Note: Descriptions are shown in the official language in which they were submitted.
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1
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 mammals, including humans, in order to provide them with
improved reactions and resistance to stress.
The effects of stress on a mammal 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 mammal 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
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organ by 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 mammalian
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
mammalian 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 mammalian patient, potentially useful in
treating conditions such as high blood pressure in mammalian
patients.
It is an object of the present invention to provide a
novel method of treating stress in a mammalian patient.
It is a further object to provide a process of
preconditioning a mammalian patient to improve the patient's
resistance and reaction to subsequently encountered stress.
According to one aspect of the present invention, there
is provided a process of treating a mammalian 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
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oxidative environment, W radiation 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 mammalian 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.
Thus, from another aspect, the present invention
provides the use for preparing a medicament for treating stress
in a patient and for preconditioning a patient to better
withstand the adverse effects of subsequently encountered stress,
of an aliquot of blood which has been subjected extracorporeally
to at least one stressor selected from an oxidative environment,
W radiation and elevated temperature.
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 subj ect the blood aliquot
to all three of the aforementioned stressors (elevated
temperature, an oxidative environment such as a mixture of ozone
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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
membranes of the white cells of the blood are caused to be
5 disrupted, or other irreversible damage is caused to an excessive
number of the cells in the blood.
The temperature stressor must keep the aliquot in the
liquid phase and should not heat it above about 45°C.
The term "elevated temperature" as used herein means
a temperature higher than that which the blood attains at the
start of the subjection of the blood to the stressors in the
process of the invention. Depending upon the precise method of
handling the blood aliquot, its temperature at the start of the
process could be as low as 15°C. Whilst the blood aliquot is at
body temperature (c. 37°C) when first extracted from the patient,
the act of extraction, the addition of anticoagulant, the
introduction into the treatment apparatus and the storage of the
blood aliquot may all exercise a cooling effect on the blood, to
bring its temperature down to as low as 15°C when the process
starts. Accordingly the "elevated temperature stressor" applied
in the process of the invention is a heating above this
introductory temperature. Any suitable source of heat known in
the art may be employed to heat the blood, preferably one or more
infrared lamps. Thus, in a preferred process of the present
invention, the blood aliquot is subjected to infra-red radiation
as a stressor, alone or in combination with the other stressors
namely W radiation and an oxidative environment, the infra-red
radiation normally but not necessarily causing heating of the
blood aliquot.
The temperature stressor preferably warms the aliquot
being treated to a temperature above normal body temperature,
i.e. to about 38-44°C, and most preferably from about 38-43°C,
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e.g. about 42.5°C. Preferably the temperature of the blood
aliquot is maintained at this elevated temperature during the
treatment with UV and ozone. Alternatively, however, the blood
sample can be heated while being subjected to UV radiation, until
the blood reaches a predetermined temperature (preferably about
42.5°C), at which point bubbling of oxygen-ozone gas through the
blood is commenced. The concurrent UV/oxygen/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 0.06-0.30 litres per minute (STP).
The ultraviolet radiation stressor is suitably applied
by irradiating the aliquot under treatment from an appropriate
source of UV 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 UV-C source of ultraviolet radiation, namely UV lamps
emitting primarily in the C-band wavelengths, i . a . at wavelengths
shorter than about 280 nm. Ultraviolet radiation corresponding
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to standard UV-A and UV-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 UV radiation,
applied simultaneously with the aforementioned temperature and
oxidative environment stressors, is obtained from lamps with a
power output of from about 5 to about 25 watts, preferably about
5 to about 10 watts, at the chosen UV wavelength, arranged to
surround the sample container holding the aliquot. Each such
lamp provides an intensity, at a distance of 1 meter, of from
about 40-80 micro watts per square centimetre. Several such
lamps surrounding the sample container, with a combined output
at 253.7 nm of 15-40 watts, preferably 20-40 watts, operated at
maximum intensity, may advantageously be used. At the incident
surface of the blood, the UV energy supplied may be from about
0.25 - 4.5 J/cm2 during a 3-minute exposure, preferably 0.9-1.8
J/cmz. 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 UV
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, UV-radiation-
transmissive container, which is then fitted into the machine.
The temperature of the aliquot is adjusted to the predetermined
value, e.g. 42.5~1°C, by the use of a suitable heat source such
as an IR lamp, and the UV 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.
Whilst the preferred process of the present invention
uses the mammalian patient's own blood as the source of the
aliquot for treatment, it is possible to use another patient's
blood as the source provided that it has the required very high
degree of compatibility.
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 mammalian
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,
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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 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, thus reducing the time of
hospitalization.
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
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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
5 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
10 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 demonstrated 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:
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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;
Figure 4 is a graphical presentation of the diastolic
blood pressure results obtained according to Example 2 described
below;
Figure S 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; and
Figures 9, 10, 11 and 12 are graphical presentations
of results obtained according to Example 6 below.
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EXAMPhE 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 bubbled through the blood sample at a rate of flow over a
range from 60 mls/min increasing eventually to about 180-240
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 UV,
heat and ozone treated blood. Each animal of group B received
at the same time a similar daily injection of the untreated
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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.
Continuous daily recordings of body temperature, blood
pressure and heart rate were made from each animal, 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 animals 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
mammalian 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
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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 l,
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 animals 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, 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 animals in each group) show that the group
CA 02230836 1998-02-27
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
5 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
10 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.
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 inj ections of ,
respectively, 150 ~Cl of the treated blood, 150 ~1 of the
untreated blood, and 150 ~1 of physiological saline. The course
of inj ection started when the animals were 12 weeks old, i . a .
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
CA 02230836 1998-02-27
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curve Cb derived from Group Cb animals and consistently lower
than curve Cc derived from Group Cc animals.
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
~1 of, respectively, the treated blood, the untreated blood and
the physiological saline. The course of injections started when
the animals were 12 weeks old. The telemetry probes were left in
place from Example 1, so that the surgery did not need to be
repeated. 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
CA 02230836 1998-02-27
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of the course of treatment, absent a second "booster" treatment
according to the invention.
EXAMPLE 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 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 animals 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.
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EXAMPhE 6
In further demonstrations of the use of the process of
the present invention in preconditioning mammalian body organs
and tissues against the adverse effects of ischemic stress and
subsequent reperfusion, experiments were performed on genetically
hypertensive rats (SHRs), of the type previously described, by
inducing ischemia in the animals' kidneys after they had been
treated by the process of the invention. The procedure was as
follows:
A group of 63 SHRs was divided into two approximately
equal sub-groups, A and B. Sub-group A was given two courses of
injections of blood from the pool described in Example l, the
injected blood having been treated with ultraviolet light, ozone-
oxygen gas and elevated temperature stressors simultaneously,
also as described in Example 1. The first course of injections
started at age 7 weeks, and comprised 10 injections, over a
period of 10 days, of a 150 ~.l aliquot of the treated blood
intragluteally injected. The second course of injections
commenced at age 12 weeks, and comprised 10 injections,
administered daily,. of the same volumes of treated blood
administered in the same manner. The animals of sub-group B were
given injections of physiological saline, at the same times and
in the same quantities, and thus acted as controls.
One day following the second course of injections, the
rats were anaesthetized under light gas anaesthesia, and the
right kidney of each animal was removed through back incision.
An occlusive clip was placed on the remaining renal artery and
vein, to expose the left kidney to transient ischemia, for 60
minutes. The skin was temporarily closed by clips, and the
animals were kept freely moving for the period of the whole
ischemia. The animals were then followed with respect to the
degree of injury resulting from the ischemia and/or the
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subsequent reperfusion, by taking blood samples for determining
serum creatinine and blood urea nitrogen (BUN) and by determining
survival rates. After taking the initial blood sample, the skin
was definitively closed.
The survival rate was monitored by daily count of
survivors. After 14 days, 55% of the sub-group A animals which
had received the treated blood injections were surviving,
compared with only 32~ of the sub-group B, saline-saline treated
animals. The survival rates are shown in more detail on
accompanying Figure 9, a plot of percentage of survivors as
ordinate against days after renal ischemia as abscissa. On Fig.
9, the solid line derives from the results from the animals of
sub-group A, and the dotted line derives from the results from
the animals of sub-group B.
The results show a significant (p<0.05, log rank
test), improvement in animals which received treatment according
to the invention, indicating successful preconditioning against
injury resulting from the ischemia and/or the subsequent
reperfusion, by the process of the invention.
Blood samples of volume 100-200 ~1 were taken from the
surviving re-perfused animals at days 1, 3, 6 and 12 after the
ischemia, from the tail artery, and were analysed for creatinine
content and for blood urea nitrogen (BUN) content. High
creatinine content indicates impaired renal function, in that
kidneys are not functioning to clear creatinine content from the
blood adequately. BUN content is similarly a measure of the
efficiency of renal function - the lower the BUN and creatinine
content of the blood, the more efficient the renal function.
Both tests are standard determinations of renal function in
mammalian patients.
CA 02230836 1998-02-27
Fig. 10 of the drawings is a graphical presentation of
the results of the BUN content determinations, averaged over each
group of animals. Each curve is a plot of BUN values against
days after ischemia. Curve lOB is derived from control sub-group
5 B animals, curve l0A from sub-group A animals which received the
treated blood. At day 6, the average BUN value for sub-group A
animals was significantly lower (p = <0.025).
Fig. 11 of the accompanying drawings similarly gives
10 a graphical presentation of the results of serum creatinine
measurements, with the averaged curve 11A from the treated, sub-
group A animals being consistently and significantly lower than
the averaged curve from the control sub-group B animals, at days
1,3 and 6 (p=<0.025). These results show consistently lower
15 creatinine levels and lower BUN levels in samples from animals
receiving treatment according to the invention, demonstrating
that the treatment effectively preconditions the individual
organs and tissues of the body for better resistance to
subsequent ischemia stress and/or reperfusion injury.
The increase in creatinine and BUN in each sub-group
peaked at day 3, with a return to basal values at day 12. This
peak increase in the sub-group A animals is significantly lower.
Six days after ischemia/reperfusion, the levels of both
creatinine and BUN in the samples from the sub-group A animal are
about half those in the samples from the control animals. In the
first 24 hours after ischemia, 83% of the rats in control sub-
group B were oliguric (less than 2 ml of urine passed in 24
hours) whereas oliguria was present in only 42% of the sub-group
A rats (p<0.01). This is shown in accompanying Figure 12, a bar
plot of number of animals in each group passing urine during the
24 hours after renal ischemia/reperfusion in volume appropriate
to classify the animals as oliguric, mildly oliguric or normal.
The solid bars on Fig. 12 derive from the animals of sub-group
A, the open bars from the animals of sub-group B.
CA 02230836 1998-02-27
21
Injury to an organ subjected to ischemia can occur as
a consequence of the ischemia alone (e.g. in cases where ischemia
is caused by a blood clot which is subsequently dissolving, but
blood does not flow back into the affected area of the organ
after the clot is dissolved - the so-called "no-reflow"
phenomenon), or by the subsequent reperfusion of the organ with
blood after ischemia, when leukocytes or free radicals or the
like may damage the blood vessels and cells of the organ. The
term "injury resulting from ischemia and/or reperfusion" and the
term "ischemia/reperfusion injury" as used herein cover both
types of injury, occurring alone or in combination.
The above results of the procedure of the present
invention can protect the kidney from injury resulting from
ischemia and/or reperfusion, as measured by survival rate, blood
urea and creatinine levels and urinary flow in the SHR. This
indicates use of the procedure in protecting body organs in
general from ischemia/reperfusion injury, including the heart,
the liver, the brain, the spinal cord and other vital organs and
tissues as well as the kidney, and indicates practical
application of the procedure on patients scheduled to undergo
surgical procedures involving ischemia/reperfusion of a body
organ, such as surgical repair, removal or transplantation of a
body organ.
In particular, ischemia acute renal failure is an
important clinical problem with high morbidity and high
mortality. The process of the present invention presents a novel
approach to combatting this disorder. It can be adopted prior
to kidney transplantation, on either or both the donor or
recipient. It can be adopted prior to kidney revascularization.
It can be adopted prior to invasive evaluation in high risk
subjects, e.g. angiography in diabetics. It can be adopted prior
to abdominal aortic surgery and renal bench surgery (i.e. where
CA 02230836 1998-02-27
22
the kidney is temporarily removed and operated on ex vivo, and
then re-implanted).
As regards its use in connection with procedures
involving the heart, the procedure of the invention can be
conducted prior to coronary angioplasty, and bypass, or prior to
transplantation, as in the case of the kidney. It is indicated
for use with patients about to undergo open heart surgery with
cardio-pulmonary bypass for coronary artery bypass grafting,
valve replacement or surgical repair of congenital or acquired
cardiac structural abnormalities. In the case of the brain or
other vital organs and tissues including the intestines, the
kidneys and limbs, the procedure of the invention can be used
prior to angioplasty or endarterectomy, in high risk subjects.
The use of the process of the present invention prior
to general anaesthesia in connection with major surgery can be
viewed as general pre-conditioning of the body, to better
withstand ischemic-reperfusion injuries to which the major organs
will later be subjected. It is indicated for use prior to
conducting major surgical procedures involving general
anaesthesia in patients known to have or likely to have a
significant degree of underlying atherosclerosis in the arteries
supplying the brain, heart, liver, intestine, spinal cord,
kidneys or limbs, the atherosclerosis rendering them more
susceptible to a thrombo-ischemic event in the operative or post-
operative period. Such similar general pre-conditioning of the
body by the process of the invention is also indicated for use
in alleviating the effects of subsequently encountered shock,
leading to under-perfusion of vital organs and tissues through
failure to cardiac action loss of blood or other body fluids,
excessive dilation of blood vessels and excessively low blood
pressure. Examples include major blood loss, trauma, sepsis and
cardiogenic shock. Individuals likely to be exposed to such
hazards, including patients awaiting surgery, troops about to
CA 02230836 1998-02-27
23
enter combat, rescue and relief crews for natural disasters,
would be beneficiaries of the process of the invention.
Other areas of utility of the procedure of the present
invention, in connection with preconditioning of a patient body
and body organ prior to subjection to stress, e.g. ischemic
stress as part of a surgical procedure, general psychological
stress or physiological stress, as part of surgery or other
foreseeable stress-situations, will be apparent to those skilled
in the art.
