Note: Descriptions are shown in the official language in which they were submitted.
842
RAN 4300/9
T~YMO -1
;~ The importance of the thymus gland in the development
and senescence of immunological competence in animals and man is
now generally accepted. Although there is little knowledge of the
molecular events by which the thymus gland exerts control ovex
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 hoxmones 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 immuno-
potentiating preparation and can act in lieu of the thymus gland
to xeconstitute immune functions in thymic deprived and/or
immunodeprived individuals. Ongoing clinical trials with fraction
5 suggest that thymosin is effecti~e in increasing T cell num-
bers and normalizing immune function in children with thymic
dependent primary immunodeficiency diseases and can increase T
cell numbers in immunodepressed cancer patients.
Mez/5.9.1977 ~
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Analytical polyacrylamide yel electrophoresis and iso-
electric focusing have demonstrated that fraction 5 consists of
10-15 m~jor components and 20 or more minor components with
molecular weights ranging form 1,000 to 15,000.
The present invention rela~es to the isolation and
first complete structural determination of an acidic polypeptide
isolated form thymosin fraction 5. Thi~; peptide has been termed :
thymosin 1 Thymosin ~1 has been ound to be 10 to 1,000 times
more active than fraction S in several in vltro and in vivo
assay systems designed to measure T cell differentiation and :~
function.
Thus the present invention provides a method of pre~
paring thymosin ~1 from thymosin fraction 5. The inventive
method comprises the :Eollowing steps in combination: - ;~
(a) chromatographing lyophilized thymosin fraction 5 on a ~:
column of carboxymsthyl-cellulose in a sodium acetatej2-mer-
captoethanol buffer of p~l 5.0, washing the column 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 step (a) on a .
column of a dextran gel (Sephadex G-25); :
(c) Chromatographing the second protein peak from step (b)
on a 2-diethylaminoethyl-cellulose column equilibrated with
tris/2-mercaptoethanol buffer of pH 8.0, eluating with said
buffer alone followed by a l.inear gradient of said buffer and :~
said buffer plus 0.8 M NaCl;
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(d) passing the first sixth of the eluted protein peak from
step (c) through a column o a dextran gel (Sephadex G-7S) in
quanidine-hydrochlor.ide/tris bufer of pH 7.5 and
(e) taking a single narrow cut from the protein peak of step
(d) and desalting on a column of a dextran gel (Sephadex G-10).
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 o~ 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-Glu-Val-Val-Glu-Glu-
Ala-Glu-Asn-OH.
Thymosin al was isolated from fraction 5 by a combination ~.
of ion-exchange chromatography and gel filtration in the follo~
wing way:
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Lyophylized thymosin fraction 5 was chromatographed on ~ .
.~ a column of carboxymethyl-cellulose in 10 mM sodium acetate
buffer, pH 5.0, containing 1.0 mM 2 mercaptoethanol. The column
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was washed first with the buffer followed by a linear gradient
of 2 liters each of starting buffer ancl the same buffer con-
taining 1.0 M NaCl. The first protein i-raction was filtered
on a oolumn of a dextran gel ~Sephadex G-25) in sterile water.
The second protein peak from the dextran gel colu~n was applied
on a 2-diethylaminoethyl-cellulose col~ (DE-3~) equilibrated
with 50 mM tris-buffer containing 1.0 mM 2-mercaptoethanol
~pH 800). The colu~n 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 calumn 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 na~row cut was made ~rom the protein
lS peak and desalted on a column of a dextran gel (Sephadex G-10)
in sterile water. The purified sample so obtained was identifled
as thymosin al The yield of thymosin al from fraction 5 was
about 0.6%. The preparation was free of carbohydrate and
nucleotide. ~`
The structure and complete amino acid sequence of thymosin
al was determined using conventional methods such as enzymatic
digestion by trypsin, chymotrypsin, thermolysin or subtilisin,
separation of the digests by paper electrophoresis and/or chroma-
tography and Edman degradation of the separated peptide fragments.
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The following table demonstrates that thlymosin al is
from 10 to 1,000 times more active than thymosin fraction 5 in an
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in vivo mouse mitogen assay, an in vitro ]ymphokine assay
measuring MIF production, and an in vitro human E-rosette assay.
Thymosin Activity (~g) in Various Bioassays
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MI~ E-~osette Mitogen*
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Thymosin Fraction 5 1-5 1-10 1-10
Thymosin 1 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 about 1 to 100 ~g/kg
of body weight per day for intravenous administration. Obviously -
~the required dosage will vary with the particular condition
being treated, the severity o~ the condition and the duration
of the treatment. A suitable dosage form for pharmaceutical use
is 1 mg. of lyophilized thymosin cl per vial to be reconstituted
prior to use by the addition of sterile water or saline.
Also included within the scope of the present inventlon
are the pharmaceutically acceptable acid addition and base
salts of thymosin a1 such as the sodium or potasslum salt or salts
with strong organic bases such as guanidine. Examples o~ acid
addition salts are the hydrochloride, hydrobromide, sulfate,
phosphate, maleate, acetate, citrate, benzoate,isuccinate,
malate and ascorbate.
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