerve injury to reduce the degree of pain such as phantom limb pain.

  In addition, when nerve cells are locally damaged, overexpression of NR2B-NMDAR is induced in the vicinity thereof, and the nerve cells are weakened by stimulation and susceptible to cell death (apoptosis). When NFkB decoy is administered, the overexpression of NR2B-NMDAR in peripheral nerve cells is suppressed and the sensitivity to stimulation is reduced, and as a result, apoptosis of nerve cells can be suppressed. It is known that activation of extrasynaptic NMDAR (mostly including NR2B) only causes transient CREB phosphorylation, so that CRE-dependent gene expression does not occur, resulting in neuronal cell death. (Nature neuroscience vol.5, p405-, 2002). Therefore, by suppressing the expression of NR2B by decoy, it becomes possible to suppress neuronal cell death and neuronal loss due to neurological disease or stroke associated with glutamate toxicity.

  Furthermore, regarding pain, the following is known.

  For example, with regard to the recognition of “pain” at the center, mice overexpressing NR2B in the anterior cingulate cortex and insular cortex of the mouse front increased the response to inflammatory pain stimuli (Wei F et al., Nature neuroscience, vol. 4 , p164-, 2001). In addition, it is known that NMDAR is involved in the transmission of “pain” in the periphery from the sensory nerve to the spinal cord (Pharmaceutical Journal, 123 (7), 533-546, 2003) ). In the drug of the present invention, decoy can suppress pain, particularly neuropathic pain, by suppressing re-expression of NR2B-NMDAR.

  In addition, overexpression of NR2B-NMDAR is induced when nerves are damaged by trauma, etc., but not limited to this, when nerve cells are damaged by diseases including infections such as postherpetic pain In this case, the active ingredient of the present invention (NFkB decoy) is also used for the re-expression of NR2B-NMDAR, pain, nerve cell death, and establishment of memory related to trauma. It is useful for the suppression.

  As a result of searching various molecules regarding suppression of re-expression of NR2B-NMDAR, the present inventor suppresses the expression of this receptor molecule by using NFkB decoys (particularly, ribbon-type decoys (cyclic decoys)). Succeeded. And by suppressing the expression of this receptor molecule, the formation of an excess / abnormal network of neural circuits was prevented, and the memory related to pain, nerve cell death, and trauma was successfully suppressed.

  Conventionally, pain has been controlled by systemic administration of analgesics, nerve block, etc., but such conventional methods are accompanied by side effects, particularly for a relatively long period of time such as neuropathic pain. The application to pain was not appropriate.

  On the other hand, in the method of the present invention, for example, when an injury such as an accident (for example, a traffic accident, a fall, a fall, etc.) or an injury occurs, the inhibitor or prevention or treatment of the present invention is relatively quickly performed from the time of the occurrence of the injury. Administering the drug prevents the establishment of memory associated with pain, neuronal cell death, and traumatic injury, or reduces the degree of pain, neuronal cell death, and memory establishment even when it cannot be completely prevented can do. It can also have preventive or therapeutic effects on the development of pain due to nerve damage due to infections such as herpes virus, and pain such as cancer and arthritis.

  In the present specification, “decoy” refers to a site on the chromosome to which NF-κB binds, or a site on the chromosome to which another transcription factor of a gene controlled by NF-κB binds (hereinafter referred to as a target binding site). A compound that binds and antagonizes NF-κB (transcription factor) and binding to these target binding sites. The decoy is typically composed of a nucleic acid and an analog thereof, and is usually a double-stranded DNA, preferably a circular (ribbon-type) decoy.

If the decoy is present in the nucleus of a neuronal cell, the biological function provided by the decoy competing with the transcription factor for binding to the target binding site, resulting in binding of the transcription factor to the target binding site That is, re-expression of NR2B-NMDAR is suppressed, and the construction of an excessive neural network is suppressed.
The decoy includes at least one nucleic acid sequence that can bind to a target binding sequence. As long as it has binding activity to the target binding sequence, the decoy can be used as an active ingredient of the agent of the present invention.

  Examples of preferred NF-κB decoys include the following.

Double means that the decoy includes two binding factors for transcription factors, and single means that the decoy includes one binding factor for transcription factors.
The oligonucleotide may be DNA or RNA, or may contain a nucleic acid modification and / or pseudo-nucleic acid within the oligonucleotide. In addition, these oligonucleotides, variants thereof, or compounds containing these in the molecule are double-stranded and may be linear or circular. A variant is a portion of the above sequence that has been mutated, substituted, inserted, or deleted, and is specific to a nucleic acid binding site to which NF-κB or other transcription factors of genes controlled by NF-κB bind A nucleic acid that antagonizes the target. Further preferred decoys of NF-κB or other transcription factors of genes controlled by NF-κB include double-stranded oligonucleotides containing one or more of the above-described nucleic acid sequences or variants thereof.

  The oligonucleotide used in the present invention is an oligonucleotide having a thiophosphoric diester bond (S-oligo) in which the oxygen atom of the phosphodiester bond is substituted with a sulfur atom, or a methyl phosphate having no phosphodiester bond. Oligonucleotides modified to make the oligonucleotides less susceptible to degradation in vivo, such as oligonucleotides substituted with groups.

  As a method for producing the decoy used in the present invention, a chemical synthesis method or a biochemical synthesis method known in the art can be used. For example, when a nucleic acid is used as a decoy, a nucleic acid synthesis method generally used in genetic engineering can be used. For example, a target decoy nucleic acid may be directly synthesized using a DNA synthesizer, or these A nucleic acid, a nucleic acid containing it, or a part thereof may be synthesized and then amplified using a PCR method or a cloning vector. Furthermore, the nucleic acid obtained by these methods may be cleaved using a restriction enzyme or the like, and bound using DNA ligase or the like to produce the target nucleic acid. Furthermore, in order to obtain a more stable decoy nucleic acid in the cell, the base, sugar, and phosphate portion of the nucleic acid may be subjected to chemical modification such as alkylation or acylation.

  The present invention can use the above decoys alone or in combination with other agents such as stabilizing compounds, diluents, carriers, or other ingredients or agents.

  The drug of the present invention is used in such a form that the decoy is taken into the cells of the affected area or the cells of the target tissue.

  The agents of the present invention may be administered in any sterile biocompatible pharmaceutical carrier, including but not limited to saline, buffered saline, dextrose, and water. Any of these molecules can be administered to a patient alone or in combination with other agents in agents mixed with suitable excipients, adjuvants, and / or pharmaceutically acceptable carriers. In an embodiment of the invention, the pharmaceutically acceptable carrier is pharmaceutically inert.

  Administration of the agents of the present invention is accomplished orally or parenterally. Parenteral delivery methods include topical (intramuscular, subcutaneous, etc.), intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, or intranasal administration. For example, in the case of peripheral nerve or spinal cord injury, administration into the spinal cord is preferable.

  The agents of the invention for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosage forms suitable for administration. Such carriers allow the drug to be formulated into tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc. suitable for ingestion by the patient.

  Drugs for oral use combine the active compound with solid excipients and, if desired, add the appropriate further compounds to break up the resulting mixture and, if desired, to obtain tablets or dragee cores Later, it can be obtained via processing the mixture of granules. Suitable excipients include, but are not limited to: sugars including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; methylcellulose, hydroxypropylmethylcellulose, Or cellulose such as sodium carboxymethylcellulose; and gums including gum arabic and tragacanth; and proteins such as gelatin and collagen. If desired, disintegrating or solubilizing agents can be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof such as sodium alginate.

  Dragee cores are provided with suitable coatings, such as concentrated sugar solutions. It may also contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and / or titanium dioxide, a lacquer solution, and a suitable organic solvent or solvent mixture. Dyestuffs or pigments may be added to the tablets or dragees for product identification or to characterize the quantity of active compound (ie, dose).

  Pharmaceutical preparations that can be used orally include, for example, gelatin capsules, soft sealed capsules made of gelatin and a coating, such as glycerol or sorbitol. Gelatin capsules may contain the active ingredient in admixture with a filler or binder such as lactose or starch, a lubricant such as talc or magnesium stearate, and optionally a stabilizer. In soft capsules, the decoy can be dissolved or suspended in a suitable liquid such as fatty oil, liquid paraffin or liquid polyethylene glycol with or without stabilizers.

  The agents of the present invention for parenteral administration comprise an aqueous solution of the active compound. For injection, the active ingredients (decoys) of the invention can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or buffered saline. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. In addition, suspensions of the active compounds can be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty acids such as sesame oil, or synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes. If desired, the suspension may contain stabilizers or suitable agents or reagents that increase the solubility of the compounds that allow for the formulation of highly concentrated solutions.

  For topical or nasal administration, penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

  The drug of the present invention is in a form used in a commonly used gene transfer method, for example, a membrane-fused liposome preparation using Sendai virus or the like, a liposome preparation such as a liposome preparation utilizing endocytosis, lipofectamine (Lifetech) It is advantageous to use a preparation containing a cationic lipid (such as Oriental) or a virus preparation using a retrovirus vector, an adenovirus vector or the like, and a membrane fusion liposome preparation is particularly preferable.

  The liposome preparation may be a large unilamellar liposome (LUV), multilamellar liposome (MLV), or small unilamellar liposome (SUV). The size can also be a particle system of 200 to 1000 nm for LUV, 400 to 3500 nm for MLV, and 20 to 50 nm for SUV. In the case of a membrane fusion liposome preparation using Sendai virus or the like, an MLV with a particle system of 200 to 1000 nm is used. It is preferable to use it.

  The method for producing the liposome is not particularly limited as long as the decoy is retained, and a conventional method such as reverse phase evaporation (Szoka, F et al., Biochim. Biophys. Acta, Vol. 601 559 ( 1980)), ether injection method (Deamer, DW: Ann. NY Acad. Sci., Vol. 308 250 (1978)), surfactant method (Brunner, J et al .: Biochim. Biophys. Acta). , Vol.455 322 (1976)), ethanol injection method or the like.

  As lipids for forming the liposome structure, phospholipids, cholesterols, nitrogen lipids and the like are used. Generally, phospholipids are preferable, and phosphatidylcholine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, phosphatidine. Acid, cardiolipin, sphingomyelin, egg yolk lecithin, soybean lecithin, lysolecithin, etc. Palmitoylphosphatidylserine, eleostearoylphosphatidylcholine, eleostearoylphosphati Ethanolamine, it can be employed including synthetic phospholipids such Jer osteoprotegerin aroyl phosphatidylserine.

  Lipids containing these phospholipids can be used alone, but two or more can be used in combination. At this time, the binding rate of an electrically negative decoy nucleic acid can be increased by using a substance having an atomic group having a positive group such as ethanolamine or choline in the molecule. In addition to the main phospholipid at the time of liposome formation, additives such as cholesterols, stearylamine, α-tocopherol and the like that are generally known as additives for liposome formation can also be used.

  The liposomes thus obtained are purified from membrane fusion promoters such as Sendai virus, inactivated Sendai virus and Senda virus in order to promote uptake of affected cells or cells of the target tissue into cells. Membrane fusion promoting protein, polyethylene glycol and the like can be added.

  An example of a method for producing a liposome preparation will be specifically described. For example, the above-mentioned liposome-forming substance is dissolved in an organic solvent such as tetrahydrofuran, chloroform, ethanol and the like together with cholesterol, and this is placed in a suitable container and the solvent is removed under reduced pressure. To form a liposome-forming substance film on the inner surface of the container. A buffer solution containing decoy is added and stirred, and the membrane fusion promoting substance is further added to the obtained liposome as desired, and then the liposome is isolated. The liposomes containing the decoy thus obtained can be suspended in a suitable solvent, or once lyophilized, re-dispersed in a suitable solvent and used for treatment. The membrane fusion promoting substance may be added after liposome isolation and before use.

  The agent or preventive or therapeutic agent of the present invention includes a composition contained in an amount effective to achieve the purpose for which the decoy is intended. “Therapeutically effective amount” or “pharmacologically effective amount” is a term well recognized by those skilled in the art and refers to the amount of an agent effective to produce the intended pharmacological result. Thus, a therapeutically effective amount is an amount sufficient to reduce symptoms of the disease to be treated. One useful assay to ascertain an effective amount (eg, a therapeutically effective amount) for a given application is to measure the extent of recovery of the target disease. The amount actually administered will depend on the individual to whom the treatment is to be applied, and is preferably an amount optimized to achieve the desired effect without significant side effects. Determination of a therapeutically effective dose is well within the ability of those skilled in the art.

  For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays or in any appropriate animal model. The animal model is also used to achieve a desired concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.

A therapeutically effective amount refers to the amount of decoy that reduces the sign or condition of the disease. The therapeutic efficacy and toxicity of such compounds is comparable to standard pharmaceutical procedures in cell cultures or laboratory animals (eg, ED ₅₀ , doses therapeutically effective in 50% of the population; and LD ₅₀ , 50% of the population). Doses that are lethal to). The dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as ratio ED ₅₀ / LD _50. Agents that exhibit large therapeutic indices are preferred. Data obtained from cell culture assays and animal experiments are used to formulate a range of quantities for human use. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED ₅₀ with little or no toxicity. This dose will vary within this range depending on the mode of administration used, the sensitivity of the patient, and the route of administration. As an example, the dose of decoy is appropriately selected depending on the age and other patient conditions, the type of disease, the type of decoy used, etc., for example, in blood administration, intramuscular administration, intraarticular administration, 1 μg to 100 mg can be administered once to several times a day.

  The exact dose is chosen by the individual clinician in view of the patient to be treated. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Additional factors that may be considered include the severity of the disease state (eg, tumor size and location; patient age, weight, and gender; food restriction time and frequency of administration, drug combination, response sensitivity, and resistance to therapy. / Response). Depending on the half-life and clearance rate of the particular formulation, the long acting drug may be administered every 3-4 days, weekly, or once every two weeks. Guidance on specific doses and methods of delivery is provided in literature known in the art.

According to the present invention, it is possible to fix memory that causes trauma due to an accident, pain caused by damage or compression of a nerve due to virus or bacterial infection, cancer, arthritis, nerve cell death, or trauma. It can be prevented or alleviated, and conventional analgesic treatments such as administration of analgesics and nerve blocks, tranquilizers and antidepressants can be eliminated or alleviated. In addition, by suppressing nerve cell death, it is possible to prevent a decrease in nervous system function.

Hereinafter, the present invention will be described in more detail based on examples.
Example 1
Materials and methods
I. Dispersion culture of hippocampal neurons. The hippocampus was excised from an 18-day-old rat (Sprague-Dawley rat), dissociated by trypsinization, and covered on a cover glass (15 mm in diameter) coated with 0.01% polyethyleneimine (SIGMA). Culture was performed. The cell density is 6 × 10 ⁴ cells / cm ² , 5% fetal bovine serum, 5% horse serum, 50 U / ml penicillin, 50 μg / ml streptomycin, 1 mM L-glutamine (Gibco), 50 μg / ml insulin (Sigma) Was cultured in an environment of 37 ° C. and 95% carbon dioxide in DMEM (Gibco). TTX (1 μM (WaKo)) was added to the medium on days 17-18 of culture. After treatment for 48-72 hours, it was used for electrophysiological experiments.
II Electrophysiological methods ・ The composition of the solution and the solution outside the drug record is as follows.
124 mM NaCl, 5 mM KCl, 25 mM Hepes (Dojin), 30 mM glucose, 1 mM CaCl2,
pH 7.4, 310 mOsmol.
Moreover, according to the objective, the channel inhibitor was added to the external solution at the following concentrations.
100 μM picrotoxin (PTX), 1 μM tetrodotoxin (TTX, Wako), 100 μM D, L-2-amino-5-phoshponovaleric acid (APV, Sigma-Aldrich), 1 μM ifenprodil (Tocris.). The pH was adjusted to 7.4, and the osmotic pressure was adjusted to 310 mOsmol.
The electrode liquid composition is as follows.
110 mM CsMS, 10 mM CsCl2, 20 mM Hepes, 4 mM MgCl2, 10 mM EGTA-Cs
0.3 mM GTP-Na, 4 mM ATP-Na, pH 7.3, 300 mOsmol.
When the current was fixed, 110 mM CsMS was changed to KMS.

1. Evaluation of synaptic connections
Two adjacent neurons within 100μm are patched in a whole cell, each cell is given 100mV, 1ms depolarization stimulus, the other cell is fixed at -60mV and 40mV, response to the stimulus The current was recorded. Experiments in the opposite direction were also performed for each cell.

2. NMDA response current recording and analysis Whole cell recording was performed, and membrane current was measured by fixing membrane potential. Cells at each developmental stage from the 4th day to the 30th day of culture (4DIV to 30DIV) were whole-cell patch clamped to -60mV, and 20μM NMDA (added to the final concentration of 20μM in the non-recording solution) was N2 gas pressure It was administered using an injector picopump (WPI) and the current in response was recorded. In order to investigate the proportion of NMDA glutamate receptors containing NR2B subunits among the NMDA glutamate receptors functioning in these cells, 1 μM ifenprodil, a specific inhibitor of receptors containing NR2B subunits, was refluxed. The NMDA response current was recorded. In addition, in order to confirm that this current was NMDA receptor-mediated current, 100 μM APV was administered by reflux, and it was confirmed that most responses were inhibited. The perfusion was performed when the fluid in the chamber was completely replaced when 5 times the volume of the 2 ml of the chamber had been flowed.
Five traces recorded from the cells were averaged to obtain a waveform for each condition. The current that is inhibited by ifenprodil is shown as a percentage by subtracting the NMDA response current in the presence of APV from the NMDA response current in the case of only the non-recording solution.

3. Evaluation of NFkB decoy for NR2B-NMDAR expression TTX was added to 1 uM on the 18th day of cultured rat hippocampal cells to inhibit neural activity. At the same time, NFkB ribbon-type decoy was added to the medium, and evaluation was made after 2 days on the re-expression of NR2B-NMDAR. The arrangement and concentration of the ribbon-type decoy used are as follows.

result)
The hippocampus is an important organ for memory. The purpose of this study is to elucidate the mechanism of the reorganization of neural circuits using the cultured hippocampal neurons. In the hippocampal neuronal cell culture system that we have developed, it has been clarified that synapse formation occurs according to the number of culture days, and that the activity as a network is enhanced. Moreover, when the expression pattern of the receptor is examined, among NMDA type receptors (NMDARs), NMDAR (NR2B-NMDARs) containing the NR2B subunit is abundant in the early stage of development, and gradually decreases. In contrast, the NR2A subunit It is clear that NMDARs with units increase. This is similar to changes in hippocampus and cerebral cortical pyramidal cells in the in vivo system so far, and considering these as criteria, hippocampal neurons taken out from embryonic day 18 Above day (18 DIV), it is clear that it has fully matured synapses and neural circuits. Using this experimental system, we investigated the nature of synapses and functional changes as neural circuits induced when neural activity was reduced.
NFκB is a transcription factor and is involved in regulating the expression of many molecules. In order to investigate whether it is involved in the regulation of the expression of NR2B-NMDARs induced in the suppression of neural activity, we attempted to suppress it using NFκB circular DNA (ribbon-type decoy). A ribbon-type decoy (double) having two NFκB recognition sequences was added to the medium simultaneously with TTX administration. Two days later, as a result of evaluating the properties of NMDAR, it was revealed that the expression of NR2B-NMDAR was suppressed (FIG. 1).
Furthermore, even in the case of a ribbon-type decoy (Single) having only one NFκB recognition site, expression suppression was also observed. In order to investigate whether this effect is specific, a decoy (scramble) with the same base composition and a different sequence was used, but this scrambled decoy does not show a suppressive effect and is induced when nerve activity is inhibited. It was revealed that the expression of NR2B-NMDARs is suppressed by the NFκB ribbon-type decoy (FIG. 2).

Effect of NFκB decoy on re-expression of NR2B-NMDAR. When NFκB ribbon-type t-decoy (Double) was added to the medium at the same time as nerve activity, the expression of NR2B-NMDAR was suppressed. Effect of NFκB decoy on re-expression of NR2B-NMDAR. Compared with the control, re-expression of NR2B-NMDAR is induced by TTX treatment and suppressed by ribbon-type decoy (Double). In addition, the ribbon-type decoy (Single) having only one NFκB recognition site was suppressed, but the ribbon-type decoy (Scramble) having the same base component and different sequence did not.

Claims (7)

A re-expression inhibitor of NR2B-NMDA receptor, comprising NF-κB decoy as an active ingredient. A preventive or therapeutic agent for pain comprising NF-κB decoy as an active ingredient. The preventive or therapeutic agent according to claim 2, wherein the pain is neuropathic pain. An inhibitor of fixing of memory related to trauma, comprising NF-κB decoy as an active ingredient. A neuronal cell death inhibitor comprising NF-κB decoy as an active ingredient. The inhibitor according to claim 5, wherein the neuronal cell death is a continuous neuronal cell death accompanying the damage of another neuronal cell. The inhibitor or preventive or therapeutic agent according to any one of claims 1 to 6, wherein the NF-κB decoy is a cyclic decoy.

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