U.S. patent application number 09/766686 was filed with the patent office on 2001-12-27 for synthetic, statistic thymic peptide combination and its use as a preparation with immunological and/or endocrinological effect.
Invention is credited to Klett-Loch, Lore Maria.
Application Number | 20010056061 09/766686 |
Document ID | / |
Family ID | 23683288 |
Filed Date | 2001-12-27 |
United States Patent
Application |
20010056061 |
Kind Code |
A1 |
Klett-Loch, Lore Maria |
December 27, 2001 |
Synthetic, statistic thymic peptide combination and its use as a
preparation with immunological and/or endocrinological effect
Abstract
Immunologically and/or endocrinologically active preparation
containing as an active ingredient short-chain peptides with a
weighted quantity ratio and a pattern of amino acids characteristic
of thymus tissue.
Inventors: |
Klett-Loch, Lore Maria;
(Mannheim, DE) |
Correspondence
Address: |
ALSTON & BIRD LLP
BANK OF AMERICA PLAZA
101 SOUTH TRYON STREET, SUITE 4000
CHARLOTTE
NC
28280-4000
US
|
Family ID: |
23683288 |
Appl. No.: |
09/766686 |
Filed: |
January 22, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09766686 |
Jan 22, 2001 |
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09424625 |
Nov 24, 1999 |
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09424625 |
Nov 24, 1999 |
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PCT/DE97/01061 |
May 26, 1997 |
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Current U.S.
Class: |
514/9.7 |
Current CPC
Class: |
A61P 37/04 20180101;
Y10S 930/18 20130101; A61K 35/26 20130101; A61K 38/00 20130101 |
Class at
Publication: |
514/2 |
International
Class: |
A61K 038/22 |
Claims
That which is claimed:
1. Preparation containing synthetic thymic peptide combinations
without the risk of BSE, available by lining up in steps
individual, chemically suitably derivatized amino acids to
short-chain peptides, and releasing these synthetic peptides from
their N-terminal group and form derivatized on side functions,
while maintaining the weighted quantity ratio and pattern of amino
acids characteristic of thymus tissue.
2. Method of producing an immunologically and/or endocrinologically
active preparation containing as an active ingredient short-chain
peptides with a weighted quantity ratio and pattern of amino acids
characteristic of thymus tissue, characterized by lining up in
steps individual, chemically suitably derivatized amino acids to
short-chain peptides, and releasing these synthetic peptides from
their N-terminal group and form derivatized on side functions,
while maintaining the weighted quantity ratio and pattern of amino
acids characteristic of thymus tissue.
3. Use of a preparation of claim 1 for producing a substance with
immunologically and/or endocrinologically active properties.
4. Use of claim 3 in the case of weak immunity and
immunodeficiencies.
5. Use of claim 3 in the case of hormonal disorders.
6. Use of claim 3 as a nutrient for skin, hair, nails.
7. Use of claim 3 in the field of cosmetics.
8. Use of claim 3 in the field of food supplements.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 09/424,625, filed Nov. 24, 1999 which is a U.S. National Phase
application of PCT/DE97/01061, filed May 26, 1997. The contents of
the above applications are incorporated herein by reference in
their entirety.
FIELD OF THE INVENTION
[0002] The invention relates to a synthetic, statistic thymic
peptide combination and its use as an immunologically and
endocrinologically active preparation.
BACKGROUND OF THE INVENTION
[0003] From an increasing number of scientific publications, it
becomes obvious that a communication exists by means of
low-molecular signaling substances between the brain, the organs of
the immune system (bone marrow, thymus, lymph nodes, spleen,
intestinal mucosa, pulmonary epithelium, and body skin) and the
endocrine system of the hormonal control. From the thymic tissue,
low-molecular proteins, so-called signaling peptides, were
identified, which function both on a neuronal level as
neurotransmitters and on an immunological level as differentiation
factors.
[0004] It was possible to prove in animal experiments that
low-molecular signaling peptides are capable of inducing a
differentiation cascade in T and B lymphocytes from pluripotent
parent cells, when such active peptide substances are
subcutaneously supplied to the organism. Moreover, is has been
found that such signaling molecules, whose composition corresponds
to the proteinic elements of the thymus gland, are capable of
protecting the organism against opportunistic infections, when same
has previously been harmed, for example, by X-rays, cancer
chemotherapy, or a latent virus infection. Such early damage to the
regulatory equilibrium of the organism is accompanied by hormonal
disorders, which express themselves by changed fertility, reduced
mental and physical performance, sleep disorders, and loss of
appetite. Moreover, humans exhibit a reduced protective function of
the skin as well of the quality of hair and nail growth.
[0005] From experience, medical science has countered this syndrome
for a long time in that preparations of organic extracts are used
as medicines or food supplements. Such preparations of the thymic
tissue from animals, in particular thymus of the calf, have found
wide use, even when a molecularly defined principle of action of
such preparations has not been described until now. Positive data
from a series of biological studies in vivo with such thymus
preparations permit concluding that substances contained in the
thymus tissue exert synergistic effects both on the neuronal and on
the immune system. However, in the last five years the risk has
come to the fore that such preparations of organic extracts from
animals are contaminated with prion proteins. These prion proteins
are associated with transmitting the symptoms of BSE (bovine
spongiform encephalopathy, "bovine madness").
[0006] It is therefore the object of the present invention to make
available an immunologically and/or endocrinologically active
preparation, which permits on the one hand extrapolating the
positive experience with thymic peptide combinations, but totally
excludes on the other hand the risk of BSE. Furthermore, it is the
object to specify a synthetic, statistic thymic peptide combination
and its use as an immunologically and/or endocrinologically
preparation with corresponding advantages.
SUMMARY OF THE INVENTION
[0007] The present invention accomplishes the foregoing object by
the characteristic features of claim 1. Accordingly, an
immunologically and/or endocrinologically active preparation
contains as an active ingredient short-chain peptides with a
weighted quantity ratio and pattern of amino acids characteristic
of thymic tissue.
[0008] Claim 2 relates to corresponding method, namely the stepwise
lineup of individual, chemically suitable derivatized amino acids
to short-chain peptides, and release of these synthetic peptides
from their N-terminal and form derivatized on side functions, while
maintaining the weighted quantity ratio and pattern of amino acids
characteristic of the thymus tissue.
[0009] Claim 3 is relates to the use of corresponding preparations,
i.e., synthesized, short-chain peptides with a weighted quantity
ratio and pattern of amino acids characteristic of thymus tissue
with immunologically and/or endocrinologically active properties,
namely preparations containing synthetic, statistic thymic peptide
combinations. The further dependent claims 4 to 8 relate to quite
particular uses, namely in the case of weak immunity and
immunodeficiencies, hormonal disorders, as a nutrient for skin,
hair, and nails, in the cosmetic field, and finally in the field of
food supplements.
[0010] Proceeding from partial hydrolysates of thymic protein,
protein mixtures from short-chain thymic peptides and amino acids,
which result from an enzymatic and/or chemical partial hydrolysis
of thymus tissue, such combinations were clarified by way of
chromatography and amino-acid analysis to that extent that the such
partial hydrolysates consist of short-chain peptides with a
quantity ratio and pattern of amino acid characteristic of thymus
tissue. When such amino acids are reacted with one another in
chemically suitably derivatized form, with the quantity ratio of
the amino acids characteristic of the thymus tissue being
maintained, when such synthetic combinations are released from
their N-terminal protective groups and coupled again and repeatedly
with such weighted combinations of amino acids, which are
characteristic of thymus tissues, one will obtain synthetic,
statistic thymic peptide combinations to the total exclusion of the
prion protein risk.
[0011] Thus, the invention discloses relatively simple ways of
purposefully producing immunologically and endocrinologically
active preparations with an effect of the preparation that is
standardizable for the first time.
[0012] It is possible to produce such thymic peptide combinations
in a pharmaceutically reproducible sterile manner, so that a
preparation results in this manner, which depending on the number
of repeated statistic synthesis operations consists of a family of
signaling thymic peptides characteristic of neurotransmitters and
immunologically active signaling molecules. Based on analytical
tests in comparison with partial hydrolysis mixtures from natural
thymus tissue, it was possible to demonstrate that, within the
margin of error of applied analytical methods, synthetic, statistic
thymic peptide combinations are comparable in their composition
with the natural preparation. From this follows that it was
possible to accomplish the object of the present invention to
eliminate the BSE risk by synthesizing a thymic peptide
combination.
[0013] Surprisingly, it has shown that the synthetic preparation of
the present invention exhibits in the biological test on human
lymphocytes a superior activity with respect to mitogenic
co-stimulation (induction of cell proliferation) in comparison with
a commercial thymic preparation produced by partial hydrolysis.
[0014] Furthermore, it comes as a surprise that the synthetic
preparation is capable of binding constituents of the extracellular
matrix, a property that had previously to be ascribed only to
scleroproteins.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] Having thus described the invention in general terms,
reference will now be made to the accompanying drawings, which are
not necessarily drawn to scale, and wherein:
[0016] FIG. 1 is a graphic representation of data from the
mitogenic co-stimulation of human peripheral blood lymphocytes
(PBL's) obtained from a first donor with phytohemagglutinin (PHA)
in the presence of a positive control (REF01), a commercial thymic
peptide combination (GKL01) and the statistic thymic peptide
combination (SP/01/211096).
[0017] FIG. 2 is a graphic representation of data from the
mitogenic co-stimulation of human PBLs obtained from a second donor
with PHA in the presence of a positive control (REF01), a
commercial thymic peptide combination (GKL01) and the statistic
thymic peptide combination (SP/01/211096).
[0018] FIG. 3 is a graphic representation of data from the
mitogenic co-stimulation of human PBLs obtained from a third donor
with PHA in the presence of a positive control thymosin fraction 5
(TF5), a commercial thymic peptide combination (GKL01) and the
statistic thymic peptide combination (SP/01/211096).
[0019] FIG. 4 is a graphic representation of data from a second
mitogenic co-stimulation of human PBLs obtained from the third
donor with PHA in the presence of a positive control thymosin
fraction 5 (TF5), a commercial thymic peptide combination (GKL01)
and the statistic thymic peptide combination (SP/01/211096).
DETAILED DESCRIPTION OF THE INVENTION
[0020] The present invention now will be described more fully
hereinafter with reference to the accompanying drawings, in which
preferred embodiments of the invention are shown. This invention
may, however, be embodied in many different forms and should not be
construed as limited to the embodiments set forth herein; rather,
these embodiments are provided so that this disclosure will be
thorough and complete, and will fully convey the scope of the
invention to those skilled in the art. Like numbers refer to like
elements throughout.
[0021] Many modifications and other embodiments of the invention
will come to mind to one skilled in the art to which this invention
pertains having the benefit of the teachings presented in the
foregoing descriptions and the associated drawings. Therefore, it
is to be understood that the invention is not to be limited to the
specific embodiments disclosed and that modifications and other
embodiments are intended to be included within the scope of the
appended claims. Although specific terms are employed herein, they
are used in a generic and descriptive sense only and not for
purposes of limitation.
[0022] The invention is described in greater detail with reference
to the following examples.
EXAMPLE 1
[0023] The statistic thymic peptide combinations of the present
invention were examined for bioactivity in the test system of the
mitogenic co-stimulation in comparison with a commercial thymic
preparation from partial hydrolysis. Tested were end concentrations
of 100, 50, and 25 .mu.g/ml.
[0024] In the case of two donors (KFG and WH) the statistic thymic
peptide combination shows a statically significantly higher
bioactivity than a commercial thymic preparation from partial
hydrolysis. While at the high concentration of 100 .mu.g/ml, an
inhibition of cell proliferation was observed for a commercial
thymic preparation from partial hydrolysis, the statistic thymic
peptide combination led to a dose-dependent increase in the
proliferation of human lymphocytes.
[0025] From two blood donations, human peripheral blood lymphocytes
were isolated, and the proliferation rate of these cells was
measured, after a suboptimal prestimulation (0.5 .mu.g/ml
phytohemagglutinin (PHA)) under the action of different
concentrations of a commercial thymic preparation from partial
hydrolysis, and the statistic thymic peptide combination in
comparison with the positive control REF 01 (thymosin fraction
5).
[0026] In terms of the measuring technique, the proliferation rate
was determined by the following method: During the
active-ingredient dependent induction of the T-cell proliferation,
the DNA synthesis is stimulated for purposes of cell division.
Therefore, it is possible to measure via incorporation of a
radioactive DNA component (3H-thymidine) the T-cell growth with
reference to incorporated radioactivity.
[0027] FIGS. 1 and 2 show results from the stimulation of the blood
lymphocytes of donor KFG and donor WH. The test data are to be
related to the result of the control formulation after adding
phytohemagglutinin (Kontr.+PHA). In the test, the reference thymus
preparation was used as positive control in a concentration of 100
.mu.g/ml; a commercial thymic preparation from partial hydrolysis
and the statistic thymic peptide combination were tested in the
three concentrations of 100, 50, and 25 .mu.g/ml.
[0028] FIG. 1 shows the results for donor KFG. The reference thymus
preparation used a positive control led to a very obvious increase
in the proliferation rate. For a commercial thymus preparation
produced by partial hydrolysis, however, it was possible to observe
a dose-dependent inhibition. Only in the case of the lowest
concentration of 25 .mu.g/ml was the value of the control
formulation reached. By the action of the statistic thymic peptide
preparation, the cell proliferation rate was statistically
significantly higher in the case of the three tested concentrations
than in the control formulation.
[0029] According to FIG. 2, the lymphocytes of donor WH showed
comparable results. Again, the reference thymus preparation used as
positive control resulted in a considerable increase in the
proliferation of human lymphocytes. A commercial thymus preparation
produced by partial hydrolysis led in the concentrations of 50 and
25 .mu.g/ml to a slight increase in the cell proliferation rate,
but not in the high concentration of 100 .mu.g/ml. However, for the
statistic thymic peptide combination SP/01/211096, it was possible
to observe a clear and dose-dependent increase in the DNA synthesis
rate.
[0030] The object of these tests according to Example 1 was the
comparative testing of the synthetic, statistic thymic peptide
preparation with a commercial thymus preparation produced by
partial hydrolysis in the test system of the mitogenic
co-stimulation. The intent was to test the capability of the test
substance of increasing the cell proliferation rate of human blood
lymphocytes.
[0031] For the statistic thymic peptide combination, it was
possible to find in the case of two donors a clearly higher
bioactivity in comparison with the commercial thymus preparation
from partial hydrolysis. In addition, donor WH showed a
dose-dependent increase in the DNA synthesis rate. Surprisingly,
the addition of a commercial thymus preparation from partial
hydrolysis to the cell culture formulations led rather to an
inhibition of the cell proliferation. Only in the case of low
concentrations of 50 or 25 .mu.g/ml was the cell proliferation rate
slightly above the value of the control formulation in the case of
donor WH.
EXAMPLE 2
[0032] The following example relates to the stimulation of the
blood lymphocytes of donor DB. The test data are to be related with
reference to FIGS. 3 and 4 to the result of the control formulation
after adding phytohemagglutinin at PHA. The thymosin fraction No. 5
(TF5) was used as positive control in two concentrations (third and
fourth columns from the left). For a comparison, a commercial
partial thymic hydrolysate (fifth and sixth columns) was also used
in the same concentration. As a control PHA was used alone.
[0033] In the mitogenic co-stimulation with suboptimal stimulation
of the blood cells with PHA of 0.05 .mu.g/ml, the
synthetic-statistic thymic peptide combination shows a surprisingly
distinct activity both in the case of a dose of the claimed
synthesized product of 100 .mu.g/ml/cell culture formulation and in
the case of 0.4 .mu.g/ml/cell culture formulation.
[0034] Likewise, in the case of a slight prestimulation of the
blood cells with PHA in an amount of 0.025 .mu.g/ml/formulation,
one can clearly notice the effects of the synthetic-statistic
thymic peptide combination according to the invention, even though
less distinct than in the case of a prestimulation of the blood
cells with 0.05 .mu.g/ml PHA/formulation.
[0035] Lastly, one has in this instance a typical, in vitro,
two-phase activity, which shows a maximum respectively in the range
of 100 .mu.g/ml and 0.4 .mu.g/ml, and is in each case better than
the commercial partial thymic hydrolysate that is used as
comparison preparation. In any event, this two-phase activity is a
proof for the wide scope of the immunological, in vitro activity,
wherein the great extent of activity in the case of the extremely
low concentration of 0.4 .mu.g/ml comes no doubt as a surprise and
is of great economic interest due to its really very low
concentration.
[0036] The analytical data of the synthetic-statistic thymic
peptide combination are as follows:
1 Certificate of Analysis Data Sheet Synthetic-Statistic Thymic
Peptides Specifications #SP/01/211096 Specified Found Description:
yellowish powder yellowish powder Residual Solvents (GC): <10%
Water <0.1% 9% .+-. 0.5% DMF <0.1% n.d.* < 200 ppm**
Methanol <0.1% 130 ppm < 130 ppm Toluol <0.1% n.d. < 40
ppm Ethylisopropylamine <0.1% n.d. < 200 ppm Acetic ester
<0.1% n.d. < 20 ppm Acetates: <3% 1.6% .+-. 0.2% Inorganic
salts: <0.5% <0.2% Specific rotation: -30.0.degree. .+-.
5.degree. -30.2.degree. .+-. 0.2.degree. (20.degree. C. c = 0.2;
H2O) Free amino acid content: GKL01 .+-. 5% complies (%, w/w) Total
amino acid composition: GKIL01 .+-. 5% complies (%, w/w) Peptide
content: 50% .+-. 5% complies
* * * * *