JP4261121B2 - Antiepileptic and therapeutic agents - Google Patents

Description

【００２８】
【表２】

【００２９】
【表３】

【００３０】
【表４】

【００３１】
ＡＤの伝播については、Ａ群（コントロール群）及びD群（ＭＫ８０１投与群）では、標本動物作成時と同強度の刺激で、前頭葉皮質、刺激反対側海馬の両方で必ずＡＤを誘発したが、被検物質投与時（Ｂ群及びＣ群）にはＡＤが誘発しない場合があった。
表１に示すように前頭葉皮質ではＡＤが誘発しなかった刺激回数は投与したテアニンの濃度に依存して有意に増加した。一方、表３に示すように刺激反対側海馬ではテアニンの投与がＡＤの誘発にはほとんど影響していなかった。
特に、Ｂ群（１００μｍｏｌ／ｋｇオーダー）に比べ、Ｃ群（ｍｍｏｌ／ｋｇオーダー）の効果は顕著であった。
【００３２】
以上に示したように、テアニンの投与によりＡＤの出現又はその発展が抑制されることが明らかになった。
また、てんかんモデル動物の作成プロセスが、ＡＤの出現⇒ＡＤの発展⇒痙攣というプロセスを経て完成することを考慮すると、脳内でＡＤの異常放電が生じ易い状態に陥った患者にテアニンを投与することによって、ＡＤ様の異常放電の出現又はその発展が抑制され、その結果、後遺症として考えられる痙攣を伴う続発性てんかんが予防できるものと考察できる。
脳内でＡＤ様の異常放電が生じ易い状態としては、分娩時の障害や頭部外傷などを挙げることができる。
【００３３】
[実験２]
本実験では、実験動物を用いた電気キンドリングモデル作成時の後発射（「ＡＤ」）出現に及ぼすテアニンの抑制作用、特にテアニンの脳室内投与の影響について検討した。
【００３４】
（標本動物の作成）
１０週齢のＷｉｓｔａｒ系雄ラットを、下記の環境下で予備飼育した後、体重２８０ｇ以上に達したものを選択して下記の要領で慢性電極及び脳室内投与用カニューレの植え込み手術を施行した。
【００３５】
＝予備飼育環境＝
飼育室：小動物管理室（ラット飼育室）
温度：２０±３℃
湿度：５０±１０％
明暗サイクル：０８：００〜２０：００まで点灯
使用ケージ：ポリカーボネイト製ケージ（床敷に電気かんなチップを使用）
ケージ交換：２回／週
給餌：固形飼料（ＣＥ−１、日本クレア）を自由摂取
給水：水道水をミリポアフィルタで濾過したものを自由摂取
【００３６】
＝慢性電極及び脳室内投与用カニューレ植え込み手術＝
麻酔：ベントバルビタール麻酔
植込部位：左右の前頭部硬膜上、左右の海馬背側部、第三脳室
使用電極：硬膜外電極…ステンレススチール製ビス電極
深部用電極…ステンレススチール製並列型双極電極（記録・刺激兼用）
投与用カニューレ…ステンレススチール製パイプ（φ０．３ｍｍ）
術後処置：縫合時に切開部にサルファ剤を内包し、術後５日間は感染予防のため３０００単位のペニシリンＧカリウム溶液を筋肉内投与
手術終了後の動物の頭部を図７に示す。
【００３７】
（ＡＤ閾値の確定）
電極植え込み手術後１０日以上経過した動物を測定ボックス内に移動し、アイソレータを介して周波数６０Ｈｚ、持続時間１秒の矩形波の刺激を右海馬背側部に２４時間間隔で与えた。通電量は、初回を１００μＡとし、ＡＤが認められるまで通電量を５０μＡずつ増加させＡＤ閾値を決定し、以降の刺激はこのＡＤ閾値で行い、三日間連続してＡＤを誘発した動物を試験に使用した。
三日間の間に最初に設定したＡＤ閾値でＡＤが発現しなかった場合は、翌日より通電量を５０μＡ増加させて刺激し、この操作を繰り返した。
【００３８】
刺激前後の脳波（皮質、刺激側または反対側海馬）を有線で導出し、増幅後に紙面に記録するとともに、サンプリング周波数２００Ｈｚで光磁気ディスク上に保存した。記録条件は、時定数０．１秒、高域遮断周波数６０Ｈｚ、減衰特性２４ｄＢ／オクターブで、記録時間は刺激前１分、刺激後４分の計５分である。また、刺激前後の動物の反応を観察するために測定ボックスに取り付けたＣＣＤカメラによる画像をデジタルビデオレコーダを用いて、脳波と同時に記録した。
【００３９】
＝使用機器＝
刺激装置：電気刺激装置（SEN-7203、日本光電工業）
アイソレータ：アイソレータ（SS-201J、日本光電工業）
増幅器：生体電気用アンプ（AB621G、日本光電工業）
記録器：熱ペン式記録器（8R-33、ＮＥＣメディカルシステムズ）
ＡＤ変換器：ＡＤ変換ボード（ADM-5298BPC、マイクロサイエンス）
その他：パーソナルコンピュータ、オシロスコープ、光磁気ディスクドライブ、ＣＣＤカメラ、デジタルビデオテープレコーダ
【００４０】
（投与実験）
被検物質（Ｌ−テアニン（東京化成社製）を滅菌生理食塩水に溶解したものを事前に固定したカニューレより、第三脳室内に０．１ｍｌ投与した（容量一定）。
【００４１】
モデル動物７匹を一群とし、コントロール群、テアニン投与群（５群）の計６群について、投与実験を行った。３日間以上のウォッシュアウト期間をおいて、１個体につき３回の投与を行った。
Ａ群： ０μｍｏｌ／ｋｇ（生理食塩水投与群、コントロール群）
Ｂ群： ２０μｍｏｌ／ｋｇ
Ｃ群： ４０μｍｏｌ／ｋｇ
Ｄ群： ６０μｍｏｌ／ｋｇ
Ｅ群： ８０μｍｏｌ／ｋｇ
Ｆ群：１００μｍｏｌ／ｋｇ
【００４２】
被検物質投与１５分後にＡＤ誘発閾値の通電量で刺激した。刺激前１分と刺激後２分の脳波と行動をＡＤ閾値確認時と同様の条件で記録した。３日間のウォッシュアウト期間をおき、同一濃度の投与を３回行った。
【００４３】
記録した脳波からＡＤ誘発の有無を確認し、振幅が大きい１０放電の平均振幅（平均最大振幅）とＡＤ持続時間を測定した。ＡＤの平均最大振幅と持続時間について、Kruskal−Wallis検定をＡＤの有無についてはカイ２乗検定を用いた。
【００４４】
（結果）
表５は皮質・海馬におけるＡＤの平均最大振幅の変化を示し、図８は皮質における各群のＡＤの平均最大振幅を示し、図９は海馬における各群のＡＤの平均最大振幅を示す。また、表６は皮質におけるＡＤ平均最大振幅の各群間の有意差の有無を示し、表７は海馬におけるＡＤ平均最大振幅の各群間の有意差の有無を示す。
投与したテアニンの濃度に依存して、皮質・海馬の両部位ともにＡＤ平均最大振幅は低下した。各群の標準偏差が大きいのはテアニン投与濃度に依存してＡＤをまったく誘発しない個体（ＡＤの振幅が０μＶ）が多くなったためである。
【００４５】
【表５】

【００４６】
【表６】

【００４７】
【表７】

【００４８】
表８は皮質・海馬におけるＡＤ持続時間の変化を示し、図１０は皮質における各群のＡＤ持続時間を示し、図１１は海馬における各群のＡＤ持続時間を示す。また、表９は皮質におけるＡＤ持続時間の各群間の有意差の有無を示し、表１０は海馬におけるＡＤ持続時間の各群間の有意差の有無を示す。
振幅と同様に、投与したテアニンの濃度に依存して、皮質・海馬の両部位ともにＡＤ持続時間は短縮した。各群の標準偏差が大きいのも振幅と同様にＡＤを全く誘発しない個体（ＡＤ持続時間が０ｓｅｃ）が多くなったためである。
【００４９】
【表８】

【００５０】
【表９】

【００５１】
【表１０】

【００５２】
表１１はＡＤ誘発の有無を示し、表１２は各群間のＡＤ誘発の有無の有意差を示す。
投与したテアニンの濃度に依存して、皮質・海馬ともにＡＤを誘発しない個体が増えていった。また、ＡＤを誘発するときは皮質・海馬の両部位ともに誘発し、誘発しないときは両部位ともに誘発しなかった。
【００５３】
【表１１】

【００５４】
【表１２】

【００５５】
今回の脳室内投与による実験では、４０μｍｏｌ／ｋｇの投与で７匹中３匹は完全にＡＤの誘発を抑制し、１００μｍｏｌ／ｋｇの投与で７匹中６匹で完全にＡＤの誘発を抑制した。また、誘発するＡＤの振幅、持続時間とも４０μｍｏｌ／ｋｇ以上のテアニン投与で濃度に依存して減少していった。脳室内投与では、完全にＡＤを誘発しなかった個体が多く標準偏差が大きくなったため、４０μｍｏｌ／ｋｇではＡＤの振幅と持続時間についてはコントロール群と有意差は認められなかったものの、その実際の効果は２ｍｍｏｌ／ｋｇ静脈内投与より強いと考えられる。
【図面の簡単な説明】
【図１】 実施例のＡＤ平均最大振幅の計測法（誘発されたＡＤの中から振幅が大きい１０放電の振幅を計測し、その平均値を算出する方法）を示す説明図であり、この図において上段（ＣＯＲＴＥＸ）は前頭葉皮質、下段（ＨＩＰＰ）は刺激反対側海馬を示している。
【図２】 実施例のＡＤ持続時間の計測法（刺激後最初に出現したＡＤから最後に出現したＡＤまでの時間をＡＤ持続時間とする方法）を示す説明図であり、この図において上段（ＣＯＲＴＥＸ）は前頭葉皮質、下段（ＨＩＰＰ）は刺激反対側海馬を示している。
【図３】 実施例の被検物質の投与が、前頭葉皮質においてＡＤ平均振幅に与える影響を示したグラフである。
【図４】 実施例の被検物質の投与が、刺激反対側海馬においてＡＤ平均振幅に与える影響を示したグラフである。
【図５】 実施例の被検物質の投与が、前頭葉皮質においてＡＤ持続時間に与える影響を示したグラフである。
【図６】 実施例の被検物質の投与が、刺激反対側海馬においてＡＤ持続時間に与える影響を示したグラフである。
【図７】 電極・脳室投与用パイプ埋め込み術１週間後の標本動物の頭部を示した写真である。
【図８】 皮質における各群のＡＤの平均最大振幅を示したグラフである。
【図９】 海馬における各群のＡＤの平均最大振幅を示したグラフである。
【図１０】 皮質における各群のＡＤ持続時間を示したグラフである。
【図１１】 海馬における各群のＡＤ持続時間を示したグラフである。[0001]
[Industrial application fields]
The present invention relates to an epilepsy preventive agent and a therapeutic agent for use in a prophylactic and therapeutic agent for “epilepsy” whose main symptom is seizure caused by excessive firing of cerebral neurons.
[0002]
[Prior art and problems to be solved by the invention]
“Epileptic” is, according to the definition of WHO (World Health Organization), “a chronic brain disorder caused by various etiologies, which is characterized by recurrent seizures (epileptic seizures) resulting from excessive firing of cerebral neurons. "With various clinical symptoms and laboratory findings".
The epileptic seizure symptom takes various forms such as disturbance of consciousness, convulsions, automatism, etc., depending on the initial site and the spread of sudden cerebral rhythm abnormalities. In addition to epileptic seizures, epilepsy often has periodic moodiness, interstitial mental disorders, personality changes, and intellectual disability.
[0003]
Antiepileptic drugs such as barbital compounds such as phenobarbital, benzodiazepine clonazepam, and valuroic acid are currently used to suppress such epilepsy attacks, and about 80% of epilepsy patients are treated with drugs. It is said that seizures are completely suppressed by administration.
[0004]
However, these currently used antiepileptic agents are extremely effective in suppressing seizures, but have a problem that they have many side effects. For example, side effects such as central nervous system depression, cardiovascular collapse, aplastic anemia, hallucinations, congestive heart failure, ataxia, personality change, psychosis, aggressive behavior, dizziness, sedation, etc. have been reported, and there are no side effects The development of epilepsy agents has been awaited.
[0005]
[Means for Solving the Problems]
Therefore, as a result of diligent research on a compound having few side effects and having an effect of preventing or treating epilepsy, the present inventors have found that theanine, which is a kind of amino acid contained in tea, has an effect of preventing and treating epilepsy. The present invention has been obtained and the present invention has been made based on such knowledge. Specifically, the present inventors have found that administration of theanine has an epileptic seizure inhibitory effect on epilepsy model rats, and have arrived at the present invention based on such findings.
[0006]
That is, the present invention embodies an epilepsy preventive agent and a therapeutic agent containing or prescribed at least one of theanine and its derivatives as an active ingredient, compared with conventional epilepsy preventive agents and therapeutic agents, In addition to being able to obtain excellent epilepsy preventive and therapeutic effects, it has features with few side effects. Theanine is a substance that is ingested daily through tea and is now approved as a food additive, and it is certain that there are few side effects.
[0007]
The disease “epilepsy” is not a single disease but a collective term for several disease units whose main symptoms are seizures caused by excessive firing of cerebral neurons. According to the international classification of epilepsy according to seizure type, it can be broadly divided into partial seizures and total seizures, and the former partial seizures can be further divided into simple partial seizures, complex partial seizures, and secondary generalization. The latter total seizure can be further divided into absence seizure, myoclonic seizure, clonic seizure, tonic seizure, tonic clonic seizure, and weakness seizure. On the other hand, if epilepsy is classified according to the cause, it can be divided into organic causes with abnormalities such as wounds in the brain and functional causes that are not, and the former causes of head trauma during labor, Inborn errors of metabolism, congenital malformations, infancy ischemia, infections, degenerative diseases, brain tumors, cerebrovascular disorders, etc., the latter is called idiopathic epilepsy And those in which genetic alterations are observed.
The epilepsy preventing agent and therapeutic agent of the present invention can be expected to have a prophylactic effect and a therapeutic effect for all types of epilepsy (partial seizures and total seizures). It can also be expected to have preventive and therapeutic effects on all causes of epilepsy (organic and functional causes), but it is particularly effective for temporal lobe epilepsy (psychomotor seizures). is there.
[0008]
Regarding the physiological functions of theanine, various studies have been conducted on the action on the central nervous system. For example, in Japanese Patent Application Laid-Open No. 9-286727, it was found that when theanine was administered to cultured neurons, it had an action of suppressing the toxicity of glutamate and an action of blocking the glutamate receptor. “A glutamate antagonism containing theanine as an active ingredient” And a neuronal cell death preventing agent characterized by a glutamate receptor blocking action ”. In addition, JP-A-2000-229854 has found that when theanine is administered to transient cerebral ischemic gerbils, it has a protective effect on ischemic delayed neuronal cell death. Intraventricular agents, intraventricular agents for the treatment and prevention of vascular dementia, and intracerebral surgical agents ”are disclosed. However, there has been no disclosure about the epilepsy preventive and therapeutic effects of theanine and derivatives.
[0009]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, embodiments of the component, dosage form, method of use and the like of the epilepsy preventing agent and therapeutic agent of the present invention will be specifically described.
[0010]
The epilepsy preventing agent and therapeutic agent of the present invention can be prepared by containing or prescribing at least one of theanine and derivatives thereof as an active ingredient.
[0011]
Theanine and its derivatives include L-glutamic acid-γ-ethylamide (L-theanine), L-glutamic acid-γ-methylamide, D-glutamic acid-γ-ethylamide (D-theanine), D-glutamic acid-γ-methylamide, etc. Consists of derivatives containing L- or D-glutamic acid-γ-alkylamide, L- or D-glutamic acid-γ-alkylamide in the basic structure (for example, glycosides of L- or D-glutamic acid-γ-alkylamide, etc.) One type of compound selected from the group or a mixture of two or more types of compounds can be used. Among these, L-theanine is not only obtainable from natural products, but is also recognized as a food additive, and is particularly preferred from the standpoint of availability and safety.
[0012]
Theanine and its derivatives can be obtained by various known methods. Specifically, it can be biosynthesized by culturing methods such as plants or microorganisms, extracted from tea leaves, or fermented or chemically synthesized. For example, in order to synthesize industrially, L-pyrrolidonecarboxylic acid obtained by heating L-glutamic acid can be made into a copper salt, reacted with anhydrous ethylamine, and finally copper removed.
[0013]
In the present invention, theanine and its derivatives can be used alone or in combination with other active ingredients.
When used alone, for example, theanine can be prepared by dissolving it in purified water or physiological saline in the form of a purified product, a crude product, or a tea extract.
When mixed with other active ingredients, for example, barbital compounds such as phenobarbital, benzodiazepine clonazepam, valuroic acid, etc. In addition, any one of components having known preventive effects, or two or more of these components can be added to a theanine aqueous solution and prepared and used by a known method.
[0014]
The agent for preventing and treating epilepsy of the present invention is an orally or parenterally administered agent (for intramuscular injection, intravenous injection, subcutaneous administration, rectal administration, transdermal administration, nasal administration, intraventricular administration, etc.) It can be used as a pharmaceutical product. Among them, an excellent effect when used as an intracerebroventricular agent, particularly an effect of suppressing induced post-launch (“AD”) has been found.
In addition to pharmaceuticals, it can be used in various ways as quasi-drugs, cosmetics, health drinks (cans, PET bottles, bottle drinks, etc.), health foods, food additives, and the like.
[0015]
As a form of use, it can be dried by freeze drying or spray drying, etc. and provided as a dry powder, or it can be provided as a liquid, tablet, powder, granule, dragee, capsule, suspension, emulsion, ampoule, injection, etc. You can also. When provided as a pharmaceutical, for example, theanine can be dissolved in purified water or physiological saline as it is and prepared for intraventricular administration. Moreover, the preventive agent which is easy to ingest can be provided by preparing as a quasi-drug and making this into drink forms, such as a bottle drink, or forms, such as a tablet, a capsule, and a granule. Furthermore, as health foods and health drinks, for example, the active ingredients of the present invention include dairy ingredients, carbonic acid, excipients (including granulating agents), diluents, or sweeteners, flavors, flours, starches, sugars, fats and oils. For example, sports drinks, fruit drinks, milk drinks, tea drinks, vegetable juices, milk drinks, alcoholic drinks, jelly , Jelly beverages, carbonated beverages, chewing gum, chocolate, candy, biscuits, snacks, bread, dairy products, fish paste products, livestock meat products, frozen desserts, dried foods, supplements and the like.
[0016]
In addition, although content of the active ingredient in this invention changes with uses, it is preferable to mix | blend 0.001-90 weight% in dry weight conversion, especially 0.01-90 weight%. For example, the active ingredient of the present invention is blended at a concentration of 0.01 to 1% by weight, added with a flavoring agent, sweetening agent, preservative, etc., and packed in a 100 ml brown bottle container for health foods and drinks and foods for specified health use. It can be commercialized as a product or special nutrition food or drink. Moreover, it can also be made into a tablet or a capsule form, and it can mix | blend with 1-90 weight% density | concentration per tablet or 1 capsule, and can also be commercialized as a pharmaceutical.
The intake of theanine and derivatives is preferably about 0.05 to 2000 mg / kg · day, more preferably 0.2 to 1000 mg / kg · day.
[0017]
Hereinafter, the effects of the present invention will be described in detail based on experiments.
[0018]
[Experiment 1]
In this experiment, the inhibitory action of theanine on the appearance of after discharge (hereinafter referred to as “AD”) at the time of creating an electric kindling model using experimental animals, particularly the influence of intravenous administration of theanine was examined.
[0019]
At this time, if the development of AD that appears after electrical stimulation is suppressed, it can be determined that it has acted in a suppressive manner on the construction and expansion of epileptic neural circuits.
In the electric kindling model, a part of the brain is repeatedly electrically stimulated under the convulsance threshold value to acquire convulsability readiness. The convulsability of the electric kindling model is introduced by an increase in the excitability of nerve cells by excitatory transmitters released from nerve cells by electrical stimulation. By repeating electrical stimulation below the stimulation threshold, the excitability of a wide range of nerve cells communicating with the stimulator via the synapse changes, and the convulsion threshold decreases, making it an excellent secondary generalization model. ing. In addition, when stimulation is repeated from the start of stimulation to the acquisition of convulsivity, the amplitude of AD, which is excessive excitement of the nerve cell group, increases, and the duration thereof is slightly extended.
[0020]
(Creation of specimen animals)
Eight 10-week-old Wistar male rats were used. After pre-breeding for one week, those that reached a body weight of 280 g or more were subjected to intracerebral electrode implantation under pentobarbital anesthesia (50 mg / kg, intraperitoneal administration). Enforced. At this time, the electrode embedding site was on the bilateral hippocampal CA1 region and the frontal dura mater, a stainless concentric bipolar deep electrode was used for the former hippocampus, and a stainless steel screw electrode was used for the latter dura mater.
Each electrode was introduced into a 6-pole microconnector and fixed on the skull with dental cement.
In order to prevent infection, a sulfa drug was encapsulated in the incision at the time of suture, and 3,000 units of penicillin G potassium solution was administered intramuscularly for 5 days after the operation.
[0021]
After the electrode implantation surgery, animals that have passed 10 days or more are transferred into a shield box, and the right hippocampal CA1 region is electrically stimulated (SEN-7203, Nihon Kohden Industry) via an isolator (SS-201J, Nihon Kohden Industry), A short wave stimulus of 60 Hz and a duration of 1 second was applied. The electrical stimulation was given exactly once a day at 24 hour intervals, the energization amount on the first day was set to 100 μA, and the energization amount was increased by 50 μA until an AD spike appeared to determine the AD induction threshold. After the determination of the AD induction threshold, a specimen animal in which the induction of AD was observed for 3 consecutive days by stimulation at 24 hour intervals according to the AD induction threshold was used in the experiment.
[0022]
(Recording system)
The brain waves (cortex, hippocampus on the opposite side of stimulation) of the sample animal were derived by wire, amplified by a bioelectric amplifier (AB621G, Nihon Kohden Industry), and recorded and stored on a magneto-optical disk at a sampling frequency of 200 Hz. The recording conditions were a time constant of 0.1 second, a high-frequency cutoff frequency of 60 Hz, an attenuation characteristic of 24 dB / oct, 1 minute before stimulation, and 2 minutes after stimulation for 3 minutes in total. Moreover, in order to observe the reaction of the animal before and after stimulation, images from a CCD camera attached to the measurement box were recorded simultaneously with the electroencephalogram using a digital video recorder.
[0023]
(Administration method and analysis)
Group A: L-theanine 0 μmol / kg (control)
Group B: L-theanine 100 μmol / kg
Group C: L-theanine 2000 μmol / kg
Group D: MK801 50 μmol / kg
In the four groups, the effects of administration of theanine or MK801 (dizocilpine), which is said to have an antiepileptic effect, on AD induction were examined.
[0024]
The administration method is a constant volume (0.5 ml) tail vein administration, and 15 minutes after administration, the AD induction threshold stimulation is given to the right hippocampal CA1 region using the same system as the preparation of the sample animal, and stimulation is performed for 1 minute before the stimulation. After 2 minutes, EEG was recorded. A 3-day washout period was given, and the same concentration was administered three times in total. The presence or absence of AD induction was confirmed from the recorded brain waves, and the average amplitude (average maximum amplitude) of 10 discharges AD having a large amplitude as shown in FIG. 1 and the AD duration as shown in FIG. 2 were measured. Significant differences were tested for the mean maximum amplitude and duration of AD using the Kruskal-Wallis test. The chi-square test was used for the presence or absence of AD induction.
[0025]
(result)
The results of the AD average maximum amplitude are shown in FIG. 3 and FIG.
Group C (theanine 2000 μmol / kg administration group) of both frontal cortex and hippocampus opposite to stimulation showed significantly lower values than group A (control group) (p <0.05 and p <0.01, respectively). ).
As shown in FIGS. 5 and 6, the AD duration was significantly shortened in the C group compared to the A group in both the frontal cortex and the hippocampus on the opposite side of stimulation (both p <0.05).
In any case, it is also clear that the effects of the group C (order of mmol / kg) are more remarkable than those of the group B (order of 100 μmol / kg).
[0026]
Table 1 below shows the number of times AD induced in each group in the frontal cortex and the number of stimulations that AD did not induce, and Table 2 shows the significant difference from other groups in that case. Table 3 shows the number of times that AD was induced in each group in the hippocampus on the opposite side of stimulation and the number of times that AD was not induced, and Table 4 shows the significant difference with respect to other groups in that case.
In addition, the numerical value in Table 2 and Table 4 has shown the significance level.
[0027]
[Table 1]

[0028]
[Table 2]

[0029]
[Table 3]

[0030]
[Table 4]

[0031]
Regarding propagation of AD, group A (control group) and group D (MK801 administration group) always induced AD in both frontal cortex and non-stimulated hippocampus with the same intensity of stimulation as in the preparation of specimen animals. There was a case where AD was not induced when the test substance was administered (Group B and Group C).
As shown in Table 1, the number of stimulations that AD did not induce in the frontal cortex increased significantly depending on the concentration of theanine administered. On the other hand, as shown in Table 3, in the hippocampus on the opposite side of stimulation, administration of theanine had little effect on the induction of AD.
In particular, the effect of Group C (on the order of mmol / kg) was significant compared to Group B (on the order of 100 μmol / kg).
[0032]
As described above, it has been clarified that administration of theanine suppresses the appearance or development of AD.
In addition, considering the completion of the process of creating epilepsy model animals through the process of the appearance of AD ⇒ development of AD ⇒ convulsions, theanine is administered to patients who are prone to abnormal discharge of AD in the brain Thus, the appearance or development of AD-like abnormal discharge is suppressed, and as a result, it can be considered that secondary epilepsy accompanied by convulsions considered as a sequela can be prevented.
Examples of the state in which an abnormal discharge like AD is likely to occur in the brain include a delivery failure and a head injury.
[0033]
[Experiment 2]
In this experiment, we investigated the inhibitory action of theanine on the post-launch ("AD") appearance when creating an electrical kindling model using experimental animals, particularly the effect of infusion of theanine into the ventricles.
[0034]
(Creation of specimen animals)
Ten-week-old Wistar male rats were preliminarily raised in the following environment, then those that reached a body weight of 280 g or more were selected, and implantation of a chronic electrode and a cannula for intracerebroventricular administration was performed as described below.
[0035]
= Preliminary rearing environment =
Breeding room: Small animal management room (rat breeding room)
Temperature: 20 ± 3 ° C
Humidity: 50 ± 10%
Light / dark cycle: Lighting from 08: 00 to 20:00 Cage used: Polycarbonate cage (using an electric planer chip on the floor)
Cage exchange: 2 times / week feeding: Free intake of solid feed (CE-1, Nippon Claire) Water supply: Free intake of tap water filtered through Millipore filter [0036]
= Chronic electrode and cannula implantation for intraventricular administration =
Anesthesia: Bent barbital anesthesia implantation site: Left and right frontal dura mater, left and right hippocampal dorsal part, third ventricle electrode: epidural electrode ... stainless steel screw electrode deep electrode ... stainless steel parallel Type bipolar electrode (for both recording and stimulation)
Cannula for administration ... Stainless steel pipe (φ0.3mm)
Post-operative treatment: The sulfa drug is included in the incision at the time of suturing, and the head of the animal after the intramuscular administration of 3000 units of penicillin G potassium solution is shown in FIG.
[0037]
(Determination of AD threshold)
Animals that had passed 10 days or more after electrode implantation surgery were moved into the measurement box, and a rectangular wave stimulus with a frequency of 60 Hz and a duration of 1 second was applied to the dorsal part of the right hippocampus at 24 hour intervals via an isolator. The energization amount is 100 μA for the first time, and the AD threshold is determined by increasing the energization amount by 50 μA until AD is recognized. The subsequent stimulation is carried out at this AD threshold, and animals that have induced AD for 3 consecutive days are tested. used.
When AD did not develop at the initially set AD threshold during the three days, stimulation was performed by increasing the energization amount by 50 μA from the next day, and this operation was repeated.
[0038]
Electroencephalograms before and after stimulation (cortex, stimulation side or opposite hippocampus) were derived by wire, recorded on paper after amplification, and stored on a magneto-optical disk at a sampling frequency of 200 Hz. The recording conditions were a time constant of 0.1 second, a high-frequency cutoff frequency of 60 Hz, an attenuation characteristic of 24 dB / octave, and a recording time of 1 minute before stimulation and 4 minutes after stimulation for a total of 5 minutes. Moreover, in order to observe the reaction of the animal before and after stimulation, an image from a CCD camera attached to a measurement box was recorded simultaneously with an electroencephalogram using a digital video recorder.
[0039]
= Equipment used =
Stimulator: Electrical stimulator (SEN-7203, Nihon Kohden Industry)
Isolator: Isolator (SS-201J, Nihon Kohden Industry)
Amplifier: Bioelectric amplifier (AB621G, Nihon Kohden Industry)
Recorder: Thermal pen recorder (8R-33, NEC Medical Systems)
AD converter: AD conversion board (ADM-5298BPC, microscience)
Other: Personal computer, oscilloscope, magneto-optical disk drive, CCD camera, digital video tape recorder
(Dose experiment)
A test substance (L-theanine (manufactured by Tokyo Chemical Industry Co., Ltd.) dissolved in sterilized physiological saline was administered in an amount of 0.1 ml into the third ventricle through a fixed cannula (constant volume).
[0041]
Seven model animals were made into one group, and administration experiments were conducted on a total of 6 groups, a control group and a theanine administration group (5 groups). Three doses were administered per individual with a washout period of 3 days or more.
Group A: 0 μmol / kg (saline administration group, control group)
Group B: 20 μmol / kg
Group C: 40 μmol / kg
Group D: 60 μmol / kg
Group E: 80 μmol / kg
Group F: 100 μmol / kg
[0042]
Stimulation was performed with an energization amount of the AD induction threshold 15 minutes after administration of the test substance. The electroencephalogram and behavior for 1 minute before stimulation and 2 minutes after stimulation were recorded under the same conditions as when the AD threshold was confirmed. A 3-day washout period was given, and the same concentration was administered three times.
[0043]
The presence or absence of AD induction was confirmed from the recorded brain waves, and the average amplitude (average maximum amplitude) of 10 discharges having a large amplitude and the AD duration were measured. The Kruskal-Wallis test was used for the average maximum amplitude and duration of AD, and the chi-square test was used for the presence or absence of AD.
[0044]
(result)
Table 5 shows changes in the average maximum amplitude of AD in the cortex / hippocampus, FIG. 8 shows the average maximum amplitude of AD in each group in the cortex, and FIG. 9 shows the average maximum amplitude of AD in each group in the hippocampus. Table 6 shows the presence or absence of a significant difference between the groups in the AD average maximum amplitude in the cortex, and Table 7 shows the presence or absence of a significant difference between the groups in the AD average maximum amplitude in the hippocampus.
Depending on the concentration of theanine administered, the AD mean maximum amplitude decreased in both the cortex and hippocampus. The reason why the standard deviation of each group is large is that the number of individuals (AD amplitude is 0 μV) that does not induce AD at all increases depending on the theanine administration concentration.
[0045]
[Table 5]

[0046]
[Table 6]

[0047]
[Table 7]

[0048]
Table 8 shows changes in AD duration in the cortex / hippocampus, FIG. 10 shows AD duration of each group in the cortex, and FIG. 11 shows AD duration of each group in the hippocampus. Table 9 shows the presence or absence of a significant difference between groups in AD duration in the cortex, and Table 10 shows the presence or absence of a significant difference between groups in AD duration in the hippocampus.
Similar to the amplitude, the duration of AD was reduced in both cortical and hippocampal regions depending on the concentration of theanine administered. The reason why the standard deviation of each group is large is that the number of individuals that do not induce AD at all (AD duration 0 sec) has increased as in the case of amplitude.
[0049]
[Table 8]

[0050]
[Table 9]

[0051]
[Table 10]

[0052]
Table 11 shows the presence or absence of AD induction, and Table 12 shows a significant difference in the presence or absence of AD induction between each group.
Depending on the concentration of theanine administered, an increasing number of individuals did not induce AD in both the cortex and hippocampus. When AD was induced, both the cortex and hippocampus were induced, and when not induced, neither was induced.
[0053]
[Table 11]

[0054]
[Table 12]

[0055]
In this experiment by intracerebroventricular administration, 3 out of 7 animals completely suppressed the induction of AD by administration of 40 μmol / kg, and 6 out of 7 animals completely suppressed the induction of AD by administration of 100 μmol / kg. . In addition, the amplitude and duration of the induced AD decreased with theanine administration of 40 μmol / kg or more depending on the concentration. With intraventricular administration, there were many individuals who did not induce AD completely, and the standard deviation increased. Therefore, although the amplitude and duration of AD were not significantly different from those of the control group at 40 μmol / kg, the actual difference was not observed. The effect is thought to be stronger than 2 mmol / kg intravenous administration.
[Brief description of the drawings]
FIG. 1 is an explanatory diagram showing a method of measuring an AD average maximum amplitude according to an embodiment (a method of measuring an amplitude of 10 discharges having a large amplitude from induced AD and calculating an average value thereof). , The upper row (COREX) shows the frontal cortex and the lower row (HIPP) shows the hippocampus on the contralateral side of stimulation.
FIG. 2 is an explanatory diagram showing an AD duration measurement method of the embodiment (a method in which the time from the first AD that appears after stimulation to the AD that appears last is the AD duration); CORTEX) indicates the frontal cortex, and the lower part (HIPP) indicates the hippocampus on the contralateral side of stimulation.
FIG. 3 is a graph showing the effect of administration of a test substance of an example on AD average amplitude in the frontal cortex.
FIG. 4 is a graph showing the influence of administration of a test substance of an example on AD average amplitude in the hippocampus on the contralateral stimulation side.
FIG. 5 is a graph showing the effect of administration of a test substance of an example on AD duration in the frontal cortex.
FIG. 6 is a graph showing the effect of administration of a test substance of an example on AD duration in the hippocampus on the contralateral stimulation side.
FIG. 7 is a photograph showing the head of a specimen animal one week after electrode / ventricular administration pipe implantation.
FIG. 8 is a graph showing the average maximum amplitude of AD in each group in the cortex.
FIG. 9 is a graph showing the average maximum amplitude of AD in each group in the hippocampus.
FIG. 10 is a graph showing AD duration of each group in the cortex.
FIG. 11 is a graph showing AD duration of each group in the hippocampus.

Claims (1)

Epilepsy prophylactic or therapeutic agent containing L- Teani in as an active ingredient.

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