CH633258A5 - Process for preparing thymosin alpha 1 - Google Patents

Description

The present invention relates to an acid polypeptide occurring in the thymosin fraction 5, which was called thymosin cu and whose amino acid sequence could be elucidated, and to its isolation and purification from this thymus fraction 5. In various in vitro and in vivo tests, thymosin ai has which are a measure of T cell differentiation and function compared to the thymosin fraction 5 which is 10-1000 times the activity.

Thymosin cu has the molecular weight 3108, a pI in the range 4.0-4.3 (determined by isoelectric focusing in a gel at pH 3-5) and 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-Glu-Val- Val-Glu-Glu-Ala-Glu-Asn-OH.

The isolation and purification of thymosin ai from thymosin fraction 5 according to the invention was carried out by a combination of ion exchange chromatography and gel filtration in the following manner:

(a) The lyophilized thymosin fraction 5 was extracted from a column of carboxymethyl cellulose in one. Chromatograph 10 mM sodium acetate and 1.0 mM 2-mercaptoethanol buffer pH 5.0, the column being washed first with this buffer and then with a linear gradient from this buffer and a mixture of this buffer and a 1.0 M sodium chloride solution was eluted.

(b) The first protein peak obtained in (a) was fractionated on a dextran gel column (Sephadex G-25) using sterile water.

(c) The second protein peak obtained according to (b) was placed on a column of 2-diethylaminoethyl cellulose (DE-32) equilibrated with 50 mM Tris / 1.0 mM 2-mercaptoethanol buffer of pH 8.0. It was eluted first with this buffer and then with a linear gradient from 1.31 of this buffer and 1.31 of this buffer containing 0.8 M sodium chloride.

(d) Those fractions which contained the first sixth of the protein peak obtained in (c) were combined and further on a dextran gel column (Sephadex G-75) in a buffer of 6.0 M guanidine hydrochloride and 10 mM Tris, pH 7 , 5 cleaned.

(e) Finally, the middle fractions of the protein peak obtained in (d) were desalted on a dextran gel column (Sephadex G-10) using sterile water.

The purified protein fraction obtained in this way (yield 0.6%) was thymosin ai with the amis indicated above

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nos acid sequence identified. The preparation was free of carbohydrates and nucleotides. The structure elucidation and determination of the amino acid sequence was carried out according to the generally customary methods, i.e. by enzymatic degradation with trypsin, chymotrypsin, thermolysin or subti-lisin, separation of the fragments by paper electrophoresis and / or chromatography and Edman degradation of the individual peptide fragments.

3 633258

The following table shows the biological activity of thymosin ai greater by a factor of 10-1000 compared to that of thymus fraction 5 in three known bioassays, namely the mitogen test in mice, the lymphokine 5 test (an in vitro test with which measures the production of the macrophage migration inhibition factor (MIF)) and the E-rosette test with human lymphocytes.

Activity ((ig)

Lymphokine E-Rosette Mitogen Test Test Test

Thymosin fraction 5 1-5 1-10 1-10

Thymosin ai 0.01-0.1 0.001-0.01 0.01-0.1

20 solution can be brought into solution.

Thymosin ai can be administered parenterally to humans and mammals, intravenously, subcutaneously or intramuscularly. With intravenous administration, the daily dose is usually in the range of 1-100 (ig / kg body weight. It goes without saying that it varies from case to case and depends on the nature of the disease, its strength and duration. A suitable pharmaceutical dosage unit is 1 mg lyophilized thymosin ai, which is added shortly before use by adding sterile water or saline

The invention also includes the physiologically acceptable salts of thymosin ai, i.e. Salts with acids and bases. Examples of such salts are the sodium or 2s potassium salt, salts with strong organic bases such as guanidine or acid addition salts such as hydrochloride, hydrobromide, sulfate, phosphate, malate, maleate, acetate, citrate, sucinate, benzoate and ascorbate.

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Claims (5)

633258 1. A process for the preparation of thymosin cu from the thymosin fraction 5, characterized in that (a) Chromatograph the lyophilized thymosin fraction 5 on a carboxymethyl cellulose column in a pH 5.0 sodium acetate / 2-mercaptoethanol buffer, washing the column first with this buffer and then with a linear gradient from this buffer and a Mixture eluted from this buffer and 1.0 M sodium chloride solution, (b) fractionating the first protein peak obtained according to (a) on a dextran gel column, (c) 'the second protein peak obtained according to (b) is chromatographed on a column of 2-diethylaminoethyl cellulose in Tris / 2-mercaptoethanol buffer of pH 8.0, first using this buffer and then using a linear gradient therefrom Buffer and a mixture of this buffer and 0.8 M sodium chloride solution, (d) passing the first sixth of the protein peak obtained according to (c) over a column of dextran gel in a guanidine hydrochloride / Tris buffer of pH 7.5, and finally (e) the middle fractions of the protein peak obtained in (d) are desalted on a dextran gel column. 2. The method according to claim 1, characterized in that the buffer used in process step (a) consists of 10 mM sodium acetate and 1.0 mM 2-mercaptoethanol. 2nd PATENT CLAIMS 3. The method according to claim 1 or 2, characterized in that the buffer used in process step (c) consists of 50 mM Tris hydrochloride and 1.0 mM 2-mercaptoethanol. 4. The method according to any one of claims 1-3, characterized in that the buffer used in process step (d) consists of 6.0 M guanidine hydrochloride and 10 mM Tris. 5. The method according to any one of claims 1-4, characterized in that process steps (b) and (e) are carried out with sterile water. It is now a generally accepted fact that the thymus is of central importance in the development and aging of the human and animal immune system. Although there is still very little knowledge of the molecular processes by which the thymus controls the development of T cells, it appears that a substantial part of this mechanism is' hormonally controlled. The thymus gland produces a group of polypeptides called thymosin, and perhaps a number of other thymus hormones and / or thymus factors that play an important role in T cell maturation, differentiation and function. It was found that thymosin induces T cell differentiation and enhances immunological functions in genetically athymic mice, adult thymectomized mice, NZB mice with strong autoimmune reactions, in tumor-containing mice and in mice with casein-induced amyloidosis. It is known that the thymus fraction 5 is an effective immunopotentiating preparation which is used in the Provision of immune functions in individuals without a thymus gland and / or without an immune system as the thymus gland itself can act. Ongoing clinical trials with the thymus fraction 5 seem to support the assumption that the thymosin increases the number of T cells both in children suffering from a thymus-dependent primary immunodeficiency and in cancer patients with impaired immune defense. With analytical methods it could be shown that the thymus fraction 5 consists of 10-15 main and 20 or more secondary components with a molecular weight between 1000 and 15,000.