Title: Method for treating
mammals with modified mammalian blood
United States Patent: 6,986,888
Issued: January 17, 2006
Inventors: Bolton; Anthony E. (Ontario,
Assignee: Vasogen Ireland Limited (IE)
Appl. No.: 564583
Filed: May 5, 2000
Executive MBA in Pharmaceutical Management, U. Colorado
An improved method for treating or
preventing a pathological condition in a mammalian subject in which
modified mammalian blood is administered to the subject. The method
comprises administering to the subject from two to four aliquots of
modified mammalian blood, with the administration of any pair of
consecutive treatments either being on consecutive days or being separated
by a rest period of l to 21 days. The treatment is useful for treating or
preventing a wide range of pathological conditions in which modified
mammalian blood is effective, including preconditioning a mammalian
subject to better withstand the adverse effects of ischemic stress,
atherosclerosis and rheumatoid arthritis.
OF PREFERRED EMBODIMENTS
According to a preferred process of the
present invention, an aliquot of blood is extracted from a mammalian
subject, preferably a human, and the aliquot of blood is treated ex vivo
with certain stressors, described in more detail below. The teens
"aliquot", "aliquot of blood" or similar terms used herein include whole
blood, separated cellular fractions of the blood including platelets,
separated non-cellular fractions of the blood including plasma, plasma
components, and combinations thereof. The effect of the stressors is to
modify the blood, and/or the cellular or non-cellular fractions thereof,
contained in the aliquot. The modified aliquot is then re-introduced into
the subject's body by any suitable method, most preferably intramuscular
injection, but also including subcutaneous injection, intraperitoneal
injection, intra-arterial injection, intravenous injection and oral, nasal
or rectal administration.
The stressors to which the aliquot of blood is subjected ex vivo according
to the method of the present invention are selected from temperature
stress (blood temperature above or below body temperature), an oxidative
environment and an electromagnetic emission, individually or in any
combination, simultaneously or sequentially. Suitably, in human patients,
the aliquot has a sufficient volume that, when re-introduced into the
patient's body, has the desired effect.
Preferably, the volume of the aliquot is up to about 400 ml, preferably
from about 0.1 to above 100 ml, more preferably from about 5 to about 15
ml, even more preferably from about 3 to about 12 ml, and most preferably
about 10 ml.
It is preferred, according to the invention, to apply all three of the
aforementioned stressors simultaneously to the aliquot under treatment, in
order to ensure the appropriate modification to the blood. It may also be
preferred in some embodiments of the invention to apply any two of the
above stressors, for example to apply temperature stress and oxidative
stress, temperature stress and an electromagnetic emission, or an
electromagnetic emission and oxidative stress. Care must betaken to
utilize an appropriate level of the stressors to thereby effectively
modify the blood to achieve the desired effect.
The temperature stressor warms the aliquot being treated to a temperature
above normal body temperature or cools the aliquot below normal body
temperature. The temperature is selected so that the temperature stressor
does not cause excessive hemolysis in the blood contained in the aliquot
and so that, when the treated aliquot is injected into a subject, the
desired effect will be achieved. Preferably, the temperature stressor is
applied so that the temperature of all or a part of the aliquot is up to
about 55° C., and more preferably in the range of from about -5° C. to
about 55° C.
In some preferred embodiments of the invention, the temperature of the
aliquot is raised above normal body temperature, such that the mean
temperature of the aliquot does not exceed a temperature of about 55° C.,
more preferably from about 40° C. to about 50° C., even more preferably
from about 40° C. to about WC, and most preferably about 42.5° C.
In other preferred embodiments, the aliquot is cooled below normal body
temperature such that the mean temperature of the aliquot is within the
range of front about -5° C. to about 36.5° C., even more preferably from
about 10° C. to about 30° C., and even more preferably from about 15° C.
to about 25° C.
The oxidative environment stressor can be the application to the aliquot
of solid, liquid or gaseous oxidizing agents. Preferably, it 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 content of the gas stream and the flow
rate of the gas stream are preferably selected such that the amount of
ozone introduced to the blood aliquot, either on its own or in combination
with other stressors, does not give rise to excessive levels of cell
damage such that the treatment is rendered ineffective Suitably, the gas
stream has an ozone content of up to about 300 μg/ml, preferably up to
about 100 μg/ml, more preferably about 30 μg/ml even more preferably up to
about 20 μg/ml, particularly preferably from about 10 μg/ml to about 20 μg/ml,
and most preferably about 14.5±1.0 μg/ml. The gas stream is suitably
supplied to the aliquot at a rate of up to about 2.0 liters/min,
preferably up to about 0.5 liters/min, more preferably up to about 0.4
liters/min, even more preferably up to about 0.33 liters/min, and most
preferably about 0.24±0.024 liters/min. The lower limit of the flow rate
of the gas stream is preferably not tower than 0.01 liters/min more
preferably not lower than 0.1 liters/min, and even more preferably not
lower than 0.2 liters/min.
The electromagnetic emission stressor is suitably applied by irradiating
the aliquot under treatment from a source of an electromagnetic emission
while the aliquot is maintained at the aforementioned temperature and/or
while the oxygen/ozone gaseous mixture is being bubbled through the
aliquot. Preferred electromagnetic emissions are selected from photonic
radiation, more preferably ultraviolet (UV), , visible and infrared light,
and even more preferably UV light. The most preferred sources of U V light
are UV lamps emitting primarily UV-C band wavelengths, i.e. wavelengths
shorter than about 280 nm. Such lamps may also emit amounts of visible and
infrared light. Sources of W light corresponding to standard UV-A
(wavelengths from about 315 to about 400 nm) and U V-B (wavelengths from
about 280 to about 315) can also be used. For example, an appropriate
dosage of such UV light, applied simultaneously with one or both of the
aforementioned temperature and oxidative environment stressors, can be
obtained from up to eight lamps arranged to surround the sample container
holding the aliquot, operated at an intensity to deliver a total UV light
energy at the surface of the blood of from about 0.025to about 10
joules/cm2, preferably from about 0.1to about 3.0 joules/cm2.
Preferably, four such lamps are used.
The time for which the aliquot is subjected to the stressors is normally
within the time range of up to about 60 minutes. The time depends to some
extent upon the chosen intensity of the electromagnetic emission, the
temperature, the concentration of the oxidizing agent and the rate at
which it is supplied to the aliquot. Some experimentation to establish
optimum times may be necessary on the part of the operator, once the other
stressor levels have been set. Under most stressor conditions, preferred
times will he in the approximate range of from about 2 to about 5 minutes,
more preferably about 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 subject to subject. Such a treatment provides a
modified blood aliquot which is ready for injection into the subject.
In one preferred embodiment of the present invention, the aliquot of blood
is stressed by being simultaneously subjected to all three of the above
stressors using an apparatus of the type described in aforementioned U.S.
Pat. No. 4,969,483, issued on Nov. 6, 1990 to Mueller. The aliquot is
placed in a suitable, sterile, UV light-transmissive container, which is
fitted into the machine. 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. The UV lamps are
typically on while the temperature of the aliquot is adjusted to the
predetermined value, e.g. 42.5±1° C. Then the oxygen ozone gas mixture, of
known composition and controlled flow rate, is applied to the aliquot, for
the predetermined duration of up to about 60 minutes, preferably to
5minutes and most preferably about 3 minutes as discussed above, so that
the aliquot experiences all three stressors simultaneously. In this way,
blood is appropriately modified according to the present invention to
achieve the desired effects.
In the preferred method of the invention, each course of treatment
comprises the administration to a mammalian subject of from two to four
aliquots of mammalian blood which has been modified as discussed above.
For optimum effectiveness of the treatment, it is preferred that no more
than one aliquot of modified blood be administered to the subject per day.
In some preferred embodiments of the invention, at least one rest period
is provided during the course of treatment. As used herein, the term "rest
period" is defined as the number of days between consecutive aliquots on
which no aliquots of modified blood are administered to the subject. The
maximum rest period between any two consecutive aliquots during the course
of treatment is preferably no greater than about 21 days, and is more
preferably from about 3 to about 15 days. .
In some preferred embodiments, aliquots of modified blood are administered
to the subject on consecutive days, i.e. without an intervening rest
period. Some preferred embodiments of the invention (comprising
administration of 3 or 4 aliquots) include administration of a pair of
aliquots on consecutive days and also provide for a rest period between a
pair of consecutive aliquots.
More preferably, a course of treatment comprises administration of two or
three aliquots to the subject, the course of treatment optionally
including at least one rest period, with the longer (in the case of three
aliquots) or the only (in the case of two aliquots) rest period between
consecutively administered aliquots being from about 5 to 15 days.
Where the course of treatment comprises the administration of two aliquots
to the subject, it is most preferred that the aliquots be administered on
consecutive days without an intervening rest period.
Where the course of treatment comprises the administration of three
aliquots to the subject, it may be preferred to provide two rest periods,
including a longer rest period having a length of 9 to 13 days, more
preferably about 10 to 12 days, and most preferably about 11 days; and a
shorter rest period of 1 to 3 days.
However, where the course of treatment comprises three injections, it is
most preferred to administer two of the three aliquots on consecutive
days, and also to provide a rest period between two aliquots having a
length of 9 to 13 days, more preferably about 10 to 12 days, and most
preferably about 11 days. 1n a particularly preferred example, the
first and second aliquots are administered on consecutive days without an
intervening rest period, and the second and third aliquots are separated
by a rest period of 11 days.
Although it may be sufficient to administer only one course of treatment
as described above to the subject, it may be preferred in some
circumstances to administer two or more courses of treatment, or to follow
the above-described course of treatment by periodic "booster" treatments,
if necessary, to maintain the desired effects of the present invention. In
particular, it may be preferred to administer booster treatments or to
administer a second course of treatments to the subject following a time
period of several weeks or months. For example, it may be preferred to
provide a tune period of from about 1 to about 4 months between
consecutive courses of treatment, with 6 weeks being particularly
preferred in some embodiments of the invention.
In one preferred embodiment, it is preferred to administer a fast course
of therapy comprising three injections, with the first and second aliquots
being administered on consecutive. days without an intervening rest
period, and the second and third aliquots are separated by a rest period
of 11 days. Following a period of six weeks from the end of the first
course of therapy, an identical second course of therapy is administered
to the subject.
Claim 1 of 6 Claims
1. A method for inhibiting
injury arising from atherosclerosis or ischemia in a mammalian subject which
identifying a mammalian subject at risk of injury from atherosclerosis or
administering to said subject three aliquots of modified mammalian blood
according to the following regimen:
a) two aliquots of modified mammalian blood in a volume of about 0.1 ml to
100 ml on consecutive days; and
b) a third aliquot of modified mammalian blood in a volume of 0.1 ml to 100
ml, after a rest period of 11 days after administration of the second
wherein said blood is modified extracorporeally by exposure for a period of
from about 2 to 5 minutes to a combination of stressors selected from the
group consisting of a concentration of ozone from about 10 to 20 μg/ml, UV
light at a dosage of from about 0.1 to about 3 Joules/cm2; and a
temperature from about 0° to about 50° C.
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