Note: Descriptions are shown in the official language in which they were submitted.
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This invention relates to the field of medicine and
medical treatments. In particular, it relates to stress treatment
and more specifically to a method and composition for treating
m~mm~l S, including humans, in order to provide them with improved
reactions and resistance to stress.
The effects of stress on a m~mm~l normally manifest
themselves in an increase in body temperature, along with a change
in hemodynamic parameters, including an increase in heart rate and
an increase in blood pressure. For patients already suffering from
elevated blood pressure (hypertension), the effects of stress can
therefore be particularly dangerous, since hypertension is a major
risk factor for cardiovascular disease.
Stresses to which a m~mm~l may be subjected, and which
can result in these effects, can take a wide variety of physical
forms. Psychological stresses induced by restraint, confinement,
sudden exposure to danger, shock and the like translate into
physical stresses affecting one or more organs of the body.
Similarly, physical stress such as exposure to heat or cold, injury
including surgical injury, over-exertion and the like, result in
abnormal functioning of body organs. Stress is now recognized as
a major detrimental factor in many diseases such as cardiovascular
disease, cancer, and immunological dysfunction. One common
physiological factor which appears to underlie all stress responses
is the induction and upregulation of synthesis, in all body cells,
of a group of specialized intracellular proteins known as heat
stress proteins or heat shock proteins (HSP's). These HSPs function
to protect the cells from potential damage caused by whatever form
of stress is being applied.
One particular species of physical stress is ischemia,
which is the deprivation of blood flow. Ischemia in a body organ,
if severe enough, causes the eventual death of cells in the organ,
primarily by necrosis. Re-perfusion of the ischemic organ by
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resumption of blood flow thereto often results in further injury to
the organ, and does not re-invigorate already necrosed cells.
Repeated application of mild ischemic stress to an organ often
leads to an increased ability to withstand stress ischemia, an
effect thought to be partially related to upregulated synthesis of
HSPs. Ischemia may occur as a pathological condition, e.g. as the
result of spasm, thrombosis, or other blood vessel obstruction.
Ischemia may be deliberately induced by clamping of blood vessels
during surgery.
It is known to precondition the body of a m~mm~l ian
patient by subjecting it to controlled stresses, so as better to
equip the body for subsequent encounters with uncontrolled stresses
of the same type. Physical exercise and training, for example,
equips a body for better handling of physical exertion stresses.
Heating a body or a body organ repeatedly under controlled
conditions has been shown to provide the body or body organ with
preconditioning for the better handling of subsequent heat
stresses. Even in respect of ischemia, a body organ such as the
heart which has previously suffered mild ischemia is better able to
resist the effects of later ischemia, of the type causing
myocardial infarction. As stated by Gersh et al., "Preconditioning
is an important phenomenon, probably with clinical implications,
because repetitive anginal episodes in patients may develop into
full fledged infarction. Patients with pre-infarction angina may
suffer from a less severe infarct than those thought to undergo
sudden coronary occlusion without the opportunity for
preconditioning. In contrast, patients with multiple short-lived
attacks of ischemia might become tolerant through the development
of protective preconditioning, according to animal data."1
Preconditioning by subjection to heat or ischemia is however
clearly impractical in respect of most m~mm~l ian bodies and body
organs.
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U.S. Patent 4,968,483 Mueller et al., describes an
apparatus for oxygenating blood, by treating an aliquot of a
patient's blood, extracorporeally, with an oxygen/ozone mixture and
ultraviolet light, at a controlled temperature. The apparatus is
proposed for use in hematological oxidation therapy.
U.S. Patent 5,591,457 Bolton, discloses a method of
inhibiting the aggregation of blood platelets in a human, a method
of stimulating the immune system and a method of treating
peripheral vascular diseases such as Raynaud's disease, by
extracting an aliquot of blood from a patient, subjecting it to
ozone/oxygen gas mixture and ultraviolet radiation at a temperature
in the range of about 37-43~C, and then reinjecting the treated
blood into the human patient.
International Patent Application PCT/GB93/00259 Bolton,
describes a similar process for increasing the content of nitric
oxide in the blood of a m~mm~l ian patient, potentially useful in
treating conditions such as high blood pressure in m~mm~l ian
patients.
It is an object of the present invention to provide a
novel method of treating stress in a m~mm~l ian patient.
It is a further object to provide a process of
preconditioning a m~mm~l ian patient to improve the patient's
resistance and reaction to subsequently encountered stress.
According to the present invention, there is provided a
process of treating a m~mm~l ian patient to counteract the adverse
effects of stress and/or to precondition the patient for improved
resistance and reaction to subsequently encountered stress, which
comprises extracting from the patient an aliquot of blood,
subjecting the extracted blood aliquot extracorporeally to at least
one stressor selected from an oxidative environment, W radiation
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and elevated temperature, and reinjecting at least a portion of the
treated blood aliquot into the patient.
The method of the invention involves the extraction of an
aliquot of blood from the patient, the subjection of the blood
aliquot extracorporeally to stressors, and the reinjection of the
treated blood aliquot into the patient. The treatment counteracts
the effects of stress from which the patient is suffering at the
time and shortly after the patient receives the treatment. More
significantly and importantly, as a result of the treatment,
preferably a series of treatments, the patient is better equipped
to withstand the adverse effects of subsequently encountered
stress. The treatment process according to the present invention
causes the m~mm~lian patients, when subsequently stressed, to
exhibit decreased stress response as detected by smaller rises in
body temperature, smaller increases in heart rate and/or smaller
increases in diastolic blood pressure.
The size of the blood aliquot to be treated is, in the
case of human patients, generally from about 0.1 ml to about 400
ml, preferably from about 0.1-100 ml and most preferably 5-15 ml,
with suitable prorating according to relative body weight for non-
human patients. It is preferred to subject the blood aliquot to
all three of the aforementioned stressors (blood temperature above
body temperature, an oxidative environment such as a mixture of
ozone and oxygen introduced into the blood aliquot, and ultraviolet
radiation), simultaneously. Care must be taken not to utilize an
excessive level of the stressors, to the extent that the cell
mem~branes of the white cells of the blood are caused to be
disrupted.
The temperature stressor must keep the aliquot in the
liquid phase and should not heat it above about 45~C. Any suitable
source of heat known in the art may be employed to heat the blood,
preferably one or more infrared lamps. The temperature stressor
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preferably warms the aliquot being treated to a temperature above
normal body temperature, i.e. to about 38-45~C, and most preferably
from about 38-43~C, e.g. about 42.5~C. Preferably the temperature
of the blood aliquot is maintained at this elevated temperature
during the treatment with W and ozone. Alternatively, however,
the blood sample can be heated while being subjected to W
radiation, until the blood reaches a predetermined temperature
(preferably about 42.5~C), at which point bubbling of ozone gas
through the blood is commenced. The concurrent W /ozone treatment
is then maintained for a predetermined period of time from about
to about 10 minutes, and preferably about 1-5 minutes, most
preferably about 3 minutes.
The application of the oxidative stressor preferably
involves exposing the aliquot to a mixture of medical grade oxygen
and ozone gas, most preferably by bubbling through the aliquot, at
the aforementioned temperature range, a stream of medical grade
oxygen gas having ozone as a minor component therein. The ozone
gas may be provided by any conventional source known in the art.
Suitably the gas stream has an ozone content of from about 1.0-100
~g/ml, preferably 3-70 ~g/ml and most preferably from about 5-50
~g/ml. The gas stream is supplied to the aliquot at a rate of from
about 0.01-2 litres per minute, preferably 0.05-1.0 litres per
minute, and most preferably at about 0.06-0.18 litres per minute
(STP).
The ultraviolet radiation stressor is suitably applied
by irradiating the aliquot under treatment from an appropriate
source of W radiation, while the aliquot is maintained at the
aforementioned temperature and while the oxygen/ozone gaseous
mixture is being bubbled through the aliquot. The ultraviolet
radiation may be provided by any conventional source known in the
art, for example by a plurality of low-pressure ultraviolet lamps.
The method of the invention preferably utilizes a standard W-C
source of ultraviolet radiation, namely W lamps emitting primarily
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in the C-band wavelengths, i.e. at wavelengths shorter than about
280 nm. Ultraviolet radiation corresponding to standard W-A and
W-B sources can also be used. Preferably employed are low-
pressure ultraviolet lamps that generate a line spectrum wherein at
least 90~ of the radiation has a wavelength of about 253.7 nm. An
appropriate dosage of such W radiation, applied simultaneously
with the aforementioned temperature and oxidative environment
stressors, is obtained from lamps with a power output of from about
15 to about 25 watts, at the chosen W wavelength, arranged to
surround the sample container holding the aliquot, each lamp
providing an intensity, at a distance of 1 meter, of from about 35
- 75 mW/sq.cm. Several such lamps surrounding the sample bottle,
with a combined output at 253.7 nm of 15 - 25 watts, operated at
maximum intensity, may advantageously be used. At the incident
surface of the blood, the W energy supplied is typically 0.25
Joules per cm2. Such a treatment provides a blood aliquot which is
appropriately modified according to the invention ready for re-
injection into the patient.
The time for which the aliquot is subjected to the
stressors can be from a few seconds to about 60 minutes. It is
normally within the time range of from about 0.5 - 60 minutes.
This depends to some extent upon the chosen intensity of the W
irradiation, the temperature and the concentration of and rate at
which the oxidizing agent is supplied to the aliquot. Some
experimentation to establish optimum times and dosages may be
necessary on the part of the operator, once the other stressor
levels have been set. Under most stressor conditions, preferred
times will be in the approximate range of about 0.5 - 10 minutes,
most preferably 2 - 5 minutes, and normally around 3 minutes. The
starting blood temperature, and the rate at which it can be warmed
or cooled to a predetermined temperature, tends to vary from
patient to patient.
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In the practice of the preferred process of the present
invention, the blood aliquot (or the separated cellular fractions
of the blood, or mixtures of the separated cells, including
platelets, these various leucocyte-containing combinations, along
with whole blood, being referred to collectively throughout as the
"aliquot") may be treated with the stressors using an apparatus of
the type described in U.S. patent 4,968,483 Mueller. The aliquot
is placed in a suitable, sterile, W-radiation-transmissive
container, which is then fitted into the machine. The temperature
of the aliquot is adjusted to the predetermined value, e.g. 42.5~C,
by the use of a suitable heat source such as an IR lamp, and the W
lamps are switched on for a fixed period before the gas flow is
applied to the aliquot providing the oxidative stress, to allow the
output of the UV lamps to stabilize. Then the oxygen/ozone gas
mixture, of known composition and controlled flow rate, is applied
to the aliquot, for the predetermined duration of 0.5 - 60 minutes,
preferably 2-5 minutes and most preferably about 3 minutes as
discussed above, so that the aliquot experiences all three
stressors simultaneously. In this way, the blood aliquot is
appropriately modified according to the present invention
sufficient to achieve the desired effects.
The process of the present invention shows utility both
in treating a patient's stress symptoms evident at the time the
treatment is administered, and in preconditioning a m~mm~l ian
patient against the adverse effects of subsequently encountered
stress, of any of the aforementioned types. It is not specific to
providing tolerance to a specific stress or type of stress, but
appears to be of general application. A patient who has undergone
a treatment or a series of treatments according to the process of
the present invention will exhibit notably reduced adverse
reactions to subsequently encountered stress, such as a notably
reduced rise in body temperature and/or a reduced increase in heart
rate and/or a reduced increase in diastolic blood pressure, in
response to stress, as compared with a similar but untreated
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patient. The process is thus particularly useful for patients who
are scheduled to undergo stress such as surgery at a predetermined
future date. They can precondition their bodies to be ready for
surgery by undergoing a treatment or a series of treatments
according to the invention prior to surgery, with the result that
they will withstand the surgery better and will recover from it
more quickly.
Another preferred use of the present invention is in
protecting tissues and organs from stress-induced damage, in a
manner similar to ischemic preconditioning. As noted previously,
repetitive mild ischemic (anginal) episodes can render tissues and
organs less susceptible to stress-induced damage, by ischemic
preconditioning, although application of ischemic preconditioning
by current methods is largely impractical. The process of the
present invention can take the place of ischemic preconditioning,
ischemia being a species of physical stress. Accordingly, the
process of the present invention offers potential for treatment of
unstable angina and decrease of infarct size, a treatment not
effectively addressed by available therapies.
Similarly, the process of the present invention is
applicable in the protection of body organs destined for
transplantation. Treatment of the donor body by the process of the
present invention serves to protect body organs against damage
resulting from the inevitable ischemia which the organ will suffer
on removal from the donor body, transportation and subsequent
surgical introduction into the recipient body. The treatment
according to the invention extends the useful life of the
transplant organ between its removal from the donor body and its
surgical introduction into the recipient body, thereby reducing
losses of viable transplant organs due to transportation delays.
A further, specific clinical application of the process
of the invention is in treatment of patients suffering from
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transient ischemic attacks (TIA's, pre-strokes), which are due to
temporary obstruction of blood flow to certain areas of the brain.
They commonly indicate the likelihood of suffering a major stroke
in the near future. Subjection of such patients to treatment
according to the process of the invention, at the onset of TIA's,
will precondition the brain to avoid or at least to lessen the
severity of the effects of the forthcoming major stroke.
The beneficial effects of the present invention have been
~emo~strated in vivo by clinical experiments on juvenile and adult
rats, specifically rats of an inbred strain of genetically
hypertensive rats (SHR's). Genetically hypertensive rats (SHR's)
are the most widely used animal model for hypertension research,
and are well known and readily available to researchers in this
field. SHR's have several genetic defects, one of the most
important being failure to produce appropriate amounts of HSPs when
subjected to stress. SHR's develop hypertension rapidly and exhibit
exaggerated increase in heart rate, blood pressure and body
temperature in response to stress. They represent a model of
hypersensitivity to stress. The results obtained using them
provide reliable indications of potential results obtainable with
human patients.
In the accompanying drawings:
Figure 1 is a graphical representation of the results
obtained according to specific Example 1 described below;
Figure 2 is a graphical presentation of the body
temperature results obtained according to Example 2 described
below;
Figure 3 is a graphical presentation of the heart rate
results obtained according to Example 2 described below;
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Figure 4 is a graphical presentation of the diastolic
blood pressure results obtained according to Example 2 described
below;
Figure 5 is a graphical presentation of the body
temperature results obtained according to Example 3 described
below;
Figure 6 is a graphical presentation of the body
temperature results obtained according to Example 4 described
below;
Figure 7 is a graphical presentation of the body
temperature results obtained according to Example 5 described
below; and
Figure 8 is a graphical presentation of the heart rate
results obtained according to Example 5 described below.
EXAMPLE 1
Blood from sacrificed SHR's of the same strain as the
test animals was collected, treated with sodium citrate
anticoagulant and pooled. A portion of the blood was then placed
in a sterile container, and subjected simultaneously to the W
radiation, ozone/oxygen gas oxidative environment and elevated
temperature stressors, in an apparatus as generally described in
the aforementioned Mueller Patent U.S. 4,968,483. More
specifically, the blood sample in the sterile, W-transparent
container was heated using infrared lamps to 42.5~C, and whilst
being maintained at that temperature, it was subjected to W
radiation of wavelength 253.7 nm under the preferred conditions
previously described. Simultaneously, a gaseous mixture of medical
grade oxygen and ozone, of ozone content 13.5-15.5 ~g/ml, was
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bubbled through the blood sample at a rate of flow over a range
from 60 mls/min increasing eventually to about 180 mls/min. The
time of simultaneous W exposure and gas mixture feed was 3
minutes.
A further portion of the pooled blood, to act as a
control, was similarly placed in a sterile container and placed in
the aforementioned apparatus, but was not heated, nor subjected to
W radiation nor subjected to application of any ozone/oxygen gas
mixture. In addition, to provide a further control, a sterile
aqueous physiological saline solution was prepared.
A total of 44 seven week old SHR's were selected and
divided into three groups, group A containing 15 animals, group B
containing 15 animals, and group C containing 14 animals. For a
period of 10 days (at 7-9 weeks of age), each animal of group A
received a daily intragluteal injection of 150 ~l of the W, heat
and ozone treated blood. Each animal of group B received at the
same time a similar daily injection of the untreated blood. Each
animal of group C received at the same time a similar injection of
physiological saline.
At the age of 9 weeks, 4 days after completion of the
injections the animals were anaesthetized and, a telemetry probe
was inserted surgically into the femoral artery of each animal.
The telemetry probe (trade-mark DATAQUEST LABPRO, available from
Data Sciences International) is a commercially available probe
equipped with a radio transmitter, to permit heartbeat, systolic
blood pressure, diastolic blood pressure and other signals to be
received and recorded without further handling of the animals,
which might induce further, uncontrolled stress reactions. An
additional probe was surgically inserted into the peritoneal cavity
of each animal, to measure body temperature.
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Continuous daily recordings of body temperature, blood
pressure and heart rate were made from each ~n;m~l, during the 10
day period following the surgery, readings being taken during the
inactive, at-rest portion of the animals' daily cycle, i.e. the
daylight ("light-on") portion, and during the activity time
(night). Group A animals which received injections of blood
treated according to the process of the invention demonstrated a
significantly more rapid recovery of normal body temperature
following surgery (6 days vs. 10 days), as compared with group B
10 ~n;m~l S which received untreated blood and group C animals which
received saline injections, as shown especially by the readings
taken during the resting periods. The differences are less evident
from night-time, activity phase readings, suggesting that the
higher cortisol levels present during activity may have an
influence on the results. This demonstrates a significant effect of
the treatment of the present invention on lessening the m~mm~l ian
body's response to the stress of surgery.
The results of these experiments are presented
graphically on Figure 1, a plot of measured body temperatures
against days after surgery, each plotted value being the mean of
values obtained from the whole group during the at-rest periods.
Curve A is derived from group A animals, curve B from group B
animals and curve C from group C animals.
EXAMPLE 2
The 44 animals treated as described in Example 1, namely
the Group A of 15 animals which had received injections of blood
treated according to the process of the invention, Group B of 15
~n;m~l S which had received injection of untreated blood, and Group
C which had received injection of saline, 10 days after the probe
implantation surgery described in Example 1, were subjected to
psychological stress through standard immobilization stress test,
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by placing them in small restraint cages for a period of 30 minutes
(age of animals - 11 weeks). During this immobilization period,
readings of body temperature, blood pressure and heart rate, at one
minute intervals, were recorded.
Accompanying Fig. 2 of the drawings is a graphical
presentation of the results of the body temperature measurements of
the three groups, namely a plot of time as ordinate against body
temperature as abscissa over the 30 minute duration of the
immobilization stress test. As Fig. 2 shows, curve 2A derived from
experimental Group A is consistently and significantly below curve
2B obtained from control Group B and curve 2C obtained from control
Group C. Statistical analysis of all the data obtained confirms
the high significance of the differences in the figures obtained
from experimental Group A.
Figure 3 of the accompanying drawings presents
graphically the results of heart rate measurements on the three
groups, with heart rate (beats per minute, bpm) plotted as ordinate
against time of the stress test. Again, the results (averaged over
the ~nim~l s in each group) show that the group which received the
injections of blood treated according to the invention, Group A,
had a lower increase in heart rate, as compared with the other two
groups, over substantially the entire duration of the test. The
differences between the respective groups are statistically
significant.
Figure 4 of the accompanying drawings presents
graphically the results of the measurements of diastolic blood
pressure of each of the three test groups, with diastolic blood
pressure (mm/Hg) plotted as ordinate against time of the stress
test. Again, the values from Group A (averaged), the group which
had received injections of blood treated according to the
invention, are consistently and significantly lower than those from
the other two groups.
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EXAMPLE 3
The Group C animals from Example 2, i.e. the control
group which had, at age 7-9 weeks, received injections of
physiological saline, were divided into three sub-groups Ca, Cb,
and Cc. Each group was given a course of 10 daily injections of,
respectively, 150 ~l of the treated blood, 150 ~l of the untreated
blood, and 150 ~l of physiological saline. The course of injection
started when the animals were 12 weeks old, i.e. fully matured
adults. The telemetry probes remained in place. The same 30
minute immobilization stress test was performed on each animal, at
age 16 weeks, and measurement of heart rate, blood pressure and
body temperature were taken.
In body temperature response, the group Ca injected with
blood treated according to the process of the invention showed a
significantly more blunted increase during the stress period. This
is illustrated in Fig. 5, a graphical presentation of the results
similar to Fig. 2. It can be seen that curves Ca, derived from the
Group Ca animals, is consistently lower than curve Cb derived from
Group Cb animals and consistently lower than curve Cc derived from
Group Cc ~n;m~l S .
EXAMPLE 4
The Group A experimentally treated animals from Example
2 which had received injections of blood treated according to the
invention at age 7-9 weeks were divided into three sub-groups,
labelled Aa, Ab and Ac, five animals in each group. They were then
subjected to a second series of 10 daily injections of 150 ~l of,
respectively, the treated blood, the untreated blood and the
physiological saline. The course of injections started when the
~nlm~l S were 12 weeks old. The telemetry probes were left in place
from Example 1, so that the surgery did not need to be repeated.
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The animals were then subjected again, at 16 weeks of age, to the
same immobilization stress test as described in Example 2 for 30
minutes, and measurements taken at 1 minute intervals of heart
rate, blood pressure and body temperature.
The differences between the groups with regard to body
temperature rise were very significant. They are illustrated on
Fig. 6, a plot of body temperature against time, based on averages,
similar to Fig. 2. After about the 12th minute of the test, group
Aa which had received 2 courses of injection with the blood treated
according to the invention exhibited consistently and significantly
the lowest rise in body temperature. Curve Aa derived from Group
Aa is consistently below curve Ab derived from Group Ab and curve
Ac derived from Group Ac. It will be observed that the values on
curve Aa are also lower than those on curve A of Fig. 2, indicating
that a second treatment according to the invention has additional
benefits on conditioning the animals for tolerance of stress. In
contrast, the values on curve Ac are higher than the values on
curve A of Fig. 2, and lower than the values on curve C of Fig. 2,
indicating that the effects of the treatment tend to be lost after
about 24 days from the conclusion of the course of treatment,
absent a second "booster" treatment according to the invention.
EXANPLE 5
The stress responses of animals from Example 1 which had
been given two courses of injection with the same fluid (saline
followed by saline, untreated blood followed by untreated blood,
and treated blood followed by treated blood), at 7 weeks of age and
12 weeks of age, were measured during a second immobilization
stress test, conducted as previously described, on animals aged 16
weeks. The results obtained from body temperature measurements are
shown on Fig. 7, a graph of body temperature against time during
the 30-minute stress test, similar to Fig. 2. Curve AA is derived
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from the measurements (average of 5 animals) of animals which had
received two courses of ten daily injections of blood treated
according to the invention. Curve BB is derived from the
measurements (average of 5 animals) of animals which received two
such courses of injections of untreated blood. Curve CC is derived
from the measurements (average of 5 animals) of animals which
received two such courses of saline injections. As the Figure
shows, the values obtained from ~n;m~l S treated according to the
process of the present invention are consistently and significantly
lower than those derived from the other two groups.
Fig. 8 of the accompanying drawings similarly presents
the heart rate measurements for the three groups, during the stress
test. Again, Curve AA derived from animals which had received two
courses of injection of blood treated according to the invention is
significantly lower than the other two curves.
