JP6670922B2 - Ampa受容体を阻害する活性ペプチド、ならびにその調製方法およびその使用 - Google Patents
Ampa受容体を阻害する活性ペプチド、ならびにその調製方法およびその使用 Download PDFInfo
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- JP6670922B2 JP6670922B2 JP2018500399A JP2018500399A JP6670922B2 JP 6670922 B2 JP6670922 B2 JP 6670922B2 JP 2018500399 A JP2018500399 A JP 2018500399A JP 2018500399 A JP2018500399 A JP 2018500399A JP 6670922 B2 JP6670922 B2 JP 6670922B2
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- 108090000765 processed proteins & peptides Proteins 0.000 title claims description 39
- 102000003678 AMPA Receptors Human genes 0.000 title claims description 24
- 108090000078 AMPA Receptors Proteins 0.000 title claims description 24
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- 238000002360 preparation method Methods 0.000 title claims description 13
- 102000004196 processed proteins & peptides Human genes 0.000 title claims description 11
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- MGRVRXRGTBOSHW-UHFFFAOYSA-N (aminomethyl)phosphonic acid Chemical compound NCP(O)(O)=O MGRVRXRGTBOSHW-UHFFFAOYSA-N 0.000 description 20
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- HOKKHZGPKSLGJE-GSVOUGTGSA-N N-Methyl-D-aspartic acid Chemical compound CN[C@@H](C(O)=O)CC(O)=O HOKKHZGPKSLGJE-GSVOUGTGSA-N 0.000 description 10
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
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- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 description 1
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- HLCFGWHYROZGBI-JJKGCWMISA-M Potassium gluconate Chemical compound [K+].OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C([O-])=O HLCFGWHYROZGBI-JJKGCWMISA-M 0.000 description 1
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- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/10—Tetrapeptides
- C07K5/1021—Tetrapeptides with the first amino acid being acidic
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/08—Antiepileptics; Anticonvulsants
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/12—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by hydrolysis, i.e. solvolysis in general
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/22—Cysteine endopeptidases (3.4.22)
- C12Y304/22002—Papain (3.4.22.2)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Description
1)サケ皮を浸漬して破砕し、水を加えてかき混ぜた(beat)後、pHを6.5〜7.5に調整してスラリーを得る工程;
2)中性プロテアーゼを用いてスラリーを第1の酵素分解に供し、第1の酵素加水分解物を得る工程;
3)パパイン酵素を用いて第1の酵素加水分解物を第2の酵素分解に供し、その後酵素を失活させて、第2の酵素加水分解物を得る工程;ならびに
4)第2の酵素加水分解物を遠心分離した後、遠心分離した上清を膜ろ過、濃縮、および脱色して、活性ペプチドを調製する工程
を含む、活性ペプチドを調製する方法を提供する。
中性プロテアーゼ:Novozymes biotechnology Co.,Ltd製;
TTX、PTX、CNQX、APV、NMDA、およびAMPA:TOCRIS Bioscience製;
パパイン、PTZ:Sigma Aldrich製;ならびに
合成EGAR:Shanghai Qiang Yao Co.,Ltd.製。
移動相A:V(水):V(トリフルオロ酢酸)=100:0.1;
移動相B:V(アセトニトリル):V(水):V(トリフルオロ酢酸)= 80:20:0.1;
検出波長:UV220nm;
流速:0.6mL/分;
カラム温度:32℃;
注入量:50μL;
勾配プログラム:0−10分、移動相B:0%−5%;10〜20分、移動相B:5%−5%;20−35分、移動相B:5%−9%;35−45分、移動相B:9%−13%;45−60分、移動相B:13%−13%;60−70分、移動相B:13%〜70%;70−90分、移動相B:70%−70%。
Claims (10)
- 活性ペプチドを調製する方法であって、以下の工程:
1)サケ皮を浸漬して破砕し、水を加えてかき混ぜた後、pHを6.5〜7.5に調整してスラリーを得る工程;
2)中性プロテアーゼを用いてスラリーを第1の酵素分解に供し、第1の酵素加水分解物を得る工程;
3)パパイン酵素を用いて第1の酵素加水分解物を第2の酵素分解に供した後、酵素を失活させて、第2の酵素加水分解物を得る工程;ならびに
4)第2の酵素加水分解物を遠心分離した後、遠心分離した上清を膜ろ過、濃縮、および脱色して、活性ペプチドを得る工程;
を含み、
前記活性ペプチドがGlu−Gly−Ala−Argのアミノ酸配列を有するテトラペプチドを含み;
中性プロテアーゼの量が50〜500U/gであり、パパインの量が100〜1000U/gであり、かつ、中性プロテアーゼ:パパインの量比が1:(1〜3)であり;
第1の酵素分解の温度を30〜60℃に制御し、第1の酵素分解の時間を4〜6時間とし、かつ、第2の酵素分解の温度を30〜60℃に制御し、第2の酵素分解の時間を1〜3時間とする、方法。 - サケ皮を浸すために質量含有率0.1〜0.5%のアルカリ溶液を使用し、サケ皮対アルカリ溶液の質量/体積比を1:(2〜4)に制御し、浸漬時間は5〜20時間とする、請求項1に記載の方法。
- 膜ろ過を、孔径50〜1000nmのセラミック膜で行う、請求項1に記載の方法。
- 逆相高速液体クロマトグラフィーを用いて活性ペプチドを分離精製し、Glu−Gly−Ala−Argのアミノ酸配列を有するテトラペプチドを得る工程をさらに含む、請求項1に記載の方法。
- 抗てんかん食物の調製における活性ペプチドの使用であって、前記活性ペプチドが、請求項1〜4のいずれか一項に記載の方法に従って調製されることを特徴とする、活性ペプチドの使用。
- 抗てんかん食物の調製におけるAMPA受容体を阻害するテトラペプチドの使用であって、
前記テトラペプチドが、Glu−Gly−Ala−Argのアミノ酸配列を有する、テトラペプチドの使用。 - AMPA受容体を阻害するテトラペプチド含む抗てんかん食物であって、前記テトラペプチドがGlu−Gly−Ala−Argのアミノ酸配列を有する、抗てんかん食物。
- 抗てんかん薬剤の調製における活性ペプチドの使用であって、前記活性ペプチドが、請求項1〜4のいずれか一項に記載の方法に従って調製されることを特徴とする、活性ペプチドの使用。
- 抗てんかん薬剤の調製におけるAMPA受容体を阻害するテトラペプチドの使用であって、
前記テトラペプチドが、Glu−Gly−Ala−Argのアミノ酸配列を有する、テトラペプチドの使用。 - AMPA受容体を阻害するテトラペプチド含む抗てんかん薬剤であって、前記テトラペプチドがGlu−Gly−Ala−Argのアミノ酸配列を有する、抗てんかん薬剤。
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