Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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USE OF CROSSLINKED HEMOGLOBIN PRODUCT IN THE TREATMENT OF
VASCULAR DISEASE
FIELD OF THE INVENTION
This invention relates to the use of blood substitutes to treat the effects of
peripheral
vascular disease. In particular, it relates to the use of blood substitutes
based on crosslinked
human hemoglobin product to treat ischemia.
BACKGROUND OF THE INVENTION
Atherosclerosis may occur by any one of several mechanisms including renal
failure,
degenerative plaque, thrombosis, fat embolus, clot, and may occur in many
tissues and locations
of the body. Atherosclerotic lesions occur primarily at vascular branch points
and impede the flow
of red blood cells. The initial symptom of intermittant claudication occurs as
oxygen tension in
the tissues (Pa02) falls below 40mmHg and is a result of a deficient oxygen
delivery due to a
reduction in blood flow to an exercising muscle. In these patients their skin
is dry and scaly with
poor nail and hair growth and their exercise tolerance is poor. As the partial
pressure of oxygen
(POZ) in the tissue falls below 20mmHg and the ischemia worsens, skin
ulcerations may appear,
particularly at sites of trauma, and wound healing is poor. At the extreme,
there is a compromise
of viable tissue that can lead to necrosis or gangrene requiring amputation of
the limb. In
situations where the ai~ected vessel is a coronary artery or an artery which
serves a vital brain or
other organ function, the blockage may be life-threatening.
Where blood flow to the tissues is reduced due to vascular obstuctions, wound
healing
improves dramatically if levels can be brought above 30mmHg, even for short
periods of time
(Wutshert R, Diabetes Care 20:1315-1318, 1997).
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Hemoglobin comprises a tetramer of four sub-units, namely two alpha sub-units
each
comprised of a globin peptide chain and two beta sub-units also having globin
peptide chains. The
tetramer has a molecular weight of approximately 64 kilodaltons, and each
subunit has
approximately the same molecular weight. The tetrameric hemoglobin in dilute
aqueous solution
readily dissociates into alpha-beta dimers, and even further under some non-
physiological
conditions to alpha-sub-unit monomers and beta-sub-unit monomers. The dimers
and monomers
have too low a molecular weight for retention in the circulatory system of the
body, and are
filtered by the kidneys for excretion with the urine. This results in an
unacceptably short half life
of such a product in the body.
Crosslinked human hemoglobin product is a hemoglobin based oxygen carrier
(HBOC)
which was developed for use in trauma and surgery, where blood may need to be
given in an
emergency but is not available. It carries oxygen, like blood, but is broken
down and does not
stay in the system for more than a few days. Hemoglobins have been alpha alpha
crosslinked as
disclosed in U.S. Pat. Nos. 4,600,531 and RE 34,271 (Walder), and virus
inactivated and purified
as taught in U.S. Pat. No. 4,861,012 (Estep). Modification by pyridoxylation,
carbamylation,
carboxymethylation, are also known, as are chemical schemes for both cross-
linking and
polymerizing, as by glutaraldehyde. A summary of these chemistries is
contained in R. M.
Winslow ( "Hemoglobin-based Red Cell Substitutes", The Johns Hopkins
University Press, 1992).
While some hemoglobin based oxygen products have been shown to improve oxygen
delivery to ischemic organs in animal models (Horn EP, Surgery 121:411-418,
1997; US Patent
No. 5,877,146 to McKenzie et al) it is not known whether a crosslinked human
hemoglobin
product can improve oxygen delivery to a limb compromised by poor circulation,
often the site of
ischemic skin ulcers. We are not aware of any attempts reported to improve
distal oxygen
delivery with a crosslinked human hemoglobin product (Scott, MG et al, a
review in Clinical
Chemistry 43:1724-1731, 1997). There are some reports that suggest that due to
vasoactive
properties, systemic oxygen delivery may actually be impaired by these
products (Kasper, SM et
al, Anesthesia & Analgesia 83:921-927, 1996).
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SUMMARY OF THE INVENTION
This invention provides a method for treating obstruction of a blood vessel,
which
comprises administering, generally by intravenous infusion, crosslinked human
hemoglobin
product to a patient in need thereof. In a preferred embodiment, the
hemoglobin product
comprises a mixture of 95-97% crosslinked hemoglobin species consisting
essentially of about
40% or less tetrameric hemoglobin units of molecular weight about 64,000
daltons, and the
balance being oligomeric hemoglobin units of molecular weight up to about
600,000 daltons, and
containing up to 6% polymeric hemoglobin species of molecular weight greater
than 600,000
daltons. Preferrably, the hemoglobin product comprises tetrameric and
oligomeric hemoglobin
units having chemical crosslinks between respective beta globin chains. In a
further preferred
embodiment, the chemical crosslinks are between respective beta globin chains
at position lysine-
82 in the 2,3-diphosphoglycerate binding (DPG) cleft of hemoglobin. These
preferred
embodiments of hemoglobin product may be produced by crosslinking hemoglobin
by reaction
with 0-raffinose, followed by chemical reduction to secondary amine groups of
the crosslinks so
formed.
The invention teaches providing the crosslinked human hemoglobin product in a
physiologically acceptable solution containing from about 10 milligrams
crosslinked human
hemoglobin product per kilogram body weight to about 2,500 milligrams
crosslinked human
hemoglobin product per kilogram body weight. Preferrably, the crosslinked
human hemoglobin
product is a physiologically acceptable solution having a total volume of
about 75 to 500
millilitres.
In one embodiment, the hemoglobin product is administered to patients with
peripheral
vascular disease. In another embodiment, crosslinked human hemoglobin product
is used as a
temporary red blood cell substitute, following the removal of a quantity of
the patient's own
blood. In these embodiments, the crosslinked human hemoglobin product is in a
physiologically
acceptable solution having a total volume of about 100 to 2000 millilitres.
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DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT
As used herein, less than 95-97% crosslinked human hemoglobin product is
defined as
comprising a mixture of hemoglobin species derived from human blood and
consisting essentially
of about 30-40% tetrameric hemoglobin units of molecular weight about 64,000
daltons, and the
balance being oligomeric hemoglobin units of molecular weight up to about
600,000 daltons, the
mixture containing up to 6% polymeric hemoglobin species of molecular weight
greater than
600,000 daltons. The crosslinked human hemoglobin product is produced by
crosslinking
hemoglobin by reaction with 0-rai~nose, followed by chemical reduction to
secondary amine
groups of the Schift-base crosslinks so formed.
The preferred crosslinked human hemoglobin product is maintained in stable
oxygen-
releasing conformation by crosslinking. The preferred method of crosslinking
involves crosslinks
between respective beta globin chains at position lysine-82 in the
diphosphoglycerate site, as
disclosed in U.S. Pat. No. 5,770,727 hereby incorporated by reference. The
crosslinked human
hemoglobin product is produced by crosslinking hemoglobin by reaction with 0-
raffinose,
followed by chemical reduction to secondary amine groups of the crosslinks so
formed, disclosed
in co-owned U.S. patent. No.5,532,352 hereby incorporated by reference. The
hemoglobin for
use in the process of the present invention is preferably human hemoglobin,
derived from red
blood cells.
The crosslinked human hemoglobin product utilized in therapy may be any type
for which
the indicator pressor effect is observed and which has the following general
properties: normal or
near-normal oxygen carrying and release properties, stroma-free, non-antigenic
and non-
pyrogenic (i.e. less than 0.25 endotoxin units per milliliter), and be free of
bacterial and viral
contamination. In addition, the crosslinked human hemoglobin product
preparation should have
suitable electroyte, osmotic and oncotic properties.
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The therapeutically efficacious dose which may be administered is defined
herein as an
amount of crosslinked human hemoglobin product which is suffcient to suppress
or reduce
ischemic injury to the tissue whose nourishment has been disrupted by the
blockage. That amount
may be defined as an amount which minimally raises mean arterial blood
pressure 1 to 15 percent
above the preadministration base line value. The dosage of crosslinked human
hemoglobin
product utilized in reperfusion therapy varies from patient to patient, but
generally will fall in the
range from 10 to 2500 mg/kg of body weight. Preferably, the dosage of
crosslinked human
hemoglobin product is 100m1 at a hemoglobin product concentration of lOg%.
Crosslinked human hemoglobin product may also be used as a temporary red blood
cell
substitute, following the removal of the patient's own blood. Doses of up to
1000 ml of
crosslinked human hemoglobin product will be infused during the procedure. It
is anticipated that
this procedure, in conjunction with other blood conservation techniques, may
facilitate a
significant reduction in the need for allogeneic blood.
Ideally, a physician will administer an amount of crosslinked human hemoglobin
product
which confers the desired effect of enhancing oxygen supply to the previously
ischemic tissue,
preventing permanent cellular damage and enhancing healing of ischemic ulcers.
This amount falls
within a range of between 10 and 2500 mg/kg of body weight. As a practical
matter, the physician
can administer crosslinked human hemoglobin product in increments until the
mean arterial blood
pressure has attained a value about 1 to 15 percent above the crosslinked
human hemoglobin
product preadministration base line. Increase in perfusion and the well-known
pressor effect of
crosslinked human hemoglobin product are not necessarily causally linked,
because suppression of
the pressor effect by drugs such as prazosin does not impair the observed
increase in perfusion.
However, pressor activity can be used as a surrogate indicator to ensure that
the patient has
received enough crosslinked human hemoglobin product to achieve the desired
perfusion increase,
because hemoglobin delivery is difficult to measure directly in the clinic.
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The timing of administration should preferably be as soon as possible after
the condition of
blockage is first diagnosed.
Reperfusion therapy utilizing crosslinked human hemoglobin product is
effective when
some degree of blood flow is restored, or in situations in which collateral
blood flow can take
advantage of the increase in perfusion resulting from crosslinked human
hemoglobin product
administration. Where occlusion of the blood vessel is essentially complete,
restoration of flow
may occur spontaneously, may be supported by administration of thrombolytic
enzymes such as
streptokinase or tissue plasminogen activator, or by surgical intervention and
angioplasty.
Crosslinked hemoglobin products have a greater ability to release oxygen to
the tissues
than do red blood cells. Unlike red blood cells, blood substitutes can be
pasteurized, filtered and
chemical-cleansed to make them sterile. These procedures remove microorganisms
responsible for
diseases such as AIDS and hepatitis. Because the substitutes do not have cell
membranes with
blood-group antigens, cross-matching and typing are not required before use.
This saves time and
decreases the cost of transfusions. Furthermore, blood substitutes can be
stored for more than one
year, as compared with about one month for donor blood stored using standard
methods.
Other oxygen-carrying therapeutics include hemoglobin-based products,
classified
according to the source of the hemoglobin - human or bovine blood, or that of
bacterial origin
developed through genetic engineering and second-generation synthetic
perfluorocarbon
emulsions. Among these products, crosslinked human hemoglobin products are
emerging as the
products that are the furthest advanced and which provide the greatest
potential for success
because (1) they are produced from human red blood cells which are extensively
tested for
infectious agents and controlled by stringent regulation; and (2) human
hemoglobin is not a
foreign substance in the body and is less likely to induce immune responses.
The benefits and objects of administering crosslinked human hemoglobin product
as a
treatment for blood vessel blockage are that it increases perfusion to the
area at risk, it stabilizes
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the circulatory system, and may act directly or indirectly to lower levels of
free oxygen radicals
and other molecular species associated with tissue damage. It is believed that
crosslinked human
hemoglobin product increases perfilsion pharmacologically to achieve its
primary beneficial
effects. It may also be effective for physical reasons, by reducing viscosity
and "bypassing"
incomplete obstructions.
The nature and composition of the crosslinked product of the present invention
is not only
controllable and reproducible, but also advantageous. The product contains few
impurities. It is
free from high molecular weight (greater than 600,000 daltons) hemoglobin
polymers. It consists
essentially of about 30-40% tetrameric hemoglobin, with the balance being
oligomers with
molecular weights between 64,000 and 500,000.
In extensive animal studies, the crosslinked human hemoglobin product of the
present
invention has proven safe and effective in a broad range of applications and
favourable safety
results in humans have also been reported in clinical trials (Hemosol Inc.,
Annual Report, 1998).
The crosslinked human hemoglobin products of the present invention are small
in size and
travel in the plasma. Due to this and other characteristics, their use can
facilitate oxygen delivery
to compromised tissues. The result is a relief of the signs and symptoms of
peripheral vascular
disease and an improvement in tissue metabolism, allowing for improved muscle
function and
wound healing.
A further advantageous feature of the preferred crosslinked human hemoglobin
product of
the present invention is that the preferred crosslinked human hemoglobin
product described herein
can be formed with hemoglobin crosslinking reactions conducted within the pH
range 5.0-7.0
without excessive formation of methemoglobin (see U.S. patent. No.5,532,352).
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Hemoglobin covalently bridged across the polyphosphate binding site. Biochem
Biophys Res
Commun 63:1123, 1975). However, the preferred crosslinked human hemoglobin
product
described herein, namely O-raf~nose polymerized hemoglobin, has good P50
without the need for
additional 2,3-DPG analogue (Pliura DH, Human Hemoglobin-based Blood
Substitutes. Artificial
Cells, Blood Substitutes and Immobilization Biotechnology, An International
Journal, 22: A146,
1994).
Other advantages of the present invention will be apparent from the Example,
which
follows.
EXAMPLE
Experiments were conducted to determine whether small doses of crosslinked
human
hemoglobin product could increase distal tissue oxygen delivery in patients
with severe peripheral
vascular disease. The goal was to increase oxygen delivery to distal limbs
compromised by
ischemia, to promote wound healing and new blood vessel growth.
Patients seated on hemodialysis were assessed by transcutaneous oxygen (Tc02)
monitoring with a TINA instrument (Radiometer Medical A/S, Denmark). This
method has
been shown to be reliable in determining limb transcutaneous oxygen pressure
measurements
under baseline and hyperbaric conditions (Nose, Y, Artificial Organs 22:618-
622, 1998).
Crosslinked human hemoglobin product was administered toward the end of
dialysis first at a
volume of 50 ml and then on another dialysis one week later at 100 ml. Only
the results found
with respect to the 100m1 trial are presented herein.
Using tissue or transcutaneous oxygen tension detection (PtcOz), an estimate
of tissue
oxygenation was obtained. The measure Ptc02 was used as an estimate of the
adequacy of
oxygen delivery to the extremities and the response to dii~erent
interventions. Blood pressure
elevation is a secondary effect of crosslinked human hemoglobin product
administration, but it is
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oxygen delivery to the extremities and the response to different
interventions. Blood pressure
elevation is a secondary effect of crosslinked human hemoglobin product
administration, but it is
useful as a surrogate measure for monitoring doses. Tc02 was measured after
equilibration at
baseline and then for 30 minutes after crosslinked human hemoglobin product
was infused. A
range of patients was tested, some with and some without peripheral vascular
disease.
While the therapy should be applicable to all patients with peripheral
vascular disease, we
tested the hypothesis in patients on hemodialysis receiving crosslinked human
hemoglobin
product. Patient JW had Type I diabetes mellitus, severe peripheral vascular
disease with
claudication, and a skin ulcer present. Patient BB had Type I diabetes
mellitus and severe
peripheral vascular disease with claudication. Patient IM had Hypertive
nephrosclerosis and
renovascular disease with diffuse vasculopathy and claudication. Patient RT
had a history of
radiation therapy and atrophy of arteries supplying the legs.
Results:
Table 1 shows the results for patients with peripheral vascular disease. In
all patients thigh
TcOz is compared with foot Tc02 to demonstrate the difference between central
and more distal
tissues. Baseline results are compared with levels at the end of the
measurement period, before
the patient is removed from dialysis. Table 1 shows trial runs with patients
treated with 100 ml of
crosslinked human hemoglobin product (CHHP).
Table 1
PatientThigh Thigh 30minChange Foot Foot 30minChange
BL BL
JW003 58 72 14 37 46 9
OOSBB 31 31 0 24 31 7
006IM 49 54 5 66 69 3
008RT 22 44 22 34 45 11
Average 7.0 7.5
Change
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The results set out in Table 1 show that the addition of crosslinked human
hemoglobin
product was associated with a rise in transcapillary oxygen pressure in
patients receiving the 100
ml infusion during hemodialysis. The data suggests that a modest dose of
crosslinked human
hemoglobin product can increase tissue oxygenation in patients with peripheral
vascular disease.
It thus appears that crosslinked human hemoglobin product can carry oxygen in
the
absence of red cells, and therefore essentially dissolved within plasma,
should be able to reach
anywhere in the vascular tree, with little if any resistance. Crosslinked
human hemoglobin
product thus appears to be carned everywhere that plasma goes so that as long
as the limb is
perfused there is the potential to improve oxygen delivery.
