KR820000188B1 - Process for the preparation of thymosin - Google Patents
Process for the preparation of thymosin Download PDFInfo
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- KR820000188B1 KR820000188B1 KR7702475A KR770002475A KR820000188B1 KR 820000188 B1 KR820000188 B1 KR 820000188B1 KR 7702475 A KR7702475 A KR 7702475A KR 770002475 A KR770002475 A KR 770002475A KR 820000188 B1 KR820000188 B1 KR 820000188B1
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- 108010046075 Thymosin Proteins 0.000 title claims description 12
- 102000007501 Thymosin Human genes 0.000 title claims description 12
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- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 9
- 102000004169 proteins and genes Human genes 0.000 claims description 8
- 108090000623 proteins and genes Proteins 0.000 claims description 8
- 108700016958 thymosin fraction 5 Proteins 0.000 claims description 6
- 239000011780 sodium chloride Substances 0.000 claims description 5
- 239000012507 Sephadex™ Substances 0.000 claims description 4
- 239000007983 Tris buffer Substances 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 3
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 3
- WFIYPADYPQQLNN-UHFFFAOYSA-N 2-[2-(4-bromopyrazol-1-yl)ethyl]isoindole-1,3-dione Chemical compound C1=C(Br)C=NN1CCN1C(=O)C2=CC=CC=C2C1=O WFIYPADYPQQLNN-UHFFFAOYSA-N 0.000 claims description 2
- 229920002134 Carboxymethyl cellulose Polymers 0.000 claims description 2
- 229920001425 Diethylaminoethyl cellulose Polymers 0.000 claims description 2
- 239000001768 carboxy methyl cellulose Substances 0.000 claims description 2
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- 238000004587 chromatography analysis Methods 0.000 claims description 2
- 229960000789 guanidine hydrochloride Drugs 0.000 claims description 2
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 claims description 2
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- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims 1
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- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
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- BJEPYKJPYRNKOW-UHFFFAOYSA-N malic acid Chemical compound OC(=O)C(O)CC(O)=O BJEPYKJPYRNKOW-UHFFFAOYSA-N 0.000 description 2
- 239000008223 sterile water Substances 0.000 description 2
- -1 tallymosin Proteins 0.000 description 2
- 230000002992 thymic effect Effects 0.000 description 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- 102000011632 Caseins Human genes 0.000 description 1
- 108010076119 Caseins Proteins 0.000 description 1
- 108090000317 Chymotrypsin Proteins 0.000 description 1
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 1
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- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
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- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical compound OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 description 1
- 238000004166 bioassay Methods 0.000 description 1
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- 239000005018 casein Substances 0.000 description 1
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 1
- 235000021240 caseins Nutrition 0.000 description 1
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- 238000006243 chemical reaction Methods 0.000 description 1
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- 238000002523 gelfiltration Methods 0.000 description 1
- 150000002357 guanidines Chemical class 0.000 description 1
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- 229930195733 hydrocarbon Natural products 0.000 description 1
- 150000002430 hydrocarbons Chemical class 0.000 description 1
- 239000000677 immunologic agent Substances 0.000 description 1
- 229940124541 immunological agent Drugs 0.000 description 1
- 238000005462 in vivo assay Methods 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 230000001926 lymphatic effect Effects 0.000 description 1
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- 102000039446 nucleic acids Human genes 0.000 description 1
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- 150000007530 organic bases Chemical class 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000013014 purified material Substances 0.000 description 1
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- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
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- 229940086735 succinate Drugs 0.000 description 1
- KDYFGRWQOYBRFD-UHFFFAOYSA-L succinate(2-) Chemical compound [O-]C(=O)CCC([O-])=O KDYFGRWQOYBRFD-UHFFFAOYSA-L 0.000 description 1
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- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
내용 없음.No content.
Description
본 발명은 흉선 질환에 의한 면역 결핍증에 유효한 티모신 α1(티모신 획분 5에서 분리된 산성 폴리펩타이드)의 완전한 구조 결정 및 그의 분리방법에 관한 것이다.The present invention relates to the complete structure determination of thymosin a 1 (acidic polypeptide isolated from thymosin fraction 5) effective for immunodeficiency caused by thymic disease and to a method for its isolation.
동물과 사람의 면역성의 발달과 노쇠현상에서 흉선의 중요성이 현재 일반적으로 시인되어 있다.The importance of the thymus in the development and senescence of animals and humans is now generally acknowledged.
T세포의 발달을 조절하는 흉선의 분자의 성질에 관한 연구가 거의 없지만 호르몬 기전을 통해서 진행 과정의 중요한 부분을 알 수 있다. 흉선은 티모신이라는 폴리 펩타이드군을 생성하고 그리고 여러가지 다른 흉선 호르몬과 T세포의 기능, 분화 및 숙성에 중요한 역할을 하는 요인들을 생성시킨다. 티모신은 유전적으로 무흉선증인 새앙쥐, 흉선 제거된 성숙한 새앙쥐, 격렬한 자동 면역작용을 가진 NZB 새앙쥐, 암이 있는 새앙쥐, 그리고 유전분증을 유발시키는 카제인이 있는 새앙쥐에서 면역작용을 증진시킨다는 것과 T세포 분화를 유발시킨다는 것이 밝혀진다.There is little research on the properties of the thymus molecules that control the development of T cells, but the hormonal mechanisms reveal important parts of the process. The thymus produces a family of polypeptides called thymosin and produces factors that play an important role in the function, differentiation and maturation of several different thymic hormones and T cells. Thymosin promotes immunity and T-cell differentiation in genetically athymic rats, thymus-depleted mature rats, NZB rats with intense autoimmune reactions, rats with cancer, and rats with casein-induced hereditary It turns out.
티모신 획분 5는 면역성 제제이고, 흉선 대신에 흉선을 제거시키거나 면역성을 제거시킨 개체에서 면역 기능을 재생시킨다. 임상적 치료에서 획분 5는 티모신 T세포수를 증가시켜 주로 흉선 질환에 의한 면역 결핍증을 가진 어린이에게서 면역작용을 정상화시켜서 면역성이 억제된 환자에게서 T세포수를 증가시킬 수 있는 기능을 나타낸다.Thymosin fraction 5 is an immunological agent and regenerates immune function in individuals who have removed the thymus instead of the thymus or have been immunized. In clinical treatment, fraction 5 demonstrates the ability to increase thymosin T-cell numbers, thereby normalizing the immune function in children with immunodeficiency caused primarily by thymic disease, thereby increasing T-cell numbers in patients with suppressed immunity.
분석용인 폴리아크릴아마이드 겔 전기영동법과 등전점법은 획분 5가 10 내지 15개로 된 주성분과 20개 또는 그 이상의 보조성분으로 되어 있으며 분자량의 범위가 1000에서 15000 정도로 구성되어 있음을 시사한다.The analytical polyacrylamide gel electrophoresis and isoelectric point method suggest that the fraction consists of 10 to 15 main components and 20 or more auxiliary components and has a molecular weight in the range of 1000 to 15000.
본 발명은 티모신 획분 5에서 분리된 산성 폴리펩타이드를 완전한 구조결정과 함께 분리하는 방법에 관한 것이다. 이 펩타이드는 티모신 α1으로 불려진다. 티모신 α1은 세포의 분화와 기능을 측정할 수 있는 여러가지 생체내와 생체외의 분석시험에서 획분 5보다 10배에서 1000배의 활성이 있는 것으로 나타난다.The present invention relates to a method for separating acidic polypeptide isolated from thymosin fraction 5 with complete structural determination. This peptide is called thymosin a 1 . Thymosin α 1 is 10 to 1000 times more active than fraction 5 in various in vivo and ex vivo assays that can measure cell differentiation and function.
티모신 α1은 분자량이 3108이고 pH 3내지 5의 범위에서 겔 등전점법에 의해 측정했을 때 등전점은 40 내지 4.3 사이에 있다. 그 화합물은 다음의 아미노산 연결순서로 되어 있다.The thymosin α 1 has a molecular weight of 3108 and has an isoelectric point between 40 and 4.3 as measured by the gel isoelectric point method in the range of pH 3 to 5. The compound has the following amino acid linking sequence.
참조 : (N-acetyl)-Ser-Asp-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-OH.Reference: (N-acetyl) -Ser-Asp-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val- Val-Glu-Glu-Ala-Glu-Asn-OH.
티모신 α1은 다음에 설명하는 이온-교환 크로마토그라피와 겔 여과의 연속방법으로 획분 5에서 분리된다.Thymosin a 1 is separated in fraction 5 by a continuous method of ion-exchange chromatography and gel filtration described below.
동결건조된 티모신 획분 5는 2-멜켑토에탄올 1.0mM을 함유하는 pH 5.0인 나트륨 아세테이트 완충액 10mM의 카복시메틸-셀루로즈의 칼럼에서 크로마토그라프 처리된다. 이 컬럼은 처음에 완충액으로 세척한 후 계속해서 선형구배(linear gradient)를 갖는 출발 완충액 및 1.0M 염화나트륨을 함유하는 동일한 완충액 2ℓ로 세척한다. 첫째번 단백질 획분은 멸균증류수 데스트란 겔칼럼(Sephadex G-25)에서 여과한다.Lyophilized thymosin fraction 5 is chromatographed on a column of carboxymethyl-cellulose in 10 mM sodium acetate buffer pH 5.0 containing 1.0 mM 2-meltotoethanol. This column is first washed with buffer and then washed with 2 liters of starting buffer with linear gradient and the same buffer containing 1.0 M sodium chloride. The first protein fraction is filtered on sterile distilled water Destran gel column (Sephadex G-25).
덱스트란 겔 칼럼에서 얻은 두번째 단백질 피크는 1.0mM 2-멜켑토에탄올을 함유하는 50mM 트리스-완충액(pH 8.0) 50mM로 채워진 2-디에틸아미노에틸-셀루로즈 칼럼(DE-32)에 통과시킨다.The second protein peak obtained on the dextran gel column is passed through a 2-diethylaminoethyl-cellulose column (DE-32) filled with 50 mM 50 mM Tris-buffer (pH 8.0) containing 1.0 mM 2-meltotoethanol.
이 칼럼은 출발 완충액으로 용출시킨 후 계속해서 선형 구배를 갖는 출발 완충액 및 0.8M 염화나트륨을 함유하는 동일한 완충액 1.3ℓ로 용출시킨다. DE-32 칼럼에서 용출한 단백질 획분 처음 6분 1(the first sixth) 획분은 6.0M 구아니딘-하이드로 클로라이드와 트리스 10mM(pH 7.5)을 함유하는 완충액의 덱스트란 겔 칼럼(Sephadex G-75)을 통해서 더욱 정제시킬 수 있다. 단일한 협단부는 단백질 피크에서 만들어지고 멸균수의 덱스트란 겔(Sephadex G-10)의 칼럼에서 탈염시킨다. 이렇게 얻어진 정제시킨 물질은 티모신 α1이다. 획분 5에서의 티모신 α1의 수율은 약 0.6%이다. 이 제제는 탄화수소와 핵산염이 없는 것이다.This column is eluted with starting buffer and then eluted with 1.3 liters of starting buffer with a linear gradient and the same buffer containing 0.8 M sodium chloride. The first sixth fraction of the protein fraction eluted from the DE-32 column was passed through a dextran gel column (Sephadex G-75) in a buffer containing 6.0 M guanidine-hydrochloride and 10 mM Tris (pH 7.5). It can be further purified. Single narrowing points are made on protein peaks and desalted on a column of dextran gel (Sephadex G-10) in sterile water. The purified material thus obtained is thymosin a 1 . The yield of thymosin a 1 in fraction 5 is about 0.6%. This agent is free of hydrocarbons and nucleic acids.
티모신 α1의 구조와 완전한 아미노산의 연결순서가 트립신, 키모트립신, 털모리신 혹은 서브틸리신에 의한 효소 소화법 즉 종이 전기영동 및 크로마토그라피 및 분리된 펩타이드 획분의 에드만 분해에 의한 분리 소화법인 전통적인 방법을 사용해서 결정할 수 있다.The sequence of the structure of thymosin α 1 and the complete amino acid sequence was determined by enzyme digestion with trypsin, chymotrypsin, tallymosin, or subtilisin, ie, paper electrophoresis and chromatography, and isolated digestion by edman decomposition of isolated peptide fractions. You can decide using the corporate traditional method.
다음 표는 새앙쥐의 아미토젠 분석의 생체내 실험, 생성을 측정하는 임파 분석의 생체의 실험, 및 사람의 E-로젯트 분석의 생체의 실험에서 티모신 α1이 티모신 획분 5보다 10배에서 1000배의 활성이 있다는 것을 표시한다.The following table shows that thymosin α 1 is 10 times greater than thymosin fraction in in vivo experiments of amitogen assay in piglets, in vivo experiments in lymphatic assays measuring production, and in vivo experiments in human E-Roset assays. Indicates that there is 1000 times activity.
여러가지 생물 검사에서 티모신의 활성(㎍)Activity of thymosin in various bioassays
*생체내 실험으로 14일간 주사. * 14 days injection in vivo experiment.
티모신 α1은 비경구적으로 정맥, 피하 및 근육주사로 투여할 수 있다. 이 화합물은 정맥 주사할 때 1일 용량 체중당 1 내지 100㎍/㎏의 면역성을 가지고 있는 제제이다. 명백히 필요되는 용량은 특별한 치료상태, 상태의 심각성 및 치료기간에 따라서 변화될 것이다. 약학적 사용에 적당한 바이알 제형은 멸균수나 식염수 첨가전에 재조성시킬 때 1개당 동결건조된 티모신 α11mg을 사용한다.Thymosin α 1 may be administered parenterally, intravenously, subcutaneously, and intramuscularly. This compound is an agent having an immunity of 1 to 100 μg / kg / day body weight when injected intravenously. Obviously the required dose will vary depending on the particular treatment state, severity of the condition and duration of treatment. Vial formulations suitable for pharmaceutical use use 1 mg of lyophilized thymosin α 1 per preparation when reconstituted prior to addition of sterile water or saline.
또한 본 발명물의 범주에 속하는 것은 약학적으로 무독한 나트륨 및 카리염 같은 티모신 α1의 산부가염과 염기 부가염 혹은 구아니딘 같은 강한 유기 염기를 가진 염이 있다. 산부가염의 예로는 염산염, 하이드로브로마이드, 황산염, 인산염, 말레이트, 아세테이트, 시트레이트, 벤조에이트, 석시네이트, 말레이트와 아스코베이트가 있다.Also within the scope of the present invention are acid addition salts of thymosin α 1 , such as sodium and cari salt, which are pharmaceutically toxic and salts with strong organic bases, such as base addition salts or guanidines. Examples of acid addition salts are hydrochloride, hydrobromide, sulfate, phosphate, malate, acetate, citrate, benzoate, succinate, malate and ascorbate.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR7702475A KR820000188B1 (en) | 1977-10-27 | 1977-10-27 | Process for the preparation of thymosin |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR7702475A KR820000188B1 (en) | 1977-10-27 | 1977-10-27 | Process for the preparation of thymosin |
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- 1977-10-27 KR KR7702475A patent/KR820000188B1/en not_active Expired
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