WO2020184676A1 - Method for high-throughput evaluation of nmda receptor inhibition activity - Google Patents

Method for high-throughput evaluation of nmda receptor inhibition activity Download PDF

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WO2020184676A1
WO2020184676A1 PCT/JP2020/010909 JP2020010909W WO2020184676A1 WO 2020184676 A1 WO2020184676 A1 WO 2020184676A1 JP 2020010909 W JP2020010909 W JP 2020010909W WO 2020184676 A1 WO2020184676 A1 WO 2020184676A1
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drebrin
nmda receptor
test substance
nerve cells
cultured
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French (fr)
Japanese (ja)
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智明 白尾
俊成 光岡
健次 花村
紀子 小金澤
祐子 関野
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国立大学法人群馬大学
国立大学法人 東京大学
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Priority to JP2021505138A priority Critical patent/JP7536240B2/en
Publication of WO2020184676A1 publication Critical patent/WO2020184676A1/en

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    • C—CHEMISTRY; METALLURGY
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor

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  • the present invention relates to a high-throughput evaluation method of NMDA receptor inhibitory activity using the linear density along the dendrites of the dendrite clusters of cultured nerve cells as an index.
  • NPS neuroactive protein
  • Non-Patent Documents 2 to 5 etc. NPS having NMDA receptor inhibitory activity such as methoxetamine and diphenidine have been reported (Non-Patent Documents 2 to 5 etc.).
  • NMDA receptors are receptors that are primarily involved in learning and memory of the central nervous system, and it is known that reduced activity of NMDA receptors causes forgetfulness, perceptual changes, hallucinations and delusions.
  • NMDA receptor inhibitors can be used as substances of abuse, and NMDA receptor inhibitors are similar to narcotics. It has been suggested that it is likely to have a dangerous effect (Non-Patent Document 6).
  • Non-Patent Documents 7 and 8 Two methods have been proposed as methods for detecting and measuring NMDA receptor inhibitory activity.
  • the first is a calcium imaging method (Non-Patent Documents 7 and 8), and the second is a substitution assay using a tritium-labeled MK801 (Non-Patent Document 9).
  • these methods cannot directly measure the action on the NMDA receptor, are complicated to operate, and may require the use of radioisotopes, so that the action on the NMDA receptor is direct and simple. , Not suitable for rapid measurement.
  • UNODC UNODC report-Global Synthetic Drugs Assessment-Amphetamine-type stimulatets and new psychoactive substances. (2017). Https://www.unodc.org/unodc/en/scientists/global-synthetic-drugs-assessment-2017.html .. Kikura-Hanajiri, R., Uchiyama, N., Kawamura, M., & Goda, Y. (2014). Changes in the prevalence of new psychoactive substances before and after the introduction of the generic scheduling of synthetic cannabinoid Testing and Analysis, 6, 832-839. Https://do.org/10.1002/dta.1584. Kikura-Hanajiri, R. (2017).
  • NMDA receptor inhibitors As mentioned above, a direct, rapid, and simple method for evaluating the inhibitory activity of NMDA receptor inhibitors has not yet been established, and new NMDA receptor inhibitors have not been regulated from the functional aspect. The current situation.
  • the present invention has been made in view of the above circumstances, and by establishing a direct, rapid, and simple high-throughput evaluation method for NMDA receptor inhibitory activity, rapid regulation of NMDA receptor inhibitor and novelty. It is intended to enable screening and activity evaluation of NMDA receptor inhibitors as therapeutic agents.
  • PCP phencyclidine
  • a high-throughput evaluation method for NMDA receptor inhibitory activity which comprises sequentially providing the following steps (A) to (E).
  • E The linear density along the dendrites of the drebrin cluster is measured, and when the linear density is higher than the linear density of the cultured nerve cells that are not in contact with the test substance, the test substance is present.
  • Steps to determine that it has an NMDA receptor inhibitory effect (2) The method according to (1) above, wherein the following step (F) is provided after the step (E). (F) A step of calculating the IC 50 of the test substance based on the correlation between the concentration of the test substance brought into contact with the cultured nerve cells in step (A) and the linear density of the drebrin cluster measured in step (E). ; (3) The method according to (1) or (2) above, wherein the following step (G) is provided after the step (F).
  • NMDA receptor inhibitory activity it is possible to provide a high-throughput evaluation method for NMDA receptor inhibitory activity, which can be carried out easily and in a short time.
  • the method for evaluating the high throughput of the NMDA receptor inhibitory activity in the present invention is (A) a step of contacting a cultured nerve cell with a test substance; (B) a step of contacting the cultured nerve cell with a glutamate solution; (C). The steps of immobilizing the cultured neurons; (D) the steps of visualizing the dendrites spine drebrin clusters of the cultured neurons; and (E) measuring the linear density along the dendrites of the cultured neurons.
  • This method is sequentially provided with a step of determining that the test substance has an NMDA receptor inhibitory action when the linear density is higher than the linear density of cultured nerve cells that are not in contact with the test substance.
  • the method of the present invention is a method for evaluating the NMDA receptor inhibitory activity of a test substance using the change in linear density of drebrin clusters formed along the dendrites of cultured nerve cells as an index.
  • the cultured nerve cell may be a cell in which the drebrin cluster is reduced by activation of the NMDA receptor, for example, a primary cultured nerve cell, a hybridoma of neuroblastoma and glioma, a neuroblastoma, or the like.
  • Cultured neurons derived from pluripotent stem cells embryonic stem cells, induced pluripotent stem cells
  • primary cultured neurons derived from Kaiba can be preferably used.
  • the origin of the cultured nerve cell is preferably one that can be used for evaluation of the inhibitory activity of the human NMDA receptor, and examples thereof include mammal origin, more preferably primate origin, and further preferably human origin.
  • cultured neurons derived from rodents such as mice and rats can also be used.
  • the cultured nerve cell may be a cultured nerve cell derived from a human fetus.
  • the test substance may be any compound that can have an inhibitory activity on the NMDA receptor, and candidate compounds for NPS and drug candidate compounds such as anesthesia can be exemplified.
  • Phencyclidine, methoxetamine, etc. It may be a known NPS analog compound such as diphenylidine, or an NMDA receptor antagonist analog compound such as ketamine.
  • the final concentration of the test substance in the above step (A) can be determined based on a conventional method at a concentration suitable for calculating the IC 50 , but for example, a concentration having an inhibition rate of 50% can be obtained. It is desirable to use a dilution series in which the test substance is serially diluted.
  • the contact time in step (A) may be a time sufficient for the test substance to reach the NMDA receptor, for example, 2 to 20 minutes, preferably 5 to 15 minutes, and more preferably 8 to 12. Minutes can be exemplified.
  • the step (B) is a step of contacting a glutamate solution in order to activate the NMDA receptor and cause a decrease in drebrin clusters.
  • the final concentration and contact time of the glutamic acid solution may be any concentration and contact time that cause appropriate NMDA receptor activation for calculating the IC 50 of the test substance.
  • the concentration may be, for example, 1 ⁇ M to 500 ⁇ M, preferably 10 ⁇ M to 300 ⁇ M, more preferably 50 ⁇ M to 200 ⁇ M, still more preferably 80 ⁇ M to 150 ⁇ M
  • the contact time may be, for example, 2 to 20 minutes, preferably 5 to 5 to. 15 minutes, more preferably 8-12 minutes can be exemplified.
  • any compound capable of inducing NMDA receptor activation may be used instead of the glutamate solution.
  • any known method may be used as long as the conditions can efficiently visualize the drebrin cluster after fixation, and examples of the fixation reagent include methanol, acetone, and the like.
  • the fixation reagent include methanol, acetone, and the like.
  • Formaldehyde, paraformaldehyde (PFA), ethanol, glutaraldehyde, dimethyl suberiminoate can be mentioned, and the fixative can be one cooled in a freezer (-20 ⁇ 2 ° C) or a refrigerator (4 ⁇ 2 ° C), or at room temperature. Can be used.
  • the visualization of the drebrin cluster of the cultured nerve cells may be visualized by any known method, and a method of visualization using a molecule capable of specifically binding to the drebrin can be exemplified. it can.
  • the molecule capable of specifically binding to the above-mentioned drebrin include anti-drebrin antibody, which is easy to detect, co-staining using another cultured neuronal marker, and 4', 6 From the viewpoint of distinguishability when cells are stained with -diamidino-2-phenylindole (DAPI), it is preferable to use a fluorescently labeled anti-drebrin antibody.
  • fluorescent label examples include fluorescent substances such as FITC, Cy3, Cy5, Rhodamine, and Alexafluor (registered trademark, manufactured by Invitrogen).
  • Anti-drebrin antibodies may be labeled directly or may be detected using secondary antibodies against them.
  • the antibody used in the present invention may be either a monoclonal antibody or a polyclonal antibody, and a commercially available antibody can be used, or it can be produced by a conventional method.
  • cultured neurons may be co-stained using other cultured neurons markers such as anti-MAP2 antibody.
  • visualization of drebrin clusters can also be performed, for example, by expressing an anti-drebrin antibody (anti-drebrin camel VHH antibody, etc.) in cultured neurons and detecting the accumulation of the anti-drebrin antibody.
  • the anti-drebrin antibody may be detected by a secondary antibody, but live cells are used by expressing the anti-drebrin antibody to which a labeling substance such as a fluorescent protein is bound in cultured nerve cells and detecting the labeling substance.
  • Real-time imaging can be performed.
  • a drebrin or a drebrin fragment to which a labeling substance such as a fluorescent protein is bound in advance is expressed in a cultured nerve cell
  • the expressed labeled drebrin or the drebrin fragment forms a drebrin cluster.
  • the drebrin cluster can be detected without additional visualization steps.
  • the cultured nerve cells in step (C) may or may not be fixed.
  • the number of drebrin positive signals visualized in the step (D) is manually or automatically counted, and the number of drebrin positive signals per unit length of the dendrite is calculated. It is measured by calculation. It is preferable to automatically measure the linear density using a known image analysis software such as IN Cell Developer Toolbox (manufactured by GE Healthcare).
  • the method of the present invention is based on the correlation between the concentration of the test substance brought into contact with the cultured nerve cells in the step (A) after the step (E) and the linear density with the drebrin cluster measured in the step (E).
  • the step (F) for calculating the IC 50 of the test substance may be provided.
  • the IC 50 can be calculated based on a conventional method. For example, an inhibition curve in which the inhibitory activity value with respect to the concentration of the test substance is plotted can be created and calculated.
  • IC 50 obtained by the method of the present invention because it is in good correlation with K i values, the IC 50 of the resultant test substance in step (F), a known NMDA receptor inhibitors By calculating the inhibitory activity ratio in comparison with the IC 50 , the activity of the test substance can be evaluated by a rapid and simple method.
  • the method of the invention can also be used for screening and evaluating activity of substances that cause NMDA receptor activation.
  • the NMDA receptor activation ability of the test substance is evaluated based on the correlation between the concentration of the test substance and the decrease in the linear density of the drebrin cluster.
  • a change in the expression level of drebrin A and / or drebrin E may be used in the activity evaluation of the test substance instead of the linear density of the drebrin cluster or together with the linear density of the drebrin cluster.
  • Such an embodiment may be useful in assessing the long-term effects of a test substance on nerve cells.
  • the present invention can evaluate the NMDA receptor inhibitory activity quickly and easily, with good quantitativeness and reproducibility, and with high accuracy as compared with the conventional method for measuring the NMDA receptor inhibitory activity. Therefore, it is expected to be applied to rapid regulation of NPS, which has been difficult to evaluate and regulate from the functional aspect, and high-throughput screening of therapeutic agents targeting NMDA receptors.
  • Cytosine ⁇ -D-arabinofuranoside (manufactured by Sigma) was added to 4DIV (in vitro culture days) to a final concentration of 0.2 ⁇ M to suppress glial cell proliferation. After 21 DIV cultures of neurons, they were treated pharmacologically.
  • test substance PCP, 3-MeO-PCMo, and 3-MeO-PCP (manufactured by Cayman Chemical Co., Ltd.) were diluted to a predetermined concentration with a 0.1% DMSO solution, and the cultured neurons in the 96-well plate were used. It was administered to the predetermined well in which the cells were cultured.
  • a glutamic acid solution (manufactured by Wako Pure Chemical Industries, Ltd.) was prepared so that the final concentration in the well was 100 ⁇ M 10 minutes after the administration of the test substance, and the solution was administered 10 minutes after the administration of the drug solution.
  • the final glutamate concentration was 1, 3, 10, 30, 50 or 100 ⁇ M for experiments to obtain a dose-response curve for the drebrin cluster density.
  • Some cultured neurons were treated with D-2-amino-5-phosphonovaleric acid (APV; 50, 100 or 500 ⁇ M), a competitive antagonist to the NMDA receptor, for 10 minutes prior to glutamate treatment.
  • AMV D-2-amino-5-phosphonovaleric acid
  • DAPI 6-diamidino-2-phenylindole dihydrochloride
  • the segmentation function localized the phosphors based on the fluorescence intensity of the MAP2-positive signal via a contrast-based segmentation algorithm with different kernel sizes and sensitivities.
  • Cell bodies of cultured neurons were identified using Erosion, Sieve, Dilation commands and DAPI-positive regions.
  • the segmentation function, as well as the Dilation and Erosion commands were used with separate parameters for MAP2-positive signals, after which the cell body region of the cultured neurons was subtracted.
  • the segmentation feature localized fluorescent objects based on the fluorescence intensity of the drebrin-positive signal via contrast-based segmentation algorithms with different kernel sizes and sensitivities.
  • the Sieve command was used to eliminate false positive background signals. In addition, only drebrin-positive spots located within a defined distance to the dendrites, as determined by the Dilation command, were counted as drebrin clusters along the dendrites. After identifying the nerve cell bodies, dendrites and dendrite clusters, number of cells per visual field, and dendrite length per visual field, the linear density of the dendrite clusters was automatically calculated.
  • PCP which is a non-competitive antagonist of the NMDA receptor
  • FIG. 2C shows that 3-MeO-PCP significantly inhibited the reduction of drebrin clusters by 100 ⁇ M glutamate at concentrations above 333 nM.
  • FIG. 2D shows that 3-MeO-PCMo significantly inhibited the reduction of drebrin clusters by 100 ⁇ M glutamate at a concentration of 3.33 ⁇ M or higher.
  • both 3-MeO-PCP and 3-MeO-PCMo inhibited the reduction of drebrin clusters in a dose-dependent manner.
  • 3-MeO-PCMo and quantitative reliability of an IC 50 value analysis of 3-MeO-PCP created the dose-response curves curve approximation based on the measured values (Figure 3). From FIG. 3, it was shown that the inhibitory activity of 3-MeO-PCP was similar to that of PCP, and the inhibitory activity of 3-MeO-PCMo was lower than that of PCP.
  • NMDA receptor inhibitory activity it is possible to provide a high-throughput evaluation method for NMDA receptor inhibitory activity, which can be carried out easily and in a short time.
  • This evaluation method enables rapid regulation of NPS, which was difficult to evaluate and regulate from the functional aspect in the past, and high-throughput screening and functional evaluation of therapeutic agents targeting NMDA receptors. Is extremely useful.

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Abstract

The present invention addresses the problem of establishing a method for the direct, rapid and simple high-throughput evaluation of an NMDA receptor inhibition activity. An NMDA receptor inhibition activity can be evaluated with high throughput by a method comprising the following steps in this order: (A) bringing a substance of interest into contact with cultured neurons; (B) bringing the cultured neurons into contact with a glutamate solution; (C) immobilizing the cultured neurons; (D) visualizing drebrin clusters of dendritic spines of the cultured neurons; and (E) measuring the line density along the dendrites of the drebrin clusters, and determining that the substance of interest has an NMDA receptor inhibition activity when the line density is higher than the line density of the cultured neurons that are not contacted with the substance of interest.

Description

ＮＭＤＡ受容体阻害活性のハイスループット評価方法High-throughput evaluation method for NMDA receptor inhibitory activity
　本発明は、培養神経細胞の樹状突起スパインのドレブリンクラスターの樹状突起に沿った線密度を指標として用いる、ＮＭＤＡ受容体阻害活性のハイスループット評価方法に関する。 The present invention relates to a high-throughput evaluation method of NMDA receptor inhibitory activity using the linear density along the dendrites of the dendrite clusters of cultured nerve cells as an index.
　近年、新精神作用物質（New psychoactive substances；ＮＰＳ、日本では「危険ドラッグ」とも呼ばれる）が、「リーガル・ハイ」、「バスソルト」、「リサーチケミカル」等の用語を使って販売されている。しかしながら、それらは国際的に規制されている薬物と同様の効果を持つ乱用物質である。ＮＰＳの出現は世界中の１００以上の国と地域で報告されており、ＮＰＳの広がりが、多くの国で公衆衛生上の懸念を高めている（非特許文献１）。したがって、リスク評価プロセスを通じてこれらの物質を速やかに規制するために、ＮＰＳに対して利用可能な関連データ及び分析結果を蓄積していくことが極めて重要である。 In recent years, new psychoactive substances (NPS, also called "dangerous drugs" in Japan) have been sold using terms such as "legal high", "bath salts", and "research chemicals". However, they are abusers that have the same effect as internationally regulated drugs. The emergence of NPS has been reported in more than 100 countries and territories around the world, and the spread of NPS has raised public health concerns in many countries (Non-Patent Document 1). Therefore, it is extremely important to accumulate relevant data and analytical results available for the NPS in order to expedite the regulation of these substances through the risk assessment process.
　メトキセタミンやジフェニジン等の、ＮＭＤＡ受容体阻害活性を有するＮＰＳが報告されている（非特許文献２～５等）。ＮＭＤＡ受容体は、中枢神経系の学習と記憶に主に関与する受容体であり、ＮＭＤＡ受容体の活動が低下すると、健忘症、知覚の変化、幻覚及び妄想が起こることが知られている。このように、ＮＭＤＡ受容体は、知覚的及び精神的現象に大いに関連しているため、ＮＭＤＡ受容体阻害剤が乱用薬物として使用される可能性、及び、ＮＭＤＡ受容体阻害剤が麻薬と同様の危険な効果を持つ可能性が高いことが示唆されている（非特許文献６）。しかしながら、中枢神経系に対して作用する化合物の安全性薬理試験法は、ネズミ個体を用いた行動解析のみであり、コスト、時間がかかるという問題点がある。そのため、規制当局は、新規化合物についてインシリコで構造の類似性を判定し、規制するか否かを判断しているのが現状である。 NPS having NMDA receptor inhibitory activity such as methoxetamine and diphenidine have been reported (Non-Patent Documents 2 to 5 etc.). NMDA receptors are receptors that are primarily involved in learning and memory of the central nervous system, and it is known that reduced activity of NMDA receptors causes forgetfulness, perceptual changes, hallucinations and delusions. Thus, because NMDA receptors are highly associated with perceptual and psychological phenomena, NMDA receptor inhibitors can be used as substances of abuse, and NMDA receptor inhibitors are similar to narcotics. It has been suggested that it is likely to have a dangerous effect (Non-Patent Document 6). However, the safety pharmacological test method for compounds that act on the central nervous system is only behavioral analysis using individual mice, and has the problem of cost and time. Therefore, the current situation is that regulators judge the structural similarity of new compounds with in silico and decide whether or not to regulate them.
　上述の状況から、ＮＭＤＡ受容体阻害活性を有するＮＰＳを、機能的側面から既存の薬物との類似度を評価して迅速に規制できるようにするため、ＮＭＤＡ受容体阻害活性を検出、測定する方法が求められている。現状では、ＮＭＤＡ受容体阻害活性の検出、測定方法として、２つの方法が提案されている。第一はカルシウムイメージング法であり（非特許文献７、８）、第二はトリチウム標識したＭＫ８０１を用いた置換アッセイである（非特許文献９）。しかしながら、これらの方法は、ＮＭＤＡ受容体に対する作用を直接測定することができないうえ、操作が煩雑であり、放射性同位体を使用しなければならない場合もあり、ＮＭＤＡ受容体阻害作用の直接的且つ簡便、迅速な測定には不向きである。 From the above situation, a method for detecting and measuring NMDA receptor inhibitory activity in order to evaluate the similarity with existing drugs from a functional aspect and rapidly regulate NPS having NMDA receptor inhibitory activity. Is required. At present, two methods have been proposed as methods for detecting and measuring NMDA receptor inhibitory activity. The first is a calcium imaging method (Non-Patent Documents 7 and 8), and the second is a substitution assay using a tritium-labeled MK801 (Non-Patent Document 9). However, these methods cannot directly measure the action on the NMDA receptor, are complicated to operate, and may require the use of radioisotopes, so that the action on the NMDA receptor is direct and simple. , Not suitable for rapid measurement.
　培養神経細胞は、培養を行うと樹状突起を徐々に伸ばし、ニューロン間の成熟シナプスを形成する。その後、培養３週間で、きのこ型の頭部を持つ「樹状突起スパイン」と呼ばれる構造が完成する。樹状突起スパインにおいて、ドレブリンはアクチンフィラメントに高濃度で蓄積され、樹状突起スパインにおける受容体及び足場タンパク質の動態を安定化させる（非特許文献１０、１１）。このように樹状突起スパインに蓄積されたドレブリンは、ドレブリンクラスターと呼ばれる。培養神経細胞に対するグルタミン酸処理は、樹状突起スパインから樹状突起への急速なドレブリン流出を誘導する（非特許文献１２、１３）。投与されたグルタミン酸は、ＡＭＰＡ型グルタミン酸レセプター（ＡＭＰＡＲ）とＮＭＤＡ受容体の両方を活性化するが、ドレブリンクラスターからのドレブリンの流出は、ＮＭＤＡ受容体活性化によって誘導され、定量的である（非特許文献１３～１５）。 Cultured nerve cells gradually extend dendrites when cultured and form mature synapses between neurons. Then, in 3 weeks of culturing, a structure called "dendritic spine" having a mushroom-shaped head is completed. In dendritic spines, drebrin accumulates in high concentrations in actin filaments and stabilizes the dynamics of receptors and scaffold proteins in dendritic spines (Non-Patent Documents 10 and 11). The drebrin accumulated in the dendrite spine in this way is called a drebrin cluster. Glutamic acid treatment of cultured neurons induces rapid outflow of drebrin from dendrite spines to dendrites (Non-Patent Documents 12 and 13). Administered glutamate activates both AMPA-type glutamate receptors (AMPAR) and NMDA receptors, but drainage of drebrin from drebrin clusters is induced by NMDA receptor activation and is quantitative (non-patent). Documents 13 to 15).
　上述したとおり、ＮＭＤＡ受容体阻害剤の、直接的且つ迅速、簡便な阻害活性の評価方法は未だ構築されておらず、機能面からの新規ＮＭＤＡ受容体阻害剤の規制は行われていないのが現状である。本発明は、以上の事情に鑑みなされたものであり、ＮＭＤＡ受容体阻害活性の直接的且つ迅速、簡便なハイスループット評価方法を確立することにより、ＮＭＤＡ受容体阻害剤の迅速な規制、及び新規治療薬としてのＮＭＤＡ受容体阻害剤のスクリーニング及び活性評価を可能にすることを目的とするものである。 As mentioned above, a direct, rapid, and simple method for evaluating the inhibitory activity of NMDA receptor inhibitors has not yet been established, and new NMDA receptor inhibitors have not been regulated from the functional aspect. The current situation. The present invention has been made in view of the above circumstances, and by establishing a direct, rapid, and simple high-throughput evaluation method for NMDA receptor inhibitory activity, rapid regulation of NMDA receptor inhibitor and novelty. It is intended to enable screening and activity evaluation of NMDA receptor inhibitors as therapeutic agents.
　本発明者らは、鋭意検討を重ねた結果、既知のＮＭＤＡ受容体阻害剤であるフェンシクリジン（ＰＣＰ）と、ＰＣＰアナログ２種（３－ＭｅＯ－ＰＣＭｏ及び３－ＭｅＯ－ＰＣＰ）を用い、これらの化合物を成熟した培養神経細胞に接触させることで、グルタミン酸処理によるドレブリンクラスターの線密度減少が抑制されることを見いだした。さらに、かかるドレブリンクラスターの線密度減少が、化合物濃度と相関すること、並びに該相関に基づいて算出した５０％阻害濃度（ＩＣ５０）の比が、これらの化合物の阻害定数（Ｋｉ）の比と良好な相関関係を示すことを見いだした。本発明は、これらの知見により完成したものである。 As a result of diligent studies, the present inventors have used phencyclidine (PCP), which is a known NMDA receptor inhibitor, and two types of PCP analogs (3-MeO-PCMo and 3-MeO-PCP). It was found that contacting these compounds with mature cultured neurons suppresses the decrease in linear density of drebrin clusters due to glutamate treatment. Furthermore, the linear density reduction of such drain Brin cluster, the ratio of to correlate with compound concentration, and 50% inhibitory concentrations were calculated based on the correlation (IC 50), the ratio of inhibition constants for these compounds (K i) It was found that it shows a good correlation with. The present invention has been completed based on these findings.
　すなわち、本発明は以下の事項により特定されるとおりのものである。
（１）以下のステップ（Ａ）～（Ｅ）を順次備えたことを特徴とするＮＭＤＡ受容体阻害活性のハイスループット評価方法。
（Ａ）培養神経細胞に、被験物質を接触させるステップ；
（Ｂ）前記培養神経細胞に、グルタミン酸溶液を接触させるステップ；
（Ｃ）前記培養神経細胞を固定するステップ；
（Ｄ）前記培養神経細胞の樹状突起スパインのドレブリンクラスターを可視化するステップ；
（Ｅ）前記ドレブリンクラスターの樹状突起に沿った線密度を測定し、前記線密度が、前記被験物質に接触させていない培養神経細胞の線密度と比較して高い場合に、前記被験物質がＮＭＤＡ受容体阻害作用を有すると判定するステップ；
（２）以下のステップ（Ｆ）を、ステップ（Ｅ）の後に備えたことを特徴とする上記（１）に記載の方法。
（Ｆ）ステップ（Ａ）で培養神経細胞に接触させた被験物質の濃度と、ステップ（Ｅ）で測定したドレブリンクラスターの線密度との相関に基づいて、前記被験物質のＩＣ５０を算出するステップ；
（３）以下のステップ（Ｇ）を、ステップ（Ｆ）の後に備えたことを特徴とする上記（１）又は（２）に記載の方法。
（Ｇ）ステップ（Ｆ）で算出した被験物質のＩＣ５０を、既知のＮＭＤＡ受容体阻害剤のＩＣ５０と比較することにより、前記被験物質と、前記既知のＮＭＤＡ受容体阻害剤とのＮＭＤＡ受容体阻害活性比を算出するステップ；
（４）培養神経細胞が、海馬神経細胞由来であることを特徴とする上記（１）～（３）のいずれかに記載の方法。
（５）培養神経細胞が、げっ歯類由来であることを特徴とする上記（１）～（４）のいずれかに記載の方法。
（６）ステップ（Ｄ）の可視化が、抗ドレブリン抗体による免疫染色であることを特徴とする上記（１）～（５）のいずれかに記載の方法。 That is, the present invention is as specified by the following matters.
(1) A high-throughput evaluation method for NMDA receptor inhibitory activity, which comprises sequentially providing the following steps (A) to (E).
(A) Step of contacting the cultured nerve cells with the test substance;
(B) A step of contacting the cultured nerve cells with a glutamic acid solution;
(C) Step of fixing the cultured nerve cells;
(D) A step of visualizing the drebrin cluster of the dendritic spine of the cultured nerve cell;
(E) The linear density along the dendrites of the drebrin cluster is measured, and when the linear density is higher than the linear density of the cultured nerve cells that are not in contact with the test substance, the test substance is present. Steps to determine that it has an NMDA receptor inhibitory effect;
(2) The method according to (1) above, wherein the following step (F) is provided after the step (E).
(F) A step of calculating the IC 50 of the test substance based on the correlation between the concentration of the test substance brought into contact with the cultured nerve cells in step (A) and the linear density of the drebrin cluster measured in step (E). ;
(3) The method according to (1) or (2) above, wherein the following step (G) is provided after the step (F).
The IC 50 of the test substance calculated in (G) Step (F), by comparing the IC 50 of the known NMDA receptor inhibitors, NMDA receptor of said analyte, and the known NMDA receptor inhibitors Steps to calculate body inhibitory activity ratio;
(4) The method according to any one of (1) to (3) above, wherein the cultured nerve cells are derived from hippocampal nerve cells.
(5) The method according to any one of (1) to (4) above, wherein the cultured nerve cells are derived from rodents.
(6) The method according to any one of (1) to (5) above, wherein the visualization in step (D) is immunostaining with an anti-drebrin antibody.
　本発明によれば、簡便且つ短時間で実施することのできる、ＮＭＤＡ受容体阻害活性のハイスループット評価方法を提供することができる。 According to the present invention, it is possible to provide a high-throughput evaluation method for NMDA receptor inhibitory activity, which can be carried out easily and in a short time.
実施例のセクション２－１の結果を示す図である。It is a figure which shows the result of section 2-1 of an Example. 実施例のセクション２－２の結果を示す図である。It is a figure which shows the result of Section 2-2 of an Example. 実施例のセクション２－３の結果を示す図である。It is a figure which shows the result of Section 2-3 of an Example.
　本発明におけるＮＭＤＡ受容体阻害活性のハイスループット評価方法とは、（Ａ）培養神経細胞に、被験物質を接触させるステップ；（Ｂ）前記培養神経細胞に、グルタミン酸溶液を接触させるステップ；（Ｃ）前記培養神経細胞を固定するステップ；（Ｄ）前記培養神経細胞の樹状突起スパインのドレブリンクラスターを可視化するステップ；及び（Ｅ）前記ドレブリンクラスターの樹状突起に沿った線密度を測定し、前記線密度が、前記被験物質に接触させていない培養神経細胞の線密度と比較して高い場合に、前記被験物質がＮＭＤＡ受容体阻害作用を有すると判定するステップ；を順次備えた方法である。 The method for evaluating the high throughput of the NMDA receptor inhibitory activity in the present invention is (A) a step of contacting a cultured nerve cell with a test substance; (B) a step of contacting the cultured nerve cell with a glutamate solution; (C). The steps of immobilizing the cultured neurons; (D) the steps of visualizing the dendrites spine drebrin clusters of the cultured neurons; and (E) measuring the linear density along the dendrites of the cultured neurons. This method is sequentially provided with a step of determining that the test substance has an NMDA receptor inhibitory action when the linear density is higher than the linear density of cultured nerve cells that are not in contact with the test substance.
　本発明の方法は、培養神経細胞の樹状突起に沿って形成されたドレブリンクラスターの線密度変化を指標として、被験物質のＮＭＤＡ受容体阻害活性を評価する方法である。ここで、培養神経細胞としては、ＮＭＤＡ受容体の活性化によりドレブリンクラスターが減少する細胞であればよく、例えば、初代培養神経細胞、神経芽細胞腫と神経膠腫のハイブリドーマ、神経芽細胞腫や、多能性幹細胞（胚性幹細胞、人工多能性幹細胞）に由来する培養神経細胞等を挙げることができ、海馬由来の初代培養神経細胞を好適に使用することができる。また、培養神経細胞の由来は、ヒトのＮＭＤＡ受容体の阻害活性評価に用いることができるものであることが好ましく、例えば哺乳類由来、より好ましくは霊長類由来、さらに好ましくはヒト由来を挙げることができるが、マウスやラット等、げっ歯類由来の培養神経細胞を用いることもできる。また、上記培養神経細胞は、ヒト胎児由来の培養神経細胞であってもよい。 The method of the present invention is a method for evaluating the NMDA receptor inhibitory activity of a test substance using the change in linear density of drebrin clusters formed along the dendrites of cultured nerve cells as an index. Here, the cultured nerve cell may be a cell in which the drebrin cluster is reduced by activation of the NMDA receptor, for example, a primary cultured nerve cell, a hybridoma of neuroblastoma and glioma, a neuroblastoma, or the like. , Cultured neurons derived from pluripotent stem cells (embryonic stem cells, induced pluripotent stem cells) and the like can be mentioned, and primary cultured neurons derived from Kaiba can be preferably used. Further, the origin of the cultured nerve cell is preferably one that can be used for evaluation of the inhibitory activity of the human NMDA receptor, and examples thereof include mammal origin, more preferably primate origin, and further preferably human origin. However, cultured neurons derived from rodents such as mice and rats can also be used. Further, the cultured nerve cell may be a cultured nerve cell derived from a human fetus.
　本発明において、被験物質とは、ＮＭＤＡ受容体の阻害活性を有し得る化合物であればよく、ＮＰＳの候補化合物や、麻酔等の医薬品候補化合物を例示することができ、フェンシクリジン、メトキセタミン、ジフェニジン等の既知のＮＰＳの類縁化合物や、ケタミン等のＮＭＤＡ受容体拮抗薬の類縁化合物であってもよい。上記ステップ（Ａ）における被験物質の終濃度は、ＩＣ５０を算出するために適した濃度を常法に基づいて決定することができるが、例えば阻害率５０％を挟む濃度が得られるように、被験物質を段階希釈した希釈系列を用いることが望ましい。また、上記ステップ（Ａ）における接触時間は、被験物質がＮＭＤＡ受容体まで到達するために十分な時間であればよく、例えば２～２０分間、好ましくは５～１５分間、より好ましくは８～１２分間を例示することができる。 In the present invention, the test substance may be any compound that can have an inhibitory activity on the NMDA receptor, and candidate compounds for NPS and drug candidate compounds such as anesthesia can be exemplified. Phencyclidine, methoxetamine, etc. It may be a known NPS analog compound such as diphenylidine, or an NMDA receptor antagonist analog compound such as ketamine. The final concentration of the test substance in the above step (A) can be determined based on a conventional method at a concentration suitable for calculating the IC 50 , but for example, a concentration having an inhibition rate of 50% can be obtained. It is desirable to use a dilution series in which the test substance is serially diluted. The contact time in step (A) may be a time sufficient for the test substance to reach the NMDA receptor, for example, 2 to 20 minutes, preferably 5 to 15 minutes, and more preferably 8 to 12. Minutes can be exemplified.
　上記ステップ（Ｂ）は、ＮＭＤＡ受容体を活性化させ、ドレブリンクラスターの減少を引き起こすために、グルタミン酸溶液を接触させるステップである。ここで、グルタミン酸溶液の終濃度及び接触時間は、被験物質のＩＣ５０を算出するために適切なＮＭＤＡ受容体活性化を引き起こす濃度及び接触時間であればよい。上記濃度としては例えば１μＭ～５００μＭ、好ましくは１０μＭ～３００μＭ、より好ましくは５０μＭ～２００μＭ、さらに好ましくは８０μＭ～１５０μＭを挙げることができ、上記接触時間としては、例えば２～２０分間、好ましくは５～１５分間、より好ましくは８～１２分間を例示することができる。また、グルタミン酸溶液に代えて、ＮＭＤＡ受容体活性化を引き起こすことのできるいかなる化合物を用いてもよい。 The step (B) is a step of contacting a glutamate solution in order to activate the NMDA receptor and cause a decrease in drebrin clusters. Here, the final concentration and contact time of the glutamic acid solution may be any concentration and contact time that cause appropriate NMDA receptor activation for calculating the IC 50 of the test substance. The concentration may be, for example, 1 μM to 500 μM, preferably 10 μM to 300 μM, more preferably 50 μM to 200 μM, still more preferably 80 μM to 150 μM, and the contact time may be, for example, 2 to 20 minutes, preferably 5 to 5 to. 15 minutes, more preferably 8-12 minutes can be exemplified. Also, instead of the glutamate solution, any compound capable of inducing NMDA receptor activation may be used.
　上記ステップ（Ｃ）における培養神経細胞の固定は、固定後のドレブリンクラスターの可視化が効率的に行える条件であればいかなる公知の方法を用いてもよく、固定化試薬としては、例えばメタノール、アセトン、ホルムアルデヒド、パラホルムアルデヒド（ＰＦＡ）、エタノール、グルタルアルデヒド、スベルイミノ酸ジメチルを挙げることができ、固定化試薬は、冷凍庫（－２０±２℃）又は冷蔵庫（４±２℃）で冷却したものや、室温のものを使用することができる。 For the fixation of the cultured nerve cells in the above step (C), any known method may be used as long as the conditions can efficiently visualize the drebrin cluster after fixation, and examples of the fixation reagent include methanol, acetone, and the like. Formaldehyde, paraformaldehyde (PFA), ethanol, glutaraldehyde, dimethyl suberiminoate can be mentioned, and the fixative can be one cooled in a freezer (-20 ± 2 ° C) or a refrigerator (4 ± 2 ° C), or at room temperature. Can be used.
　上記ステップ（Ｄ）において、培養神経細胞のドレブリンクラスターの可視化は、いかなる公知の方法により可視化してもよく、ドレブリンに対して特異的に結合し得る分子を用いて可視化する方法を例示することができる。上記ドレブリンに対して特異的に結合し得る分子としては、例えば抗ドレブリン抗体を挙げることができ、検出の簡便性や、他の培養神経細胞マーカーを用いて共染色した場合や、４’，６－ジアミジノ－２－フェニルインドール（ＤＡＰＩ）を用いて細胞を染色した場合における識別性の観点から、蛍光標識された抗ドレブリン抗体を用いることが好ましい。ここで、蛍光標識としては、ＦＩＴＣ、Ｃｙ３、Ｃｙ５、Rhodamine、Alexa fluor（登録商標、インビトロジェン社製）等の蛍光物質を挙げることができる。抗ドレブリン抗体は、直接標識してもよく、これらに対する二次抗体を用いて検出してもよい。本発明で用いる抗体は、モノクローナル抗体、ポリクローナル抗体のいずれであってもよく、市販のものを使用できる他、常法により作製することもできる。また、抗ＭＡＰ２抗体等の他の培養神経細胞マーカーを用いて、培養神経細胞を共染色してもよい。 In the above step (D), the visualization of the drebrin cluster of the cultured nerve cells may be visualized by any known method, and a method of visualization using a molecule capable of specifically binding to the drebrin can be exemplified. it can. Examples of the molecule capable of specifically binding to the above-mentioned drebrin include anti-drebrin antibody, which is easy to detect, co-staining using another cultured neuronal marker, and 4', 6 From the viewpoint of distinguishability when cells are stained with -diamidino-2-phenylindole (DAPI), it is preferable to use a fluorescently labeled anti-drebrin antibody. Here, examples of the fluorescent label include fluorescent substances such as FITC, Cy3, Cy5, Rhodamine, and Alexafluor (registered trademark, manufactured by Invitrogen). Anti-drebrin antibodies may be labeled directly or may be detected using secondary antibodies against them. The antibody used in the present invention may be either a monoclonal antibody or a polyclonal antibody, and a commercially available antibody can be used, or it can be produced by a conventional method. In addition, cultured neurons may be co-stained using other cultured neurons markers such as anti-MAP2 antibody.
　一態様では、ドレブリンクラスターの可視化は、例えば培養神経細胞において抗ドレブリン抗体（抗ドレブリンラクダＶＨＨ抗体等）を発現させ、該抗ドレブリン抗体の集積を検出することにより行うこともできる。抗ドレブリン抗体は、二次抗体で検出してもよいが、蛍光タンパク質等の標識物質を結合させた抗ドレブリン抗体を培養神経細胞内で発現させ、標識物質を検出することにより、生細胞を用いたリアルタイムイメージングを行うことができる。また、一態様において、あらかじめ蛍光タンパク質等の標識物質を結合させたドレブリン又はドレブリン断片を培養神経細胞内で発現させると、発現した標識ドレブリン又はドレブリン断片がドレブリンクラスターを形成する。この態様においては、該標識ドレブリン又はドレブリン断片を検出することにより、追加の可視化ステップなしにドレブリンクラスターを検出することができる。これらの態様においては、ステップ（Ｃ）における培養神経細胞の固定は行っても行わなくてもよい。 In one aspect, visualization of drebrin clusters can also be performed, for example, by expressing an anti-drebrin antibody (anti-drebrin camel VHH antibody, etc.) in cultured neurons and detecting the accumulation of the anti-drebrin antibody. The anti-drebrin antibody may be detected by a secondary antibody, but live cells are used by expressing the anti-drebrin antibody to which a labeling substance such as a fluorescent protein is bound in cultured nerve cells and detecting the labeling substance. Real-time imaging can be performed. Further, in one embodiment, when a drebrin or a drebrin fragment to which a labeling substance such as a fluorescent protein is bound in advance is expressed in a cultured nerve cell, the expressed labeled drebrin or the drebrin fragment forms a drebrin cluster. In this embodiment, by detecting the labeled drebrin or drebrin fragment, the drebrin cluster can be detected without additional visualization steps. In these embodiments, the cultured nerve cells in step (C) may or may not be fixed.
　上記ステップ（Ｅ）におけるドレブリンクラスターの線密度は、上記ステップ（Ｄ）により可視化されたドレブリン陽性シグナルの個数を手動又は自動で計数し、樹状突起の単位長さ当たりのドレブリン陽性シグナルの個数を算出することにより測定される。かかる線密度の測定は、IN Cell Developer Toolbox（ＧＥヘルスケア社製）等の公知の画像解析ソフトウェアを用いて自動測定することが好ましい。 For the linear density of the drebrin cluster in the step (E), the number of drebrin positive signals visualized in the step (D) is manually or automatically counted, and the number of drebrin positive signals per unit length of the dendrite is calculated. It is measured by calculation. It is preferable to automatically measure the linear density using a known image analysis software such as IN Cell Developer Toolbox (manufactured by GE Healthcare).
　本発明の方法は、上記ステップ（Ｅ）の後に、上記ステップ（Ａ）で培養神経細胞に接触させた被験物質の濃度と、ステップ（Ｅ）で測定したドレブリンクラスターとの線密度の相関に基づいて、前記被験物質のＩＣ５０を算出するステップ（Ｆ）を備えてもよい。ＩＣ５０の算出は、常法に基づいて行うことができ、例えば、被験物質の濃度に対する阻害活性値をプロットした阻害曲線を作成して算出することができる。また、本発明の方法により得られたＩＣ５０は、Ｋｉ値と良好な相関関係にあるため、上記ステップ（Ｆ）で得られた被験物質のＩＣ５０を、既知のＮＭＤＡ受容体阻害剤のＩＣ５０と比較して阻害活性比を算出することで、前記被験物質の活性を迅速且つ簡便な方法にて評価することができる。 The method of the present invention is based on the correlation between the concentration of the test substance brought into contact with the cultured nerve cells in the step (A) after the step (E) and the linear density with the drebrin cluster measured in the step (E). The step (F) for calculating the IC 50 of the test substance may be provided. The IC 50 can be calculated based on a conventional method. For example, an inhibition curve in which the inhibitory activity value with respect to the concentration of the test substance is plotted can be created and calculated. Further, IC 50 obtained by the method of the present invention, because it is in good correlation with K i values, the IC 50 of the resultant test substance in step (F), a known NMDA receptor inhibitors By calculating the inhibitory activity ratio in comparison with the IC 50 , the activity of the test substance can be evaluated by a rapid and simple method.
　本発明の一態様において、本発明の方法は、ＮＭＤＡ受容体活性化を引き起こす物質のスクリーニング及び活性評価に用いることもできる。かかる態様において、被験物質の濃度とドレブリンクラスターの線密度の減少との相関に基づいて、被験物質のＮＭＤＡ受容体活性化能が評価される。 In one aspect of the invention, the method of the invention can also be used for screening and evaluating activity of substances that cause NMDA receptor activation. In such an embodiment, the NMDA receptor activation ability of the test substance is evaluated based on the correlation between the concentration of the test substance and the decrease in the linear density of the drebrin cluster.
　本発明の一態様において、ドレブリンクラスターの線密度に代えて、或いはドレブリンクラスターの線密度と共に、ドレブリンＡ及び／又はドレブリンＥの発現量変化を、被験物質の活性評価に用いてもよい。かかる態様は、被験物質が神経細胞に与える長期的影響を評価するのに有効である可能性がある。 In one aspect of the present invention, a change in the expression level of drebrin A and / or drebrin E may be used in the activity evaluation of the test substance instead of the linear density of the drebrin cluster or together with the linear density of the drebrin cluster. Such an embodiment may be useful in assessing the long-term effects of a test substance on nerve cells.
　本発明は、従来のＮＭＤＡ受容体阻害活性の測定方法と比較して迅速且つ簡便に、定量性、再現性良く、高精度にＮＭＤＡ受容体阻害活性の評価を行うことができる。そのため、従来は機能面からの評価及び規制が困難であったＮＰＳの迅速な規制や、ＮＭＤＡ受容体をターゲットとした治療薬のハイスループットスクリーニングへの応用が期待される。 The present invention can evaluate the NMDA receptor inhibitory activity quickly and easily, with good quantitativeness and reproducibility, and with high accuracy as compared with the conventional method for measuring the NMDA receptor inhibitory activity. Therefore, it is expected to be applied to rapid regulation of NPS, which has been difficult to evaluate and regulate from the functional aspect, and high-throughput screening of therapeutic agents targeting NMDA receptors.
　以下、実施例により本発明をより具体的に説明するが、本発明の技術的範囲はこれらの例示に限定されるものではない。 Hereinafter, the present invention will be described in more detail with reference to Examples, but the technical scope of the present invention is not limited to these examples.
１．方法
１－２　動物実験
　動物実験は、群馬大学昭和キャンパス（群馬県前橋市）の動物実験委員会によって設定されたガイドラインに従って行い、研究における動物の使用に関するＮＩＨガイドラインに準拠した。動物の苦しみを最小限に抑え、使用される動物の数を減らすためにあらゆる努力を払った。妊娠ウィスターラットは、日本チャールス・リバー株式会社から入手した。動物は標準的な動物施設条件下で飼料と水を自由に摂取させて飼育した。 1. 1. Method 1-2 Animal Experiments Animal experiments were conducted in accordance with the guidelines set by the Animal Experiment Committee of Gunma University Showa Campus (Maebashi City, Gunma Prefecture), and were based on the NIH guidelines on the use of animals in research. Every effort has been made to minimize animal suffering and reduce the number of animals used. Pregnant Wistar Rats were obtained from Charles River Laboratories, Japan. Animals were bred with free feed and water under standard animal facility conditions.
１－２　海馬神経細胞培養
　１８日目胚のラットの海馬から調製した凍結ニューロンを、ポリリジンであらかじめコーティングした９６ウェルプレート（Thermo 96 Well Black Poly Bottom Poly-Lysine、Thermo Fisher Scientific社製）に、３．０×１０４細胞／ｃｍ２の密度で播種した。Ｂ２７サプリメント、ペニシリン／ストレプトマイシン、及びＬ－アラニル－Ｌ－グルタミン（Glutamax-I；Thermo Fisher Scientific社製）を含むNeurobasal Medium（Thermo Fisher Scientific社製）を使用して、３７℃、５％ＣＯ２環境下でインキュベートした。
４ＤＩＶ（インビトロでの培養日数）に、シトシンβ－Ｄ－アラビノフラノシド（Sigma社製）を最終濃度０．２μＭになるように加えてグリア細胞の増殖を抑制した。ニューロンを２１ＤＩＶ培養した後、それらを薬理学的に処理した。 1-2 Hippocampal neuron culture Day 18 Frozen neurons prepared from rat hippocampus were placed on a 96-well plate (Thermo 96 Well Black Poly Bottom Poly-Lysine, manufactured by Thermo Fisher Scientific) pre-coated with polylysine. Seeded at a density of 0.0 × 10 4 cells / cm 2 . 37 ° C, 5% CO 2 environment using Neurobasal Medium (Thermo Fisher Scientific) containing B27 supplement, penicillin / streptomycin, and L-alanyl-L-glutamine (Glutamax-I; Thermo Fisher Scientific) Incubated below.
Cytosine β-D-arabinofuranoside (manufactured by Sigma) was added to 4DIV (in vitro culture days) to a final concentration of 0.2 μM to suppress glial cell proliferation. After 21 DIV cultures of neurons, they were treated pharmacologically.
１－３　被験物質の投与
　ＰＣＰ、３－ＭｅＯ－ＰＣＭｏ、及び３－ＭｅＯ－ＰＣＰ（Cayman Chemical社製）を０．１％ＤＭＳＯ溶液により所定の濃度に希釈し、９６ウェルプレートのうち、培養神経細胞が培養されている所定のウェルに投与した。被験物質の投与から１０分後に、ウェル内の最終濃度が１００μＭとなるようにグルタミン酸溶液（和光純薬株式会社製）を調製し、薬液投与１０分後に投与した。ドレブリンクラスター密度の用量反応曲線を得るための実験のために、最終グルタメート濃度は１、３、１０、３０、５０又は１００μＭであった。いくつかの培養神経細胞は、グルタミン酸塩処理の前に、ＮＭＤＡ受容体に対する競合アンタゴニストであるＤ－２－アミノ－５－ホスホノバレリン酸（ＡＰＶ；５０、１００又は５００μＭ）で１０分間処理した。 1-3 Administration of test substance PCP, 3-MeO-PCMo, and 3-MeO-PCP (manufactured by Cayman Chemical Co., Ltd.) were diluted to a predetermined concentration with a 0.1% DMSO solution, and the cultured neurons in the 96-well plate were used. It was administered to the predetermined well in which the cells were cultured. A glutamic acid solution (manufactured by Wako Pure Chemical Industries, Ltd.) was prepared so that the final concentration in the well was 100 μM 10 minutes after the administration of the test substance, and the solution was administered 10 minutes after the administration of the drug solution. The final glutamate concentration was 1, 3, 10, 30, 50 or 100 μM for experiments to obtain a dose-response curve for the drebrin cluster density. Some cultured neurons were treated with D-2-amino-5-phosphonovaleric acid (APV; 50, 100 or 500 μM), a competitive antagonist to the NMDA receptor, for 10 minutes prior to glutamate treatment.
１－４　免疫細胞化学
　培養神経細胞を、リン酸緩衝液中４％パラホルムアルデヒドで固定した。リン酸緩衝生理食塩水（ＰＢＳ）中０．１％トリトンＸ－１００で５分間浸透処理した後、培養神経細胞をＰＢＳ中３％ウシ血清アルブミン（ＰＢＳＡ） で室温（ＲＴ）にて１時間ブロッキングし、次いで一次抗体と共に４℃で一晩インキュベートした。抗ドレブリン抗体（マウスモノクローナル、Ｍ２Ｆ６、１：１）及び抗ＭＡＰ２抗体（ウサギポリクローナル、１：２０００）（Merck Millipore社製）を一次抗体として使用した。ＰＢＳで洗浄した後、細胞を二次抗体及び４’，６－ジアミジノ－２－フェニルインドール二塩酸塩（ＤＡＰＩ、１：１０００、Thermo Fisher Scientific社製）と共に室温で１時間インキュベートした。使用した二次抗体は、Alexa Fluor 488ロバ抗マウスＩｇＧ（１：２５０）及びAlexa Fluor 594ロバ抗ウサギＩｇＧ（１：２５０、Jackson Immuno Research Laboratories社製）であった。 1-4 Immunocytochemistry Cultured neurons were fixed with 4% paraformaldehyde in phosphate buffer. After permeation with 0.1% Triton X-100 in phosphate buffered saline (PBS) for 5 minutes, cultured neurons are blocked with 3% bovine serum albumin (PBSA) in PBS for 1 hour at room temperature (RT). Then, it was incubated with the primary antibody at 4 ° C. overnight. Anti-drebrin antibody (mouse monoclonal, M2F6, 1: 1) and anti-MAP2 antibody (rabbit polyclonal, 1: 2000) (manufactured by Merck Millipore) were used as primary antibodies. After washing with PBS, cells were incubated with secondary antibody and 4', 6-diamidino-2-phenylindole dihydrochloride (DAPI, 1: 1000, Thermo Fisher Scientific) at room temperature for 1 hour. The secondary antibodies used were Alexa Fluor 488 donkey anti-mouse IgG (1: 250) and Alexa Fluor 594 donkey anti-rabbit IgG (1: 250, manufactured by Jackson Immuno Research Laboratories).
１－５　画像の自動取得と解析
　９６ウェルプレートで培養された海馬神経細胞の三重染色像は、IN Cell Analyzer 2200（GE Healthcare社製）の自動焦点機能を使用することによって自動的に取得された（２０倍レンズ、開口数０．４５）。全てのデータは１６ビット／ピクセルで２０４８×２０４８の解像度で収集された。画像内の単一の画素は、標本面内の３２５ｎｍの正方形に対応していた。Ｚスタック画像は、ハイコンテント画像ソフトウェアであるIN Cell Developer Toolbox v1.9（GE Healthcare社製）を用いて拡張焦点変換プロトコル（extended focus transformation protocol）により処理され、Ｚシリーズスタックからの焦点があった単一の２次元画像を生成した。培養神経細胞の細胞体は、ＭＡＰ２及びＤＡＰＩ染色に基づいて認識された。培養神経細胞の細胞体を特定するために、セグメンテーション機能により、異なるカーネルサイズ及び感度を有するコントラストベースのセグメンテーションアルゴリズムを介して、ＭＡＰ２陽性シグナルの蛍光強度に基づいて蛍光体を局在化させた。Erosion（収縮）、Sieve（ふるい）、Dilation（拡張）のコマンド及びＤＡＰＩ陽性領域を用いて、培養神経細胞の細胞体を同定した。培養神経細胞の樹状突起を識別するために、セグメンテーション機能、並びにDilation及びErosionコマンドを、ＭＡＰ２陽性シグナルについての別個のパラメーターと共に使用し、その後培養神経細胞の細胞体領域を差し引いた。ドレブリンクラスターを同定するために、セグメンテーション機能により、異なるカーネルサイズ及び感度を有するコントラストベースのセグメンテーションアルゴリズムを介して、ドレブリン陽性シグナルの蛍光強度に基づいて蛍光物体を局在化させた。偽陽性のバックグラウンドシグナルを除去するためにSieveコマンドを使用した。加えて、Dilationコマンドによって決定される、樹状突起までの定義された距離内に位置するドレブリン陽性斑点のみを、樹状突起に沿ったドレブリンクラスターとしてカウントした。神経細胞体、樹状突起及びドレブリンクラスター、視野当たりの細胞数、視野当たりの樹状突起長を同定した後、ドレブリンクラスターの線密度が自動的に算出された。 1-5 Automatic acquisition and analysis of images Triple-stained images of hippocampal neurons cultured in 96-well plates were automatically acquired by using the autofocus function of IN Cell Analyzer 2200 (GE Healthcare). (20x lens, numerical aperture 0.45). All data was collected at 16 bits / pixel with a resolution of 2048 x 2048. A single pixel in the image corresponded to a 325 nm square in the specimen plane. Z-stack images were processed by the extended focus transformation protocol using IN Cell Developer Toolbox v1.9 (GE Healthcare), a high-content image software, and had focus from the Z-series stack. A single 2D image was generated. Cell bodies of cultured neurons were recognized based on MAP2 and DAPI staining. To identify the cell bodies of cultured neurons, the segmentation function localized the phosphors based on the fluorescence intensity of the MAP2-positive signal via a contrast-based segmentation algorithm with different kernel sizes and sensitivities. Cell bodies of cultured neurons were identified using Erosion, Sieve, Dilation commands and DAPI-positive regions. To identify the dendrites of cultured neurons, the segmentation function, as well as the Dilation and Erosion commands, were used with separate parameters for MAP2-positive signals, after which the cell body region of the cultured neurons was subtracted. To identify drebrin clusters, the segmentation feature localized fluorescent objects based on the fluorescence intensity of the drebrin-positive signal via contrast-based segmentation algorithms with different kernel sizes and sensitivities. The Sieve command was used to eliminate false positive background signals. In addition, only drebrin-positive spots located within a defined distance to the dendrites, as determined by the Dilation command, were counted as drebrin clusters along the dendrites. After identifying the nerve cell bodies, dendrites and dendrite clusters, number of cells per visual field, and dendrite length per visual field, the linear density of the dendrite clusters was automatically calculated.
１－６　統計
　データは、ＡＮＯＶＡ（エクセル統計Statcel4、有限会社オーエムエス出版製）に従ってｔ検定又はターキー－クレイマー検定によって統計的に分析した。全てのデータは平均値±平均値の標準誤差（ＳＥＭ）として示した。市販のソフトウェア（GraphPad Prism 8、GraphPad Software社製）を用いて用量適合曲線を描き、ＩＣ５０を計算した。　 1-6 Statistical data was statistically analyzed by t-test or turkey-kramer test according to ANOVA (Excel Statistics Statcel4, manufactured by OS Publishing Co., Ltd.). All data are shown as mean ± standard error of mean (SEM). A dose matching curve was drawn using commercially available software (GraphPad Prism 8, manufactured by GraphPad Software), and the IC 50 was calculated.
２．結果
２－１　グルタミン酸処理は、ＮＭＤＡ受容体活性化を介してドレブリンクラスター密度を減少させた
　まず、グルタミン酸がドレブリンクラスター密度に及ぼす影響を、ハイスループットイメージングシステムを用いて調べた。２１インビトロ培養日数（ＤＩＶ）のラット海馬培養神経細胞を、いくつかの濃度のグルタミン酸溶液（１μＭ、Ｎ＝６１；３μＭ、Ｎ＝９７；１０μＭ、Ｎ＝８８；３０μＭ、Ｎ＝９７；５０μＭ、Ｎ＝２０；１００μＭ、Ｎ＝１０６）で処理した。上記１－４の免疫細胞化学の処理を行った後、画像取得及び分析は自動的に行われた。得られたドレブリンクラスター密度のグルタミン酸用量反応曲線を図１Ａに示す。ＥＣ５０は１０．４μＭであり、９５％信頼区間（ＣＩ）は６．２９μＭ～１７．４μＭの範囲であった。 2. 2. Results 2-1 Glutamic acid treatment reduced drebrin cluster density through NMDA receptor activation First, the effect of glutamate on drebrin cluster density was investigated using a high-throughput imaging system. 21 In vitro culture days (DIV) rat hippocampal cultured neurons were subjected to several concentrations of glutamate solution (1 μM, N = 61; 3 μM, N = 97; 10 μM, N = 88; 30 μM, N = 97; 50 μM, N. = 20; 100 μM, N = 106). After the immunocytochemistry treatment of 1-4 above, image acquisition and analysis were performed automatically. The glutamic acid dose-response curve of the obtained drebrin cluster density is shown in FIG. 1A. The EC 50 was 10.4 μM and the 95% confidence interval (CI) ranged from 6.29 μM to 17.4 μM.
　また、グルタミン酸刺激によるドレブリンクラスター密度の減少は、ＡＰＶ（５０μＭ、Ｎ＝４；１００μＭ、Ｎ＝７；５００μＭ、Ｎ＝６）によって阻害されることを確認した（図１Ｂ）。ここでの結果により、当該実験系が、グルタミン酸刺激によるドレブリンクラスター密度の減少、及び、ＮＭＤＡ受容体競合的アンタゴニストによるドレブリンクラスター密度減少の阻害を検出することができることが示された。 It was also confirmed that the decrease in drebrin cluster density due to glutamate stimulation was inhibited by APV (50 μM, N = 4; 100 μM, N = 7; 500 μM, N = 6) (Fig. 1B). The results here indicate that the experimental system can detect glutamate-stimulated reduction in drebrin cluster density and inhibition of NMDA receptor-competitive antagonist-induced reduction in drebrin cluster density.
２－２　グルタミン酸誘発ドレブリンクラスター密度の減少に対するＰＣＰ、３－ＭｅＯ－ＰＣＭｏ及び３－ＭｅＯ－ＰＣＰの阻害効果
　まず、本発明者らは、１００μＭグルタミン酸溶液を用いてドレブリンクラスターの密度をどの程度減少させるかを決定した（対照、Ｎ＝９５；グルタミン酸１００μＭ、Ｎ＝７７）。図２Ａに示されるように、１００μＭグルタミン酸溶液処理により、対照群と比較してドレブリンクラスター密度が約４０％減少した（対照０．５３８±０．０１４３；１００μＭグルタミン酸０．３１９±０．００３５）。 2-2 Inhibitory effect of PCP, 3-MeO-PCMo and 3-MeO-PCP on reduction of glutamate-induced drebrin cluster density First, we use a 100 μM glutamate solution to reduce the density of drebrin clusters. (Control, N = 95; glutamic acid 100 μM, N = 77). As shown in FIG. 2A, treatment with a 100 μM glutamate solution reduced the drebrin cluster density by about 40% compared to the control group (control 0.538 ± 0.0143; 100 μM glutamate 0.319 ± 0.0035).
　次に、グルタミン酸を用いたＮＭＤＡ受容体刺激によるドレブリンクラスターの密度の低下に対する、ＮＭＤＡ受容体の非競合的拮抗薬であるＰＣＰの抑制効果を調べた（０～１０　μＭ、Ｎ＝１８）。対照群に対する各投与群の平均阻害率を計算することにより、用量依存的効果を分析した。図２Ｂより、ＰＣＰが１００μＭグルタメートのＮＭＤＡＲ刺激によって１μＭ以上の濃度でドレブリンクラスターの減少を有意に抑制し、ＰＣＰが用量依存的にドレブリンクラスターの減少を抑制することが示された。 Next, the inhibitory effect of PCP, which is a non-competitive antagonist of the NMDA receptor, on the decrease in the density of drebrin clusters by NMDA receptor stimulation with glutamate was investigated (0 to 10 μM, N = 18). Dose-dependent effects were analyzed by calculating the mean inhibition rate of each treatment group relative to the control group. From FIG. 2B, it was shown that PCP significantly suppressed the decrease of drebrin cluster at a concentration of 1 μM or more by NMDAR stimulation of 100 μM glutamate, and PCP suppressed the decrease of drebrin cluster in a dose-dependent manner.
　同様の方法により、ＰＣＰアナログである３－ＭｅＯ－ＰＣＭｏ及び３－ＭｅＯ－ＰＣＰの阻害作用を調べた（０～１０μＭ；３－ＭｅＯ－ＰＣＭｏ、Ｎ＝２９；３－ＭｅＯ－ＰＣＭｏ、Ｎ＝３０）。図２Ｃは、３－ＭｅＯ－ＰＣＰが、３３３ｎＭ以上の濃度で１００μＭグルタミン酸によるドレブリンクラスターの減少を有意に阻害したことを示す。図２Ｄは、３ －ＭｅＯ－ＰＣＭｏが、３．３３μＭ以上の濃度で１００μＭグルタミン酸によるドレブリンクラスターの減少を有意に阻害したことを示す。さらに、３－ＭｅＯ－ＰＣＰ及び３－ＭｅＯ－ＰＣＭｏの両方が、用量依存的にドレブリンクラスターの減少を阻害した。 The inhibitory effect of the PCP analogs 3-MeO-PCMo and 3-MeO-PCP was investigated by the same method (0 to 10 μM; 3-MeO-PCMo, N = 29; 3-MeO-PCMo, N = 30. ). FIG. 2C shows that 3-MeO-PCP significantly inhibited the reduction of drebrin clusters by 100 μM glutamate at concentrations above 333 nM. FIG. 2D shows that 3-MeO-PCMo significantly inhibited the reduction of drebrin clusters by 100 μM glutamate at a concentration of 3.33 μM or higher. In addition, both 3-MeO-PCP and 3-MeO-PCMo inhibited the reduction of drebrin clusters in a dose-dependent manner.
２－３　ＰＣＰ、３－ＭｅＯ－ＰＣＭｏ及び３－ＭｅＯ－ＰＣＰのＩＣ５０値
　分析の定量的信頼性を高めるために、測定値に基づいて曲線近似した用量作用曲線を作成した（図３）。図３より、３－ＭｅＯ－ＰＣＰの阻害活性がＰＣＰの阻害活性と同程度であり、３－ＭｅＯ－ＰＣＭｏの阻害活性がＰＣＰのそれよりも低いことが示された。ＩＣ５０は、ＰＣＰ＝２．０２μＭ（９５％ＣＩ：１．３９～２．９７μＭ）、３－ＭｅＯ－ＰＣＰ＝１．５１μＭ（９５％ＣＩ：１．１６～１．９９μＭ）、３－ＭｅＯ－ＰＣＭｏ＝２６．７μＭ（９５％ＣＩ：２０．０～３７．３μＭ）であった。 To increase the 2-3 PCP, 3-MeO-PCMo and quantitative reliability of an IC 50 value analysis of 3-MeO-PCP, created the dose-response curves curve approximation based on the measured values (Figure 3). From FIG. 3, it was shown that the inhibitory activity of 3-MeO-PCP was similar to that of PCP, and the inhibitory activity of 3-MeO-PCMo was lower than that of PCP. The IC 50 has PCP = 2.02 μM (95% CI: 1.39 to 2.97 μM), 3-MeO-PCP = 1.51 μM (95% CI: 1.16 to 1.99 μM), 3-MeO-. PCMo = 26.7 μM (95% CI: 20.0 to 37.3 μM).
３．考察
　上記２－３で算出したＩＣ５０に基づく阻害活性の順は、活性が大きい方から３－ＭｅＯ－ＰＣＰ＞ＰＣＰ＞３－ＭｅＯ－ＰＣＭｏであった。最近なされたＮＭＤＡ受容体に対する結合アッセイの報告によれば、ＰＣＰの阻害定数（Ｋｉ）値は２２．１ｎＭ（Colestock et al. (2018)、Drug Testing and Analysis, 10, 272-283）及び５７．９ｎＭ（Wallach, J. (2014). Structure activity relationship (SAR) studies of arylcyclohexylamines as N-methyl-D-aspartate receptor antagonists. PhD dissertation. University of the Sciences, Philadelphia）であると報告されており、３－ＭｅＯ－ＰＣＰのＫｉ値は３８．１ｎＭであると報告されている（Wallach, J. (2014)）。これは、３－ＭｅＯ－ＰＣＰが、ＰＣＰの平均よりもＮＭＤＡ受容体に対してわずかに高い親和性を有することを示す。一方で、３－ＭｅＯ－ＰＣＭｏのＫｉ値は２５２．９ｎＭであり、ＰＣＰのＫｉ値と比べて１／１２であると報告されている（Colestock et al. (2018)）。 3. 3. Discussion The order of inhibitory activity based on IC 50 calculated in 2-3 above was 3-MeO-PCP>PCP> 3-MeO-PCMo in descending order of activity. According to reports of the binding assay on recently made the NMDA receptor, PCP inhibition constant (K i) values 22.1nM (Colestock et al. (2018 ), Drug Testing and Analysis, 10, 272-283) and 57 . 9nM (Wallach, J. (2014). Structure activity relationship (SAR) studies of arylcyclohexylamines as N-methyl-D-aspartate receptor antagonists. PhD dissertation. University of the Sciences, Philadelphia). K i values of -MeO-PCP has been reported to be 38.1nM (Wallach, J. (2014) ). This indicates that 3-MeO-PCP has a slightly higher affinity for the NMDA receptor than the average of PCP. On the other hand, K i values of 3-MeO-PCMo is 252.9NM, compared to the K i values of PCP has been reported to be 1/12 (Colestock et al. (2018 )).
　上記のＫｉ値からＰＣＰに対する親和性比を計算したところ、ＰＣＰ＝１としたときに、３－ＭｅＯ－ＰＣＰ＝０．７３（Wallach, J. (2014)のＫｉ値からの再計算）、３－ＭｅＯ－ＰＣＭｏ＝１１．４（Colestock et al. (2018)のＫｉ値からの再計算）であった。まとめると、親和性の順序は、３－ＭｅＯ－ＰＣＰ＞ＰＣＰ＞３－ＭｅＯ－ＰＣＭｏで、本発明の方法により得られたＩＣ５０に基づく活性の順序と同じであった。 Was calculated affinity ratio for PCP from the K i values of, when the PCP = 1, (recalculated from K i value of Wallach, J. (2014)) 3 -MeO-PCP = 0.73 was 3-MeO-PCMo = 11.4 (recalculated from K i value of Colestock et al. (2018)) . In summary, the affinity of the order, in 3-MeO-PCP>PCP> 3-MeO-PCMo, was the same as the order of activity based on IC 50 obtained by the method of the present invention.
　同一の作用機序を有する阻害剤の場合、それらのＩＣ５０値を比較することにより、相対的な阻害活性を評価するのに有用であるとの報告がある（Cheng＆Prusoff (1973) Biochemical Pharmacology, 22(23), 3099-3108；Cer et al. (2009) Nucleic Acids Res. 1(37): W441-W445）。ＰＣＰ及び他の高親和性ＮＭＤＡ受容体アンタゴニストは、ＮＭＤＡＲと同様の阻害メカニズムを有することが示唆されている（Lodge＆Mercier (2015) Br. J. Pharmacol, 172, 4254-4276）。かかる知見に基づいて、本研究で得られたＩＣ５０値からＰＣＰに対する効力比を計算したところ、ＰＣＰ＝１としたときに、３－ＭｅＯ－ＰＣＰ＝０．７５、３－ＭｅＯ－ＰＣＭｏ＝１３．２であり、上記Ｋｉ値から計算した効力比と類似していた。この結果より、本発明によって得られるＩＣ５０値は、ＮＭＤＡ受容体阻害作用を反映しており、ＮＭＤＡ受容体阻害活性を有する化合物（ＮＰＳ候補化合物や、新規治療剤等）の薬理学的評価（毒性評価）に使用することができることが明らかになった。 For inhibitors which have the same mechanism of action, by comparing their IC 50 values, it has been reported to be useful to assess the relative inhibitory activity (Cheng & Prusoff (1973) Biochemical Pharmacology, 22 (23), 3099-3108; Cer et al. (2009) Nucleic Acids Res. 1 (37): W441-W445). PCP and other high-affinity NMDA receptor antagonists have been suggested to have an inhibitory mechanism similar to NMDAR (Lodge & Mercier (2015) Br. J. Pharmacol, 172, 4254-4276). Based on such findings, the calculated efficacy ratio PCP from IC 50 values obtained in this study, when a PCP = 1, 3-MeO- PCP = 0.75,3-MeO-PCMo = 13 It was .2, which was similar to the efficacy ratio calculated from the above Ki value. This result, IC 50 values obtained by the present invention reflects the NMDA receptor inhibitory action, (or NPS candidate compounds, novel therapeutic agents, and the like) compounds having an NMDA receptor inhibitory activity Pharmacological Evaluation of ( It became clear that it can be used for toxicity evaluation).
　本発明によれば、簡便且つ短時間で実施することのできる、ＮＭＤＡ受容体阻害活性のハイスループット評価方法を提供することができる。かかる評価方法により、従来は機能面からの評価及び規制が困難であったＮＰＳの迅速な規制や、ＮＭＤＡ受容体をターゲットとした治療薬のハイスループットスクリーニング及び機能評価が可能になるため、産業上の有用性は極めて高い。 According to the present invention, it is possible to provide a high-throughput evaluation method for NMDA receptor inhibitory activity, which can be carried out easily and in a short time. This evaluation method enables rapid regulation of NPS, which was difficult to evaluate and regulate from the functional aspect in the past, and high-throughput screening and functional evaluation of therapeutic agents targeting NMDA receptors. Is extremely useful.

Claims (6)

  1. 　以下のステップ（Ａ）～（Ｅ）を順次備えたことを特徴とするＮＭＤＡ受容体阻害活性のハイスループット評価方法。
    （Ａ）培養神経細胞に、被験物質を接触させるステップ；
    （Ｂ）前記培養神経細胞に、グルタミン酸溶液を接触させるステップ；
    （Ｃ）前記培養神経細胞を固定するステップ；
    （Ｄ）前記培養神経細胞の樹状突起スパインのドレブリンクラスターを可視化するステップ；
    （Ｅ）前記ドレブリンクラスターの樹状突起に沿った線密度を測定し、前記線密度が、前記被験物質に接触させていない培養神経細胞の線密度と比較して高い場合に、前記被験物質がＮＭＤＡ受容体阻害作用を有すると判定するステップ；     A high-throughput evaluation method for NMDA receptor inhibitory activity, which comprises sequentially providing the following steps (A) to (E).
    (A) Step of contacting the cultured nerve cells with the test substance;
    (B) A step of contacting the cultured nerve cells with a glutamic acid solution;
    (C) Step of fixing the cultured nerve cells;
    (D) A step of visualizing the drebrin cluster of the dendritic spine of the cultured nerve cell;
    (E) The linear density along the dendrites of the drebrin cluster is measured, and when the linear density is higher than the linear density of the cultured nerve cells that are not in contact with the test substance, the test substance is present. Steps to determine that it has an NMDA receptor inhibitory effect;
  2. 　以下のステップ（Ｆ）を、ステップ（Ｅ）の後に備えたことを特徴とする請求項１に記載の方法。
    （Ｆ）ステップ（Ａ）で培養神経細胞に接触させた被験物質の濃度と、ステップ（Ｅ）で測定したドレブリンクラスターの線密度との相関に基づいて、前記被験物質のＩＣ５０を算出するステップ；     The method according to claim 1, wherein the following step (F) is provided after the step (E).
    (F) A step of calculating the IC 50 of the test substance based on the correlation between the concentration of the test substance brought into contact with the cultured nerve cells in step (A) and the linear density of the drebrin cluster measured in step (E). ;
  3. 　以下のステップ（Ｇ）を、ステップ（Ｆ）の後に備えたことを特徴とする請求項１又は２に記載の方法。
    （Ｇ）ステップ（Ｆ）で算出した被験物質のＩＣ５０を、既知のＮＭＤＡ受容体阻害剤のＩＣ５０と比較することにより、前記被験物質と、前記既知のＮＭＤＡ受容体阻害剤とのＮＭＤＡ受容体阻害活性比を算出するステップ；     The method according to claim 1 or 2, wherein the following step (G) is provided after the step (F).
    The IC 50 of the test substance calculated in (G) Step (F), by comparing the IC 50 of the known NMDA receptor inhibitors, NMDA receptor of said analyte, and the known NMDA receptor inhibitors Steps to calculate body inhibitory activity ratio;
  4. 　培養神経細胞が、海馬神経細胞由来であることを特徴とする請求項１～３のいずれかに記載の方法。 The method according to any one of claims 1 to 3, wherein the cultured nerve cells are derived from hippocampal nerve cells.
  5. 　培養神経細胞が、げっ歯類由来であることを特徴とする請求項１～４のいずれかに記載の方法。 The method according to any one of claims 1 to 4, wherein the cultured nerve cells are derived from rodents.
  6. 　ステップ（Ｄ）の可視化が、抗ドレブリン抗体による免疫染色であることを特徴とする請求項１～５のいずれかに記載の方法。 The method according to any one of claims 1 to 5, wherein the visualization in step (D) is immunostaining with an anti-drebrin antibody.
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