Note: Descriptions are shown in the official language in which they were submitted.
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A Biological Active Blood Serum, Methods for Producing it and Uses thereof
The present invention relates to a method for preparing a blood serum product,
the blood se-
rum product, and a pharmaceutical composition comprising said blood serum
product as well
as uses thereof in the treatment of various diseases and conditions including
epileptic seizures
and apoplexy.
Background of the Invention
Methods for the preparation of active substances from blood serum are known,
in the art. One
is based on the withdrawal of blood from humans or animals, the subsequent
incubation as
well as the separation of the active substance and finally the preservation of
the substance
(see, for example, JP 2123287, EP 0 542 303, RU 2096041, RU 2120301). The
prior art
method concerns the preparation of a blood serum which improves the resistance
of the body
in respect of exogenic and endogenic factors like air pressure, air
temperature, gravity, light
etc. as well as hunger, thirst, sleeping and sexual desires etc. The blood
serum is drawn from
the donor who has previously been brought into a certain functional state and
according to the
length of the application of the functional state and the type of functional
state, e.g. sleep dep-
rivation, alcohol abuse, nicotine abuse etc., blood serum with different
biological activity can
be obtained which shows mitogenic, somnogenic, opthalmogenic, audio active,
thermo active,
diatary active, sexually active, anti-hypoxic, anti-alcohol and anti-nicotine
activity.
A different method is disclosed in EP 1 283 047 and concerns the treatment of
animal blood
serum by gamma irradiation with the aim to increase the biological activity of
the blood se-
rum product.
Presently, a lot of research focuses on the mechanisms, which regulate
cellular proliferation
of different human tissues. In this respect both stimulators and inhibitors of
proliferation of
normal and pathologically abnormal somatic cells, including nerve cells, are
investigated (see,
for example Aschmarin, LP. "Neurochemistry", Moskwa, Publishers of the
Biomedical
Chemical Institute of the Russian Academy of Science", 1996).
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It has been observed that peptide growth factors apart from their general
activating functions,
e.g. stimulation of mitosis, cell differentiation and cell growth of different
types of normal
tissue, increased wound healing, can cause tumor formation and proliferation
(see, for exam-
ple, Bouneres, P. (1993), Horm. Res. 40: 31; Robinson, C., (1993) Ann. Med 25:
535;
Dignez, C. and Casanueva F. (1995) Trends Endoc~in. Metab. 6: 55, Menster D.
et al. (1995)
Clin. Exp. Metastasis 13: 67).
Such peptides as, for example, the parathyroid peptides, gastrin or bombesin
foster the devel-
opment of tumor cells as well as the development of breast, bone and colon
cancer (see, for
example, Kitazawa S. and Maeda S. (1995) Clin. Oy~thop. 312: 45-50 and I~aji
et al. (1995)
Endocrinology 136: 842).
Although some peptides facilitate normal cell division and are stimulators for
human and
animals there is the danger that the use will lead to tumor cell development
and eventually to
the development of cancer.
Earlier experiments have shown that stimulation of animals with electricity
leads to an in-
crease of the (3-endorphin level in the blood (see, for example, Litvinova
S.V. et al. (1990)
Biomed. Sci. 5: 471). In a reference work by Udovitschenko, W.I. numerous data
is provided
with respect to the results of stimulation or shock due to various causes. It
has been shown
that, for example, electroshock leads to a marked increase of the
concentration of (3-
endorphines, meta- and leu-encephalines within the blood (see Udowitschenko,
W.I. (1989)
"Xenogenic Opioid System in Shock" Pathiological Physiology and Experimental
Therapy"
6: 72-77).
Summary of the Invention
An object of the present invention was the development of a novel method for
the preparation
of a biological active blood serum from animal blood. Surprisingly it was
found that the bio-
logical active blood serum prepared according to the method of the invention
exhibited new
therapeutic properties.
Consequently, one aspect of the present invention is a method for producing a
biological ac-
tive blood serum comprising the steps of:
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a) electrostimulation of a non-human animal
b) withdrawal of blood from said animal,
c) isolation of serum from said blood, and
d) gamma irradiation of said serum.
In a preferred embodiment the non-human animal is selected form the group
consisting of
mammals and birds, preferably from poultry, e.g. chicken, dug, goose, ostrich,
and quail.
Although the electrostimulation can be employed to any part of the body it is
preferred that
step a) of the method of the present invention is applied to the head, the
neck, the body and/or
one or more limbs of the animal. Out of those it is preferred that the head of
the animal is
electrostimulated. In the context of the present invention the terms
electrostimulation and
electroshock are used interchangeably.
In a preferred embodiment of the method of the present invention the
electrostimulation is
carried out for a time period of between 1 and 60 seconds, preferably between
1 and 30 sec-
onds, and more preferably between 2 and 10 seconds. It is also preferred that
the electrostimu-
lation is carried out with a voltage in the range of between 50 V and 150 V,
preferably in the
range of between 80 V to 120 V, and more preferably in the range of between
110 V and 120
V. During the performance of the electrostimulation certain currents are
preferred and pref
erably the electrostimulation is carried out with a current in the range of
between 0.01 A and
0.4 A, preferably in the range of between 0.02 A and 0.1 A, and more
preferably in the range
of between 0.04 A and 0.06 A.
In a preferred embodiment of the method of the present invention the
electrostimulation is
carried out with a frequency in the range of between 10 and 200 Hz, preferably
in the range of
between 20 to 100 Hz and more preferably in the range of between 45 to 65 Hz.
In a further preferred embodiment of the method of the present invention the
gamma irradia-
tion is administered with an adsorbed radiation dose of between 10 to 40 kGy,
preferably 15
to 35 kGy and more preferably of between 20 and 30 kGy. The gamma radiation
source can
be any source, however, a preferred source of gamma radiation is selected from
the group
consisting of 6°Co, 137Cs, 67Cu, 67Ga, lllIn,192Ir, 99mTc and
17°Tm.
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In a further preferred embodiment of the method of the present invention the
method further
comprises the step of incubating said blood prior to step c).
In a further preferred embodiment of the method of the present invention the
method further
comprises the step of lyophilization of said serum prior to step d).
In a preferred embodiment of the method of the present invention the blood is
arterial and/or
venous blood.
Another aspect of the present invention is the biological active blood serum
which is produc-
ible according to a method of the present invention.
A further aspect of the present invention is a pharmaceutical composition
comprising a blood
serum according to the present invention and one or more pharmaceutically
acceptable dilu-
ents; carriers; excipients, including fillers, binders, lubricants, glidants,
disintegrants, adsorb-
ents; and/or preservatives.
In a preferred embodiment of the pharmaceutical composition of the present
invention the
composition is formulated as a syrup, an infusion or injection solution, a
tablet, a capsule, a
capslet, lozenge, a liposome, a suppository, a plaster, a band-aid, a retard
capsule, a powder,
or a slow release formulation. Preferably the diluent is water, a buffer, a
buffered salt solution
or a salt solution and the carrier preferably is selected from the group
consisting of cocoa but-
ter and vitebesole.
A further aspect of the present invention is the use of a blood serum of the
present invention
or of a pharmaceutical composition of the present invention for the
production' of a medica-
ment for the treatment of a disease or condition, which can be affected by an
increase of cy-
clic adenosine monophosphoric acid contents in the brain of the subject
requiring treatment.
Another aspect of the present invention is the use of a blood serum of the
present invention or
of a pharmaceutical composition of the present invention for the production of
a medicament
for the improvement of cognitive and/or learning skills in particular
improvement of the long
term memory.
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Another aspect of the present invention is the use of a blood serum of the
present invention or
of a pharmaceutical composition of the present invention for the production of
a medicament
for the treatment of seizures, in particular epileptic seizures.
A further aspect of the present invention is a use of a blood serum of the
present invention or
of a pharmaceutical composition of the present invention for the production of
a medicament
for the treatment of proliferative diseases and apoplexy.
In a preferred embodiment of the use of the present invention the
proliferative disease is se-
lected from the group consisting of malignomas of the gastrointestinal or
colorectal tract, the
liver, the pancreas, the kidney, the bladder, the thyroid, the prostate, the
endometrium, the
cervix, the ovary, the uterus, the testes, the skin, the oral cavity;
melanoma; dysplastic oral
mucosa; invasive oral cancers; small cell and non-small cell lung carcinomas;
mammary tu-
mors, in particular hormone-dependent breast cancers and hormone independent
breast can-
cers; transitional and squamous cell cancers; neurological malignancies
including neuroblas-
tomas, gliomas, astrocytomas, osteosarcomas, meningiomas; soft tissue
sarcomas; heman-
gioamas and endocrinological tumors, in particular pituitary adenomas,
pheochromocytomas,
paragangliomas, haematological malignancies , in particular lymphomas and
leukemia.
And in a further preferred embodiment of the use of the present invention the
proliferative
disease comprises cells similar to the human T cell lymphoma cell line Jurkat,
the human B
cell lymphoma cell line Raji, the human melanoma cell line Bro, the human
cervical cancer
cell line HeLa, the human adenocarcinoma cell line MCF-7, the osteosarcoma
cell line Mg63,
the fibrosarcoma cell line HT1080, the neuroblastoma cell line IMR-32 and the
hepatocarci-
noma cell line HepG2.
In a further preferred embodiment of the use of the present invention the
medicament is ad-
ministered to a subject in need of treatment in an amount ranging from 50 to
150 mg/kg body
weight, preferably ranging from 90 to 100 mg/kg body weight.
Detailed Description of the Invention
The present inventors have surprisingly found that the stimulation of animals,
in particular
chickens with electric currents and the further treatment of blood serum
obtained from the
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blood with y-radiation leads to a significant increase of the biological
activity of the blood
serum product. The resulting products are capable of positively influencing
various body
functions, conditions and diseases of a patient.
The beneficial effect of blood serum treated with electricshock and y-
radiation is surprising
for two reasons. Firstly, it is known in the prior art that electric shocks
cause severe distur-
bances of all vital functions and systems within the organism in particular
the central nervous
system, the blood and circulatory system and the respiratory system (see, for
example, Orlow,
A.N. et al. (1977) Medicine). Secondly, it was also discovered that blood and
serum prepared
from blood is sensitive to radiation and is unstable and deactivated under
ionizing radiation
(see, for example, Radiomedicine - M. Atomisdat (1972) 123-125 and Gergely, J.
et al.
(1967) Radioste~ilizatioh of Medical Products 115-124).
These studies did not lead to the results of the present invention and would
have suggested
that it would not be possible to obtain a biological active blood serum
through the combina-
tion of electroshock and y-radiation treatment. Thus, it was surprising that
arterial and/or ve-
nous blood drawn from an animal, in particular from a chicken treated with an
electroshock of
grade II to grade III and with gamma radiation resulted in a biological active
blood serum.
The specific activities of the thus obtained blood serum will be set out in
more detail below.
Accordingly, a first aspect of the present invention is a method for producing
a biological
active blood serum comprising the steps of:
a) electrostimulation of a non-human animal,
b) withdrawal of blood from said animal,
c) isolation of serum from said blood, and
d) gamma irradiation of said serum.
Various animals can be used in the method of the present invention, however,
it is preferred
that the non-human animal is selected from the group consisting of mammals and
birds. Be-
cause of their easy availability it is particularly preferred to use farm
animals like poultry, e.g.
chicken, dug, goose, ostrich and quail. A particular preferred animal which
can be used in the
method of the present invention is a chicken. The type of mammal that can be
used in the
method of the present invention is not particularly restricted and comprises
without limitation
rodents, e.g. mouse, hamster and rat, cats, dogs, horses, donkeys, sheep,
cows, and goats.
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It is envisioned that the electrostimulation leads to the release of certain
compounds within
the animal which cause and/or contribute to the surprising therapeutic effect
of the biological
active blood serum of the present invention. The non-human animal can be
stimulated in dif
ferent regions of the body. Preferably the electrostimulation is carried out
at the head, the
neck, the body and/or on one or more of the limbs. It is possible to stimulate
the body only at
one position or at several positions at once. A particular preferred body part
for the elec-
trostimulation is the head of the respective animal. When stimulating birds,
in particular
chicken it is preferred that the head is electrostimulated.
The electrostimulation can be carried out by art known methods, preferably
using metal elec-
trodes or water baths as used, for example, during culling of cattle or
electrocution of poultry.
Preferably, the electrostimulation is carried out for a time period of between
1 and 60 sec-
onds, preferably between 1 and 30 seconds, more preferably between 2 and 10
seconds, and
most preferably between 3 and 4 seconds. The length of a time period will be
longer in case
that a large animal is electrostimulated and can be shorter in cases were
small animals are
electrostimulated. For example for the stimulation of chicken heads a
particular preferred time
period of the electrostimulation is between 2 and 10 seconds and more
preferably between 3
and 4 seconds. The other variables which can be adapted during the
electrostimulation of the
animal is the voltage, the current and the frequency of the current and the
present inventors
have defined certain preferred ranges for these parameters. The actual
parameter chosen will
depend in part on the size of the animal as well as on the region of the
animal to be stimu-
lated. In general larger animals and larger regions will require a higher
voltage and current.
Thus, the electro stimulation is preferably carried out with a voltage in the
range of between
50 Volt and 150 Volt, preferably 80 Volt to 120 Volt and more preferably
between 110 Volt
and 120 Volt. The ranges for the currents that can be applied are between 0.01
A and 0.4 A,
preferably between 0.02 A and 0.1 A, more preferably between 0.04 A and 0.06 A
and most
preferably about 0.05 A. Voltage, current and application time are preferably
chosen to ad-
minister energy in the range of between 1 and 1,000 Ws, preferably in the
range of 10 to 200
Ws and even more preferably in the range of 15 to 100 Ws.
For the stimulation of the preferred animals, i.e. chicken, it is preferred
that the electrostimu-
lation is carried out with a voltage in the range of between 80 Volt to 120
Volt and more pref
erably between 110 Volt and 120 Volt. Furthermore, a current in the range of
between 0.04 A
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and 0.06 A, in particular of 0.05 A is preferred in the context of the
electrostimulation of
birds, in particular of chicken.
In a particular preferred embodiment of the method of the present invention
the electrostimu-
lation of a bird, in particular a chicken, is carried out for between 3 and 4
seconds at a voltage
of between 80 V and 120 V, in particular 110 V and 120 V. In this preferred
embodiment the
current is preferably between 0.04 A and 0.06 A and most preferably about 0.05
A.
The frequency of the electrostimulation does not appear to be particularly
critical but is pref
erably in the range of between 10 and 200 Hertz, more preferably in the range
of between 45
to 65 Hz and most preferably around 50 Hz.
The gamma irradiation of the serum during step d) of the method of the present
invention can
be carried out with any gamma source including X-ray sources and
radionuclides. Preferably
the gamma radiation source is a radionuclide with a defined gamma radiation
pattern. Pre-
ferred sources for the gamma radiation are selected from the group consisting
of 6°Co, 137Cs,
67Cu, 67Ca, 111In, l9alr, 99mTc and 17°Tm. Out of those 6°Co,
137Cs, l9aIr and 17°Tm are particular
preferred with 6°Co being the most preferred gamma radiation source,
for us in the method of
the present invention. The radiation dose adsorbed by the serum is in the
range of between 10
to 40 kGy preferably in the range of between 15 to 35 kGy and more preferably
in the range
of between 20 and 30 kGy, i.e. 25 ~ 5 kGy.
The withdrawal of the blood from the animal can be effected by any art known
method and
includes syringes as well as puncturing of arteries or veins or decapitation
in particular in the
context of the withdrawal of blood from birds. It is possible to withdraw only
a part of the
blood or to completely with draw the blood of the animal. The later is
preferably used, if a
lethal dose of electricity has been applied to the animal. The withdrawn blood
can be arterial
and/or venous blood.
The serum can be isolated from the blood by any known method including
filtration, sedimen-
tation and centrifugation. It is, however, preferred that the blood is
incubated for between 4
and 72 h at a low temperature, e.g. between 2° and 10°C,
preferably between 4-8°C to allow
clotting of the blood which leads to the release of additional factors into
the blood. Thus, in a
preferred embodiment to the method of the present invention the method further
comprises
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the step of incubating the blood after the withdrawal of the blood from the
animal and prior to
the isolation of the serum from the blood, e.g. for between 4 and 72 h at a
low temperature,
e.g. between 2° and 10°C, preferably between 4-8°C.
In a further preferred embodiment of the method of the present invention the
method com-
prises the further step of lyophilization of the serum prior to the
irradiation step d). The ly-
ophilization allows easier handling of the serum during irradiation and
optimizes absorption
of the radiation by the serum components.
A further aspect of the present invention is the biological active blood serum
itself, which is
producible according to a method of the present invention. It is distinct from
prior art blood
serums which do not employ the steps of the method of the present invention,
which is evi-
denced by its particular therapeutic effects, which are not exhibited by prior
art blood serum
products.
As it has been surprisingly found that the biological active blood serum of
the present inven-
tion provides certain therapeutical effects, e.g. an anti-proliferative
acitivty or an anti epileptic
activity, a further aspect of the present invention is a pharmaceutical
composition, comprising
a biological active blood serum producible according to the method of the
present invention.
Such pharmaceutical composition can further comprise one or more
pharmaceutically accept-
able diluents; carriers; excipients, including fillers, binders, lubricants,
glidants, disintegrants,
and adsorbents; and/or preservatives.
The pharmaceutical composition of the present invention can be administered by
various well
known routs, including oral and parenteral administration, e.g. intravenous,
intramuscular,
intranasal, intradermal, subcutaneous and similar administration routes.
Parenteral administra-
tion and particular intravenous administration is preferred. Depending on the
route of admini-
stration different pharmaceutical formulations are required and some of those
may require that
protective coatings are applied to the drug formulation to prevent degradation
of the biologi-
cal active serum in, for example, the digestive tract.
Thus, preferably the pharmaceutical composition of the present invention is
formulated as a
syrup, an infusion solution, or injection solution, a tablet, a capsule, a
capslet, a lozenge, lipo-
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some, a suppository, a plaster, a band-aid, a retard capsule, a powder or a
slow release formu-
lation.
Particular preferred pharmaceutical forms are forms suitable for injectionable
use and include
sterile aqueous solutions or dispersions and sterile powders for the
extemporaneous prepara-
tion of sterile injectable solutions or dispersion. In all cases the final
solution or dispersion
form must be sterile and fluid. Typically, such a solution or dispersion will
include a solvent
or dispersion medium, containing, for example, water-buffered aqueous
solutions, e.g. bio-
compatible buffers, ethanol, polyol, such as glycerol, propylene glycol,
polyethylene glycol,
suitable mixtures thereof, surfactants or vegetable oils. The biological
active blood serum of
the present invention can also be formulated into liposomes, in particular for
parenteral ad-
ministration. Liposomes provide the advantage of increased half life in the
circulation, if
compared to the free drug and a prolonged more even release of the enclosed
drug.
Sterilization of infusion or injection solutions can be accomplished by any
number of art rec-
ognized techniques including but not limited to addition of preservatives like
anti-bacterial or
anti-fungal angents, e.g. parabene, chlorobutanol, phenol, sorbic acid or
thimersal. Further,
isotonic agents, such as sugars or salts, in particular sodium chloride may be
incorporated in
infusion or injection solutions.
Production of sterile injectable solutions containing the biological active
blood serum is ac-
complished by incorporating the biological active serum in the required amount
in the appro-
priate solvent with various ingredients enumerated above as required followed
by steriliza-
tion. To obtain a sterile powder the above solutions are vacuum-dried or
freeze-dried as nec-
essary. Preferred diluents of the present invention are water, physiological
acceptable buffers,
physiological acceptable buffer salt solutions or salt solutions. Preferred
carriers of the pre-
sent invention are cocoa butter and vitebesole. Excipients which can be used
with the various
pharmaceutical forms of the biological active blood serum can be chosen from
the following
non-limiting list:
a) binders such as lactose, mannitol, crystaline sorbitol, dibasic phosphates,
calcium phos-
phates, sugars, microcrystalline cellulose, carboxymethyl cellulose,
hydroxyethyl cellu-
lose, polyvinyl pyrrolidone and the like;
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b) lubricants such as magnesium stearate, talc, calcium stearate, zinc
stearate, stearic acid,
hydrogenated vegetable oil, leucine, glycerids and sodium stearyl furnarates,
c) disintegrants such as starches, croscaramellose, sodium methyl cellulose,
agar, bentonite,
alginic acid, carboxymethyl cellulose, polyvinyl pyrrolidone and the like.
Other suitable excipients can be found in the Handbook of Pharmaceutical
Excipients, pub-
lished by the American Pharmaceutical Association, which is herein
incorporated by refer-
ence.
A further aspect of the present invention is the use of a blood serum or of a
pharmaceutical
composition of the present invention for the production of a medicament for
the treatment of a
disease or condition which can be effected by an increase of cyclic adenosine
monophos-
phoric acid content in the brain of the treated subject.
Another aspect of the present invention is the use of the biological active
blood serum or of a
pharmaceutical composition of the present invention for the production of a
medicament for
the improvement of nootropic, cognitive and/or learning skills of the treated
subject and in
particular the improvement of the long-term memory. The usefulness for this
indication is
based on the discovery that the blood serum or the pharmaceutical composition
of the present
invention increase the learning abilities of treated subjects.
Furthermore, the blood serum or the pharmaceutical composition can be used for
the treat-
ment of seizures of any type in particular, however, for the treatment of
epileptic seizures. In
particular in severe epileptic forms and during grand mal seizures the
administration of the
biologically active blood serum or of the pharmaceutical composition of the
present invention
can prevent death that is sometimes associated with severe seizures. In
connection with the
observation that the present invention can be used for the treatment of
epileptic seizures, it has
also discovered that the blood, serum or the pharmaceutical composition of
this invention can
be used for the treatment of nervous diseases, including without limitation,
bipolar disorder,
depression, anxiety related disorders, epilepsy, Alzheimer's disease,
Parkinson's disease, pe-
ripheral neurophathy, cerebral amyloid angiopathy, neuro degenerative
disorders and spinal
cord injury.
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A further aspect of the present invention is the use of the blood serum or of
the pharmaceuti-
cal composition of the present invention for the production of a medicament
for the treatment
of proliferative diseases and apoplexy. It was particular surprising that the
biological active
blood serum or the pharmaceutical composition of the present invention did
show a marked
anti-proliferative effect, if tested on a variety of tumor cell lines in
vitro. It, therefore, appears
that the biological active blood serum or the pharmaceutical composition of
the present inven-
tion is particular suitable for the treatment of proliferative diseases and
preferred proliferative
diseases which are treatable according to the use of the present invention are
selected from the
group consisting of malignomas of the gastrointestinal or colorectal tract,
the liver, the pan-
creas, the kidney, the bladder, the thyroid, the prostate, the endometrium,
the cervix, the
ovary, the uterus, the testes, the skin, the oral cavity; melanoma; dysplastic
oral mucosa; inva-
sive oral cancers; small cell and non-small cell lung carcinomas; mammary
tumors, in particu-
lar hormone-dependent breast cancers and hormone independent breast cancers;
transitional
and squamous cell cancers; neurological malignancies including neuroblastomas,
gliomas,
astrocytomas, osteosarcomas, meningiomas; soft tissue sarcomas; hemangioamas
and endo-
crinological tumors, in particular pituitary adenomas, pheochromocytomas,
paragangliomas,
haematological malignancies , in particular lymphomas and leukemia.
Since the anti-proliferative effect of the biological active blood serum of
the present invention
or of the pharmaceutical composition of the present invention has been first
established for a
variety of tumor cell lines it is particular suitable for the treatment of
proliferative diseases
which comprise cells andlor tumor tissue comprising cells similar to the tumor
cell lines used
in those experiments. Accordingly, preferred prolifertive diseases treatable
with the biological
active blood serum or the pharmaceutical composition comprising the biological
active serum
comprise cells similar to the human T cell lymphoma cell line Jurkat, the
human B cell lym-
phoma cell line Raji, the human melanoma cell line Bro, the human cervical
cancer cell line
HeLa, the human adenocarcinoma cell line MCF-7, the osteosarcoma cell line
Mg63, the fi-
brosarcoma cell line HT1080, the neuroblastoma cell line IMR-32 and the
hepatocarcinoma
cell line HepG2. In this context the term "similar cells" are cells, which
have the same origin,
e.g. T-cell, B cell or neural lineage, as the respective cell line and which
carry a mutation in
the same or a functionally equivalent gene and wherein this mutation
contributes to the prolif
erative activity of the cell, e.g. mutation in p53, pRb, cdc 2, cdk 4, cyclin
A, cyclin B, p2lr~,
c-fos, c-jun, p107, p130 and the like; which carry the same or a functionally
similar exoge-
nous gene, e.g. human papilloma virus (HPV), E 6 or E 7, insertion into the
cyclin B promoter
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13
by hepatitis B virus and the like; or which have the same chromosomal
rearrangement or ab-
normality , i.e. deletion, chromosomal multiplicity etc.
Based on the results in animals and in cell culture certain amounts of the
biological active
blood serum are preferred for the treatment of the diseases and conditions for
which the blood
serum and pharmaceutical composition can be employed. It is, however,
understood that de-
pending on the respective condition as well as on the respective patient to be
treated, i.e. de-
pending on the severity of the disease or condition, the general health status
of the patient,
etc., different doses of the biological active blood serum or the
pharmaceutical composition
are required to elicit a therapeutic effect. The determination of the
appropriate dose lies within
the discretion of the attending physician. It is contemplated that the dosage
of the biologically
active blood serum in the therapeutic method of the invention should be in the
range of about
0.1 mg to about 200 mg serum per kg body weight. However, in a preferred use
of the present
invention the biologically active blood serum is administered to a subject in
need thereof in an
amount ranging from 50 to 150 mg/kg body weight, preferably ranging from 90 to
100 mg/kg
body weight. The duration of therapy with biologically active blood serum will
vary, depend-
ing on the severity of the disease being treated and the condition and
idiosyncratic response of
each individual patient.
The following examples are included to demonstrate preferred embodiments of
the invention.
It should be appreciated by those of skill in the art that the techniques
disclosed in the exam-
ples that follow represent techniques discovered by the inventors to function
well in the prac-
tice of the invention, and thus, can be considered preferred modes for its
practise. However,
those of skill in the art should, in light of the present disclosure,
appreciate that many changes
can be made in the specific embodiments that are disclosed without departing
from the spirit
and scope of the invention as set out in the appended claims. All references
cited are incorpo-
rated herein by reference.
Brief Description of the Figures and Drawings
Fig. 1: Cytotoxic effect of two pharmaceutical preparations on Jurkat cells.
The cytotoxic effect on the human T cell lymphoma cell line Jurkat is depicted
for two differ-
ent pharmaceutical preparations comprising biological active serum of the
invention various
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14
concentrations of the. The viability of the Jurkat cells is depicted on the y-
axis in percentages,
while the amount of the biological active serum is indicated on the x-axis in
mg/ml.
Fig. 2: Cytotoxic effect of two pharmaceutical preparations on Raji cells.
The cytotoxic effect on the human B cell lymphoma cell line Rajiis depicted
for two different
pharmaceutical preparations comprising biological active serum of the
invention various con-
centrations of the. The viability of the Raji cells is depicted on the y-axis
in percentages,
while the amount of the biological active serum is indicated on the x-axis in
mg/ml
Fig. 3: Cytotoxic effect of two pharmaceutical preparations on Bro B-19 cells.
The cytotoxic effect on the human T cell lymphoma cell line Bro B-19 is
depicted for two
different pharmaceutical preparations comprising biological active serum of
the invention
various concentrations of the. The viability of the Bro B-19 cells is depicted
on the y-axis in
percentages, while the amount of the biological active serum is indicated on
the x-axis in
mg/ml
Fig. 4: Cytotoxic effect of two pharmaceutical preparations on HeLa cells.
The cytotoxic effect on the human T cell lymphoma cell line HeLa is depicted
for two differ-
ent pharmaceutical preparations comprising biological active serum of the
invention various
concentrations of the. The viability of the HeLa cells is depicted on the y-
axis in percentages,
while the amount of the biological active serum is indicated on the x-axis in
mglml
Fig. 5: Cytotoxic effect of two pharmaceutical preparations on MCF-7 cells.
The cytotoxic effect on the human T cell lymphoma cell line MCF-7 is depicted
for two dif
ferent pharmaceutical preparations comprising biological active serum of the
invention vari-
ous concentrations of the. The viability of the MCF-7 cells is depicted on the
y-axis in per-
centages, while the amount of the biological active serum is indicated on the
x-axis in mg/ml
Fig. 6: Cytotoxic effect of two pharmaceutical preparations on IMR-32 cells.
The cytotoxic effect on the human T cell lymphoma cell line IMR-32 is depicted
for two dif
ferent pharmaceutical preparations comprising biological active serum of the
invention vari-
ous concentrations of the. The viability of the IMR-32 cells is depicted on
the y-axis in per-
centages, while the amount of the biological active serum is indicated on the
x-axis in mg/ml
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Fig. 7: Cytotoxic effect of two pharmaceutical preparations on HT1080 cells.
The cytotoxic effect on the human T cell lymphoma cell line HT1080 is depicted
for two dif
ferent pharmaceutical preparations comprising biological active serum of the
invention vari-
ous concentrations of the. The viability of the HT1080 cells is depicted on
the y-axis in per-
centages, while the amount of the biological active serum is indicated on the
x-axis in mg/ml
Fig. 8: Cytotoxic effect of two pharmaceutical preparations on HepG2 cells.
The cytotoxic effect on the human T cell lymphoma cell line HepG2 is depicted
for two dif
ferent pharmaceutical preparations comprising biological active serum of the
invention vari-
ous concentrations of the. The viability of the HepG2 cells is depicted on the
y-axis in per-
centages, while the amount of the biological active serum is indicated on the
x-axis in mg/ml
Fig. 9: "Bell" curve of the proliferative activity of cells treated with
mitogens
The curve of the lymphocytes is assayed on the basis of the amount of DNA
biosynthesis in
relation to the concentration of the mitogen proliferative activity of. The
DNA biosynthesis
activity is measured by the amount of incorporated radioactivity and the acid
insoluble counts
per minute.
Fig.10: Mitogenic activity of the pharmacological composition.
Depicted is the mitogenic effect of the pharmaceutical composition on the
amount of DNA
biosynthesis within a substance concentration range from 0.1 to 100.0 mg/ml.
The amount of
DNA synthesis is assessed on the basis of the amount of radioactivity
incorporated into the
DNA.
Fig. 11: Effect of pharmacological composition on MCF-7 cells.
Depicted is the amount of DNA synthesis in the human milk gland carcinoma cell
line MCF-7
in relation to the amount of biological active serum comprised in the
pharmacological compo-
sition.
Examules
Examine 1
Method for obtaining of chicken blood treated by electroshock
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For the preparation of serum from chicken, the chicken were treated with an
electroshock of
grade II to III (electrical voltage 80-120 V, current 0.05 A, frequency 50 Hz,
application time:
3 to 4 sec at the head). Blood was then drawn from the arteria carotis and
further incubated at
a temperature of 4-8°C for 18-24 h in polyethylene flasks. After
complete retraction of blood
clots the flasks were spun at 3.000 rpm for between 20-30 minutes. The serum
was separated
from the blood clots and lyophilized under art known conditions. The flasks
with the lyophi-
lized serum were treated on a RZ-100-M apparatus with 20-30 kGy, preferably at
around 25
kGy using 6°CO as a gamma radiation source. The treated serum was
stored at a temperature
of between 4-8°C for later use.
Evidence for the stimulating effect of chicken blood serum treated with
electroshock
The below experiments were carried out using male Wistar rats with an average
body mass of
280-300 g. The animals were randomly assigned to 4 groups of 10 rats each. The
first group
was administered with 1.0 ml of a physiological salt solution. The second
group was adminis-
tered a dose of 100 ~ 5.0 mg/kg body weight of the blood serum of the present
invention
within 1.0 ml solution. 30 minutes after the injection the rats were
decapitated. 1 ml of a
physiological salt solution was administered to the rats of the third group
and the rats of the
fourth group received biological active serum (100 ~ 5 mglkg body weight) in
an amount of
1.0 ml solution. 30 minutes after the injection the animals, which had a
weight attached to
their tail (10% of the body weight of the rat), were placed in a basin with
water (25°C). After
the first signs of agony the animals were removed from the water and
decapitated.
The brain, heart, liver (all being organs, which are exposed to an extensive
energetic strain in
processes of extreme adaptation) as well as the skeletal muscles (as mainly
effected organ)
were taken as samples for further analysis. The tissue samples of each rat
were weight, cooled
with an isotonic NaCI solution and rapidly frozen in liquid nitrogen. The
complete time which
elapsed between the application "stress" and the final processing of the
samples was in the
range of between 5 to 6 minutes maximally.
The amount of adenosine triphosphoric acid, adenosine diphosphoric acid and
adenosine mo-
nophosphoric acid in the skeletal muscles of the rats was determined.
Nucleotides were sepa-
rated by means of ionexchange chromatography on columns employing Anionit
Dowex 1.
The determination of the amount of adenosine triphosphoric acid, adenosine
diphosphoric
acid and adenosine monophosphoric acid was carried out spectophotometrically
at a 256 nm
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range (spectrophotometer Hitachi-557). The energy potential was determined
according to the
following formula
(ATP-O. SADF)/(ATP+ADP+AMP).
The determination of the amount of cyclic adenosine monophosphoric acid in the
brain, heart
and liver was carried out using known radioimmunology analysis with a
detection apparatus
of Amersham (Great Britain).
The determination of the labelled adenosine monophosphoric acid was carried
out using art
known methods employing the scintillation counter GS-8.
Results
The various amounts of adenosine triphosphoric acid, adenosine diphosphoric
acid and
adenosine monophosphoric acid and the increase of the energy potential are
apparent both
when comparing the third group with either the control group one or the second
group
(p<0.05) as well as when comparing the fourth group with either the control
group one or the
second group (p<0.05) and further when comparing the third and the fourth
group among
each other (p<O.OS) (see also Table 1).
Table 1
Amount of adenosine triphosphoric acid, adenosine diphosphoric acid and
adenosine
monophosphoric acid and the energy potential within the tissue of the skeletal
muscles
with and without the administration of the biological active serum
Group of ratsAmount of Energy poten-
nucleotides
mol/1/ tissue
ATPh Mom ADPh Mom AMPh Mom tial (condi-
tional coeffi-
cient)
I (physiologi-7,550.19 0.950.12 0.250.08 0.9170.080
cal salt solu-
tion - control)(7.28-7.85) (0.79-1.18) (0.14-0.37)
n=10
II (serum) 7,g9~0.12 1.12+0.11 0.140.05 0.9230.125
n=10
(7.65-8.02) (0.93-1.27) (0.08-0.25)
III (physio- 1.340.08 3.560.17 0.780.07 0.5490.014
logical salt
solution + (1.19-1.49) (3.21-3.81) (0.65-0.93)
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swimming)
n=10
IV (serum 4.78+0.17 2.55+0.11 0.33+0.09 0.7900.095
+
swimming)
n=10 (4.35-4.95) (2.36-2.72) (0.19-0.48)
The amounts of adenosine phosphoric acid determined during the active period
and 30 min-
utes after mild stress because of the injection (control group I) confirm that
under the influ-
ence of serum a mixing of the amount of adenosine triphosphoric acid and
adenosine diphos-
phoric acid occurs at the upper norm values in the muscle tissue, while at the
same time the
amount of adenosine monosphoric acid decreases.
These results can be interpreted in such that the biological active serum
drives the increase of
the energetic potential within the muscle tissue. The calculation of the
energy potential con-
firms this tendency.
Under the influence of an extreme swimming stress in the third group
(physiological salt solu-
tion + swimming) a decrease of adenosine triphosphoric acid and an increase of
the content of
adenosine diphosphoric and adenosine monophosphoric acid in relation to the
control group
was determined.
Under extreme stress the rats of the fourth group (serum + swimming) the
general tendencies
of the changes of the amounts of adenosine triphosphoric, diphosphoric and
monophosphoric
acid observed in the third group remained similar, however, the amount of
adenosine triphos-
phoric acid stayed significantly higher and when calculating the energy
potential an increase
in the energy potential of 43% (p<0.05) in comparison to the third group was
observed.
A comparative analysis of the cyclic adenosine monophosphoric acid in heart
and liver tissue
as well as in brain tissue of rats of the first and second group demonstrated
that under the in-
fluence of serum an increase of the amount of cyclic monophosphoric acid can
be detected in
the brain tissue (p<0.01) while in heart and liver tissue no significant
difference in compari-
son to the control group was determined (see Table 2).
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Table 2
Amount of cyclic adenosine monophosphoric acid in brain, heart and liver
tissue after
the administration of biological active serum.
Group of rats Amount of
cyclic adenosine
monophosphoric
acid
( mol/U raw
tissue)
brain Mom heart M+m liver M+m
I (physiological3,95+0.58 3.07+0.11 2.75+0.18
solution -
con-
trol n=10 (3.32-4.55) (2.98-3.32) (2.47-2.99)
II (serum) 4,58+0.23 3.26+0.16 2.98+0.99
n=10
(4.26-4.85) (3.02-3.47) (2.95-3.12)
III (physiologi-O.g2+0.13 0.49+0.17 1.01+0.07
cal solution
+
swimming) (0.66-1.05) (0.32-0.78) (0.86-1.14)
n=10
IV (serum + 1,58+0.13 0.83+0.14 1.27+0.08
swimming)
n=10 (1.38-1.76) (0.56-0.96) (1.12-1.41)
Under the influence of extreme stress a significant reduction (p<0.05) of the
amount of cyclic
adenosine monophosphoric acid was observed in all tissues of rats of the third
group (physio-
logical salt solution + swimming) as well as of the fourth group (serum +
swimming). This
decrease was, however, lessened under the influence of biological active
serum. Accordingly,
the amount of adenosine monophosphoric acid in brain tissue was 92% higher as
the respec-
tive amount of the third group (p<0.05), 96% higher in heart tissue and 25.7%
higher in liver
tissue.
Consequently, the serum of the present invention treated with electroshock and
y-radiation
increases the energy potential in skeletal muscles of rats, increases the
amount of cyclic
adenosine monophosphoric acid in brain tissue both during rest phases as well
as under ex-
treme physical stress and facilitates the increase of cyclic adenosine
monophosphoric acid in
heart and liver tissue after physical stress of rats.
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Example 2
Long-term memory.
In this experiment 150 male Wistar rats were used. All rats were previously
tested with a clas-
sical "open field" test and were assigned according to the activity into three
groups: active,
medium activity and passive.
For the establishment of a situation reflex rats were placed in a cylinder
which was located
within a thermostatically controlled basin. The cylinder was placed on three
supports and al-
lowed the animals to dive under the lower edge of the cylinder to reach a
platform located
outside of the cylinder.
The latent time for the first experiment as well as the latent time of the
final solving of the
task of "escaping" from a closed room and from the water was determinable.
When establishing the situation reflex the rats were divided into three
groups:
- "fast diver": those rats, which were able to find the exit within the first
minute
- "slow divers": those rats which only started to look for the exit after 2 to
3 minutes
rats, which were not willing to solve the problem within 10 minutes
The biological active serum was injected into the peritoneal cavity 30 minutes
prior to the
onset of the experiment in a dose of 100 mg/kg body weight. 1 ml of a
physiological salt solu-
tion was administered to the control animals. On the next day the situation
reflex behaviour
was tested and afterwards the rats were given a 40 day pause.
According to this method 120 rats - 60 control rats and 60 rats receiving
biological active
serum - were "educated". The rats were previously assigned to three groups as
another above,
i.e. active, medium activity and passive (20 animals within each group)
furthermore they were
subdivided for this analysis in "fast" and "slow" divers.
Results
Some rats did not show a "diving reflex" at all (Table 3). Within the control
group six of the
active rats and rats of medium activity and 12 animals of the passive group
did not show the
"diving reflex" at a.11. On the second day this number remained the same for
the active rats
and for rats of medium activity, however, declined for the passive animals to
7. After 42 days
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the same 11 rats of the passive group refused to dive under the edge of the
cylinder (1 rat died
during the experiment).
For the active rats of the rats receiving biological active serum there was
not a single rat that
declined to solve the problem on the second day. For all the rats of medium
activity three re-
fused to solve the problem on the first day but nevertheless solved it at the
second day and
continued to have this capability even after 42 days. Among the passive rats
of the rats receiv-
ing serum 4 rats were not willing to solve the problem, however, at the second
day this
amount was reduced to 2 and remained constant even after 42 days.
Table 3
Number of rats which did not show a "diving reflex"
Test animal 1 day (24
hours)
1 2 42
Active rats Control 6 6 6
and rats
of Serum 3 0 0
medium activ-
it
Passive ratsControl 12 7 11 *
Serum 4 2 2
* one rat died during the experiment
Within the group of the "fast divers" a single serum injection within the 40
day time period of
the experiment did not show a significant change in maintaining the situation
reflex.
For the "slow divers" there was a statistically significant capability to
maintain the ability to
quickly find an escape route from an extreme situation (Table 4). Thus, the
time of reflex
formation on the second day decreased in comparison to the first day for both
the control
group and the trial group. In the control group these reflexes were completely
gone after 42
days while for animals which had received an injection of biological active
serum the reflex
completely remained. In addition there was a difference in the time required
for the reflex,
which was 2-3-fold higher (p<0.01) for control rats.
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Table 4
Influence of blood serum on the formation and maintenance of the situation
reflex in
"slow divers" (in sec.)
Test 1 day
(24
liours)
ani-
mal 1 2 42
LP-1 (la-LP-2 LP-1 LP-2 LP-1 LP-2
tency
pe-
riod)
Ac- Control125.7+25 176.7+19,645.5+9.959.0+8.837.5+30.0181.5+15.5
tive
group Serum 111.3+39.7164.8+33.555.3+15.668.4+18.83.8+29.355.7+37.28
Grou Control144.8+21.8191.2+16.349.6+11.262+14.3 3.8+24.019.2+9.8
p of
me- Serum 118.2+71.6178.6+76.83.8+50.377+54.1 9.2+28.854.6+39.7
dium
activ-
i
Pas- Control53.3+25.7189.6+12.653+15.0 66.6+8.0149.3+24.7196.0+11.1
sive
group serum 18.2+71.6178.6+76.863.8+50.377+54.1 39.2+28.854.6+39.7
During the formation and the long-term maintenance of information a
significant role is at-
tributed to H-cholino-ceptive mechanisms. 30 animals who were taught for five
days how to
dive under a "bell" were subdivided into three groups.
A physiological solution of zytisin (a H-cholino-ceptive antagonist of the
brain) was injected
with a dose of 1 mg/kg body weight into the peritoneal cavity 30 minutes prior
to the test of
the animals of the first group (ten rats).
Biologically active serum was administered in a dose of 100 mg/kg body weight
to the ani-
mals of the second group (ten rats) and 30 minutes later a zytisin solution
was administered
with a dose of 1 mg/kg body weight.
The third animal group (control - ten rats) received a 1 ml injection of a
physiological salt
solution.
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The complex behaviour of the animals was tested according to above-indicated
method 48
hours after the injection of the indicated substances. The biotests showed 48
hours after the
application of zytisin a marked slowing of the "escape reaction" from the
closed room namely
a 3-fold slowing in comparison to control animals.
The administration of the serum prior to the zytisin injection not only
alleviated the effect of
this antagonist but also led to an increase in the "escape" reaction by 20% in
comparison to
the control group and the overall effect of the biological active serum
exceeded the effect of
the antagonist by 5-fold.
Table 5
Latency period for the reaction of rats to the situation in connection with
the admini-
stration of zytisin and biologically active serum (sec.)
Time parameter Control (third Administration Administration
group) of of
zytisin (first serum + zytisin
group)
(second group)
After 48 h 13.330.58 40.014 8.35.5
<0.05 >0.05
<0.05
On the basis of the results it was determined that brain H-cholino receptors
play a role in the
restoration of the "escape reaction" in connection with the modelling of a
complex behaviour
of animals and that the biological active serum prevents the establishment of
attenuated learn-
ing.
The biologically active blood serum, therefore, stimulates the formation of
long-term memory
and exhibits this effect in particular in animals with a slowed down escape
reaction from a
closed room and out of water. Furthermore, it appears that the H-cholino-
ceptive brain
mechanisms play an important role in the maintenance of long-term memory and
that the ef
fects of substances negatively effecting H-cholino-ceptive brain mechanisms
can be antago-
nized by the biologically active serum of the present invention.
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Example 3
Epilepsy
It is known in the state of the art that camphor at a toxic dosage leads to
hyperactivation of the
motoric area of the central nervous system which in turn causes the
development of tonic
cramps. Because of this camphor is used besides corazole in animal models for
for the genera-
tion of cramps. Dependent on the doses of the administered camphor it is
possible to cause all
aspects of a small and large epileptic seizure including grand mal seizures.
In the experiments the influence of biologically active serum on the epileptic
activity, which
was induced by injecting camphor into the peritoneal cavity of rats was
investigated. 40 male
Wistar rats with a weight of 190-210 g were used in this experiment. A
solution of camphor
oil (20%) was injected into the peritoneal cavity in an amount of 0.25 and 0.5
ml (using 20
rats). The serum was injected at a dosage of 1005.0 mg/kg body weight (using
20 rats).
The seizures were observed and assessed by experts. The following parameters
were assessed.
The latency time of the reaction, the type of cramp reaction (tonic-klonic,
large and small sei-
zures), the length of the epileptic seizure and the time intervals between
them, the loss of
normal mobility and the final result (death of the animal or the recovery from
the pathological
condition). The results are summarized in Table 6.
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Table 6
Effect of biologically active serum on experimentally induced epilepsy
Doses of cam-Epileptic seizures Control group Test group (cam-
phor (camphor) phor+serum in
a
dosage of 100
mg/kg)
0.25 Occurrence of tonic 7'20"~10" 5'30"~15"
cramps
Occurrence of klonic
cramps in the form
of
grand mal seizures 12'30"~32" 8'45""~28"
after
Medium amount of On in 6'-7' One in 8'-9'
sei-
zures
Length of seizures ~0'-80' 15"-20"
Condition of the All survived All survived
animal
after 48 hours
0.5 ml Occurrence of tonic 5'40"~30" 5'00"-32"
cramps
Occurrence of klonic
cramps in the form
of
grand mal seizures 8'30"~27" 8'40"
after
Medium amount of One in 5' One in 7'
sei-
zures
Length of seizures 90"-120" 25"-30"
Condition of the 60% died All survived
animal
after 48 hours
The biological active serum at a dosage of 100 mg/kg was administered in the
latency period
of epileptic seizures caused by injection of camphor decreased all signs of
the seizure activity:
the latency time of the seizure activity was increased, the klonic cramps are
less severe and
without loss of normal mobility and additionally the serum treated with
electroshock pre-
vented animals in the model of severe epilepsies from death (all animals
survived the admini-
stration of 0.5 ml of a 20% camphor solution).
Example 4
Effect of the blood serum of the present invention on human cell
proliferation.
The blood serum of the present invention is a lyophilized chicken blood
treated with electro-
shock of grade II to III and Y-radiation. The biological active serum was used
in two formula-
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26
tions types: (i) serum resuspended in water at a concentration of 100 mg/ml
(trial fraction 1)
and (ii) the supernatant of the suspension of 100 mg/ml biological active
serum in water after
three minute of centrifugation of the suspension at 10.000 x g (trial fraction
2).
Cell culture
The cells of the Jurkat and Raji cell line as well as lymphocytes of the human
peripheral
blood were cultured in plastic tissue culture plates (Nunc or Falcon) in 1640-
RPMI medium
(Sigma) containing 10% fetal calf serum (FCS, Gibco), 100 unitslml penicilin
and 100 ~g/ml
streptomycin at a temperature of 37°C, at 5% C02 content and 95%
humidity. The cell lines
Bro, HeLa, MCF-7, Mg63, HT1080, IMR-32 as well as HepG2 were cultivated as
above,
however, using DMEM medium (Sigma) instead of 1640-RPMI medium.
Isolating of mononuclear leucocytes (ML) according to the method of Boyum
Mononuclear leucocytes were isolated according to the method described by
Boyum A. (Iso-
lation of mononuclear cells and granulocytes from human blood, 1968, Cand. J.
Lab. Clip.
Ihvest.,120 (97): 9-18).
15 ml Ficoll-pack-solution was placed in each of two conical test tubes
(Falcon) and subse-
quently 25 ml of blood two fold diluted in phosphate-bufferd saline (PBS) was
applied to the
Ficoll. Then the test tubes were spun at 400 x g and 20°C for 30
minutes in a Baket-Rotor.
The upper phase comprising plasma was not used.
The mononuclear leucocytes (ML) which concentrated at the interface between
plasma and
separation medium were carefully sucked off with a pipette and collected in a
centrifuge tube.
Thereafter the cells were washed twice with PBS by spinning the cells at 250 x
g for 10 min-
utes and eventually suspending them in culture medium. This fraction comprised
between 10-
30% monocytes and 80-90% lymphocytes which are in the following termed
"lymphocytes".
One part of the lymphocytes was used for studying the mitogenic activity of
the biological
active serum of the present invention and another to study proliferation. For
this purpose phy-
tohemaglutinine was added at a concentration of 20 mg/ml to cell suspension.
Evaluation of the amount of desoxyribonucleic acid synthesis (DNA) in cells
To evaluate the synthesis of DNA 3H-thymidin was added to the cells and the
acid insoluble
fraction was evaluated for incorporated counts. Briefly, lymphocytes were
incubated in 96
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27
well plates with 200 ~,1 medium comprising between 200 to 800 x 103 cells, To
each well dif
ferent concentration of the biological active serum of the present invention
was added. 3H-
thymidin (1 ~.Ci/Well, 40 mCi/mmol/1) was added two hours prior to termination
of the incu-
bation. For the radiometric assay the cells were harvested on filters with an
automatic device
for cell harvesting. The acid soluble products were washed away with 5%
trifluor acetic acid
(H20) and the radioactivity of the substances retained was measured with a
scintimeter. The
amount of DNA biosynthesis was measured in counts/min.
Determination of cell survival after incubation with different substances
using the MTT-
test
The MTT-test was carried out as described by Mosmann T. (Rapid colorimetric
assay for cel-
lular growth and survival: application to proliferation and cytotoxicity
assays (1983) J. Immu-
hol. Meth. 65: 55-63).
To carry out the test cells were collected in the log. phase (adherent cells
were collected when
they filled about half of the tissue culture plate). They were placed into
growth media in a
Gorjaew-chamber, counted and subsequently resuspended in medium at a
concentration of
50-100 x 106/m1. The cell solutions were placed into the 96 well plates after
the addition of
the various concentrations of the trial fractions in a total amount of 100
~,1. For counting of
viable cells 50 ~,1 of a solution of 3-4,5-dimethyl thiazole-2-yl)-2,5
Biphenyl tetrazolium bro-
mide (MTT) in culture medium was added to each of the different 96 well plates
after termi-
nation of incubation. For the preparation of the MTT solution 1 ml MTT stock
solution was
mixed with 4 ml culture medium. The preparation of the MTT stock solution was
carried out
by the dissolving MTT in PBS (PBS comprises 0.01 mmol/1 sodium phosphate
buffer, pH 7.4,
with 0.15 mmol/1 NaCI) at a concentration of 5 mg MTT/ml followed by
filtration through a
filter with a pore size of 0.45 Vim. The stock solution was stored at
+4°C for up to one month.
After addition of the MTT solution the tissue culture plates were further
incubated for 4 hours
in the incubator under identical conditions. As a next step culture medium was
removed by
suction with a pump and 150 ~,l dimethyl sulfoxide (DMSO) were added to each
well to dis-
solve blue formazan crystals, which had formed, and the optical density of the
solution in
each well was detected with a multichannel spectrophotometer with a microplate
reader at a
wave length of 540 nm (Labsystem). The viability of the cells in relation to
the concentration
of the added trial fraction 1 and 2, respectively, is indicated in percentages
of survival of con-
trol cells. The data was processed with the computer software "Origin".
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28
Results and discussion
The results are depicted on Fig. 1-8. From these results the following can be
derived: a high
concentration of the biological active serum of the present invention (2.5-20
mg/ml) had an
inhibiting effect on all cell lines, however, the reaction of cells from
different tissue types to
trial fraction 1 and 2, respectively, is different. The ICSO dose
(concentration of the substance
which leads to 50% inhibition of the cells) is substantially different among
the cell lines rang-
ing from 2.2 (Jurkat) to > 20 mg/ml (Mg63) for trial substance 1 and from 3.6
(lymphocytes
of the peripheral blood) to > 20 mg/ml (Mg63 and HeLa) for the soluble
fraction of the blood
serum of the present invention (trial fraction 2). For all cell lines the
toxicity of the starting
substance (trial fraction 1 ) was above the toxicity of the soluble substance
(trial fraction 2).
However, it should be mentioned that the toxicity observed for the individual
cell lines was
almost identical for both trial fractions (Mg63, Rajii, lymphocytes of the
peripheral blood)
and also for the others (Jurkat, MCG-7, IMR-32) again there were 2-3-fold
differences in the
ICSO. The sensitivity of the cells to the trial fractions in comparison to the
common cancer
agent doxorubicin is depicted in Table 7.
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29
Table 7
Sensitivity of the cell lines tested against doxorubicin
Cell line ICSa, mmol/ml
Jurkat 100
Raji 20
Bro B-19 -*'i'
HeLa 400
MCF-7 150
Mg63 -*
HT1080 -*
IMR-32 4
HepG2 100
Lymphocytes of the peripheral 40
blood
Comments:
*-mmol/1= 0.58 mg/ml
* * - the sensitivity against doxorubicin was not tested.
In addition it was observed that in a concentration range from 0.3 to 3 mg/ml
depending on
the cell line and the form of the substance (i.e. trial fraction 1 or trial
fraction 2) the agent had
a stimulating effect on some cells. A stimulating effect of the serum of the
present invention
was observed for Jurkat, Raji, Bro B-19, Mg63, HT1080 and HepG2 cells. The
stimulating
effect was insignificant, (10, 20, 40%) but present for both forms of the test
substance. No
stimulation was observed for cells of the cell line HeLa, MCF-7, JMR-32 and
for lympho-
cytes of the peripheral blood. The stimulating effect of the test substance in
its first form was
observable at a much lower concentration as with the soluble fraction (trial
fraction 2). Possi-
bly the reason for this stimulation is not the stimulation of proliferation as
such but rather an
increase of the respiration of the cells which would also be detectable with
the MTT method
used for evaluating the viability of cells. The question of whether the
observed stimulatory
effect was due to a stimulation of proliferation or due to an increase in
respiration required
some further study.
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When studying the interaction between different substances with immune
competent cells in
general at least two questions have to be discussed:
1. Does a test substance have a mitogenic activity, i.e. does it have an
ability to
stimulate lymphocyte proliferation (in this case the increase of the amount of
the
substance usually leads to an increase of the DNA biosynthesis of the
lymphocytes
which can be evaluated by the incorporation of 3H-thymidin)?
2. Does a test substance have a toxic effect (this question is usually
determined by the
inhibition of lymphocyte proliferation as assayed by the amount of
incorporated
3H-thymidin or using vital dyes of the MTT-type on lymphocytes which were pre-
viously stimulated with mitogens)?
Furthermore, it has to be mentioned that non activated lymphocytes do not
proliferate in cul-
tore and that the amount of proliferation only increases with increasing
amounts of mitogens
added to the culture medium. This is reflected in the increase in radioactivly
labelled DNA.
The effect of mitogens on lymphocytes in relation to the administered doses
cam be depicted
by a so called "bell curve" (see Fig. 9). The first section of the bell curve
reflects the range of
the mitogen concentration wherein an increase of mitogen leads to an increase
of proliferation
(as measured by DNA biosynthesis) and wherein, therefore, direct relation
between mitogen
concentration and proliferation exists. The second section of the curve (2)
shows a saturation
effect wherein a further increase of the mitogen concentration does not lead
to a further in-
crease of proliferation, i.e. the mitogen has already elicited its maximal
effect. A cytotoxic
activity is not yet observed. Section (3) shows the range of the mitogen
concentration wherein
the mitogen exerts an increasingly cytotoxic effect on the lymphocytes.
The assessment of the mitogenic activity of the trial fractions 1 and 2 was
carried out in two
experiments:
1. The subject matter of this experiment was the determination of the effect
of the
trial fractions on non-activated lymphocytes of human peripheral blood. To
this
end the correlation of the proliferative activity (i.e. the effect) with the
adminis-
tered doses was determined.
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2. The subject matter of this experiment was the determination of the effect
of the
trial substance on the amount of DNA synthesis of lymphocytes activated by phy-
tohemaglotinin (FHA 20 ~g/ml) in a second phase.
When examining the substance no mitogenic activity was observed, i.e. within a
substance
concentration range of 0.1-100 mg/ml no stimulation of DNA biosynthesis of
lymphocytes of
the peripheral blood was observed (Fig. 10).
Upon increase of the dose of the trial fractions the number of lymphocytes of
the peripheral
blood did not change significantly. When assessing the effect of the trial
fractions on lympho-
cytes, which had been subsequently activated with 20 mg/ml FHA, an inhibition
of activated
lymphocytes by the trial fractions was observed.
Within the experiments it was determined that trial fractions inhibited DNA
biosynthesis of
the stimulated lymphocytes at a concentration of 0.3 mg/ml (Fig. 11). However,
the mecha-
nism of action still needs to be determined. Further experiments to elucidate
the cytostatic and
the cytotoxic effect are therefore important.
To determine how the results obtained in cell culture relate to the treatment
of human subjects
it is important to determine the substance concentration in different organs
or tissues in ani-
mal experiments. In this context it is important to investigate which
different concentrations
of the biologically active serum of the present invention is capable to
influence DNA synthe-
sis in organs that are involved in the lymphogenesis. Thus, it is important to
determine the
concentration of the biological active serum in particular in bone marrow,
spleen and thymus
of the mouse.
Apart from lymphocytes the cytotoxic activity of the serum of the present
invention was also
investigated using cells of the human milk gland carcinoma cell line MCF-7
that were spread
into a monocellular layer. Because of contact inhibition no proliferation was
observed for the
cells. In such a model the toxic effect of a substance can be determined, i.e.
it is possible to
differentiate the cytotoxic effect from the cytostatic effect. When using this
model it was de-
termined that the serum of the present invention had a cytotoxic effect in a
concentration of
2.5 mg/ml independent of the fact of whether the insoluble fraction of the
serum of the pre-
sent invention was removed or not, i.e. independent of whether trial fraction
1 or 2 was used.
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It is notable that the toxic effect is markedly decreased when incubating the
serum of the pre-
sent invention with proliferating cells which potentially is connected with
the fact that the
effect was measured 24 hours after admiW stration and not 72 hours after
administration as
was the case for proliferating cells (a prolonged incubation of monolayer
cells can lead to
death of control cells).
The above results show that the biologically active serum of the present
invention has both
cytostatic effects and a cytotoxic activity. The cytotoxic activity was,
however, less pro-
nounced as the combined cytotoxic and cytostatic activity albeit it was
observed at the same
concentration.
Taken together, blood serum from animals treated with electroshock of grade II-
III and the
subsequently prepared biological active substance leads to a 10-40% increase
proliferation of
the human cell lines Jurkat, Raji, Bro B-19, Mg63, HT1080 and HepG2, if
compared to a
control group. In HeLa, MCF-7, JMR-32 and lymphocytes of the peripheral blood
the bio-
logically active serum of the present invention exhibited a significant
inhibition of prolifera-
tion of all tested human cancer cell lines at higher dosages (2.5-20 mg/ml).
The sensitivity of
the cells towards the biological active serum of the present invention is much
stronger than
the sensitivity against the widely used anti-cancer agent doxorubicin.
Furthermore, when us-
ing the DNA biosynthesis as assessed by 3H-thymidin incorporation as a marker
the substance
of the present invention has both a cytostatic and a cytotoxic effect.
