JPH0592928A - Therapeutic agent for dysmnesia - Google Patents

Therapeutic agent for dysmnesia

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Publication number
JPH0592928A
JPH0592928A JP3278252A JP27825291A JPH0592928A JP H0592928 A JPH0592928 A JP H0592928A JP 3278252 A JP3278252 A JP 3278252A JP 27825291 A JP27825291 A JP 27825291A JP H0592928 A JPH0592928 A JP H0592928A
Authority
JP
Japan
Prior art keywords
epo
therapeutic agent
dysmnesia
preventive
improving
Prior art date
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Granted
Application number
JP3278252A
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Japanese (ja)
Other versions
JP3156236B2 (en
Inventor
Ryuzo Sasaki
隆造 佐々木
Masaji Ueda
正次 上田
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Snow Brand Milk Products Co Ltd
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Snow Brand Milk Products Co Ltd
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Abstract

PURPOSE:To obtain a therapeutic agent for dysmnesia capable of raising the intracellular calcium level of neurons, enhancing choline acetyltransferase activity and preventing, improving or treating Alzheimer type dysmnesia. CONSTITUTION:A therapeutic agent for dysmnesia containing erythropoietin(EPO) protein which is one kind of cytokinin and a hematopoietic factor, preferably the EPO or EPO mutein protein prepared by a genetic engineering technique as an active ingredient. The above-mentioned therapeutic agent is a preventive, improving or therapeutic agent for Alzheimer type dysmnesia in the dosage form of preferably a direct infusion into intracranial septal areas. The preventive, improving and therapeutic agent acts as a neurotrophic factor for neurons and has the preventive, improving and therapeutic actions on the dysmnesia. Since toxicity is low, the agent can safely be used. Furthermore, although the dose of the above-mentioned active ingredient can suitably be changed according to the extent of damage, it is about 1000-100000 IU at a time for an adult.

Description

【発明の詳細な説明】Detailed Description of the Invention

【0001】[0001]

【産業上の利用分野】本発明は、サイトカインの一種、
造血因子であるエリスロポエチン(以下EPOと略す
る)蛋白質を有効成分として含有するアルツハイマー型
記憶障害の予防・改善・治療剤に関する。
The present invention relates to a kind of cytokine,
The present invention relates to a prophylactic / ameliorating / therapeutic agent for Alzheimer-type memory disorders, which contains an erythropoietin (hereinafter abbreviated as EPO) protein, which is a hematopoietic factor, as an active ingredient.

【0002】[0002]

【従来の技術】EPOは、分子量30〜40kDa の高度に糖
付加された蛋白質で赤血球産生を促進する造血因子とし
て発見され、すでにその全アミノ酸配列が明かにされて
いる〔Jacobs et al, Nature, 313, 806-810 (198
5)〕。また、ヒト・EPOは、遺伝子工学的手段により
高純度のものが大量に生産され貧血治療薬としてすでに
応用されている〔Urabe et al, Int. J. Cell Cloling,
6, 179-191 (1988)〕。EPOの作用は、EPOの標的
細胞表面に存在するEPOレセプターを介して行われて
おり、EPOレセプターは、サイトカインスパーファミ
リーに属することが明かにされている〔D'Andrea et a
l, J. Clin. Invest., 86, 681-687 (1990)〕。中枢神
経障害修復の観点に立ち、サイトカインの神経栄養因子
としての作用に関する研究も近年活発に行われ、IL−
3が培養ラット中隔野神経細胞やコリン作動性ニューロ
ン由来PC−12細胞のコリンアセチルトランスフェラ
ーゼ(ChAT活性)を高めること〔田平 武 他, 平
成2年度ヒューマンサイエンス研究報告、第3分野 3-4
-1-A, 288-302 (1991)〕や、IL−6がPC−12細胞
の樹状突起の進展を促進すること〔鳥居邦夫, 代謝, 2
6,臨時増刊号, 脳代謝とその異常, 195-201 (1989)〕が
報告されている。
Background Art EPO was discovered as a hematopoietic factor that promotes erythropoiesis, which is a highly glycosylated protein having a molecular weight of 30 to 40 kDa, and its entire amino acid sequence has already been clarified [Jacobs et al, Nature, 313, 806-810 (198
Five)〕. In addition, human EPO is produced in high purity in a large amount by genetic engineering means and has already been applied as an anemia treatment drug [Urabe et al, Int. J. Cell Cloling,
6, 179-191 (1988)]. The action of EPO is mediated by the EPO receptor existing on the surface of target cells of EPO, and it has been revealed that the EPO receptor belongs to the cytokine superfamily [D'Andrea et a
l, J. Clin. Invest., 86, 681-687 (1990)]. From the viewpoint of central nervous system repair, studies on the action of cytokines as neurotrophic factors have been actively conducted in recent years, and IL-
3 enhances choline acetyltransferase (ChAT activity) of cultured rat septal area neurons and PC-12 cells derived from cholinergic neurons [Takeshi Tahira, et al., Human Science Research Report, 1990, 3rd field 3-4]
-1-A, 288-302 (1991)] and that IL-6 promotes the development of dendrites of PC-12 cells [Kunio Torii, Metabolism, 2
6, Extra edition, Brain metabolism and its abnormalities, 195-201 (1989)] have been reported.

【0003】一方、アルツハイマー病患者においては、
脳や髄液中のChAT活性の著しい低下が認められる
〔中村重信, 蛋白質核酸酵素, 36, 30-40 (1991)〕。即
ち、アルツハイマー型痴呆病のような中枢退行性疾患に
おいては、前脳基底部(中隔野、ブロッカー対角帯およ
びマイネルト基底核)のコリン作動性神経細胞の変性脱
落により、神経伝達物質であるアセチルコリンが生産さ
れず顕著な低下が惹起される。アセチルコリンの低下
が、アルツハイマー型痴呆病の記憶学習障害の原因と考
えられている〔Whiteahouse et al, Science, 215, 123
7-1239 (1982); Coyle et al, Science, 219, 1184-119
0 (1983)〕。実験的には、ラットの前脳基底部のコリン
作動性神経細胞を破壊するとアセチルコリン量の低下と
記憶障害が惹起されることが報告されている〔Helper e
t al, J Neurosci, 5, 866-873 (1985)〕。1980年
代に入って、海馬の長期増強と学習・記憶の相関が注目
され、細胞内カルシウムの上昇がカルモジュリン依存性
キナーゼ ないしCキナーゼを介して長期増強を誘導す
ることによって脳を活性化すると考えられている〔中川
八郎, 代謝, 26, 臨時増刊号, 脳代謝とその異常, 37-4
4 (1989)〕。
On the other hand, in Alzheimer's disease patients,
A marked decrease in ChAT activity in the brain and cerebrospinal fluid is observed [Shigenobu Nakamura, Protein Nucleic Acid Enzyme, 36, 30-40 (1991)]. That is, in central degenerative diseases such as Alzheimer's dementia, degeneration of cholinergic neurons in the basal forebrain region (septal area, blocker diagonal zone and basal Meinert's nucleus) causes degeneration of the neurotransmitter acetylcholine. Is not produced, causing a significant decrease. Decreased acetylcholine is thought to be the cause of memory learning impairment in Alzheimer-type dementia [Whiteahouse et al, Science, 215, 123]
7-1239 (1982); Coyle et al, Science, 219, 1184-119.
0 (1983)]. Experimentally, it has been reported that destruction of cholinergic neurons in the basal forebrain of rats causes a decrease in the amount of acetylcholine and memory impairment [Helper e
t al, J Neurosci, 5, 866-873 (1985)]. In the 1980s, attention has been paid to the correlation between long-term potentiation of the hippocampus and learning / memory, and it is considered that elevation of intracellular calcium activates the brain by inducing long-term potentiation via calmodulin-dependent kinase or C kinase. [Hachiro Nakagawa, Metabolism, 26, Extra edition, Brain metabolism and its abnormalities, 37-4
4 (1989)].

【0004】以上のことからコリン作動性神経細胞に作
用してChAT活性を上昇させる薬物(例えばNGF)
や神経細胞内カルシルム濃度を上昇させる物質は、アル
ツハイマー型痴呆症のような中枢退行性疾病に対して予
防・治療の作用を有すると考えられている。尚、近年、
高齢化に伴い急増する老人性疾患の中でも、とりわけ老
人性アルツハイマー型痴呆症は、有効な治療法がなく、
社会問題となり、その有効な治療薬の開発が切望されて
いる。
From the above, drugs that act on cholinergic nerve cells to increase ChAT activity (eg NGF)
It is considered that substances that increase the concentration of calcirum in nerve cells have preventive and therapeutic actions on central degenerative diseases such as Alzheimer's dementia. In addition, in recent years
Among the senile diseases that rapidly increase with aging, there is no effective treatment for senile Alzheimer's dementia,
It has become a social problem, and there is a strong demand for the development of effective therapeutic agents.

【0005】[0005]

【発明が解決しようとする課題】本発明は、神経細胞に
作用し、ChAT活性を上昇させる薬剤をアルツハイマ
ー型記憶障害の予防、改善、治療に応用するものであ
る。すなわち、本発明の課題は、ChAT活性を上昇さ
せてアルツハイマー型記憶障害を予防、改善あるいは治
療する薬剤を提供することにある。
The present invention is to apply a drug that acts on nerve cells and increases ChAT activity to the prevention, amelioration and treatment of Alzheimer's memory impairment. That is, an object of the present invention is to provide a drug for preventing, improving or treating Alzheimer's memory impairment by increasing ChAT activity.

【0006】[0006]

【課題を解決するための手段】本発明者等は、上記の技
術背景に立ち、サイトカインの中から赤血球造血因子で
あるEPOに着目し、ヒト神経芽細胞株(コリン作動性
ニューロン由来)PC−12細胞に対するEPOの作用
を精査したところ、EPOがPC−12細胞に作用し、
細胞内Ca2+濃度を上昇させ、細胞内ChAT活性を上
昇させる作用を有することを見出し、本発明を完成し
た。
Based on the above technical background, the present inventors have focused on EPO, which is an erythropoietic factor among cytokines, and have investigated human neuroblast cell line (derived from cholinergic neurons) PC- When the action of EPO on 12 cells was scrutinized, EPO acted on PC-12 cells,
The present invention has been completed by finding that it has an action of increasing intracellular Ca 2+ concentration and increasing intracellular ChAT activity.

【0007】すなわち、本発明は、EPO蛋白質を有効
成分として含有するアルツハイマー型記憶障害の予防・
改善・治療剤に関する。上記EPO蛋白質は、EPO活
性、即ち赤血球前駆細胞の分化・増殖作用を有する物質
であればいずれでもよい。このようなEPO蛋白質とし
ては、天然のヒト尿由来のEPO蛋白質でもよく、ま
た、遺伝子工学的手法によって製造したEPO蛋白質、
あるいは、ミュテインでもよい。EPOミュテインとし
ては、例えば、特開平3−151399号公報に記載し
たミュテインが挙げられる。上記EPOミューテインと
しては、もとの糖蛋白質のペプチド鎖のAsnがGln に変
異し、結合するN結合型糖鎖の結合数が変異したものが
ある。また他に、アミノ酸変異としては、特開平2−5
9599号公報、特開平3−72855号公報記載のも
のが挙げられる。即ち、EPOの有するEPOレセプタ
ーへの作用特性を失わない限り、アミノ酸の変異、欠
失、付加は何個でもよい。
That is, the present invention is directed to the prevention of Alzheimer's memory disorder containing EPO protein as an active ingredient.
Regarding improvement / treatment agents. The EPO protein may be any substance as long as it has EPO activity, that is, an action of differentiating / proliferating erythroid progenitor cells. Such an EPO protein may be a natural human urine-derived EPO protein, or an EPO protein produced by a genetic engineering method,
Alternatively, it may be a mutein. Examples of the EPO mutein include the mutein described in JP-A-3-151399. As the EPO mutein, there is one in which Asn of the peptide chain of the original glycoprotein is mutated to Gln and the number of N-linked sugar chains to be mutated is mutated. In addition, as an amino acid mutation, Japanese Patent Laid-Open No. 2-5
Examples thereof include those described in Japanese Patent Laid-Open No. 9599 and Japanese Patent Laid-Open No. 3-72855. That is, any number of amino acid mutations, deletions, and additions may be made as long as the property of EPO acting on the EPO receptor is not lost.

【0008】本発明のEPOは貧血治療薬としてすでに
臨床に応用されており、特筆すべき副作用も報告されて
いない。このように、治療剤は低毒性であるので、安全
に使用することができる。EPOはPC−12細胞に作
用し細胞中のアセチルコリン含量を高める作用があるの
で脳の神経細胞に作用して、アセチルコリン量を上昇さ
せる作用があり、アルツハイマー型記憶障害の予防・改
善・治療に用いることができる。あるいは、脳神経細胞
に作用して神経細胞内Ca2+濃度を調節し長期増強を誘
導する。本治療剤は、公知の製剤学的製造法に準じ薬学
的に許容しうる液体に1万〜10万単位で加え分散させ
て調製することができる。また、得られる溶液を凍結乾
燥し、用時生理食塩水に溶解して用いる用時溶解型とし
てもよい。さらに、安定化剤として公知の生物学的製剤
に使用される一般的薬剤、例えば、ヒト血清アルブミン
や糖を添加することにより安定に使用することができ
る。さらに、他の配合剤として、アミノ酸や、脂肪酸を
加えることもできる。
The EPO of the present invention has already been clinically applied as a therapeutic agent for anemia, and no remarkable side effect has been reported. Thus, the therapeutic agent has low toxicity and can be safely used. EPO acts on PC-12 cells to increase the content of acetylcholine in the cells, and thus acts on nerve cells in the brain to increase the amount of acetylcholine, and is used for the prevention / amelioration / treatment of Alzheimer's memory impairment. be able to. Alternatively, it acts on cerebral nerve cells to regulate Ca 2+ concentration in nerve cells and induce long-term potentiation. This therapeutic agent can be prepared by adding and dispersing 10,000 to 100,000 units to a pharmaceutically acceptable liquid according to a known pharmaceutical manufacturing method. Alternatively, the resulting solution may be freeze-dried and dissolved in a physiological saline solution before use to prepare a dissolution type solution before use. Further, it can be stably used by adding a general drug used in biological preparations known as a stabilizer, for example, human serum albumin or sugar. Furthermore, amino acids and fatty acids can be added as other compounding agents.

【0009】本記憶障害の改善・治療剤は、投与の方法
として直接脳組織内に投与することにより、アルツハイ
マー型記憶障害の改善・治療を促進させることができ
る。投与部位としては脳内中隔野に直接注入することが
望ましい。また、投与量は障害の程度によって適宜変更
しうるが、成人の場合、一回あたり1000〜1000
00IU程度を投与する。
[0009] The present agent for improving and / or treating memory disorders can be administered directly into brain tissue as a method of administration to promote improvement / treatment of Alzheimer's type memory disorders. It is desirable to inject directly into the septal area of the brain as the administration site. Further, the dose may be appropriately changed depending on the degree of the disorder, but in the case of an adult, it is 1000 to 1000 per dose.
Administer about 100 IU.

【0010】次に実施例及び実験例を挙げて、本発明を
より具体的に説明する。しかし、本発明はこれらの実施
例及び実験例に限定されるものではない。
Next, the present invention will be described more specifically with reference to Examples and Experimental Examples. However, the present invention is not limited to these examples and experimental examples.

【実施例1】アルツハイマー型記憶障害の予防・改善・治療剤の製造 人尿由来EPO 6,000,000IU ヒト血清アルブミン 2,500mg 注射用蒸留水にて全量を1lとする。この組成を1ml
ずつアンプルに充填し、滅菌を行い、EPO 6000
IU注射液を作成した。
Example 1 Production of a preventive / ameliorating / therapeutic agent for Alzheimer-type memory disorders Human urine-derived EPO 6,000,000 IU human serum albumin 2,500 mg Distilled water for injection makes the total amount 1 l. 1 ml of this composition
Fill ampoules one by one, sterilize, EPO 6000
An IU injection solution was prepared.

【0011】[0011]

【実施例2】アルツハイマー型記憶障害の予防・改善・治療剤の製造 遺伝子組換EPO 120,000,000IU ヒト血清アルブミン 2,500mg 注射用蒸留水にて全量を1lとする。この組成液をミリ
ポアフィルターによりろ過し、滅菌バイアル瓶に1ml
づつ分注し、凍結乾燥後密封した。このバイアル瓶の内
容を1mlの生理食塩水に溶解することにより、12
0,000IUの注射液を作成した。
[Example 2] Production of preventive / ameliorating / therapeutic agent for Alzheimer's memory disorder Genetically modified EPO 120,000,000 IU human serum albumin 2,500 mg Distilled water for injection makes the total amount 1 l. This composition liquid is filtered with a Millipore filter, and 1 ml is put in a sterile vial bottle.
Each was dispensed, freeze-dried and sealed. By dissolving the contents of this vial in 1 ml of physiological saline,
An injection solution of 10,000 IU was prepared.

【0012】[0012]

【実施例3】アルツハイマー型記憶障害の予防・改善・治療剤の製造 遺伝子組換EPO原液 2.44ml(2,10
0,000IU) 20%ヒト血清アルブミン 7.75ml 注射用蒸留水 144.81ml 全量を混合溶解後、ミリポアフィルターでろ過し、滅菌
バイアル瓶に0.5mlづつ充填し、凍結乾燥後密封し
た。本バイアルは6,000IUに相当する。
[Example 3] Production of prophylactic / ameliorating / therapeutic agent for Alzheimer-type memory disorder Genetically-modified EPO stock solution 2.44 ml (2,10)
20,000 IU) 20% human serum albumin 7.75 ml Distilled water for injection 144.81 ml After mixing and dissolving the whole amount, the mixture was filtered with a Millipore filter, filled in sterile vial bottles at 0.5 ml each, lyophilized and sealed. This vial corresponds to 6,000 IU.

【0013】以下に本発明の治療剤を用いた実験例を示
す。
Experimental examples using the therapeutic agent of the present invention are shown below.

【実験例1】神経細胞由来PC−12細胞のEPOレセプター測定 レセプターアッセイ並びにレセプターの分子サイズの測
定はSasakiらの方法〔Eur. J. Biochem., 168, 43-48
(1987)〕に従い実施した。PC−12細胞(ATCC
CRL−1721)1〜8×106 細胞/150μl、
EPO濃度0.5〜10nMとし、15℃、2〜3時間
反応を行った。PC−12細胞のEPOレセプターのE
POに対する結合親和常数はKd=8nM、レセプター
数は細胞あたり約1000個と算出された。また、SD
S電気泳動法によるEPOレセプターの分子サイズは、
約66kDaと算出された。
[Experimental Example 1] Measurement of EPO receptor in nerve cell-derived PC-12 cells Receptor assay and measurement of receptor molecular size are carried out by the method of Sasaki et al. [Eur. J. Biochem., 168, 43-48].
(1987)]. PC-12 cells (ATCC
CRL-1721) 1-8 × 10 6 cells / 150 μl,
The EPO concentration was 0.5 to 10 nM, and the reaction was performed at 15 ° C for 2 to 3 hours. E of the EPO receptor of PC-12 cells
The binding affinity constant for PO was calculated to be Kd = 8 nM, and the number of receptors was calculated to be about 1000 per cell. Also, SD
The molecular size of the EPO receptor by S electrophoresis is
It was calculated to be about 66 kDa.

【0014】[0014]

【実験例2】PC−12細胞のEPOレセプターの特性 実験例1と同様に、PC−12細胞のEPOレセプター
に対するEPOの親和性と、糖、キレート剤、サイトカ
イン類、ホルモンの結合阻害性を測定した。測定は 125
I-rHuEPOを用い、この特異的結合に対する阻害能を測定
した。特異的結合は、無添加時の125I-EPOの結合阻害率
を100とした相対率で表した。数値が小さいほど125I
-EPOとPC−12細胞の結合阻害が大きい。すなわち添
加した物質のEPOレセプターへの結合親和力が強い。
このように測定したPC−12細胞のEPOレセプター
の特性を表1〜表3に記載した。EPOレセプターは、
ヒトEPO、N結合型糖鎖結合部位を欠く変異EPO並
びにマウスEPOを認識した(表1)。表1に示したr
HuEPOとは遺伝子組換EPOであり、rHuEPO
(N−グリカナーゼ処理)は、遺伝子組換EPOを津田
らの方法(Tsuda et.al., J.Biochem, vol.188, 405-41
1, 1990)に従って調製したものであり、NQ123は特
開平3−151399号公報に開示された方法に従い調
製したN結合型糖鎖を一分子当たり3本を欠くrHuE
POミュテインである。また、他の各種サイトカイン
(表3)および糖(表2)は、EPOとレセプターの結
合に影響しなかった。即ち、PC−12細胞のEPOレ
セプターは、EPOと特異的に結合した。
[Experimental Example 2] Characteristics of EPO receptor of PC-12 cells As in Experimental Example 1, the affinity of EPO for the EPO receptor of PC-12 cells and the binding inhibition of sugars, chelating agents, cytokines and hormones were measured. did. The measurement is 125
I-rHuEPO was used to measure the ability to inhibit this specific binding. The specific binding was expressed as a relative rate with the binding inhibition rate of 125 I-EPO in the absence of addition as 100. The smaller the number, the more 125 I
-Great inhibition of binding between EPO and PC-12 cells. That is, the added substance has a strong binding affinity to the EPO receptor.
The properties of the EPO receptor of PC-12 cells thus measured are shown in Tables 1 to 3. The EPO receptor is
It recognized human EPO, mutant EPO lacking the N-linked sugar chain binding site, and mouse EPO (Table 1). R shown in Table 1
HuEPO is a genetically modified EPO, which is rHuEPO.
(N-glycanase treatment) was carried out by the method of Tsuda et al. (Tsuda et.al., J. Biochem, vol. 188, 405-41).
1, 1990), and NQ123 is rHuE which lacks three N-linked sugar chains per molecule prepared according to the method disclosed in JP-A-3-151399.
It is a PO mutein. In addition, other various cytokines (Table 3) and sugars (Table 2) did not affect the binding between EPO and the receptor. That is, the EPO receptor of PC-12 cells specifically bound to EPO.

【0015】[0015]

【表1】 PC−12細胞に対する各種EPO結合の特異性 ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ 添 加 物 添 加 量 125I-rHuEPOの (ng in 150μl) 特異的結合 (%) ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ 無 添 加 ── 100 rHuEPO 15 16 rHuEPO(N-ク゛リカナーセ゛処理) 15 29 NQ123(N結合型糖鎖結合 部位を欠くrHuEPOミュテイン) 15 21 ラットEPO 15 23 ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ *rHuEPO:遺伝子組換ヒトエリスロポエチン * 125I-rHuEPOの特異的結合は、無添加時の125I-EPOと
PC−12細胞の結合量の相対値であらわした。数値が
小さいほど添加物質のEPOレセプターへの結合親和力
が強い。
[Table 1] Specificity of various EPO binding to PC-12 cells ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ Specific addition of 125 I-rHuEPO (ng in 150 μl) Specific binding (%) ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ ━━━ No addition ── 100 rHuEPO 15 16 rHuEPO (N-glycanase treatment) 15 29 NQ123 (rHuEPO mutein lacking N-linked sugar chain binding site) 15 21 rat EPO 15 23 ━━━━━━━━━━ ━━━━━━━━━━━━━━━━━━━━━━━━ * rHuEPO: genetically modified human erythropoietin * 125 I-rHuEPO specific binding is at no added 125 I- It was expressed as a relative value of the binding amount of EPO and PC-12 cells. The smaller the value, the stronger the binding affinity of the added substance to the EPO receptor.

【0016】[0016]

【表2】 PC−12細胞とEPOの結合に対する糖、キレート剤の影響 ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ 添 加 物 添加量 (mM) 125I-rHuEPO の 特異的結合 (%) ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ な し ── 100 N-アセチルガラクトサミン 10 105 ガラクトース 10 95 EDTA 40 98 (エチレンジアミンテトラ酢酸塩) EGTA 40 91 (エチレングリコール・ビス・ベータ・ アミノエチルエーテルN N N'N'-4酢酸塩) ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ * 125I-rHuEPOとPC−12細胞の特異的結合量を10
0とした相対値で表した。数値が小さいほど結合阻害が
おおきい。すなわち添加物のEPOレセプターへの結合
親和力が強い。
[Table 2] Effects of sugars and chelating agents on the binding between PC-12 cells and EPO ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ ━━━━ Additive additive amount (mM) 125 Specific binding of I-rHuEPO (%) ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ ━━━━━━━━ None ── 100 N-acetylgalactosamine 10 105 galactose 10 95 EDTA 40 98 (ethylenediaminetetraacetic acid salt) EGTA 40 91 (ethylene glycol bis beta aminoethyl ether NN N'N ' -4 acetate) ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ * 125 I-rHuEPO and PC-12 cells Specific binding of 10
The relative value was set to 0. The smaller the value, the greater the inhibition of binding. That is, the binding affinity of the additive to the EPO receptor is strong.

【0017】[0017]

【表3】 PC−12細胞に対する各種サイトカイン、ホルモンの結合特異性 ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ 添 加 物 添 加 量 125I-rHuEPO の (ng in 150μl) 特異的結合 (%) ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ 無 添 加 ─── 100 rHuEPO 125 16 ヒト・IL−2 50 102 サル・IL−3 50 117 ヒト・IL−6 200 92 マウス・G−CSF 500 92 ヒト・GM−CSF 500 82 ヒト・EDF 500 100 ヒト・TNF 500 99 ヒト・HGF 500 102 マウス・NGF 500 91 ヒト・EGF 500 97 ヒト・インシュリン 1000 102 ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ * 125I-rHuEPOの特異的結合は、無添加時の 125I-rHuE
POとPC−12細胞の結合量の相対値で表した。数値が
小さいほど添加物質のEPOレセプターへの結合親和力
が強い。
[Table 3] Binding specificity of various cytokines and hormones for PC-12 cells ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ Additive 125 I-rHuEPO (ng in 150 μl) Specific binding (%) ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ --100 rHuEPO 125 16 human IL-2 50 102 monkey IL-3 50 117 human IL-6 200 92 mouse G-CSF 500 92 human GM-CSF 500 82 human EDF 500 100 human TNF 500 99 human HGF 500 102 mouse NGF 500 91 human EGF 500 97 human insulin 1000 102 102 ━━━━━━━━━━━━━━━━━━━━━━━━━━━━ ━ * 125 specific binding of I-rHuEPO is At the time of addition of 125 I-rHuE
It was represented by the relative value of the amount of binding between PO and PC-12 cells. The smaller the value, the stronger the binding affinity of the added substance to the EPO receptor.

【0018】[0018]

【実験例3】rHuEPOのPC−12細胞内カルシウム濃度上昇活性の測
細胞内カルシウム濃度の測定は、Grynkiewiez ら〕J.Bi
o. Chem., 260, 3440-3450 (1985))方法に準じ、PC
−12細胞5×106 cell/mlで行った。EPO
20単位/mlのPC−12細胞内カルシウム濃度の
上昇は、ブラジキニン 10μM 添加時の効果に匹敵
した。EPO添加量と細胞内カルシウム濃度を表4に示
した。
[Experimental Example 3] Measurement of PC-12 intracellular calcium concentration increasing activity of rHuEPO
Measurement of constant intracellular calcium concentration, Grynkiewiez et al] J.Bi
o. Chem., 260, 3440-3450 (1985))
-12 cells were performed at 5 × 10 6 cells / ml. EPO
The increase in PC-12 intracellular calcium concentration of 20 units / ml was comparable to the effect of addition of 10 μM bradykinin. Table 4 shows the amount of EPO added and the intracellular calcium concentration.

【0019】[0019]

【表4】 PC−12細胞カルシウムレベルに対するEPOの効果 ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ 添 加 物 添 加 量 細胞内カルシウム濃度 測定回数 の相対増加率(%) (対 basal level) ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ EPO 0.3単位/ml 143±40 12 〃 3 〃 149±46 12 〃 10 〃 160±48 12 〃 20 〃 169±45 7 EPO (20年単位/ml) 添加後EGTA(1mM) 126± 9 3 を添加 ブラジキニン 10μM 169±40 10 ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ 以上の実験の結果、EPOは神経細胞の細胞内カルシウ
ム濃度を上げ、ChAT活性を上昇させることが確認さ
れた。
[Table 4] Effect of EPO on PC-12 cell calcium level ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ Additives Additives Intracellular calcium concentration Relative rate of increase in number of measurements (%) (vs. basal level) ━━━━━━━━━━━━━━━━━━━━━━━━━━━ ━━━━━━━━━━━ EPO 0.3 unit / ml 143 ± 40 12 〃 3 〃 149 ± 46 12 〃 10 〃 160 ± 48 12 〃 20 〃 169 ± 45 7 EPO (20-year unit / ml) After addition, EGTA (1 mM) 126 ± 93 was added Bradykinin 10 μM 169 ± 40 10 ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ As a result of the above experiment, it was confirmed that EPO increases intracellular calcium concentration in nerve cells and ChAT activity.

【0020】[0020]

【発明の効果】本発明のEPO蛋白質を有効成分とする
製剤は、神経細胞に対し神経栄養因子として働き、アル
ツハイマー型記憶障害の予防、改善、治療作用を有する
もので、アルツハイマー型記憶障害の予防・改善・治療
剤として有利に用いることができる。
INDUSTRIAL APPLICABILITY The preparation of the present invention containing the EPO protein as an active ingredient acts as a neurotrophic factor for nerve cells, and has the preventive, ameliorating, and therapeutic effects on Alzheimer-type memory disorders. -It can be advantageously used as an improving / therapeutic agent.

Claims (3)

【特許請求の範囲】[Claims] 【請求項1】 エリスロポエチン蛋白質を有効成分とし
て含有するアルツハイマー型記憶障害の予防・改善・治
療剤。
1. A preventive / ameliorating / therapeutic agent for Alzheimer's memory disorders, which comprises an erythropoietin protein as an active ingredient.
【請求項2】 エリスロポエチン蛋白質が遺伝子工学的
手法によって作製されたエリスロポエチンまたはエリス
ロポエチンミュテイン蛋白質である請求項1記載の予防
・改善・治療剤。
2. The preventive / ameliorating / therapeutic agent according to claim 1, wherein the erythropoietin protein is erythropoietin or an erythropoietin mutein protein produced by a genetic engineering method.
【請求項3】 剤型が脳内中隔野直接注入剤である請求
項1または2に記載の予防・改善・治療剤。
3. The preventive / ameliorating / therapeutic agent according to claim 1 or 2, wherein the dosage form is a direct injection into the septal area of the brain.
JP27825291A 1991-09-30 1991-09-30 Remedies for improving memory impairment Expired - Lifetime JP3156236B2 (en)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002020031A3 (en) * 2000-09-04 2002-08-01 Hannelore Ehrenreich Use of erythropoietin and the derivatives of the same for treating schizophrenia and related psychoses
WO2008151841A3 (en) * 2007-06-15 2009-04-09 Univ Zuerich Treatment for alzheimer' s disease
US7645733B2 (en) 2003-09-29 2010-01-12 The Kenneth S. Warren Institute, Inc. Tissue protective cytokines for the treatment and prevention of sepsis and the formation of adhesions
JP2010031017A (en) * 2000-12-29 2010-02-12 Kenneth S Warren Inst Inc Protection, restoration and enhancement of erythropoietin-responsive cell, tissue and organ
US8763746B2 (en) 2011-12-02 2014-07-01 Toyo Denso Kabushiki Kaisha Mounting structure of handle switch device

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002020031A3 (en) * 2000-09-04 2002-08-01 Hannelore Ehrenreich Use of erythropoietin and the derivatives of the same for treating schizophrenia and related psychoses
JP2004522696A (en) * 2000-09-04 2004-07-29 エーレンライヒ,ハネローレ Methods of treating schizophrenia and related psychiatric disorders and the use of erythropoietin or erythropoietin derivatives for the treatment of schizophrenia and related psychiatric disorders
JP2010031017A (en) * 2000-12-29 2010-02-12 Kenneth S Warren Inst Inc Protection, restoration and enhancement of erythropoietin-responsive cell, tissue and organ
JP2015221816A (en) * 2000-12-29 2015-12-10 ザ ケネス エス.ウォーレン インスティテュート,インコーポレーテッド Protection, restoration, and enhancement of erythropoietin-responsive cells, tissues and organs
US7645733B2 (en) 2003-09-29 2010-01-12 The Kenneth S. Warren Institute, Inc. Tissue protective cytokines for the treatment and prevention of sepsis and the formation of adhesions
WO2008151841A3 (en) * 2007-06-15 2009-04-09 Univ Zuerich Treatment for alzheimer' s disease
US20100172919A1 (en) * 2007-06-15 2010-07-08 Jan Grimm Noveltreatment for neurological disorders
JP2010534194A (en) * 2007-06-15 2010-11-04 チューリッヒ大学 Novel treatment for nervous system disorders
US8763746B2 (en) 2011-12-02 2014-07-01 Toyo Denso Kabushiki Kaisha Mounting structure of handle switch device

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