FI56317C - FOERFARANDE FOER ISOLERING AV TYMOSIN ALFA1 MED IMMUNITETPOTENTIERANDE VERKAN - Google Patents

Description

I- "Ti.. PUBLICATION _ _ _.„ ^ (11) UTLÄGGNI NGSSKRIFT 5 63 1 7 C (4S) Patent granted 10 01 1930 Patent oieddelat

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FINLAND — FINLAND OT Patent application - PatentaneBknlng 77 3221 (22) Hakemlipilvl— AnsSknlngsdag 28.10.77 ^ ^ ^ (23) Primary cloud - Glltlghetsdag 28.10.77 (41) Tullut julklaakal - Bllvlt offantflg 29 .. 78 hunttl · | R. M <Mrih.nitu. NiMMk «p« »n pm-

Patent and Registration Office Anaökan utligd och utl.skrtften publkerad 28.09.79 (32) (33) (31) Fyydstty etuoikeus - Begird prlorltat 28.10.76 08.02.77 USA (us) 736638, 766638 (71) F. Hofflnann-La Roche & Co. Aktiengesellschaft, CH-U002 Basel, Switzerland-Switzerland (CH), The University of Texas System ,. Board of Regents, Austin,

Texas, USA (72) Allan L. Goldstein, Galveston, Texas, Teresa L.K. Low, Galveston, Texas, Chun-Yen Lai, Clifton, NJ, Su-Sun Wang, Bloomfield, NJ, USA (US) (7 ^ +) 0y Roister Ab (5 ^) Method for isolating immune-enhancing thymosin CY · ^ - For the purpose of isolation from the immunological potential network

It is now a well-known fact that the thyroid gland plays a key role in the development and aging of the human and animal immune systems. Although there is still very little information on the molecular events by which the thyroid gland controls T cell development, it appears that hormones regulate an essential part of this mechanism. The thyroid gland produces polypeptides called the thymosin group and possibly a number of other hand hormones and / or thyroid factors that play an important role in T cell development, differentiation and function. It has now been found that thymosin induces T cell differentiation and enhances immunological functions genetically in thymus-deficient mice, thymus-deprived adult mice, NZB mice with strong self-immune responses, tumor-bearing mice, and mice with casein. amyloid disease caused by

56317

From Proc. Nat. Acad. Sci. USA, 725 (1977) and Annals N.Y. Acad.

Sci. 2kSt 125 (1975) it is known that thymic fraction 5 is an effective immune-boosting preparation which can act like restoring the immune function of the thyroid gland itself in individuals without a thyroid gland and / or in individuals without an immune system. Long-term clinical trials with thymus fraction 5 appear to confirm the hypothesis that thymosin increases T cell counts both in children with primary thyroid immune defect and in cancer patients with limited immunosuppression.

It has been shown by analytical methods that the thymus fraction 5 consists of the main component of IO-15 and 20 or more secondary components having a molecular weight of 1000-15,000.

The present invention relates to a process for the preparation of a pure acidic polypeptide present in the thymus fraction 5, called thymosin ditches, the amino acid sequence of which has been determined. Thymosin has 10-1,000-fold activity compared to the thymus fraction in various in vitro and in vivo tests, which are considered a measure of T cell differentiation and function 5 ·

Thymosin 0 (^) has a molecular weight of 3108, its pI is in the range of ^ 0-1 *, 3 (isoelectric focusing in the gel in the pH range 3-5) and its amino acid sequence is as follows: (N-Acetyl) -Ser-Asp Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp

Leu-Glu-Lys-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-OH.

Example

Isolation and preparation of thymosin βί β in pure form according to the invention from thymosin fraction 5 is performed by a combination of ion exchange chromatography and gel filtration as follows: (a) Lyophilized thymus fraction 5 is chromatographed on a column consisting of carboxymethylcellulose mercaptoethanol, having a pH of 5.0, the column being washed first with this buffer and then eluted with a linear gradient consisting of this buffer and a mixture of this buffer and 1.0 M sodium chloride solution.

(b) The first protein peak obtained according to (a) is fractionated on a dextran-gel column (Sephadex-G-25, manufactured by Pharmacial Fine Chemicals AB, Uppsala), using sterile water.

(e) The second protein peak obtained according to (b) is chromatographed on a column consisting of 2-diethylaminoethylcellulose (DE-32, sold by H. Reeve Angel & Co. Ltd., London) in 50 mM Tris-HCl / l. 0 mM mercaptoethanol buffer, pH 8.0, eluting first with this buffer and then with a linear gradient consisting of 1.3 liters of this buffer and 1.3 liters of this buffer, which contains 0.8 M sodium chloride.

3,56317 (d) The fractions containing the protein peak obtained according to the first sixth paragraph (c) are combined and further purified on a dextran gel column (Sephadex G-75 »manufactured by Pharmacia Fine Chemicals AB, Uppsala) in a buffer consisting of 6.0 M: guanidine hydrochloride and 10 mM tris having a pH of 7.5 · (e) Finally, desalting of the middle fractions of the protein peak obtained in (d) is carried out on a dextran gel column (Sephadex G-10, manufactured by Pharmacia Fine Chemicals AB, Uppsala). using sterile water.

The purified protein fraction thus obtained (yield 0.6%) has been identified as a thymosin having the above amino acid sequence.

The following table shows the 10-1000 higher biological activity of thymosin o (^) compared to the biological activity of thymus fraction 5 in three known animal experiments, namely the mitogen test in mice, the lymphokine test (in vitro test for the inhibition of macrophage migration factor, MIF production) and in the E-rosette (collection) test performed on human lymphocytes (lymphocytes) (see, e.g., Ann. NY Acad. Sci. 2 ^ 9, 125 Z597§7) -

Activity fyig)

Lymphokine test E-rosette test Mitogen test Thymosin fraction 1-5 1-10 1-10

Thymosin ^ 0.01-0.1 0.001-0.01 0.01-0.1

Thymosin can be administered to humans and mammals parenterally, i. intravenously (intravenously), subcutaneously (subcutaneously) or intramuscularly (intramuscularly). For intravenous administration, the daily dose is usually in the range of 1 to 100 pg / kg body weight.

Claims (2)

A method for chemically pure isolation of an immune-enhancing thymosin of the amino acid sequence of (N-Acetyl) -Ser-Asp-Ala-Ala-Val-Asp-Thr-Ber-Ser-Glu-Ile-Thr -Thr-Lys -Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-OH from thymus fraction 5, characterized in that (a) the lyophilized thymus ear fraction 5 is chromatographed on a column consisting of carboxymethyl cellulose in a sodium acetate / 2-mercaptoethanol buffer having a pH of 5 → 0, the column being washed first with this buffer and then eluted with a linear gradient consisting of this buffer and a mixture of this buffer and 1.0 M sodium chloride solution, (b ) the first protein peak obtained according to (a) is fractionated on a dextran gel column, (c) the second protein peak obtained according to (b) is chromatographed on a column consisting of 2-diethylaminoethylcellulose in tris / 2-mercaptoethanol buffer at pH 8.0, eluting first with this buffer and then with a linear gradient consisting of this buffer and a mixture of this buffer and 0.8 M sodium chloride solution, (d) the first sixth of the protein peak obtained according to (c) is passed through a dextran gel column to a guanidine hydrochloride / Tris buffer having a pH of 7, 5 and finally (e), the middle fractions of the protein peak obtained according to (d) are desalted on a dextran gel column. Process according to Claim 1, characterized in that the buffer used in step (a) consists of 10 mM sodium acetate and 1.0 mM 2-mercaptoethanol, the buffer used in step (c) consists of 50 mM tris-hydrochloride and 1.0 mM mM 2-mercaptoethanol, the buffer used in step (d) consists of 6.0 M guanidine hydrochloride and 10 mM Tris, and steps (b) and (e) are performed using sterile water.