CA2269258A1 - Use of crosslinked hemoglobin product in the treatment of vascular disease - Google Patents

Abstract

Administration of low doses of crosslinked human hemoglobin product minimizes damage to the extremities after blockage and significantly reduces ischemic injury.
Crosslinked human hemoglobin product exerts a pharmacological effect by increasing perfusion and alleviating ischemic injury.

Description

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).

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).

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.

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.

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.

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 _7_ 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).

_g_ 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 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

Average 7.0 7.5 Change -lU-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.

Claims (12)

1. A method for alleviating ischemic injury in tissue whose oxygen flow has been disrupted by obstruction of a blood vessel, comprising administering crosslinked human hemoglobin product to a patient in need thereof.

2. The method of claim 1 wherein the hemoglobin product comprises a mixture of hemoglobin species and consisting essentially of about 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.

3. The method as claimed in claim 1 wherein the hemoglobin product comprises hemoglobin that is 95-97% crosslined.

4. The method of claim 1 wherein the hemoglobin product comprises tetrameric and oligomeric hemoglobin units having chemical crosslinks between respective beta globin chains.

5. The method of claim 1 wherein the chemical crosslinks are between respective beta globin chains at position lysine-82 in the 2,3-diphosphoglycerate binding (DPG) cleft of hemoglobin.

6. The method of claim 1 wherein the 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.

7. The method of claim 1 wherein the crosslinked human hemoglobin product is a physiologically acceptable solution that will deliver a dose of 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.

8. The method of claim 1 wherein the crosslinked human hemoglobin product is a physiologically acceptable solution having a total volume of about 75 to 500 millilitres.

9. The method of claim 1 wherein the crosslinked human hemoglobin product is used as a temporary red blood cell substitute.

10. The method of claim 1 wherein the crosslinked human hemoglobin product is in a physiologically acceptable solution having a total volume of about 500 to 2000 millilitres.

11. The method of claim 1 wherein the crosslinked human hemoglobin product is a physiologically acceptable solution and is administered to a patient until the patient's mean arterial blood pressure is about 1% to 15% greater than a preadministration value.

12. The method of claim 1 wherein the hemoglobin product is administered in the course of dialysis.