GB1590457A - Thymosin a1 - Google Patents
Thymosin a1 Download PDFInfo
- Publication number
- GB1590457A GB1590457A GB45001/77A GB4500177A GB1590457A GB 1590457 A GB1590457 A GB 1590457A GB 45001/77 A GB45001/77 A GB 45001/77A GB 4500177 A GB4500177 A GB 4500177A GB 1590457 A GB1590457 A GB 1590457A
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- GB
- United Kingdom
- Prior art keywords
- glu
- buffer
- thymosin
- lys
- column
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/57581—Thymosin; Related peptides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biophysics (AREA)
- Medicinal Chemistry (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Toxicology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Gastroenterology & Hepatology (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Endocrinology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
Process for preparing thymosin alpha 1 from thymosin fraction 5 by chromatography on dextran gels as well as on carboxymethyl cellulose and 2-diethylaminoethyl cellulose using gradient elution. Thymosin alpha 1 possesses immunostimulatory properties.
Description
(54) THYMOSIN al
(71) We, F. HOFFMANN-LA ROCHE & CO. AKTIENGESELLSCHAFT a Swiss
Company of 124-184, Grenzacherstrasse, Basle, Switzerland and BOARD OF REGENTS
OF THE UNIVERSITY OF TEXAS SYSTEM, Austin, Texas, The United States of
America, do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement:
The importance of the thymus gland in the development and senescence of immunological competence in animals, including man is now generally accepted. Although there is little knowledge of the molecular events by which the thymus gland exerts control over T cell development, it appears that a vital part of the process occurs via a hormonal mechanism.
The thymus gland produces a family of polypeptides termed thymosin and perhaps several other thymic hormones and/or factors which play an important role in the maturation, differentiation and function of T cells. Thymosin has been found to induce T cell differentiation and to enhance immunological functions in genetically athymic mice, in adult thymectomized mice, in NZB mice with severe autoimmune reactions, in tumor bearing mice and in mice with casein-induced amyloidosis.
It is known that thymosin fraction 5 is a potent immunopotentiating preparation and can act in lieu of the thymus gland to reconstitute immune functions in thymic deprived and/or immunodeprived individuals; Thymosin fraction 5 is described in the article by Mesper et al,
Annals New York Acad. Sci. 249, 125 (1975). Ongoing clinical trials with fraction 5 suggest that thymosin is effective in increasing T cell numbers and normalizing immune function in children with thymic dependent primary immunodeficiency diseases and can increase T cell numbers in immunodepressed cancer patients.
Analytical polyacrylamide gel electrophoresis and iso-electric focusing have demonstrated that fraction 5 consists of 10-15 major components and 20 or more minor components with molecular weights ranging from 1,000 to 15,000.
The present invention relates to the isolation and first complete structural determination of an acidic polypeptide isolated from thymosin fraction 5. This peptide has been termed thymosin al. Thymosin al has been found to be 10 to 1,000 times more active than fraction 5 in several in vitro and in vivo assay systems designed to measure T cell differentiation and function.
Thymosin al has a molecular weight of 3,108 and a pI in the range of 4.0-4.3 as determined by gel isoelectric focusing at a pH range of 3-5. The compound has the following amino acid sequence: (N-acetyl)-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-
Lys-Glu-Lys-Lys-Ulu-Val-Val-Glu-Olu-Ala-Glu-Asn-OH.
According to one aspect of this invention we provide a method of preparing thymosin a having the amino acid sequence: (N-acetyl)-Ser-Asp-Ala-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; from thymosin fraction 5, said method comprising
(a) chromatographing lyophilized thymosin fraction 5 on a column of carboxymethylcellulose in a sodium acetate/2-mercaptoethanol buffer of pH 5.0, washing the column, after the fraction 5 has been introduced, first with this buffer and then eluting with a linear gradient of said buffer and said buffer plus 1.0 M NaCl;
(b) fractionating the first protein peak of (a) on a column of a dextran gel (diameter of dry particle = 10 to 300 Fm; volume of dry gel = 4 to 6 ml/g; water regain value = 2.5 ml/g dry gel);
(c) chromatographing the second protein peak from (b) on a 2-diethylaminoethylcellulose column equilibrated with tris/2-mercaptoethanol buffer of pH 8.0, eluting with said buffer alone followed by a linear gradient of said buffer and said buffer plus 0.8 M
NaCl;
(d) passing the first sixth of the eluted protein peak from (c) through a column of a dextran gel (diameter of dry particle = 40 to 120 llm; volume of dry gel = 12 to 15 ml/g; water regain value = 7.5 ml/g dry gel) in guanidine-hydrochloride/tris buffer of pH 7.5 and
(e) taking a single narrow cut from the protein peak of (d) and desalting on a column of a dextran gel (diameter of dry particle = 40 to 120 Fm; volume of dry gel = 2 to 3 ml/g; water regain value = 1 ml/g dry gel).
According to a further aspect of this invention we provide thymosin al having the amino acid sequence
(N-acetyl)-Ser-Asp-Ala-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 and pharmaceutically acceptable acid addition salts and base salts thereof, essentially free of other thymic polypeptides.
Thymosin al was isolated from fraction 5 by a combination of ion-exchange chromatography and gel filtration in the following way. It will be appreciated that the following experimental conditions could be varied by a skilled peptide chemist within the scope of the invention as defined in the appended claims.
Lyophilized thymosin fraction 5 was chromatographed on a column of carboxymethylcellulose in 10 mM sodium acetate buffer, pH 5.0, containing 1.0 mM 2-mercaptoethanol.
After introduction of the sample of the column, the column was washed first with the buffer followed by a linear gradient of 2 liters each of starting buffer and the same buffer containing 1.0 M Nail. The first protein fraction was filtered on a column of a dextran gel (Sephadex G-25) using sterile water. The second protein peak from the dextran gel column was applied on a 2-diethylaminoethyl-cellulose column (DE-32) equilibrated with 50 mM tris-HCl buffer containing 1.0 mM 2-mercapthoethanol (pH 8.0). The column was eluted with the starting buffer followed by a gradient of 1.3 liters each of starting buffer and the same buffer containing 0.8 M NaCl. The first sixth of the protein peak from the DE-32 column was further purified by being passed through a column of a dextran gel (Sephadex G-75) in a buffer containing 6.0 M guanidine hydrochloride and 10 mM tris (pH 7.5). A single narrow cut was made from the protein peak and desalted on a column of a dextran gel (Sephadex G-10) using sterile water. The purified sample so obtained was identified as thymosin al. The yield of thymosin a1 from fraction 5 was about 0.6%. The preparation was free of carbohydrate and nucleotide. "Sephadex" is a registered Trade Mark.
The structure and complete amino acid sequence of thymosin a1 was determined using conventional methods such as enzymatic digestion by trypsin, chymotrypsin, thermolysin or subtilisin, separation of the digests by paper electrophoresis and/or chromatography and
Edman degradation of the separated peptide fragments.
The following table demonstrates that thymosin a1 is from 10 to 1,000 times more active than thymosin fraction 5 in an in vivo mouse mitogen assay, an in vitro lymphokine assay measuring MIF production, and an in vitro human E-rosette assay.
Thymosin Activity (lug) in Various Bioassays
MIF E-Rosette Mitogen*
Thymosin Fraction 5 1-5 1-10 1-10
Thymosin a1 0.01-0.1 0.001-0.01 0.01-0.1 * In vivo 14 daily injections
Thymosin al may be administered to warm blooded mammals by parenteral application either intravenously, subcutaneously or intramuscularly. The compound is a potent immunopotentiating agent with a daily dosage in the range of 1 to 100 llg/kg of body weight per day for intravenous administration. Obviously the required dosage will vary with the particular condition being treated, the severity of the condition and the duration of the treatment. A suitable dosage form for pharmaceutical use is 1 mg. of lyophilized thymosin al per vial to be reconstituted prior to use by the addition of sterile water or saline.
The invention therefore also includes a method for reconstituting immune functions in thymic deprived or immuno deprived warm blooded non-human mammals which method comprises administering to said mammals an immunopotentiating effective amount of thymosin al.
Also included within the scope of the present invention are the pharmaceutically acceptable acid addition salts and base salts of thymosin al such as the sodium or potassium salt or salts with strong organic bases such as guanidine. Examples of acid addition salts are the hydrochloride, hydrobromide, sulfate, phosphate, maleate, acetate, citrate, benzoate, succinate, malate and ascorbate.
WHAT WE CLAIM IS:
1. A method of preparing thymosin a1 having the amino acid sequence (N-acetyl)-Ser
Asp-Ala-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 from thymosin fraction 5, said method comprising:
(a) chromatographing lyophilized thymosin fraction 5 on a column of carboxymethyl cellulose in a sodium acetate/2-mercaptoethanol buffer of pH 5.0, washing the column, after the fraction 5 has been introduced, first with this buffer and then eluting with a linear gradient of said buffer and said buffer plus 1.0 M NaCl;
(b) fractionating the first protein peak of (a) on a column of a dextran gel (diameter of particle = 100 to 300 ijm; volume of dry gel = 4 to 6 ml/g; water regain value = 2.5 ml/g dry gel)
chromatographing the second protein peak from (b) on a 2-diethylaminoethyl- cellulose column equilibrated with tris/2-mercaptoethanol buffer of pH 8.0, eluting with said buffer alone followed by a linear gradient of said buffer and said buffer plus 0.8 M
NaCl;
(d) passing the first sixth of the eluted protein peak from (c) through a column of a dextran gel (diameter of dry particle = 40 to 120 llm; volume of dry gel = 12 to 15 ml/g; water regain value = 7.5 ml/g dry gel) in guanidine hydrochloride/tris buffer of pH 7.5 and
(e) taking a single narrow cut from the protein peak of (d) and desalting on a column of a dextran gel (diameter of dry particle = 40 to 120 llm; volume of dry gel = 2 to 3 ml/g; water regain value = 1 ml/g dry gel).
2. The method of claim 1 wherein the buffer in (a) is 10 mM sodium acetate and 1.0 mM 2-mercaptoethanol.
3. The method of claim 1 or 2 wherein the buffer in (c) is 50 mM tris-HCl and 1.0 mM 2-mercaptoethanol.
4. The method of claim 1, 2 or 3 wherein the buffer in (d) is 6.0 M guanidine hydrochloride and 10 mM tris.
5. The method of any one of claims 1 to 4 wherein (b) and (e) are carried out using sterile water.
6. A method of preparing thymosin a1 having the amino acid sequence defined in claim 1 as hereinbefore described.
7. A method for reconstituting immune functions in thymic deprived or immunodeprived warm blooded non-human mammals which method comprises administering to said mammals an immunopotentiating effective amount of thymosin al having the amino acid sequence defined in claim 1.
8. The method of claim 7 wherein a daily dosage in the range of from 1 to 100 llg/kg of body weight per day is administered intravenously.
9. Thymosin a1 whenever prepared according to a method claimed in any one of claims 1 to 6.
10. Thymosin a1 having the amino acid sequence
(N-acetyl)-Ser-Asp-Ala-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 and pharmaceutically acceptable acid addition salts and base salts thereof, essentially free of other thymic polypeptides.
**WARNING** end of DESC field may overlap start of CLMS **.
Claims (10)
1. A method of preparing thymosin a1 having the amino acid sequence (N-acetyl)-Ser
Asp-Ala-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 from thymosin fraction 5, said method comprising:
(a) chromatographing lyophilized thymosin fraction 5 on a column of carboxymethyl cellulose in a sodium acetate/2-mercaptoethanol buffer of pH 5.0, washing the column, after the fraction 5 has been introduced, first with this buffer and then eluting with a linear gradient of said buffer and said buffer plus 1.0 M NaCl;
(b) fractionating the first protein peak of (a) on a column of a dextran gel (diameter of particle = 100 to 300 ijm; volume of dry gel = 4 to 6 ml/g; water regain value = 2.5 ml/g dry gel)
chromatographing the second protein peak from (b) on a 2-diethylaminoethyl- cellulose column equilibrated with tris/2-mercaptoethanol buffer of pH 8.0, eluting with said buffer alone followed by a linear gradient of said buffer and said buffer plus 0.8 M
NaCl;
(d) passing the first sixth of the eluted protein peak from (c) through a column of a dextran gel (diameter of dry particle = 40 to 120 llm; volume of dry gel = 12 to 15 ml/g; water regain value = 7.5 ml/g dry gel) in guanidine hydrochloride/tris buffer of pH 7.5 and
(e) taking a single narrow cut from the protein peak of (d) and desalting on a column of a dextran gel (diameter of dry particle = 40 to 120 llm; volume of dry gel = 2 to 3 ml/g; water regain value = 1 ml/g dry gel).
2. The method of claim 1 wherein the buffer in (a) is 10 mM sodium acetate and 1.0 mM 2-mercaptoethanol.
3. The method of claim 1 or 2 wherein the buffer in (c) is 50 mM tris-HCl and 1.0 mM 2-mercaptoethanol.
4. The method of claim 1, 2 or 3 wherein the buffer in (d) is 6.0 M guanidine hydrochloride and 10 mM tris.
5. The method of any one of claims 1 to 4 wherein (b) and (e) are carried out using sterile water.
6. A method of preparing thymosin a1 having the amino acid sequence defined in claim 1 as hereinbefore described.
7. A method for reconstituting immune functions in thymic deprived or immunodeprived warm blooded non-human mammals which method comprises administering to said mammals an immunopotentiating effective amount of thymosin al having the amino acid sequence defined in claim 1.
8. The method of claim 7 wherein a daily dosage in the range of from 1 to 100 llg/kg of body weight per day is administered intravenously.
9. Thymosin a1 whenever prepared according to a method claimed in any one of claims 1 to 6.
10. Thymosin a1 having the amino acid sequence
(N-acetyl)-Ser-Asp-Ala-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 and pharmaceutically acceptable acid addition salts and base salts thereof, essentially free of other thymic polypeptides.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US73663876A | 1976-10-28 | 1976-10-28 | |
US05/766,638 US4079127A (en) | 1976-10-28 | 1977-02-08 | Thymosin alpha 1 |
Publications (1)
Publication Number | Publication Date |
---|---|
GB1590457A true GB1590457A (en) | 1981-06-03 |
Family
ID=27113070
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
GB45001/77A Expired GB1590457A (en) | 1976-10-28 | 1977-10-28 | Thymosin a1 |
Country Status (22)
Country | Link |
---|---|
JP (1) | JPS5411220A (en) |
AR (1) | AR214903A1 (en) |
AT (1) | AT362493B (en) |
AU (1) | AU514996B2 (en) |
CA (1) | CA1101842A (en) |
CH (1) | CH633258A5 (en) |
DE (1) | DE2748213A1 (en) |
DK (1) | DK149094C (en) |
ES (1) | ES463588A1 (en) |
FI (1) | FI56317C (en) |
FR (1) | FR2369248A1 (en) |
GB (1) | GB1590457A (en) |
IL (1) | IL53218A (en) |
IT (1) | IT1195254B (en) |
LU (1) | LU78395A1 (en) |
MC (1) | MC1167A1 (en) |
NL (1) | NL188699C (en) |
NO (1) | NO143346C (en) |
NZ (1) | NZ185519A (en) |
PT (1) | PT67204B (en) |
SE (1) | SE442479B (en) |
YU (1) | YU40314B (en) |
Families Citing this family (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE2919592A1 (en) * | 1979-05-15 | 1981-01-15 | Max Planck Gesellschaft | METHOD FOR PRODUCING THYMOSINE ALPHA 1 AND DERIVATIVES THEREOF |
US4339427A (en) * | 1980-04-14 | 1982-07-13 | Hoffmann-La Roche Inc. | Radioimmunoassay of thymosinα |
FR2492663B1 (en) * | 1980-10-24 | 1985-11-08 | Vtoroi Mo G | MEDICINAL PRODUCT REGULATING THE IMMUNITY T-SYSTEM AND ITS PREPARATION METHOD |
DE3137231A1 (en) * | 1981-09-18 | 1983-04-14 | Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V., 3400 Göttingen | BIS-THYMOSINE (ALPHA) (DOWN ARROW) 1 (DOWN ARROW) CONNECTIONS |
JPH01250018A (en) * | 1988-03-30 | 1989-10-05 | Noble Sangyo Kk | Displacement detector electronic measuring instrument |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB1195980A (en) * | 1966-08-24 | 1970-06-24 | Univ Yeshiva | Hormone-Like Preparations Derived from Thymus Gland and Methods of Producing the Same. |
-
1977
- 1977-10-05 CH CH1216177A patent/CH633258A5/en not_active IP Right Cessation
- 1977-10-14 AU AU29699/77A patent/AU514996B2/en not_active Expired
- 1977-10-25 IL IL53218A patent/IL53218A/en unknown
- 1977-10-26 NZ NZ185519A patent/NZ185519A/en unknown
- 1977-10-26 CA CA289,510A patent/CA1101842A/en not_active Expired
- 1977-10-26 JP JP12766677A patent/JPS5411220A/en active Granted
- 1977-10-26 SE SE7712071A patent/SE442479B/en not_active IP Right Cessation
- 1977-10-26 MC MC771268A patent/MC1167A1/en unknown
- 1977-10-26 IT IT29020/77A patent/IT1195254B/en active
- 1977-10-26 FR FR7732259A patent/FR2369248A1/en active Granted
- 1977-10-27 NO NO773681A patent/NO143346C/en unknown
- 1977-10-27 AR AR269752A patent/AR214903A1/en active
- 1977-10-27 NL NLAANVRAGE7711814,A patent/NL188699C/en not_active IP Right Cessation
- 1977-10-27 ES ES463588A patent/ES463588A1/en not_active Expired
- 1977-10-27 DK DK478277A patent/DK149094C/en not_active IP Right Cessation
- 1977-10-27 LU LU7778395A patent/LU78395A1/xx unknown
- 1977-10-27 DE DE19772748213 patent/DE2748213A1/en active Granted
- 1977-10-27 AT AT765877A patent/AT362493B/en not_active IP Right Cessation
- 1977-10-27 YU YU2578/77A patent/YU40314B/en unknown
- 1977-10-27 PT PT67204A patent/PT67204B/en unknown
- 1977-10-28 GB GB45001/77A patent/GB1590457A/en not_active Expired
- 1977-10-28 FI FI773221A patent/FI56317C/en not_active IP Right Cessation
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Legal Events
Date | Code | Title | Description |
---|---|---|---|
PS | Patent sealed [section 19, patents act 1949] | ||
704A | Declaration that licence is not available as of right for an excepted use (par. 4a/1977) | ||
PE20 | Patent expired after termination of 20 years |
Effective date: 19971027 |