WO2013016598A2

WO2013016598A2 - Methods for reducing reperfusion injury using a hemoglobin-based carbon monoxide carrier

Info

Publication number: WO2013016598A2
Application number: PCT/US2012/048450
Authority: WO
Inventors: Mark Young; Kim D. Vandegriff
Original assignee: Sangart, Inc.
Priority date: 2011-07-27
Filing date: 2012-07-27
Publication date: 2013-01-31
Also published as: WO2013016598A3

Abstract

The present invention relates generally to methods for reducing reperfusion injury. Specifically, the present invention is directed to administering a hemoglobin-based carbon monoxide carrier to a subject, prior to or following surgery, to reduce injury resulting from reperfusion by delivering both carbon monoxide and oxygen to ischemic tissue.

Description

METHODS FOR REDUCING REPERFUSION INJURY USING A HEMOGLOBIN- BASED CARBON MONOXIDE CARRIER

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This patent application is an Internationa] Application that claims priority to United States provisional patent application serial no. 6 1 /5 1 2,335 filed July 27, 20 1 1 and entitled "Methods for Reducing Reperfusion Injury using a Hemoglobin-Based Carbon Monoxide Carrier," the contents of which are incorporated herein by reference as if set forth in their entirety.

TECHNICAL FIELD

[0002] The present invention relates generally to methods for reducing reperfusion injury. Specifically, the present invention is directed to administering a hemoglobin-based carbon monoxide carrier to a subject, prior to or following surgery to reduce injury resulting from reperfusion by delivering both carbon monoxide and oxygen to ischemic tissue.

BACKGROUND OF THE INVENTION

[0003] Ischemia is a restriction in blood supply to cells that results in damage or dysfunction of tissue. Ischemia may occur because of damage to the vessels supplying blood to the organ, such as by laceration, a blockage in the vessel, such as a blood clot, or in surgery where the supply is blocked during a procedure. In each case, cellular damage results because of the shortage of oxygen, glucose and other fuels provided by the blood.

[0004] It is well recognized that the treatment for ischemia or hypoxia is the reintroduction of blood, nutrients and oxygen to the affected tissue, as well as eliminating metabolic byproducts. However, the restoration of blood flow and oxygen to tissues is also known to cause injury to the tissue. Surprisingly, this injury often outweighs the damage resulting from ischemia. The factors believed to be responsible for reperfusion injury include the rapid introduction of reactive oxygen species ("ROS"), cellular calcium overload, loss of membrane integrity, mitochondrial membrane depolarization and inflammation. [0005] Injury resulting from reperfusion of ischemic tissues has been controversial for many years. Recently, it has been observed that certain interventions at the time of reperfusion can decrease post-ischemic damage. For example, 'post-conditioning' intervention involves multiple short bursts of reperfusion prior to continuous reperfusion. This method has been shown to reduce myocardial infarct size in animal models. Mykytenko, J. et al. found that ROS generation was reduced to levels appropriate for signaling functions, specifically by abrogating the generation of high level ROS responsible for cell injury (Basic Res. Cardiol. 103:472-484, 2008). Similar mechanisms are thought to operate with low pressure/flow reperfusion, and low pH reperfusion.

[0006] The beneficial effects of 'postconditioning' have been demonstrated by reduction of myocardial infarct size and improved cardiac function one year after coronary stenting (Thibault. H. et ah, 2008, Circulation 117: 1037-1044). Similar benefits would be expected for cardiac function following coronary bypass graft surgery, neural function following carotid stenting, surgical intervention for repair of persistent ischemic insult or for prolonged surgical

interventions where blood flow is interrupted {e.g. cardiac bypass, aortic crossclamping, endarterectomy, cerebrovascular thrombolysis or embolectomy) (Molina, C.A. and Alvarez- Sabin, J., 2009, Cerebrovasc. Dis. 27: 162-167).

[0007] While the mechanisms mediating protection from reperfusion injury are not elucidated, the preliminary success with interventions, such as postconditioning, suggests that pathways exist that can be targeted pharmacologically to reduce injury. The recent

demonstration that MP4CO reduces myocardial infarct size ( Vandegriff, K.D. et al., 2008, British Journal of Pharmacology 154: 1649-1661) suggests that delivery of CO to tissues is capable of protecting against reperfusion injury.

[0008] Postconditioning has offered some protection from reperfusion injury. However, this protection has been limited to use in coronary stenting. Consequently, there is a need for a system that can deliver oxygen and nutrients to a variety of ischemic tissues and organs with reduced reperfusion injury. SUMMARY OF THE INVENTION

[0009] This application relates to a method of reducing reperfusion injury in a subject suspected of having ischemic tissue comprising the steps of: administering a maleimidyl polyethylene glycol surface modified hemoglobin-carbon monoxide complex (MalPEG-HbCO); and allowing the MalPEG-HbCO to exchange CO for 0₂ such that both CO and 0₂ are delivered to the ischemic tissue.

[0010] In one embodiment of the present invention, the MalPEG-HbCO can be administered intravenously to a subject. In other embodiments, the MalPEG-HbCO is administered intra- arterially. Furthermore, in various aspects of the present invention, the step of administering MalPEG-HbCO is performed by positioning a catheter in the arterial circulation supplying an organ.

[0011] In exemplary embodiments of the present invention, the Mai PEG is maleimidyl- activated polyethylene glycol having an average molecular weight of about 5000 (MalPEG 5000). In other exemplary embodiments, the MalPEG-Hb has a p50 between 3 and 10 mmHg. In further embodiments of the present invention, the MalPEG-Hb has a p50 between 4 and 6 mmHg.

[0012] Various exemplary embodiments of the present invention feature a MalPEG-HbC wherein the hemoglobin is thiolated before being surfaced modified with the MalPEG.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 shows a graph depicting experimental measurements of ischemic risk area and infarct size resulting from the administration of MalPEG-HbCO (MP4CO) or MalPEG- HbO? (MP40X ), where Lactated Ringer^'s (LR) solution and preconditioning (PC) were used as controls.

[0014] Figure 2 shows a graph depicting experimental measurements of phosphorylation of extracellular signal-regulated kinase (ERK) in brain tissue resulting from the administration of cross-linked hemoglobin (acxHb), MalPEG-Hb0₂ (MP4) or MalPEG-HbCO (COMP4). DETAILED DESCRIPTION OF THE INVENTION

[0015] The present invention relates generally to methods for reducing reperfusion injury. Specifically, the present invention is directed to administering a hemoglobin-based carbon monoxide carrier to a subject, prior to or following surgery, to reduce injury resulting from reperfusion by delivering both carbon monoxide and oxygen to ischemic tissue.

[0016] In the description that follows, a number of terms used in the field of molecular biology, immunology and medicine are extensively utilized. In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given such terms, the following non-limiting definitions are provided.

[0017] When the terms "one," "a," or "an" are used in this disclosure, they mean "at least one^" or "one or more," unless otherwise indicated.

[00181 The terms "activated polyalkylene oxide" or "activated PAO" as used herein refer to a PAO molecule that has at least one functional group. A functional group is a reactive moiety that interacts with free amines, sulfhydryls or carboxyl groups on a molecule forming a covalent bond. For example, maleimide is a functional group that interacts with free sulfhydryls and succinimide is a functional group that interacts with free amines. One type of activated polyalkylene oxide is polyethylene glycol (PEG) with a single maleimide group at one terminus ( Mai PEG- Hb).

[0019] The terms "donor" or "donor patient^" as used herein refer to an animal (human or non-human) from whom an organ or tissue can be obtained for the purposes of transplantation to a recipient patient. The terms "recipient^" or ""recipient patient" refer to an animal (human or non- human) into which an organ or tissue is transplanted.

[0020] The terms "hemoglobin^" or "Hb" as used he ein refer generally to the protein within red blood cells that transports oxygen. l ib by itself refers both to native unmodified Hb as well as modified Hb. Each molecule o Hb contains 4 subunits, 2 a-chain subunits and 2 β -chain subunits that are arranged in a tetrameric structure. Each subunit also contains one heme group, which is the iron-containing center that binds ligands like 0₂, NO and CO. [0021] The term "MalPEG-Hb" as used herein refers to Hb bound to maleimidyl-activated PEG. The conjugation is performed by reacting MalPEG with surface thiol groups and to lesser extent amino groups of Hb. These thiol groups may be present in the native amino acid sequence of Hb or they may be introduced by modifying free amines in the native sequence with a thiolating reagent, such as iminothiolane.

[0022] The terms "methemoglobin" or "metHb" as used herein refer to an oxidized form of Hb that contains iron in the ferric state and cannot function as an oxygen carrier.

[0023] The terms "methoxy-PEG" or "mPEG" as used herein refer to PEG wherein the hydrogen of the hydroxyl terminus is replaced with a methyl (-C¾) group.

[0024] The terms ^''modified hemoglobin'^* or "modi fied Hb" as used herein refer to, but are not limited to, Hb that has been altered so thai it is no longer in the "native" state. Native Hb may be chemically modified, such as by inter- or intra-moiecular crosslinking or by preparing a modified I lb utilizing recombinant techniques known in the art.

[00251 The term "on average^'* as used herein refers to the sum of a set of numbers divided by the number of members in the set. For example, the average of the set of numbers 8, 10, 7, 7, 8 can be calculated as the sum of the numbers (40) divided by the number of members in the set (5) giving the average of the numbers in the set as 8.

[0026] The terms "organ" or "organs" as used herein refer to any anatomical part or tissue having a specific function in an animal. This also includes a portion of an organ, e.g., a lobe of a lung. Such organs include, but are not limited to, the kidney, liver, heart, intestine, pancreas, and lung.

[0027] The term "perfluorocarbons" as used herein refers to inert synthetic molecules that consist entirely fluorine and carbon atoms. Perfluorocarbons emulsions dissolve many times more oxygen than equivalent amounts of plasma or water and are potential substitutes for blood.

[0028] The term "plasma expander" as used herein refers to any solution that may be given to treat blood loss. [0029] The terms "polyethylene glycol" or "PEG" as used herein refer to polymers of the general chemical formula H(OCH₂CH₂)_n OH, also known as (a-Hydro-co-hydroxypoly-( oxy- 1 , 2- ethanediyl), where "n" is greater than or equal to 4. Any PEG formulation, substituted or unsubstituted, is encompassed by this term. PEGs are commercially available in a number of formulations (e.g., Carbowax™ (Dow Chemical, Midland, MI) and Poly-G® (Arch Chemicals. Norwalk, CT)).

[0030] The terms '^"polyethylene glycol-conjugated hemoglobin.^" "PEG-Hb conjugate," or ^' "PEG-Hb^" as used herein refer to Hb that has molecules of PEG covalently bound to its surface.

[0031 ] The term "reper fusion injury" as used herein refers to damage to all or part of a tissue, organ or organ system caused by the resumption of normal blood flow following a cessation or diminishment of blood flow, such as ischemia. This will include injury acquired both during the ischemic episode and in response to the reperfusion.

[0032] The terms "stroma-free hemoglobin^" or "SFH" as used herein refer to Hb from which all red blood cell membranes have been removed.

[0033] The term "surgery" as used herein refers to any invasive or non-invasive medical procedure performed on a patient, organ or tissue, including any surgical operation or medical intervention that involves a partial or complete blood flow occlusion and/or blood loss.

[0034] The term "terminal activity" as used herein, in reference to the functionalized

MalPEG. refers to the percentage of MalPEG having an active maleimide ring able to react with free sulfhydryls and amines on Hb.

[0035] The term "thiolation" as used herein refers to a process that increases the number of sul hydryl groups on a molecule. For example, reacting a protein with 2-iminothiolane ("2-ΓΤ") converts free amines on the surface of a protein to sulfhydryl groups.

[0036] The term "transplantation^" as used herein refers to the process of transferring an organ or tissue from one patient into another. The term "transplantation^" is defined in the art as the transfer of living tissues or cells from a donor to a recipient, with the intention of maintaining the functional integrity of the transplanted tissue or cells in the recipient (see, e.g., The Merck Manual, Berkow, Fletcher, and Beers, Eds., Merck Research Laboratories, Rahway, N.J., 1992).

[0037] The effector mechanisms mediating protection from reperfusion injury have not been elucidated, but the preliminary success with interventions, such as poslconditioning. suggests that pathways exist that can be targeted pharmacologically to reduce reperfusion injury. The recent demonstration that MP4CO reduces myocardial infarct size (Vandegriff, K.D. et al., 2008, British Journal of Pharmacology 154: 1649-1661) suggests that CO is capable of protecting against reperfusion injury.

Carbon Monoxide Metabolism

[0038] Carbon monoxide ("CO") functions as a vasodilator. CO is a colorless, odorless and tasteless gas which is formed in many chemical reactions and in the thermal or incomplete decomposition of many organic materials. In the atmosphere, the average global levels are estimated to be 0.19 parts per million ("ppm"), 90% of which comes from natural sources including ocean microorganism production, and 10% of which is generated by human activity. Thus, inhalation of even small quantities of CO is inevitable for living organisms. Additionally, CO is produced naturally in the body by the enzyme, hemeoxygenase, which catalyzes the metabolism of heme to the products CO, biliverdin and free iron. The CO released by this metabolism is thought to have important cell signaling properties which might he amplified by exogenous administration of CO.

[0039] Inhaled CO is potentially toxic at levels of prolonged exposure greater than 100 ppm. This is because CO binds to Hb in circulation to form carbox y he mo lob i n , which prevents the hemoglobin from carrying oxygen. This causes an overall reduction in the oxygen-carrying capacity of the blood and leads to hypoxia. Symptoms of low level exposure may include, for example, headaches, vertigo and flu-like symptoms. High levels of exposure can be toxic to the central nervous system and the heart, and can even cause death.

[0040] There are now numerous studies documenting the beneficial effects of inhaled CO in animal models. Such studies include, for example, the use of CO in the treatment of sickle cell disease (Belcher, J.D. et al, 2006, J. Clin. Invest. 116:808-816), the heart (Akamatsu, Y. et al.,

1 2004, FASEB J. 18:771-772) and also in the prevention of organ damage (Neto, J.S. et al, 2004, Am. J. Physiol. Heart Circ. Physiol. 289:H542-548; Kohmoto, J. et al, 2007, Am. J. Transplant. 7:2279-2290; Zuckerbraun, B.S. et al, 2005, Shock 23:527-532; Ryter, S.W. et al, 2007, Antioxid. Redox Signal 9:2 157-2 1 73; and Fujimoto. H. et al, 2004, Arterioscler. Thromb. Vase. Biol. 24: 1848-1854.)

[0041 ] Most of these studies utilized inhaled CO at levels between 250- 1000 ppm for extended periods, and achieved carboxyhemoglobin saturation near 20% of total Hb. These levels of carboxyhemoglobin are considered toxic during chronic exposure (Mirza, A. et al,

2005, Toxicol. Sci. 85:976-982; Durante, W. et al, 2006, J. Cell. Mol. Med. 10:672-686; and Gamier, M. et al, 2007, Am. J . Heart Circ. Physiol. 293: 11 1046-11 1052) and. at the very least, will engage acute compensatory changes due to hypoxia when arterial oxygen saturation

("Sa0₂") falls below 90% ( Koehler. R.C. et al, 1982. Am. J. Physiol. 243:H27-H32). The dose- dependency of CO-mediated cytoprotection is not completely understood.

[0042] It has been demonstrated that PAO-Hbs are effective CO carriers. {See Vandegriff, K., et al, 2008, Br. Journal of Pharmacol. 254: 1649- 1661.) Specifically, MalPEG-Hb conjugates to which CO is bound have overall CO equilibrium constants similar to that of unmodi fied Hb.

Hemoglobin Based Carbon Monoxide Carrier

[0043] In previous studies, it was observed that the molecular size of surface modified hemoglobin has to be large enough to avoid being cleared by the kidneys and to achieve the desired circulation hal - li fe. Blumenstein. J. et al, determined that this could be achieved at, or above, a molecular weight of 84,000 Daltons ("Da") ("Blood Substitutes and Plasma

Expanders," Alan R. Liss. editors. New York, N.Y., pages 205-212 ( 1978)). In that study, the authors conjugated dextran of varying molecular weight to Hb. They reported that a conjugate of Hb (with a molecular weight of 64,000 Da) and dextran (having a molecular weight of 20,000 Da) "was cleared slowly from the circulation and negligibly through the kidneys." Further, it was observed that increasing the molecular weight above 84,000 Da did not significantly alter these clearance curves. Accordingly, in the practice of the present invention, the surface modified Hb has a molecular weight of greater than 64,000 Da. a. Hemoglobin

[0044] The Hb utilized in the present methods is not limited by its source and can be derived from humans or animals, or from recombinant techniques. It may be either native (unmodified) or modified, or recombinantly engineered. Human a- and β-globin genes have both been cloned and sequenced (Liebhaber, S.A. et al, PNAS 1980, 77:7054-7058; Marotta, C.A. et al., J. Biol. Chem. 1977, 353: 5040-5053 (β-globin cDNA)). In addition, many recombinantly modi fied Hbs have now been produced using site-directed mutagenesis, {e.g., Nagai, K. et al, PNAS 1 85. 82:7252-7255). Preferably, the Hb is stroma free and endotoxin free. b. Organic Polymers

[0045] For surface decorated hemoglobin, suitable polyalkylene oxide polymers include, polyethylene oxide (-(CH₂ O F 0)_n-), polypropylene oxide (-(CH(CH₃)CH₂ 0)_n-) and a polyethylene/polypropylene oxide copolymer (-(CH₂ O F 0)_n -(CH(CH₃)CH₂ 0)_n-). Other straight, branched chain and optionally substituted synthetic polymers that would be suitable in the practice of the present invention are well known in the medical field.

[0046] The most common PAO presently used to modify the surface of Hb is PEG because of its pharmaceutical acceptability and commercial availability. In addition, PEG is available in a variety of molecular weights based on the number of repeating subunits of ethylene oxide {i.e. - OCIFCTF- ) within the molecule. PEG formulations are usually followed by a number that corresponds to their average molecular weight. For example, PEG-200 has an average molecular weight of 200 Da and may have a molecular weight range of 1 0-2 10 Da. c. Conjugation

[0047] Conjugation of organic polymers to Hb is described in the literature. In one embodiment, the organic polymers are attached via sulfhydryl groups on the Hb. One method to increase the number of available conjugation sites on Hb is to introduce sulfhydryl groups ("- SH"), which tend to be more reactive with MalPEG than free amines. This is called "thiolation." A variety of methods are known in the art for thiolation of proteins. These include, for example, thiolating free amine containing residues of the protein by reaction with succinimidyl 3-(2- pyridyldithio) propionate followed by reduction of the 3-(2-pyridyldithio) propionyl conjugate with dithiothreitol ("DTT"), or tris( 2-carboxyelhyI )phosphine ("TCEP").

[0048] Amines can also be indirectly thiolated by reaction with succinimidyl

acetylthioacetate, followed by removal of the acetyl group with 50 mM hydroxylamine, or hydrazine, at near-neutral pH. In addition, 2-iminothiolane (2-IT) can be used to convert free amine groups into thiol groups. In one embodiment, the thiolation reaction is carried out at a pH of between 7 to 9, which is below the pH at which the 2-IT hydrolyzes significantly before the reaction is completed and also below the pKa of lysine to optimize the extent of the reaction. d. Complex at ion

[0049] Complexation of the Hb with CO is accomplished using any known method for forming a complex of 0₂ and Hb. simply by substituting CO instead of 0₂ as the ligand. For example, CO gas can be introduced into a solution of Hb, and since Hb has a higher affinity for CO than for 0₂, it will readily replace the 0₂ as the ligand.

Ligand Affinity

[0050] In one embodiment, the CO carrier is a MalPEG-Hb conjugate, in which case the MalPEG-Hb conjugate may have an oxygen affinity greater than whole blood, and more specifically, twice or even thrice that of whole blood. Stated differently, the MalPEG-Hb may have an oxygen affinity greater than that of stroma free hemoglobin (SFH), when measured under the same conditions. This means that the PEG-Hb conjugate will generally have a P50 less than 10 millimeters of mercury (mmHg), but greater than 3 mmHg. In one embodiment, the MalPEG-Hb conjugate, when the ligand is O?, will have a p50 between 4 and 6 mmHg. SFH has a p50 of approximately 1 5 mmHg at 37° C, pH 7.4. whereas the p50 for wh le blood is approximately 28 mmHg under the same conditions. It was suggested that increasing oxygen affinity of a hemoglobin-based oxygen carrier ("HBOC"), and thereby lowering the p50, could enhance delivery of oxygen to tissues, but that an oxygen affinity lower than that of SFH would not be acceptable. See Winslow, R.M. et al, in "Advances in Blood Substitutes" ( 1997), Birkauser, eds. Boston, Mass., at page 167, and U.S. Pat. No. 6,054,427. This suggestion contradicts the widely held belief that HBOCs should have lower oxygen affinities, and specifically p50s that approximate that of whole blood. Hence, many researchers have used pyridoxyl phosphate to raise the p50 of SFH from 10 mmHg to approximately 20-22 mmHg, since pyridoxylated Hb more readily releases oxygen when compared to SFH.

[0051] There are many diffe ent scientific approaches to manufacturing HBOCs with high oxygen affinity (i.e. those with p50s less than SFH ). For example, studies have identified the amino acid residues that play an important role in oxygen affinity, such as β93 cysteine. Because of these findings, site-directed mutagenesis can now be easily carried out to manipulate oxygen affinity to the desired level (see, e.g., U.S. Pat. No. 5,661 , 124). The β93 cysteine residue is located immediately adjacent to the proximal β92 histidine residue, which is the only residue in the β-subunit directly coordinated to the heme iron. Attachment of the rigid, bulky maleimide group to the β93 cysteine displaces a salt bridge that normally stabilizes the low-affinity T-stale Hb conformation (Perutz M.F. et al. , Biochemistry 1974, 13:2163-2173). This shifts the quaternary conformation towards the R state, resulting in higher 0₂ affinity (Imai, K.el al., Biochemistry 1973, 12:798-807). Many other approaches are discussed in U.S. Pat. No.

6,054,427.

Formulation for Administration

[0052] The HbCO complex of the present invention is formulated in an aqueous diluent that is suitable for administration to a subject prior to or following surgery. Although the

concentration of the oxygen carrier in the diluent may vary according to the application, it does not usually exceed a concentration of 10 g/dl of Hb. More specifically, the concentration is usually between 0.1 and 8 g/dl Hb.

[0053] Suitable aqueous diluents (i.e., those that are pharmaceutically acceptable for intravenous injection) include, inter alia, aqueous solutions of proteins, glycoproteins, polysaccharides, and other col loids. It is not intended that these embodiments be limited to any particular diluent. Consequently, diluents may encompass aqueous cell-free solutions of albumin, other colloids, or other non-oxygen carrying components.

[0054] The solution properties of a PEG-Hb conjugate are influenced by the strong interaction between PEG chains and solvent water molecules. This is believed to be an important attribute for an HBOC for two reasons: 1) higher viscosity decreases the diffusion constant of the PEG-Hb molecule, and 2) higher viscosity increases the shear stress of the solution flowing against the endothelial wall, eliciting the release of vasodilators to counteract vasoconstriction. Accordingly, the formulation of PEG-Hb in the aqueous diluent usually has a viscosity of at least 2 centipoise (cP). More specifical ly, between 2 and 4 cP, and particularly around 2.5 cP. In other embodiments, the viscosity of the aqueous solution may be 6 cP or greater, but is usually not more than 8 cP.

Administration

[0055] Reperfusion injury can occur in a number of ways. For example, the injury may be enzymatic (e.g. thrombolysis), mechanical (e.g. angioplasty, stenting, or embolectomy), or the result of bypass grafting. Because reperfusion injury results from reinitiating the blood supply, it can occur in any event where such perfusion is necessary. Consequently, injury can result in organ preservation, organ transplantation, and treatment of conditions such as myocardial infarct, pulmonary hypertension, stroke as well as diseases such as malaria and multiple sclerosis. Injury can be determined by direct measurement of organ function (e.g. renal function, myocardial ejection, and cognition), the presence or absence of biomarkers (e.g. cardiac enzymes), imaging methods (e.g. MRI, MR A. PET scan) or clinical signs (e.g. length of hospital stay, ventilator days, etc.).

[0056] Reperfusion injury can be reduced using the methods of the present invention by administering HbCO directly into the affected artery undergoing reperfusion or intravenously to expose the entire circulation. In one method. HbCO is administered intravenously prior to and continuing after reperfusion (i.e. loading the peripheral circulation ). For example, HbCO is infused up to 1 20 minutes following reperfusion and continuing intravenously for 24-48 hours.

[0057] In the case where an organ is reperfused in situ, HbCO is administered intravenously and/or intra-arterially prior to the reperfusion event and continuing for 2 to 24 hours after the event. Following reperfusion, a functional evaluation is performed to determine if the treatment has achieved the desired effect. For example, if a patient has had an embolic stroke and is a candidate for thrombolysis in a cerebral artery, treatment could begin with administration of HbCO immediately by intravenous infusion. This initial administration is then followed by direct intra-cerebral artery infusion after clot lysis has been confirmed. After treatment, the patient is given a series of cognitive tests to determine the extent of damage. Similarly, a patient with occluded coronary arteries scheduled for stenting would receive HbCO intravenously during the surgical preparation. Following preparation, the patient is administered HbCO intra-arterially through the coronary artery immediately before or at the time of stent placement. Several days or weeks after the operation, the patient is given a cardiac function evaluation to determine the extent of damage. Preclinical data shows that administration of HbCO results in reduced whole body oxygen consumption, which is one mechanism by which HbCO translates into reduced reper fusion injury and preserved organ function. In the case where elective surgery might cause ischemia and reperfusion, e.g. cardiac bypass graft surgery or carotid endarterectomy, HbCO could be administered prior to the ischemic episode and continue through the time of

reperfusion.

EXAMPLES Example 1

Preparation of a Hemoglobin - Carbon Monoxide Complex

A. Thiolation of Hemoglobin

[0058] Packed red blood cells ("RBCs") are procured from a commercial source, such as from a local Blood Bank, the New York Blood Center, or the American Red Cross. The material is obtained not more than 45 days from the time of collection. All units are screened for viral infection and subjected to nucleic acid testing prior to use. Non-leukodepleted pooled units are leukodepleted by membrane filtration to remove white blood cells. Packed RBCs are pooled into a sterile vessel and stored at 2- 15"C until further processing. The volume is noted, and Hb concentration is determined using a commercially available co-o imete , or other art-recognized method.

[0059] RBCs are washed with six volumes of 0.9% sodium chloride using a 0.45-μιπ tangential flow filtration, followed by cell lysis by decreasing the concentration of salt. Hb extraction is performed using the same membrane. The cell wash is analyzed to verify removal of plasma components by a spectrophotometric assay for albumin. The lysate is processed through a 0.16-μηι membrane in the cold to purify Hb. The purified Hb is collected in a sterile depyrogenated vessel and then ultrafiltered to remove viruses. Additional viral-reduction steps, including solvent/detergent treatment, nanofiltration and anion Q membrane purification, may be performed. All steps in this process are carried out at between 2- 15° C.

[0060] Hb from lysate is exchanged into Ringer's lactate ("RL"), Ringer's acetate ("RA") or phosphate-buffered saline ("PBS"), pH 7.4 using a 30-kD membrane. The Hb is concentrated to 1 . 1 - 1 .5 mM (tetramer). Between 10 to 12 volumes o RL or PBS are used for solvent exchange. This process is carried out at 2-15° C. The pH of the solution prepared in RL is adjusted to 7.0- 7.6. The Hb is sterile-filtered through a 0.45 or 0.2-μιη disposable filter capsule and stored at 4±2° C before the chemical modification reaction is performed.

[0061 ] Thiolation is carried out using less than 8-fold molar excess of 2- IT over l ib. This ratio and reaction time were optimized to maximize the number of thiol groups for PEG yl at ion and to minimize product heterogeneity. Approximately 1 mM Hb (tetramer) in RL (pH 7.0-8.5), PBS or any similar buffer was combined with less than 8 mM 2-IT in the same buffer. This mixture was continuously stirred for less than 6 hours at 10+5° C.

B. Conjugation of Hb with Ma! PEG

[0062 ] Thiolated Hb is PEGylated with less than a 15-fold molar excess of Mai PEG based on 100% terminal activity over the starting Hb tetramer concentration. The Hb is first allowed to equilibrate with the atmosphere to oxygenate the Hb. Approximately 1 mM thiolated Hb in RL (pH 7.0-8.5), PBS or any similar buffer was combined with less than 16 mM MalPEG in the same buffer. This mixture was continuously stirred for less than 6 hours at 10+5 C°.

[0063] PEGylated-Hb is processed through a 70-kD membrane (i.e. a 20-volume filtration) to remove excess unreactcd reagents and Hb. This process is monitored by size-exclusion liquid chromatography at 540 nm and 280 nm. The protein concentration is diluted to 4 g/dl and the pH is adjusted to 7.3+0.3 using 1 N NaOH.

[0064] The final MalPEG-Hb product is sterile-filtered using a 0.2-μιη sterile disposable capsule and collected into a sterile depyrogenated vessel at 4+2° C.

[0065] PEG-Hb is diluted to 4 g/dl RL and the pH adjusted to 7.4+0.2. [0066] The final PEG-Hb is sterile-filtered (0.2-μη ) and aliquoted by weight into sterile glass vials. The vials are sealed with sterile rubber stoppers and crimped seals in a laminar flow hood. The vials are then stored at -80 C° until use.

C. Carbon Monoxide Loading of PEGylated Hemoglobin

[0067] The PEG-Hb is equilibrated with a desired concentration of CO in a chamber containing a prescribed concentration of CO.

Example 2

Perfusion of Hemoglobin-Based Carbon Monoxide Carrier

[0068] The clinical benefits of preventing reperfusion injury have been demonstrated by a reduction of myocardial infarct size and improved cardiac function one year after coronary stenting. These benefits may also be seen following coronary bypass graft surgery, neural function following carotid stenting, endarterectomy, cerebrovascular thrombolysis or embolectoniy. Benefits may also be observed in surgical intervention for repair of any persistent ischemic insult or for prolonged surgical interventions where blood flow is interrupted (e.g. cardiac bypass, renal function following aortic crossclamping for aortic vascular repai , or resuscitation following protracted cardiac arrest). During these procedures, HbCO is

administered to the patient at any time prior to, during or following surgery. For example, a patient undergoing elective reperfusion (e.g. coronary artery bypass graft, coronary stenting or cerebral embolectomy) is administered HbCO any time prior to the procedure and for 24-168 hours following the procedure. For emergency procedures, the in usion of HbCO would begin at the time of diagnosis and continue for 24- 168 hours following the procedure. The volume and rate of administration is regulated to achieve CO-hemoglobin saturation of 1-30% of total hemoglobin in the blood. Temperature of the solution infused is between room temperature and 38°C.

[0069] Administration of HbCO is performed by intravenous infusion or intra-arterial infusion via a catheter positioned in the arterial circulation supplying the organ. Functional assessment of the ischemic/reperfused organ is performed using standard clinical techniques for evaluation of organ function. For example, recovery of myocardial function following a cardiac procedure often includes electrocardiography, echocardiography for myocardial wall motion and ejection fraction. Radionuclide scintigraphy, positron emission tomography, magnetic resonance imaging and magnetic resonance angiography are utilized to assess metabolic and vascular function. Similarly, evaluation of reper used kidney function following aortic crossclamping includes measurement of serum blood urea nitrogen and creatinine, determination of glomerular filtration rate and renal plasma flow, urine concentrating ability, fractional sodium excretion, and biomarkers of renal tubular damage (e.g. neutrophil gelatinase associated lipocalin, N-acetyl glucosaminidase. glutathione-S transferase and kidney injury molecule- 1). Histologic assessment of t issue biopsies may also be performed where appropriate.

Example 3

HbCO Protection against Ischemia/Reperfusion Injury

[0070] Tissue damage, including necrosis and apoptosis, can result from prolonged periods of ischemia and reperl^'usion. This damage can result from the absence of blood flow and oxygen supply during the ischemia, and also from the re-introduction of oxygen at the time of reperl^'usion. In the past two decades strategies have emerged using pharmacologic approaches or manipulation of the blood supply to limit tissue death from i sc he mi a/ repe r fu s i on injury. The majority of these studies have been described in salvage of myocardial tissue ( Hausenloy, et ai, 2007, Heart Fail Rev . 12(3-4):217-234; Cohen et ai, 201 1 , Antioxid Redox Signal 14(5):821- 831), but the protection has been demonstrated in brain as well (Ovize et ai , 2010, Cardiovasc Res. 87(3):406-423 ). The Reperl^'usion Injury Salvage Kinase (RISK) pathway describes the role of pro-survival intracellular kinase enzymes, including Akt and ER K 1/2, in conferring powerful protection against i sc he m i a repe i f us i on injury. Examples of agents which confer protection via activation of the RIS K pathway include cytokines, G-protein coupled receptor agonists such as adenosine and bradykinin, opioids, volatile anesthetics, and vascular

manipulations such as pre-conditioning, post-conditioning, low pressure reperfusion (Hausenloy et al.) and hypothermia (Yang et al , 201 1 , Basic Res Cardiol. 106(3):421-430).

[0071] Sangart has reported that administration of MalPEG-HbCO induced significant cardioprotection and reduced myocardial infarct size (Figure 1) resulting from i sche m i a/repe i t u s ion in rats (Vandegriff et al , 2008, Br J Pharmacol. 154(8): 1649- 1661 ). As shown in Figure 1, following 30 minutes and 24 hours of reperfusion in rats, administration of MalPEG-HbCO (MP4CO) significantly reduced infarct size (bottom panel) as compared to MalPEG-Hb0₂ (MP40X), where Lactated Ringer's (LR) solution and preconditioning (PC) were used as controls . Ischemic area at risk (top panel) was not shown to be affected. In separate experiments of hemodilution in rats, MalPEG-HbCO increased phospho ERK 1/2 in the rat brain tissue ( Figure 2). As shown in Fi ure 2, following 50% exchange transfusion with MalPEG-HbCO (COMP4), phosphorylation of ERK is increased in brain tissue. Such effects are not observed in animals hemodi!uted with cross- linked hemoglobin (aaHb) or MalPEG-Hb0₂ ( MP4 ).

[0072] We propose that MalPEG-HbCO acts to induce cardioprotection. and protection of other tissues/organs undergoin ischemia reperfusion by activation of the RISK pathway, including ERK and Akt as well as other, unspeci ied intracellular signaling pathways. This is supported by recent reports of cardioprotection in rabbits with inhaled CO mediated via the Akt pathway (Fujimoto et al , 2004, Arterioscler Thromb Vase Biol. 24( 10): 1848- 1853) and neuronal protection in culture with atmospheric CO mediated by the ERK 1/2 pathway (Dallas et al, 201 1 . FASEB J. 25(5): 1519-1530).

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[0073] The examples set forth above are provided to give those of ordinary skill in the art a complete disclosure and description of how to make and use the preferred embodiments of the compositions, and are not intended to limit the scope of what the inventors regard as their invention. Modifications of the above-described modes (for carrying out the invention that are obvious to persons of skill in the art) are intended to be within the scope of the following claims. All publications, patents, and patent applications cited in this specification are incorporated herein by reference as if each such publication, patent or patent application were speci ically and individually indicated to be incorporated herein by reference.

Claims

CLAIMS We claim:

1. A method of reducing reperfusion injury in a subject suspected of having ischemic tissue comprising the steps of:

administering a maleimidyl (Mai) polyethylene glycol (PEG) surface modified hemoglobin-carbon monoxide complex (MalPEG-HbCO); and

allowing the MalPEG-HbCO to exchange CO for 0₂ such that both CO and 0₂ are delivered to the ischemic tissue.

2. The MalPEG-HbCO of claim 1, wherein the MalPEG-HbCO is administered

intravenously.

3. The MalPEG-HbCO of claim 1 , wherein the MalPEG-HbCO is administered intra- arterially.

4. The MalPEG-HbCO of claim 1 , wherein the step of administering MalPEG-HbCO is performed by positioning a catheter in the arterial circulation supplying an organ.

5. The MalPEG-HbCO of claim 1 , wherein the MalPEG is maleimidyl-activated polyethylene glycol having an average molecular weight of about 5000 ( MalPEG 5000).

6. The MalPEG-HbCO of claim 1 , wherein the MalPEG-Hb has a p50 between 3 and 10 mmHg.

7. The MalPEG-HbCO of claim 1 , wherein the MalPEG-Hb has a p50 between 4 and 6 mmHg.

8. The MalPEG-HbCO of claim 1 , wherein the hemoglobin is thiolated before being surfaced modified with the MalPEG.