MC1167A1 - THYMOSIN D 1 - Google Patents

Description

1

It is now generally accepted that the thymus plays a central role in the development and aging of the human and animal immune system. Although the molecular processes by which the thymus controls T cell development are still poorly understood, it appears that a significant portion of this mechanism is hormonally driven. The thymus produces a group of polypeptides called thymosin, and possibly several other hormones and/or thymus factors that play important roles in T cell maturation, differentiation, and function. Thymosin has been found to induce T cell differentiation and enhance immune function in genetically athymic mice, thymectomized adult mice, NZB mice with a strong autoimmune response, tumor mice, and mice with casein-induced amyloidosis.

Thymic fraction 5 is known to be an effective immunostimulatory preparation that can function like the thymus itself in restoring immunological functions in individuals lacking a thymus and/or immune system. Long-term clinical research on thymic fraction 5 appears to support the hypothesis that thymosin increases the number of T cells in both children suffering from primary thymus-dependent immunodeficiency and cancer patients with limited immune defenses.

Analytical methods have shown that thymic fraction 5 consists of 10 to 15 main components and 20 or more secondary components with a molecular weight between 1000 and 15,000.

The present invention relates to an acidic polypeptide present in the thymosin 5' fraction, which has been called thy-mosineoC, and whose amino acid sequence has been elucidated, as well as its isolation and purification from this thymic 5' fraction. ThymosineoC exhibits, in various in vitro and in vivo tests that allow measurement of T cell differentiation and function, an activity 10 to 1000 times greater than that of the thymosin 5' fraction.

2

Therefore, thymosin can be used in the treatment of children suffering from primary thymus-dependent immunodeficiency, as well as in cancer patients with limited immune defenses, at doses significantly lower than those used with the thymosin fraction.

Thymosinec-^^ has a molecular weight of 3108, a pi (isoelectric point) between 4.0 and 4.3 (determined by isoelectric concentration 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 according to the invention of thymosin 5 from the thymosin 5 fraction is carried out by a combination of ion-exchange chromatography and gel filtration as follows: (a) The lyophilized thymosin 5 fraction is chromatographed in a carboxymethylcellulose column in a pH 5.20 buffer composed of 10 mM sodium acetate and 1.0 mM 2-mercaptoethanol, during which the column is first washed with this buffer, then eluted with a linear gradient of this buffer and a mixture of this buffer and a 1.0 M sodium chloride solution. (b) The first protein peak obtained according to (a) is fractionated onto a dextran gel column (Sephadex G-25) using sterile water. (c) The second protein peak obtained according to (b) is placed in a column equilibrated with a 50 mM Tris/1.0 mM 2-mercaptoethanol buffer at pH 8 and composed of 2-diethylaminoethylcellulose (DE-32). It is eluted first with this buffer and then with a linear gradient of 1.3 L of this buffer and 1.3 L of a buffer containing 0.0 M sodium chloride. (d) The fractions containing the first sixth of the protein peak obtained according to (c) are purified and then further purified on a dextran gel column (Sephadex G~75) in a 6.0 M guanidine hydrochloride buffer and 10 mM Tris at pH 7.5. (e) Finally, the average fractions of the protein peak 40 obtained according to (d) are desalted on a dextran gel column (Sephadex 3G-10) using sterile water. The purified protein fraction thus obtained (yield 0.6%) is identified as thymosin O with the amino acid sequence given above. The preparation is an example of carbohydrates and nucleotides. The explanation of the structure is 1b. Determination of the amino acid sequence is carried out according to the usual general methods, i.e., by enzymatic decomposition with trypsin, chymotrypsin, therraolysin, or subtilisin, separation of the fragments by paper electrophoresis and/or chromatography, and Edman decomposition of the isolated peptide fragments. * The following table shows the activity of thymosin multiplied by a factor ranging from 10 to 1000 relative to

to that of the thymic fraction 5 in three bioassays, namely 15 the mitogenic test on mice, the lymphokinetic test (an in vitro test which measures the production of the macrophage migration inhibition factor, MIF) and the E rosette test with human lymphocytes,

t

20 Lymphokinetic Test

Thymosin fraction 5 1-5 Thymosin 0.01-0.1 0.001-0.01 0.01-0.1

25 Thymosin can be administered to humans and mammals parenterally, that is, intravenously, subcutaneously, or intramuscularly. For intravenous administration, the daily dose is usually in the range of 1 to 100 mg/kg of body weight. It is evident that this can vary depending on the case and the nature, severity, and duration of the illness. The unit of dose used in pharmacy practice is 1 mg of lyophilized thymosin, which can be dissolved just before use by adding sterile water or table salt solution.

35 The invention also includes pharmaceutical-useful salts of thyrnosine, that is, salts formed with acids and bases. Examples of such salts include sodium or potassium salts, salts formed with strong organic bases such as guanidine, or salts

Activity (yig)

Mitogenic rosette test E

1-10 1-10

4

of acid additions such as hydrochloride, hydrobromide, sulfate, phosphate, malate, maleate, acetate, citrate, succinate, benzoate and ascorbate.

5

Claims (1)

DEMANDS 1. A process for preparing thymosin from the thymosin 5 fraction, characterized in that: (a) The lyophilized thymosin 5 fraction is chromatographed in a carboxymethylcellulose column in a pH 5 buffer composed of sodium acetate and 2-mercaptoethanol, during which the column is first washed with this buffer and then eluted with a linear gradient of this buffer and a mixture of this buffer and a 1.0 M sodium chloride solution. (b) The first protein peak obtained according to (a) is fractionated onto a dextran gel column (Sephadex G-25) using sterile water. (c) The second protein peak obtained according to (b) is placed on a column equilibrated with a pH 8 2-mercaptoethanol tris buffer and composed of 2-diethylaminoethylcellulose. (DE-;32). First, eluting is performed with this buffer, then with a linear gradient of this buffer and a mixture of this buffer containing 0.8 M sodium chloride. (d) The first sixth of the protein peak obtained according to (c);20 is placed in a column of dextran gel (Sephadex G-75) in guanidine hydrochloride/tris buffer at pH 7.5, and finally; (e) The fractions of the protein peak obtained according to (d) are desalted on a column of dextran gel (Sephadex G-10). 2. A process according to claim 1, characterized in that in step (a) of the process, the buffer used is composed of 10 mM sodium acetate and 1.0 mM 2-mercaptoethanol. 3. A process according to any one of claims 1 and 2, characterized in that the buffer used in step (c) of the process is composed of 50 mM tris hydrochloride and 1.0 mM 2-mercaptoethanol; 4. A process according to any one of claims 1 to 3, characterized in that the buffer used in step (d) of the process is composed of 6.0 M guanidine hydrochloride and 10 mM tris; 5. A process according to any one of claims 1 to 4, 6, characterized in that steps (b) and (e) of the process are carried out with sterile water; 6. A process for manufacturing preparations with therapeutic action, characterized in that thymosin a^, 5, characterized by the 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 its pharmaceutically usable salts, free from other thymus polypeptides, are mixed, as active substances, with non-toxic, inert, and therapeutically compatible solid or liquid carriers commonly used in such preparations, and/or excipients. n„l cl ^;José CURAU;Industrial Property Consultant;26w,s, Princess Charlotte Blvd. MONTE-CARLO;procu ratio*» ^—-v ^ <=r