WO2025257801A1 - Heme-binding protein for the treatment of ischemia-reperfusion injury (iri) - Google Patents

Abstract

The present disclosure relates generally to heme-binding proteins and compositions thereof, and their use in the treatment of ischemia-reperfusion injury, in particular ischemia-reperfusion injury-mediated inflammation.

Description

HEME-BINDING PROTEIN FOR THE TREATMENT OF ISCHEMIA-REPERFUSION INJURY (IRI)

Related Applications

This application claims priority to European Provisional Application No. EP24181904.4 entitled “METHOD OF TREATMENT OF ISCHEMIA-REPERFUSION INJURY (IRI)” filed 13 June 2024, the contents of which are incorporated herein by reference in their entirety.

Technical Field

The present disclosure relates generally to heme-binding proteins and compositions thereof, and their use in the treatment of ischemia-reperfusion injury, in particular ischemia-reperfusion injury-mediated inflammation.

Background

As noted by Cowled et al. (201 1 , In: Fitridge R, Thompson M, editors. Mechanisms of Vascular Disease: A Reference Book for Vascular Specialists [Internet]. Adelaide (AU): University of Adelaide Press. 18), ischemia-reperfusion injury (IRI) is typically defined as the paradoxical exacerbation of cellular dysfunction and death, following restoration of blood flow to previously ischemic tissue. While re-establishment of blood flow is essential to salvage ischemic tissue, reperfusion paradoxically causes further damage, threatening organ function and tissue viability. IRI occurs in a wide range of organs, including the heart, lung, kidney, gut, skeletal muscle and brain. IRI may also lead to injury to distant organs, potentially leading to multisystem organ failure. Ischemia-reperfusion injury contributes to mortality and morbidity in many pathological conditions, including trauma, ischemic stroke, myocardial infarction, and postcardiac arrest syndrome, and is implicated in undesirable treatment side-effects (e.g. radiation I chemotherapy).

IRI is a major complication in organ transplantation and is frequently an inevitable consequence during the procurement of donor organs. It also contributes significantly to the development of primary-graft dysfunction (PGD).

Despite the significant clinical and economic burden of IRI, its underlying mechanisms remain ill-defined and, as such, there have been very few effective therapeutic interventions. Hence, there remains an urgent need for more effective treatments for IRI.

Summary

In an aspect of the present invention, there is provided a heme-binding protein for use in the treatment of ischemia-reperfusion injury (IRI) in a subject in need thereof.

In an embodiment, the IRI is to a tissue or organ. In an embodiment the IRI is to a transplanted tissue or organ. In an embodiment, the tissue or organ is selected from the group consisting of lung, heart, kidney, liver, pancreas, intestine, stomach, thymus, uterus, bone marrow, skin, tendon and cornea. In another embodiment, the tissue or organ is lung.

In some embodiments, the heme-binding protein is hemopexin. In an embodiment, the hemopexin is plasma-derived hemopexin. In another, the hemopexin is human hemopexin.

In some embodiments, the heme-binding protein is formulated for administration in a therapeutically effective amount of from about 0.05 mg/kg body weight to about 200 mg/kg body weight. In an embodiment, the heme-binding protein is formulated for administration in a therapeutically effective amount of from about 0.05 mg/kg body weight to about 1 mg/kg body weight.

In some embodiments, the heme-binding protein is administered i) prior to; ii) during; and/or iii) after induction of the IRI.

In some embodiments, the heme-binding protein is administered parenterally.

In some embodiments, said treatment is characterised by one or more of: reduced inflammation; reduced number of infiltrating neutrophils; reduced perivascular edema; reduced levels of heme oxygenase-1 ; and reduced number of thrombotic blood vessels.

In particular embodiments of the invention as it relates to a heme-binding protein for use in the treatment of IRI, as described above and herein, the IRI is to a lung tissue or to a lung, optionally transplanted lung tissue or transplanted lung; and the heme-binding protein is hemopexin or a functional analogue thereof, optionally human hemopexin or a functional analogue thereof; optionally wherein the heme-binding protein is administered parenterally, preferably intravenously.

In another aspect disclosed herein, there is provided a method of treating ischaemiareperfusion injury in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a heme-binding protein. In particular embodiments, the IRI is to a lung tissue or to a lung, optionally transplanted lung tissue or transplanted lung; and the heme-binding protein is hemopexin or a functional analogue thereof, optionally human hemopexin or a functional analogue thereof; optionally wherein the hemebinding protein is administered parenterally, preferably intravenously.

The present disclosure also extends to the use of a heme-binding protein in the manufacture of a medicament for treating ischaemia-reperfusion injury in a subject. In particular embodiments, the IRI is to a lung tissue or to a lung, optionally transplanted lung tissue or transplanted lung; and the heme-binding protein is hemopexin or a functional analogue thereof, optionally human hemopexin or a functional analogue thereof; optionally wherein the hemebinding protein is administered parenterally, preferably intravenously.

Brief description of the drawings

Figure 1 shows lung tissue damage in the applied mouse model of unilateral pulmonary IRI. Evaluation was carried out according to the ALI score of the American Thoracic Society. The left ischemic lung showed significantly higher levels of tissue damage as compared to the right control lung in the applied mouse model (73.7 ± 11.8 vs. 55.0 ± 7.6, respectively: p = 0.02).

Figure 2 shows (A) A model of unilateral warm ischemia/ reperfusion injury (IRI) of the left lung was established in C57BL6/J mice by unilateral pulmonary clamping of the left lung hilum for 1 , 1 .5 and 2 h after left-sided thoracotomy followed by 4 h of reperfusion (A). The ischemia times of 1 .5 h followed by 4 h of reperfusion were applied throughout this study, because signs of marked tissue injury were observed in the left lun after 1 .5 h (see Figure 1 ). (B) Perivascular edema was determined with a microscope at 200x magnification using Elastica-van-Gieson staining. Five vessels of right and left lung were assessed. The distances from tunica intima to tunica intima (Dv) were compared with the shortest diameter from tunica adventia to tunica adventia (De). The shortest distances were used as measured values in order to compensate for distortion of the results caused by different cutting planes, respectively. To compare different vessel sizes, the results were combined into one parameter using the following formula (the formula at the lower left: B).

Figure 3 shows infiltration of neutrophils as determined by PAS staining. Neutrophils as indicated by arrows. The number of neutrophils was significantly higher in the left ischemic compared to the right control lung (42.8 ± 23.5 vs. 16.4 ± 8.6, respectively: p = 0.047). In the group of Hx-treated mice significantly lower numbers of neutrophils were determined in the left ischemic lung (IRI group vs. Hx treated group: 42.8 ± 23.5 vs. 22.8 ± 15.9, respectively: p = 0.047). *: not significant (NS).

Figure 4 shows the extent of perivascular edema as determined by the cuff area-to-vessel area ratio, which was markedly higher in the left ischemic compared to the right control lung (10.3 ± 2.6 vs. 6.1 ± 2.3, respectively: p = 0.031 ). Increased levels of cuff area-to-vessel area ratio in the ischemic lung were reduced by treatment with Hx (IRI group vs. Hx treated group: 10.3 ± 2.6 vs. 6.8 ± 1.5, respectively: p = 0.032) (Outlier [35.4] in the control group was excluded) *: not significant (NS).

Figure 5 shows significantly higher numbers of thrombotic blood vessels in the left ischemic compared to the right control lung (76.5 ± 8.5% vs. 3.3 ± 4.5, respectively: p = 0.008). The number of thrombotic pulmonary vessels was significantly reduced in the group of mice treated with Hx (IRI group vs. Hx treated group: 76.5 ± 8.5% vs. 26.0 ± 18.2%, respectively: p = 0.008). *: not significant (NS).

Figure 6 shows significantly higher levels of HO-1 expression in the left ischemic relative to the right control lung (0.79 ± 0.38 vs. 0.36 ± 0.39, respectively: p = 0.005). In the group of Hx- treated mice, HO-1 expression levels were significantly reduced in the ischemic left lung (A: IRI group vs. Hx treated group: 0.79 ± 0.38 vs. 0.36 ± 0.34, respectively: p = 0.049).

Figure 7 shows lower levels of B- (CD3⁺CD19⁺ cells) and T- (CD3⁺CD8⁺ cells) lymphocytes were detected in IRI mice with relative to mice without Hx treatment (n = 5, respectively). Higher levels of dendritic cells were detected in IRI mice with Hx treatment compared to mice without Hx treatment (n = 5, respectively). The p-values were calculated using two-tailed Mann- Whitney rank sum test. A p value of < 0.06 was considered as statistically significant.

Detailed description

It is to be understood that this disclosure is not limited to the particular methodology, protocols, proteins, reagents etc. described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present disclosure that will be limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

As used herein, the term “derived from” shall be taken to indicate that a particular integer or group of integers has originated from the species specified, but has not necessarily been obtained directly from the specified source. Further, as used herein the singular forms of “a”, “and” and “the” include plural referents unless the context clearly dictates otherwise.

As used herein, the “about”, as applied to one or more values, refer to a value that is similar to a stated reference value. In certain embodiments, the term “about” refers to a range of values that fall within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value). In a particular embodiment, the term “about” means ±10% of the recited value.

In the absence of any indication to the contrary, reference made to a "%" content throughout this specification is to be taken as meaning % w/w (weight/weight). For example, a composition comprising a heme-binding protein content of at least 50% of total protein is taken to mean a composition comprising a heme-binding protein content of at least 80% w/w of total protein. The term “corresponding” as used herein in reference to a particular gene is intended to mean an analogous or equivalent or comparable gene. For example, where reference is made to a corresponding endogenous gene, it is intended to mean the analogous, equivalent or comparable naturally-occurring gene. In some embodiments, the corresponding gene has analogous or equivalent function or having sequence similarity. In one embodiment, the corresponding gene may be identical in function and/or sequence. In another embodiment, the corresponding gene may have about the same function or activity. In another embodiment, the corresponding gene may have reduced function or activity. In some embodiments, the phrase “corresponds to” or “corresponding to” is meant a nucleic acid sequence that displays substantial sequence identity to a reference nucleic acid sequence. In general, the nucleic acid sequence will display at least about 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even up to 100% sequence identity to the reference nucleic acid sequence. The terms "treating", “treatment”, "treat" and the like, are used interchangeably herein to mean relieving, minimising, reducing, alleviating, ameliorating or otherwise inhibiting ischemiareperfusion injury, including one or more symptoms thereof, as described herein. The terms "treating", “treatment” and the like are also used interchangeably herein to mean preventing an ischemia-reperfusion injury from occurring or delaying the onset or subsequent progression of ischemia-reperfusion injury in a subject that may be predisposed to, or at risk of, developing an ischemia-reperfusion injury, but has not yet been diagnosed as having it. In that context, the terms "treating", “treatment” and the like are used interchangeably with terms such as “prophylaxis”, “prophylactic” and “preventative”. It is to be understood, however, that the methods disclosed herein need not completely prevent ischemia-reperfusion injury from occurring in the subject to be treated. It may be sufficient that the methods disclosed herein merely relieve, reduce, alleviate, ameliorate or otherwise inhibit ischemia-reperfusion injury in the subject, including to the extent that there are fewer ischemia-reperfusion injury symptoms and / or less ischemia-reperfusion injury outcomes than would otherwise have been observed in the absence of treatment. Thus, the methods described herein may reduce one or more symptoms and / or the severity of ischemia-reperfusion injury outcomes in the subject.

The terms “peptide”, “polypeptide” and “protein” are to be understood as referring to a chain of amino acids linked by peptide bonds, irrespective of the number of amino acids forming said chain. Amino acids are typically represented by their one-letter or three-letters code, according to the following nomenclature: A: alanine (Ala); C: cysteine (Cys); D: aspartic acid (Asp); E: glutamic acid (Glu); F: phenylalanine (Phe); G: glycine (Gly); H: histidine (His); I: isoleucine (lie); K: lysine (Lys); L: leucine (Leu); M: methionine (Met); N: asparagine (Asn); P: proline (Pro); Q: glutamine (Gin); R: arginine (Arg); S: serine (Ser); T: threonine (Thr); V: valine (Vai); W: tryptophan (Trp) and Y: tyrosine (Tyr).

The term “recombinant”, as used herein, refers to a biomolecule, e.g., a gene or protein, or to a cell or microorganism. The term “recombinant” may be used in reference to cloned DNA isolates, chemically synthesized polynucleotides, or polynucleotides that are biologically synthesized by heterologous systems, as well as proteins or polypeptides encoded by such nucleic acids, e.g. enzymes.

Ischemia-Reperfusion Injury (IRI)

The present invention is predicated, at least in part, on the inventors' surprising finding that heme-binding proteins (e.g., hemopexin) can alleviate the deleterious effects of ischemiareperfusion injury (IRI), in particular IRI-mediated inflammation. Thus, in an aspect disclosed herein, there is provided a method of treating ischaemiareperfusion injury (IRI) in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a heme-binding protein.

Ischemia-reperfusion injury, also referred to as IRI, reperfusion injury or reoxygenation injury, typically refers to tissue damage caused when blood supply returns to tissue after a period of ischemia.

Ischemia can occur during tissue or organ injury or trauma (e.g. myocardial infarctions or other heart diseases, radiation-induced injury, vascular / circulatory disorders) or when artificially induced (i.e. application of tourniquets). Ischemia causes tissue/cellular damage, at least in part due to of the build-up of metabolic waste products, inability to maintain cell membranes and mitochondrial function, and eventual leakage of damaging proteins into the cell and surrounding tissues.

Tissue reperfusion (restoration of blood supply) is required to prevent further ischemia. However, reperfusion typically worsens the injury and often ends up being more damaging than the initial ischemic insult. Ischemia-reperfusion injury contributes to mortality and morbidity in many pathological conditions, including trauma, ischemic stroke, myocardial infarction, and post-cardiac arrest syndrome, and is implicated in undesirable treatment sideeffects (e.g. radiation / chemotherapy).

Ischaemia is a restriction in blood supply to tissue, muscle or organ, resulting in inadequate or lack of oxygen required for cellular and tissue metabolism necessary to keep tissues alive and functional. The reduced / lack of oxygen supply may also be accompanied by restriction of nutrients from blood during the ischemic period, and/or inadequate removal of metabolic waste. Ischemia can be partial (poor perfusion) or total blockage.

The absence of oxygen and nutrients from blood creates a condition in which the restoration of circulation results in inflammation and oxidative damage through the induction of oxidative stress rather than (or along with) restoration of normal function.

Upon IRI, adaptive cellular responses activate the innate immune system with its Toll-like receptors and the complement system as well as the adaptive immune system, which may result in an inflammatory tissue reaction with immune cells infiltrating the tissue. This response is understood to be mediated by various cytokines, chemokines, adhesion molecules, and compounds of the extracellular matrix. The expression of these factors is regulated, at least in part, by transcription factors such as NF-KB. Strategies to prevent or treat IRI include blockade of cytokines/chemokines, adhesion molecules, NF-KB, specific MAP kinases, metalloproteinases, induction of protective genes, and modulation of the innate immune system (see, for example, Lutz et al. 2010 Journal of Inflammation 7:27).

Repeated bouts of ischemia and reperfusion has been associated with the formation and failure to heal of chronic wounds such as pressure sores and ulcers. Reperfusion injury is a major part in the pathology of organ dysfunction / failure following reversal of cardiac arrest. IRI can occur in a wide range of organs including the heart, lung, kidney, gut, skeletal muscle and brain. The physiological damage may involve not only the ischemic organ itself but may also induce systemic damage to distant organs, potentially leading to multi-system organ failure.

There is also a risk of IRI developing during surgical procedures when tourniquets are used to provide optimal operating conditions.

Ischemia-reperfusion injury (IRI) is a significant contributor to various lung disorders and is implicated in the development of primary graft dysfunction (PGD) following organ transplantation, particularly during lung transplantation.

In an embodiment, the methods and uses disclosed herein are for treating IRI-mediated inflammation. In an embodiment, the methods and uses disclosed herein are for treating IRI- mediated sterile inflammation.

In an embodiment, the IRI is to a tissue or organ. In some embodiments, the tissue or organ is selected from the group consisting of lung, heart, kidney, liver, pancreas, intestine, stomach, thymus, uterus, bone marrow, skin, tendon and cornea. In an embodiment, the tissue or organ is lung. In another embodiment, the IRI is to a transplanted tissue or organ. In another embodiment, the IRI is to a transplanted lung, heart, kidney, liver, pancreas, intestine, stomach, thymus, uterus, bone marrow, skin, tendon and cornea. In another embodiment, the IRI is to a transplanted lung.

In an embodiment, the IRI is the result of a lung disease or disorder. In an embodiment, the IRI is a symptom of, or is associated with, a lung disease or disorder. Illustrative examples of lung disease or disorder include, but are not limited to, asbestosis, silicosis, asthma, lung collapse, lung infections, chronic obstructive pulmonary disease (COPD), lung cancers, cystic fibrosis, emphysema, pulmonary fibrosis and sarcoidosis.

In an embodiment, the IRI is associated with cardiovascular disease. Illustrative examples of cardiovascular diseases include, but are not limited to, atherosclerosis, chronic heart failure, congenital heart disease, rheumatic heart disease, peripheral artery disease, coronary heart disease, deep vein thrombosis and pulmonary embolism.

Heme-binding proteins

Heme is a ring-shaped iron-containing molecular component of hemoglobin, which is necessary to bind oxygen in the bloodstream.

Under physiological conditions, cell-free haemoglobin (also referred to as free hemoglobin) is typically bound by soluble proteins such as haptoglobin (Hp) (see Andersen et al., 2012, Nature, 489(7416) :456-459) and transported to macrophages and hepatocytes. However, where haemolysis is accelerated, the buffering capacity of Hp is overwhelmed. As a result, hemoglobin is quickly oxidised to ferri-haemoglobin, which in turn releases free heme (comprising protoporphyrin IX and iron; see Schaer et al. (2014; frontiers in PHYSIOLOGY, 5:1 -13)). Free heme may also arise from myoglobin and other hemoproteins released from damaged cells during tissue injury. Whilst heme plays a critical role as a prosthetic group in hemoproteins e.g., haemoglobin and myoglobin) that are involved in several biological processes, free heme is highly toxic. For instance, free heme is a source of redox-active iron, which in turn produces highly toxic reactive oxygen species (ROS) that damages lipid membranes (see Deuel etal. (2015; Free Radical Biology and Medicine, 89:931 -943), proteins and nucleic acids.

The requirement to maintain low levels of free heme under physiological steady-state conditions and during mild haemolysis typically involves the release of a group of plasma proteins that bind heme, including the heme scavenger Hp and heme scavenger proteins, such as hemopexin (Hx) and a1 -microglobulin (see Schaer et al. 2013; Blood, 121 (8):1276-84). After exceeding the binding capacity of the heme scavenger proteins, free heme accumulates in the plasma.

The term "heme-binding protein" is to be understood as meaning a protein or a peptide that has binding affinity for heme, in particular cell-free heme. Suitable heme-binding proteins (HBPs) include, but are not limited to, metalloproteins that contain a heme ligand (an iron- porphyrin complex) as a prosthetic group. Suitable methods for screening for heme-binding activity will be familiar to persons skilled in the art, illustrative examples of which are described elsewhere herein, and in Immenschuh et al. (2017. Front. Pharmacol. 8: 146), Karnaukhova etal. (2012. Biochim. Biophys. Acta 1820(12): 2020-2029) and Detzel etal. (2021. Biol. Chem. 402(6): 675-691 ).

Suitable heme-binding proteins will be familiar to persons skilled in the art, illustrative examples of which are described in Immenschuh etal. (2017. Front. Pharmacol. 8: 146) and include, but are not limited to, hemopexin, albumin, a1 -antitrypsin and a1 -microglobulin. In an embodiment, the heme-binding protein comprises, consists, or consists essentially of a1 -antitrypsin or a functional analogue thereof. In an embodiment, the heme-binding protein comprises, consists, or consists essentially of a1-microglobulin or a functional analogue thereof. In an embodiment, the heme-binding protein comprises, consists, or consists essentially of hemopexin or a functional analogue thereof.

It is to be understood that the term "hemopexin" (Hx), as used herein, includes native (naturally-occurring) and non-natural (synthetic) variants and non-human isoforms thereof. The Hx may be homogenous (insofar as it consists essentially of an Hx of the same isoform) or heterogeneous (insofar as it comprises a combination of different Hx isoforms, including human and non-human isoforms of Hx). Suitable methods for determining Hx isoforms that are present in an isolate will be familiar to persons skilled in the art, illustrative examples of which include high performance size exclusion chromatography (HPLC-SEC assay) and Hx ELISA. Hemopexin (Hx) is a 61 -kDa plasma p-1 B-glycoprotein composed of a single 439 amino acids long peptide chain, which is formed by two four-bladed p-propeller domains, resembling two thick disks that lock together at a 90° angle and are joined by an interdomain linker peptide. The heme, which is released into the blood as the result of intra- and extra- vascular haemolysis, is bound between the two four-bladed p-propeller domains in a pocket formed by the interdomain linker peptide. Residues His213 and His266 coordinate the heme iron atom giving a stable bis-histidyl complex, similar to haemoglobin.

Hemopexin represents the primary line of defence against heme toxicity attributed at least in part to its ability to bind heme with high affinity (K_D 10^-14) and function as a heme-specific carrier from the bloodstream to the liver. It binds heme in an equimolar ratio, but there is no evidence that heme is covalently bound to the protein. Whilst endogenous hemopexin can control the adverse effects of free heme under physiological steady-state conditions, it has little effect in maintaining steady-state heme levels under pathophysiological conditions, such as those associated with haemolysis, where a high level of heme leads to the depletion of endogenous hemopexin, causing heme-mediated oxidative tissue damage.

It is to be understood that naturally-occurring and recombinant forms of Hx are suitable for the methods described herein, as long as they are capable of binding to, and forming a complex with, cell-free heme and thereby neutralise the deleterious activity of the cell-free heme. Suitable naturally-occurring forms of Hx will be known to persons skilled in the art, illustrative examples of which are described in UniProtKB P02790; Koch et al. (2002, Clin. Chem. 48: 1377-1382) and Takahashi et al. (PNAS, 1985, 82(1 ):73-77), the entire contents of which are incorporated herein by reference.

In an embodiment, the hemopexin is human hemopexin. Illustrative examples of human Hx will be familiar to persons skilled in the art, an illustrative example of which is described in

UniProtKB P02790 and reproduced below:

MARVLGAPVA LGLWSLCWSL AIATPLPPTS AHGNVAEGET KPDPDVTERC SDGWSFDATT

LDDNGTMLFF KGEFVWKSHK WDRELISERW KNFPSPVDAA FRQGHNSVFL IKGDKVWVYP

PEKKEKGYPK LLQDEFPGIP SPLDAAVECH RGECQAEGVL FFQGDREWFW DLATGTMKER

SWPAVGNCSS ALRWLGRYYC FQGNQFLRFD PVRGEVPPRY PRDVRDYFMP CPGRGHGHRN

GTGHGNSTHH GPEYMRCSPH LVLSALTSDN HGATYAFSGT HYWRLDTSRD GWHSWPIAHQ

WPQGPSAVDA AFSWEEKLYL VQGTQVYVFL TKGGYTLVSG YPKRLEKEVG TPHGI ILDSV

DAAFICPGSS RLHIMAGRRL WWLDLKSGAQ ATWTELPWPH EKVDGALCME KSLGPNSCSA

NGPGLYLIHG PNLYCYSDVE KLNAAKALPQ PQNVTSLLGC TH ( SEQ ID NO : 1 )

In an embodiment, the Hx is a recombinant Hx.

In some embodiments, the heme-binding protein comprises, consists or consists essentially of plasma-derived Hx. In an embodiment, the hemopexin is plasma-derived hemopexin.

In another embodiment, the hemopexin is human hemopexin or a functional analogue thereof.

In an embodiment, the hemopexin or the functional analogue thereof comprises, consists or consists essentially of an amino acid sequence having at least about 50%, preferably at least about 55%, preferably at least about 60%, preferably at least about 65%, preferably at least about 70%, preferably at least about 75%, preferably at least about 80%, preferably at least about 85%, preferably at least about 90%, preferably at least about 95%, or more preferably at least about 99% sequence identity to a native hemopexin protein. In an embodiment, the human hemopexin comprises, consists or consists essentially of an amino acid sequence as shown in UniProtKB P02790 (SEQ ID NO:1 ).

As used herein, the term "plasma derived" is to be understood to mean that the heme-binding protein (e.g., hemopexin) is isolated, purified or otherwise extracted from plasma or a plasma fraction. Suitable methods for the isolation of Hx from a natural source of Hx- (e.g., blood plasma) will be familiar to persons skilled in the art, illustrative examples of which are described in WO 2014/055552 and WO 2019/030262, the entire contents of which are incorporated herein by reference.

The term "functional analogue" is to be understood to mean a variant of a native (naturally- occurring) molecule that shares substantially the same biological activity of the native molecule, insofar as that biological activity is characterised by at least the ability of the analogue to bind to, and form a complex with, cell-free heme and thereby neutralise its activity. By "substantially the same biological activity" typically means the functional analogue has a binding affinity for cell-free heme that is at least about 40% (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 85%, 90%, 95%, 100%, 105%, 100%, 105%, 1 10%, 1 15%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165% and so on) of the binding affinity of the naturally-occurring molecule, including naturally-occurring isoforms (e.g., human and/or nonhuman isoforms of the heme-binding protein). Suitable methods for determining whether an agent is a functional analogue of a heme-binding protein such as Hx will be familiar to persons skilled in the art, illustrative examples of which include are described elsewhere herein.

In an embodiment disclosed herein, the functional analogue is a functional fragment of the native heme-binding protein. A functional fragment of native heme-binding protein can be any suitable length, as long as the fragment retains the ability to bind to, and form a complex with, cell-free heme and thereby neutralise its activity. In an embodiment disclosed herein, the functional analogue is a functional fragment of native Hx. A functional fragment of native Hx can be any suitable length, as long as the fragment retains the ability to bind to, and form a complex with, cell-free heme and thereby neutralise its biological activity.

In another embodiment, the functional analogue is a peptide that has a different amino acid sequence to a naturally-occurring (native) heme-binding protein (i.e., the comparator or reference protein). The functional analogue may include a molecule that has an amino acid sequence that differs from the amino acid sequence of the native protein (e.g., Hx) by one or more (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9 or more) amino acid substitutions, wherein said difference does not, or does not completely, abolish the ability of the analogue to bind to, and form a complex with, cell-free heme and thereby neutralise its activity. In some embodiments, the functional analogue comprises amino acid substitutions that enhance the ability of the analogue to bind to, and form a complex with, cell-free heme, as compared to the native hemebinding protein. In an embodiment, the functional analogue has an amino acid sequence that differs from the amino acid sequence of native heme-binding protein by one or more conservative amino acid substitutions. As used herein, the term “conservative amino acid substitution” refers to changing amino acid identity at a given position to replace it with an amino acid of approximately equivalent size, charge and/or polarity. Examples of natural conservative substitutions of amino acids include the following 8 substitution groups (designated by the conventional one-letter code): (1 ) M, I, L, V; (2) F, Y, W; (3) K, R, (4) A, G; (5) S, T; (6) Q, N; (7) E, D; and (8) C, S.

In an embodiment, the functional analogue has at least about 70% sequence identity to an amino acid sequence of the native heme-binding protein. Reference to "at least about 70%" includes about 70%, about 71 %, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% sequence identity or similarity, for example, after optimal alignment or best fit analysis. Thus, in an embodiment, the sequence has at least about 75%, at least about 80%, at least about 85%, preferably at least about 86%, preferably at least about 87%, preferably at least about 88%, preferably at least about 89%, preferably at least about 90%, preferably at least about 91%, preferably at least about 92%, preferably at least about 93%, preferably at least about 94%, preferably at least about 95%, preferably at least about 96%, preferably at least about 97%, preferably at least about 98%, preferably at least about 99% or preferably

100% sequence identity or sequence homology with the sequences identified herein, for example, after optimal alignment or best fit analysis.

The terms “identity”, “similarity”, “sequence identity”, “sequence similarity”, “homology”, “sequence homology” and the like, as used herein, mean that at any particular amino acid residue position in an aligned sequence, the amino acid residue is identical between the aligned sequences. The term “similarity” or “sequence similarity” as used herein, indicates that, at any particular position in the aligned sequences, the amino acid residue is of a similar type between the sequences. For example, leucine may be substituted for an isoleucine or valine residue. As noted elsewhere herein, this may be referred to as conservative substitution. In an embodiment, an amino acid sequence may be modified by way of conservative substitution of any of the amino acid residues contained therein, such that the modification has no effect on the binding specificity or functional activity of the modified polypeptide when compared to the unmodified (native) heme-binding protein.

In some embodiments, sequence identity with respect to a peptide sequence relates to the percentage of amino acid residues in the candidate sequence which are identical with the residues of the corresponding peptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percentage homology, and not considering any conservative substitutions as part of the sequence identity. Neither N- or C- terminal extensions, nor insertions shall be construed as reducing sequence identity or homology. Methods and computer programs for performing an alignment of two or more amino acid sequences and determining their sequence identity or homology are well known to persons skilled in the art. For example, the percentage of identity or similarity of two amino acid sequences can be readily calculated using algorithms, for example, BLAST, FASTA, or the Smith-Waterman algorithm.

Techniques for determining an amino acid sequence "similarity" are well known to persons skilled in the art. In general, "similarity" means an exact amino acid to amino acid comparison of two or more peptide sequences or at the appropriate place, where amino acids are identical or possess similar chemical and/or physical properties such as charge or hydrophobicity. A so- termed "percent similarity" then can be determined between the compared peptide sequences. In general, "identity" refers to an exact amino acid to amino acid correspondence of two peptide sequences.

Two or more peptide sequences can also be compared by determining their "percent identity". The percent identity of two sequences may be described as the number of exact matches between two aligned sequences divided by the length of the shorter sequence and multiplied by 100. An approximate alignment for nucleic acid sequences is provided by the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981 ). This algorithm can be extended to use with peptide sequences using the scoring matrix developed by Dayhoff (Atlas of Protein Sequences and Structure, M. O. Dayhoff ed., 5 suppl. 3:353-358, National Biomedical Research Foundation, Washington, D.C., USA), and normalized by Gribskov (Nucl. Acids Res. 14(6):6745-6763, 1986). Suitable programs for calculating the percent identity or similarity between sequences are generally known in the art.

Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, Wl, USA) or by inspection and the best alignment (i.e., resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as for example disclosed by Altschul etal., (1997, Nucl. Acids F?es.25:3389. A detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et al. ("Current Protocols in Molecular Biology", John Wiley & Sons Inc, 1994-1998, Chapter 15).

In an embodiment, a functional analogue includes amino acid substitutions and/or other modifications relative to native heme-binding protein in order to increase the stability and / or solubility of the heme-binding protein.

The functional analogue may suitably be a naturally-occurring molecule or it may be synthetically produced by recombinant or chemical synthesis using methods known to persons skilled in the art.

The heme-binding protein may suitably be produced as a recombinant protein in a microorganism, which can be isolated and, if desired, further purified. Suitable microorganisms for the production of recombinant proteins, including recombinant heme-binding proteins, will be familiar to persons skilled in the art, illustrative examples of which include bacteria, yeast or fungi, eukaryote cells e.g., mammalian or an insect cells), or in a recombinant virus vector e.g., adenovirus, poxvirus, herpesvirus, Simliki forest virus, baculovirus, bacteriophage, sindbis virus or sendai virus). Suitable bacteria for producing recombinant peptides will be familiar to persons skilled in the art, illustrative examples of which include E. coli, B.subtilis or any other bacterium that is capable of expressing the peptide sequences. Illustrative examples of suitable yeast types for producing recombinant peptides include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida, Pichia pastoris or any other yeast capable of expressing peptides. Corresponding methods are well known in the art. Also, methods for isolating and purifying recombinantly produced peptide sequences are well known in the art and include, for example, gel filtration, affinity chromatography and ion exchange chromatography. To facilitate isolation of a recombinant proteins, including recombinant heme-binding proteins, as described herein, a fusion polypeptide may be made where the peptide sequence of the heme-binding protein, or functional analogue thereof, is translationally fused (covalently linked) to a heterologous polypeptide which enables isolation by affinity chromatography. Illustrative examples of suitable heterologous polypeptides are His-Tag (e.g. Hise. 6 histidine residues), GST-Tag (Glutathione-S-transferase) etc.

For preparing recombinant heme-binding proteins, phage libraries and/or peptide libraries are also suitable, for instance, produced by means of combinatorial chemistry or obtained by means of high throughput screening techniques for the most varying structures (see, for example, Display: A Laboratory Manual by Carlos F. Barbas (Editor), et al. and Willats WG Phage display: practicalities and prospects. Plant Mol. Biol. 2002 December; 50(6):837-54).

The heme-binding protein, as described herein, may be fused, coupled or otherwise attached to one or more heterologous moieties as part of a fusion protein or construct. The one or more heterologous moieties may suitably improve, enhance or otherwise extend the activity or stability of the heme-binding protein. In an embodiment, the heme-binding protein, as described herein, is suitably attached to a heterologous moiety for extending the half-life of the heme-binding protein in vivo. Suitable half-life extending heterologous moieties will be familiar to persons skilled in the art, illustrative examples of which include polyethylene glycol (PEGylation), glycosylated PEG, hydroxyl ethyl starch (HESylation), polysialic acids, elastinlike polypeptides, heparosan polymers and hyaluronic acid. Thus, in an embodiment disclosed herein, heterologous moiety is selected from the group consisting of polyethylene glycol (PEGylation), glycosylated PEG, hydroxyl ethyl starch (HESylation), polysialic acids, elastinlike polypeptides, heparosan polymers and hyaluronic acid. In other embodiments, the heterologous moiety may be a heterologous amino acid sequence fused to the heme-binding protein.

Alternatively, or in addition, the heterologous moiety may be chemically conjugated to the heme-binding protein, for example, a covalent bond. The half-life extending heterologous moiety can be fused, conjugated or otherwise attached to the heme-binding protein by any suitable means known to persons skilled in the art, an illustrative example of which is via a chemical linker. The principle of this conjugation technology has been described in an exemplary manner by Conjuchem LLC (see, e.g., US patent No. 7,256,253), the entire contents of which are incorporated herein by reference. In other embodiments, the heterologous moiety is a half-life enhancing protein (HLEP). Suitable half-life enhancing proteins will be familiar to persons skilled in the art, an illustrative example of which includes albumin or fragments thereof. Thus, in an embodiment, the HLEP is an albumin or a fragment thereof. The N-terminus of the albumin or fragment thereof may be fused to the C-terminus of the heme-binding protein, such as Hx. Alternatively, or in addition, the C-terminus of the albumin or fragment thereof may be fused to the N-terminus of the heme-binding protein, such as Hx. One or more HLEPs may be fused to the N- or C- terminal part(s) of the heme-binding protein provided that they do not abolish the ability of the heme-binding protein to bind to, and form a complex with, cell-free heme. It is to be understood, however, that some reduction in the binding of the heme-binding protein to cell-free heme may be acceptable, as long as the heme-binding protein component of the fusion protein is still capable of forming a complex with, and thereby neutralise, cell-free heme.

The fusion protein may further comprise a chemical bond or a linker sequence positioned between the heme-binding protein and the heterologous moiety. The linker sequence may be a peptidic linker consisting of one or more amino acids, in particular of 1 to 50, preferably 1 to 30, preferably 1 to 20, preferably 1 to 15, preferably 1 to 10, preferably 1 to 5 or more preferably 1 to 3 (e.g. 1 , 2 or 3) amino acids and which may be equal or different from each other. Preferably, the linker sequence is not present at the corresponding position in the wild-type or native heme-binding protein. Illustrative examples of suitable amino acids present in said linker sequence include Gly and Ser. In an embodiment, the linker sequence is substantially non- immunogenic to the subject to be treated in accordance with the methods disclosed herein. By substantially non-immunogenic is meant that the linker sequence will not raise a detectable antibody response to the linker sequence in the subject to which it is administered. Suitable linkers may be comprised of alternating glycine and serine residues. Other suitable linkers will be familiar to persons skilled in the art, illustrative examples of which are described in W02007/090584. In an embodiment, the peptidic linker between the heme-binding protein and the heterologous moiety comprises, consists or consists essentially of peptide sequences, which serve as natural interdomain linkers in human proteins. Such peptide sequences in their natural environment may be located close to the protein surface and are accessible to the immune system so that one can assume a natural tolerance against this sequence. Illustrative examples are given in WO 2007/090584. Suitable cleavable linker sequences are described, e.g., in WO 2013/120939 A1 . In an embodiment, the heterologous moiety is a half-life extending polypeptide. In an embodiment, the half-life extending polypeptide is selected from the group consisting of albumin, a member of the albumin-family or fragments thereof, solvated random chains with large hydrodynamic volume (e.g. XTEN (see Schellenberger et al. 2009; Nature BiotechnoL 27:1 186-1190), homo-amino acid repeats (HAP) or proline-alanine-serine repeats (PAS), afamin, alpha-fetoprotein, Vitamin D binding protein, transferrin or variants or fragments thereof, carboxyl-terminal peptide (CTP) of human chorionic gonadotropin-B subunit, a polypeptide capable of binding to the neonatal Fc receptor (FcRn), in particular an immunoglobulin constant region and portions thereof, e.g. the Fc fragment, polypeptides or lipids capable of binding under physiological conditions to albumin, to a member of the albumin-family or to fragments thereof or to an immunoglobulin constant region or portions thereof. The immunoglobulin constant region or portions thereof is preferably an Fc fragment of immunoglobulin G1 ( IgG 1 ), an Fc fragment of immunoglobulin G2 (lgG2) or an Fc fragment of immunoglobulin A (IgA). A half-life enhancing polypeptide, as used herein, may be a full- length half-life-enhancing protein or one or more fragments thereof that are capable of stabilizing or prolonging the therapeutic activity or the biological activity of the Hx, in particular of increasing the in vivo half-life of the Hx. Such fragments may be of 10 or more amino acids in length or may include at least about 15, preferably at least about 20, preferably at least about 25, preferably at least about 30, preferably at least about 50, or more preferably at least about 100, or more contiguous amino acids from the HLEP sequence, or may include part or all of specific domains of the respective HLEP, as long as the HLEP fragment provides a functional half-life extension of at least about 10%, preferably of at least about 20%, or more preferably of at least about 25%, compared to the respective heme-binding protein in the absence of the HLEP. Methods of determining whether a heterologous moiety provides a functional half-life extension to the heme-binding protein (in vivo or in vitro) will be familiar to persons skilled in the art, illustrative examples of which are described elsewhere herein.

The HLEP portion of the fusion protein, as described herein, may be a variant of a wild type HLEP. The term "variant" includes insertions, deletions and / or substitutions, either conservative or non-conservative, where such changes do not substantially alter the ability of the heme-binding protein to form a complex with, and thereby neutralise, cell-free heme.

The fusion proteins, as described herein, can be created by in-frame joining of at least two DNA sequences encoding the heme-binding protein and the heterologous moiety, such as a HLEP. Persons skilled in the art will understand that translation of the fusion protein DNA sequence will result in a single protein sequence. As a result of an in-frame insertion of a DNA sequence encoding a peptidic linker according to an embodiment disclosed herein, a fusion protein comprising the heme-binding protein, a suitable linker and the heterologous moiety can be obtained.

In an embodiment disclosed herein, the heme-binding protein is fused to a heterologous moiety. In an embodiment, the heterologous moiety comprises, consists or consists essentially of a polypeptide selected from the group consisting of albumin or fragments thereof, transferrin or fragments thereof, the C-terminal peptide of human chorionic gonadotropin, an XTEN sequence, homo-amino acid repeats (HAP), proline-alanine-serine repeats (PAS), afamin, alpha-fetoprotein, Vitamin D binding protein, polypeptides capable of binding under physiological conditions to albumin or to immunoglobulin constant regions, polypeptides capable of binding to the neonatal Fc receptor (FcRn), particularly immunoglobulin constant regions and portions thereof, preferably the Fc portion of immunoglobulin, and combinations of any of the foregoing. In another embodiment, the heterologous moiety is selected from the group consisting of hydroxyethyl starch (HES), polyethylene glycol (PEG), polysialic acids (PSAs), elastin-like polypeptides, heparosan polymers, hyaluronic acid and albumin binding ligands, e.g. fatty acid chains, and combinations of any of the foregoing.

The terms, "human serum albumin" (HSA) and "human albumin" (HA) and "albumin" (ALB) are used interchangeably herein. The terms "albumin" and "serum albumin" are broader and encompass human serum albumin (and fragments and variants thereof), as well as albumin from other species (and fragments and variants thereof). As used herein, "albumin" refers collectively to albumin polypeptide or amino acid sequence, or an albumin fragment or variant, having one or more functional activities (e.g., biological activities) of albumin. In particular, "albumin" refers to human albumin or fragments thereof, including the mature form of human albumin or albumin from other vertebrates or fragments thereof, or analogs or variants of these molecules or fragments thereof. In some embodiments disclosed herein, the alternative term "FP" is used to identify the HLEP, in particular to define albumin as the HLEP.

The fusion proteins described herein may suitably comprise naturally-occurring polymorphic variants of human albumin and / or fragments of human albumin. Generally speaking, an albumin fragment or variant will be at least 10, preferably at least 40, or most preferably more than 70 amino acids in length.

In an embodiment, the HLEP is an albumin variant with enhanced binding to the FcRn receptor. Such albumin variants may lead to a longer plasma half-life of the heme-binding protein or functional analogue thereof compared to the heme-binding protein or functional fragment thereof that is fused to a wild-type albumin. The albumin portion of the fusion proteins described herein may suitably comprise at least one subdomain or domain of human albumin or conservative modifications thereof.

In an embodiment, the heterologous moiety is an immunoglobulin molecule or a functional fragment thereof. Immunoglobulin G (IgG) constant regions (Fc) are known in the art to increase the half-life of therapeutic proteins (see, e.g., Dumont J A et al. 2006. BioDrugs 20:151 -160). The IgG constant region of the heavy chain consists of 3 domains (CH1 -CH3) and a hinge region. The immunoglobulin sequence may be derived from any mammal, or from subclasses lgG1 , lgG2, lgG3 or lgG4, respectively. IgG and IgG fragments without an antigenbinding domain may also be used as a heterologous moiety, including as a HLEP. The hemebinding protein or functional analogue thereof may suitably be connected to the IgG or the IgG fragments via the hinge region of the antibody or a peptidic linker, which may even be cleavable. Several patents and patent applications describe the fusion of therapeutic proteins to immunoglobulin constant regions to enhance the therapeutic proteins’ in vivo half-lives. For example, US 2004/0087778 and WO 2005/001025 describe fusion proteins of Fc domains or at least portions of immunoglobulin constant regions with biologically active peptides that increase the half-life of the peptide, which otherwise would be quickly eliminated in vivo. Fc- IFN-p fusion proteins were described that achieved enhanced biological activity, prolonged circulating half-life and greater solubility (WO 2006/000448 A2). Fc-EPO proteins with a prolonged serum half-life and increased in vivo potency were disclosed (WO 2005/063808 A1 ) as well as Fc fusions with G-CSF (WO 2003/076567 A2), glucagon-like peptide-1 (WO 2005/000892 A2), clotting factors (WO 2004/101740 A2) and interleukin-10 (U.S. Pat. No. 6,403,077), all with half-life enhancing properties.

Illustrative examples of suitable HLEP which can be used in accordance with the present invention are also described in WO 2013/120939 A1 , the contents of which are incorporated herein by reference in their entirety.

Methods of treatment

It is to be understood that a reference to a subject herein does not imply that the subject has had a ischaemia-reperfusion injury, but also includes a subject that is at risk of a ischaemiareperfusion injury. In an embodiment, the subject has (i.e., is experiencing) an ischaemiareperfusion injury, or a symptom thereof. In another embodiment, the subject has not had an ischaemia-reperfusion injury at the time of treatment, but is at risk of ischaemia-reperfusion injury. As an illustrative example, the subject has a lung disease or a cardiovascular disease or disorder that has not yet progressed to ischaemia or an ischaemia-reperfusion injury, but is at risk of ischaemia-reperfusion injury. In some instances, the subject may undergo surgical intervention to minimise the risk of disease-progression. In another illustrative example, the subject is an organ transplant recipient where the organ to be transplanted is at risk of ischaemia-reperfusion injury. The methods described herein may therefore suitably be prescribed to the subject as a prophylactic measure to minimise, reduce, abrogate or otherwise inhibit ischaemia-reperfusion injury, such as following a surgical procedure or surgical intervention.

The extent to which the methods disclosed herein provide a subjective, qualitative and / or quantitative reduction in the severity of the ischaemia-reperfusion injury may suitably be represented as a percentage reduction, for example, by at least about 10%, preferably from about 10% to about 20%, preferably from about 15% to about 25%, preferably from about 20% to about 30%, preferably from about 25% to about 35%, preferably from about 30% to about 40%, preferably from about 35% to about 45%, preferably from about 40% to about 50%, preferably from about 45% to about 55%, preferably from about 50% to about 60%, preferably from about 55% to about 65%, preferably from about 60% to about 70%, preferably from about 65% to about 75%, preferably from about 70% to about 80%, preferably from about 75% to about 85%, preferably from about 80% to about 90%, preferably from about 85% to about 95%, or most preferably from about 90% to 100% of, for example, a marker of inflammation, including sterile inflammation, in the subject; or when compared to the corresponding marker in the same subject prior to administration of the therapeutically effective amount of the hemebinding protein.

The reduction in the severity of ischaemia-reperfusion injury and/or inflammation may suitably be represented as a percentage reduction, for example, by at least 5%, preferably from about 10% to about 20%, preferably from about 15% to about 25%, preferably from about 20% to about 30%, preferably from about 25% to about 35%, preferably from about 30% to about 40%, preferably from about 35% to about 45%, preferably from about 40% to about 50%, preferably from about 45% to about 55%, preferably from about 50% to about 60%, preferably from about 55% to about 65%, preferably from about 60% to about 70%, preferably from about 65% to about 75%, preferably from about 70% to about 80%, preferably from about 75% to about 85%, preferably from about 80% to about 90%, preferably from about 85% to about 95%, or most preferably from about 90% to 100% in the number of neutrophils infiltrating the affected tissue or organ when compared to the number of neutrophils infiltrating a corresponding affected tissue or organ of a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of neutrophils infiltrating the tissue or organ of a subject prior to administration of the therapeutically effective amount of the heme-binding protein. In an embodiment, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the number of infiltrating neutrophils in the affected tissue or organ by at least about 5% when compared to the number of infiltrating neutrophils of a corresponding affected tissue or organ of a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of neutrophils infiltrating the tissue or organ of the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the number of infiltrating neutrophils by at least about 10% when compared to the number of infiltrating neutrophils of a corresponding affected tissue or organ of a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of neutrophils infiltrating the tissue or organ of the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a hemebinding protein is sufficient to reduce the number of infiltrating neutrophils by at least about 20% when compared to the number of infiltrating neutrophils of a corresponding affected tissue or organ of a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of neutrophils infiltrating the tissue or organ of the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the number of infiltrating neutrophils by at least about 25% when compared to the number of infiltrating neutrophils of a corresponding affected tissue or organ of a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of neutrophils infiltrating the tissue or organ of the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the number of infiltrating neutrophils by at least about 30% when compared to the number of infiltrating neutrophils of a corresponding affected tissue or organ of a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of neutrophils infiltrating the tissue or organ of the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a hemebinding protein is sufficient to reduce the number of infiltrating neutrophils by at least about 35% when compared to the number of infiltrating neutrophils of a corresponding affected tissue or organ of a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of neutrophils infiltrating the tissue or organ of the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the number of infiltrating neutrophils by at least about 40% when compared to the number of infiltrating neutrophils of a corresponding affected tissue or organ of a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of neutrophils infiltrating the tissue or organ of the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the number of infiltrating neutrophils by at least about 45% when compared to the number of infiltrating neutrophils of a corresponding affected tissue or organ of a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of neutrophils infiltrating the tissue or organ of the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a hemebinding protein is sufficient to reduce the number of infiltrating neutrophils by at least about 50% when compared to the number of infiltrating neutrophils of a corresponding affected tissue or organ of a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of neutrophils infiltrating the tissue or organ of the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the number of infiltrating neutrophils by at least about 60% when compared to the number of infiltrating neutrophils of a corresponding affected tissue or organ of a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of neutrophils infiltrating the tissue or organ of the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the number of infiltrating neutrophils by at least about 70% when compared to the number of infiltrating neutrophils of a corresponding affected tissue or organ of a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of neutrophils infiltrating the tissue or organ of the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a hemebinding protein is sufficient to reduce the number of infiltrating neutrophils by at least about 80% when compared to the number of infiltrating neutrophils of a corresponding affected tissue or organ of a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of neutrophils infiltrating the tissue or organ of the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the number of infiltrating neutrophils by at least about 90% when compared to the number of infiltrating neutrophils of a corresponding affected tissue or organ of a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of neutrophils infiltrating the tissue or organ of the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the number of infiltrating neutrophils by at least about 100% when compared to the number of infiltrating neutrophils of a corresponding affected tissue or organ of a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of neutrophils infiltrating the tissue or organ of the subject prior to administration of the therapeutically effective amount of the heme-binding protein. Suitable methods of identifying the number of infiltrating neutrophils in an affected tissue or organ of a subject would be familiar to persons skilled in the art, including illustrative methods of which are disclosed herein. For example, infiltrating neutrophils can be identified by the eosinophilic granular cytoplasm and the circular nucleus and neutrophils in vessels were excluded.

The reduction in the severity of ischaemia-reperfusion injury and/or inflammation may also be suitably represented as a percentage reduction in the level of perivascular edema in the affected tissue or organ, for example, by at least about 5%, preferably from about 10% to about 20%, preferably from about 15% to about 25%, preferably from about 20% to about 30%, preferably from about 25% to about 35%, preferably from about 30% to about 40%, preferably from about 35% to about 45%, preferably from about 40% to about 50%, preferably from about 45% to about 55%, preferably from about 50% to about 60%, preferably from about 55% to about 65%, preferably from about 60% to about 70%, preferably from about 65% to about 75%, preferably from about 70% to about 80%, preferably from about 75% to about 85%, preferably from about 80% to about 90%, preferably from about 85% to about 95%, or most preferably from about 90% to 100% of the cuff area-to-vessel area ratio when compared to the cuff area- to-vessel area ratio of a corresponding affected tissue or organ of a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the cuff area-to-vessel area ratio of the affected tissue or organ of the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a hemebinding protein is sufficient to reduce the cuff area-to-vessel area ratio by at least about 5% when compared to the cuff area-to-vessel area ratio of a corresponding affected tissue or organ of a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the cuff area-to-vessel area ratio of the affected tissue or organ of the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the cuff area-to-vessel area ratio by at least about 10% when compared to the cuff area-to-vessel area ratio of a corresponding affected tissue or organ of a subject to whom a therapeutically effective amount of the hemebinding protein has not been administered, or when compared to the cuff area-to-vessel area ratio of the affected tissue or organ of the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the cuff area- to-vessel area ratio by at least about 15% when compared to the cuff area-to-vessel area ratio of a corresponding affected tissue or organ of a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the cuff area-to-vessel area ratio of the affected tissue or organ of the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the cuff area-to-vessel area ratio by at least about 20% when compared to the cuff area-to-vessel area ratio of a corresponding affected tissue or organ of a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the cuff area-to-vessel area ratio of the affected tissue or organ of the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a hemebinding protein is sufficient to reduce the cuff area-to-vessel area ratio by at least about 25% when compared to the cuff area-to-vessel area ratio of a corresponding affected tissue or organ of a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the cuff area-to-vessel area ratio of the affected tissue or organ of the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the cuff area-to-vessel area ratio by at least about 30% when compared to the cuff area-to-vessel area ratio of a corresponding affected tissue or organ of a subject to whom a therapeutically effective amount of the hemebinding protein has not been administered, or when compared to the cuff area-to-vessel area ratio of the affected tissue or organ of the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the cuff area- to-vessel area ratio by at least about 35% when compared to the cuff area-to-vessel area ratio of a corresponding affected tissue or organ of a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the cuff area-to-vessel area ratio of the affected tissue or organ of the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the cuff area-to-vessel area ratio by at least about 40% when compared to the cuff area-to-vessel area ratio of a corresponding affected tissue or organ of a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the cuff area-to-vessel area ratio of the affected tissue or organ of the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a hemebinding protein is sufficient to reduce the cuff area-to-vessel area ratio by at least about 45% when compared to the cuff area-to-vessel area ratio of a corresponding affected tissue or organ of a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the cuff area-to-vessel area ratio of the affected tissue or organ of the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the cuff area-to-vessel area ratio by at least about 50% when compared to the cuff area-to-vessel area ratio of a corresponding affected tissue or organ of a subject to whom a therapeutically effective amount of the hemebinding protein has not been administered, or when compared to the cuff area-to-vessel area ratio of the affected tissue or organ of the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the cuff area- to-vessel area ratio by at least about 60% when compared to the cuff area-to-vessel area ratio of a corresponding affected tissue or organ of a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the cuff area-to-vessel area ratio of the affected tissue or organ of the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the cuff area-to-vessel area ratio by at least about 70% when compared to the cuff area-to-vessel area ratio of a corresponding affected tissue or organ of a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the cuff area-to-vessel area ratio of the affected tissue or organ of the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a hemebinding protein is sufficient to reduce the cuff area-to-vessel area ratio by at least about 80% when compared to the cuff area-to-vessel area ratio of a corresponding affected tissue or organ of a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the cuff area-to-vessel area ratio of the affected tissue or organ of the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the cuff area-to-vessel area ratio by at least about 90% when compared to the cuff area-to-vessel area ratio of a corresponding affected tissue or organ of a subject to whom a therapeutically effective amount of the hemebinding protein has not been administered, or when compared to the cuff area-to-vessel area ratio of the affected tissue or organ of the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the cuff area- to-vessel area ratio by at least about 100% when compared to the cuff area-to-vessel area ratio of a corresponding affected tissue or organ of a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the cuff area-to-vessel area ratio of the affected tissue or organ of the subject prior to administration of the therapeutically effective amount of the heme-binding protein. Suitable methods for determining the level of perivascular edema would be familiar to persons skilled in the art, illustrative examples of which are disclosed herein, and described in Conhaim, Lai-Fook et al. 1986 and Lowe, Alvarez et al. 2010, the contents of which are incorporated herein by reference in their entirety.

The reduction in the severity of ischaemia-reperfusion injury and/or inflammation may also be suitably represented as a percentage reduction in the level of thrombotic vasoocclusion in blood vessels in the affected tissue or organ, for example, by at least about 10%, preferably from about 10% to about 20%, preferably from about 15% to about 25%, preferably from about 20% to about 30%, preferably from about 25% to about 35%, preferably from about 30% to about 40%, preferably from about 35% to about 45%, preferably from about 40% to about 50%, preferably from about 45% to about 55%, preferably from about 50% to about 60%, preferably from about 55% to about 65%, preferably from about 60% to about 70%, preferably from about 65% to about 75%, preferably from about 70% to about 80%, preferably from about 75% to about 85%, preferably from about 80% to about 90%, preferably from about 85% to about 95%, or most preferably from about 90% to 100% when compared to the level of thrombotic vasoocclusion of a corresponding affected tissue or organ of a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the level of thrombotic vasoocclusion of the affected tissue or organ of the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a hemebinding protein is sufficient to reduce the level of thrombotic vasoocclusion in blood vessels by at least about 10% when compared to the level of thrombotic vasoocclusion of a corresponding affected tissue or organ of a subject to whom a therapeutically effective amount of the hemebinding protein has not been administered, or when compared to the level of thrombotic vasoocclusion of the affected tissue or organ of the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the level of thrombotic vasoocclusion in blood vessels by at least about 15% when compared to the level of thrombotic vasoocclusion of a corresponding affected tissue or organ of a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the level of thrombotic vasoocclusion of the affected tissue or organ of the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the level of thrombotic vasoocclusion in blood vessels by at least about 20% when compared to the level of thrombotic vasoocclusion of a corresponding affected tissue or organ of a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the level of thrombotic vasoocclusion of the affected tissue or organ of the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the level of thrombotic vasoocclusion in blood vessels by at least about 25% when compared to the level of thrombotic vasoocclusion of a corresponding affected tissue or organ of a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the level of thrombotic vasoocclusion of the affected tissue or organ of the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the level of thrombotic vasoocclusion in blood vessels by at least about 30% when compared to the level of thrombotic vasoocclusion of a corresponding affected tissue or organ of a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the level of thrombotic vasoocclusion of the affected tissue or organ of the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the level of thrombotic vasoocclusion in blood vessels by at least about 35% when compared to the level of thrombotic vasoocclusion of a corresponding affected tissue or organ of a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the level of thrombotic vasoocclusion of the affected tissue or organ of the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the level of thrombotic vasoocclusion in blood vessels by at least about 40% when compared to the level of thrombotic vasoocclusion of a corresponding affected tissue or organ of a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the level of thrombotic vasoocclusion of the affected tissue or organ of the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the level of thrombotic vasoocclusion in blood vessels by at least about 45% when compared to the level of thrombotic vasoocclusion of a corresponding affected tissue or organ of a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the level of thrombotic vasoocclusion of the affected tissue or organ of the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the level of thrombotic vasoocclusion in blood vessels by at least about 50% when compared to the level of thrombotic vasoocclusion of a corresponding affected tissue or organ of a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the level of thrombotic vasoocclusion of the affected tissue or organ of the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the level of thrombotic vasoocclusion in blood vessels by at least about 55% when compared to the level of thrombotic vasoocclusion of a corresponding affected tissue or organ of a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the level of thrombotic vasoocclusion of the affected tissue or organ of the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the level of thrombotic vasoocclusion in blood vessels by at least about 60% when compared to the level of thrombotic vasoocclusion of a corresponding affected tissue or organ of a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the level of thrombotic vasoocclusion of the affected tissue or organ of the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the level of thrombotic vasoocclusion in blood vessels by at least about 65% when compared to the level of thrombotic vasoocclusion of a corresponding affected tissue or organ of a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the level of thrombotic vasoocclusion of the affected tissue or organ of the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the level of thrombotic vasoocclusion in blood vessels by at least about 70% when compared to the level of thrombotic vasoocclusion of a corresponding affected tissue or organ of a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the level of thrombotic vasoocclusion of the affected tissue or organ of the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the level of thrombotic vasoocclusion in blood vessels by at least about 75% when compared to the level of thrombotic vasoocclusion of a corresponding affected tissue or organ of a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the level of thrombotic vasoocclusion of the affected tissue or organ of the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the level of thrombotic vasoocclusion in blood vessels by at least about 80% when compared to the level of thrombotic vasoocclusion of a corresponding affected tissue or organ of a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the level of thrombotic vasoocclusion of the affected tissue or organ of the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the level of thrombotic vasoocclusion in blood vessels by at least about 85% when compared to the level of thrombotic vasoocclusion of a corresponding affected tissue or organ of a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the level of thrombotic vasoocclusion of the affected tissue or organ of the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the level of thrombotic vasoocclusion in blood vessels by at least about 90% when compared to the level of thrombotic vasoocclusion of a corresponding affected tissue or organ of a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the level of thrombotic vasoocclusion of the affected tissue or organ of the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the level of thrombotic vasoocclusion in blood vessels by at least about 95% when compared to the level of thrombotic vasoocclusion of a corresponding affected tissue or organ of a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the level of thrombotic vasoocclusion of the affected tissue or organ of the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the level of thrombotic vasoocclusion in blood vessels by at least about 100% when compared to the level of thrombotic vasoocclusion of a corresponding affected tissue or organ of a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the level of thrombotic vasoocclusion of the affected tissue or organ of the subject prior to administration of the therapeutically effective amount of the heme-binding protein. The level of thrombotic vasoocclusion in blood vessels can be assessed or measured by quantifying the number of thrombotic vessels. Other suitable methods of determining and measuring the level of thrombotic vasoocclusion in blood vessels would be familiar to persons skilled in the art, illustrative examples of which are disclosed herein and described in Conhaim, Lai-Fook et al. 1986 and Lowe, Alvarez et al. 2010, the contents of which are incorporated herein by reference in their entirety.

The reduction in the severity of ischaemia-reperfusion injury and/or inflammation may also be suitably represented as a percentage reduction, for example, by at least about 5%, preferably from about 10% to about 20%, preferably from about 15% to about 25%, preferably from about 20% to about 30%, preferably from about 25% to about 35%, preferably from about 30% to about 40%, preferably from about 35% to about 45%, preferably from about 40% to about 50%, preferably from about 45% to about 55%, preferably from about 50% to about 60%, preferably from about 55% to about 65%, preferably from about 60% to about 70%, preferably from about 65% to about 75%, preferably from about 70% to about 80%, preferably from about 75% to about 85%, preferably from about 80% to about 90%, preferably from about 85% to about 95%, or most preferably from about 90% to 100%, in the level of expression of a pro-inflammatory marker in a subject when compared to the level of expression of one or more pro-inflammatory markers in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the level of expression of the one or more pro-inflammatory markers in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. Suitable pro-inflammatory markers will be familiar to persons skilled in the art, illustrative examples of which include heme-inducible enzyme (HO- 1 ), cyclooxygenase-2 (COX-2), interleukin-1 (IL-1 ), interleukin-6 (IL-6) and tumour necrosis factor (TNF) alpha. Thus, in an embodiment, the one or more pro-inflammatory marker is selected from the group consisting of heme-inducible enzyme (HO-1 ), cyclooxygenase-2 (COX-2), interleukin-1 (IL-1 ), interleukin-6 (IL-6) and tumour necrosis factor (TNF) alpha. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the level of expression of HO-1 by at least about 5% when compared to the level of expression of the HO-1 in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the level of expression of the HO-1 in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the expression of HO-1 by at least about 10% when compared to the level of expression of the HO-1 in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the level of expression of the HO-1 in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the expression of HO-1 by at least about 20% when compared to the level of expression of the HO-1 in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the level of expression of the HO-1 in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a hemebinding protein is sufficient to reduce the expression of HO-1 by at least about 25% when compared to the level of expression of the HO-1 in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the level of expression of the HO-1 in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the expression of HO-1 by at least about 30% when compared to the level of expression of the HO-1 in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the level of expression of the HO-1 in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the expression of HO-1 by at least about 35% when compared to the level of expression of the HO-1 in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the level of expression of the HO-1 in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a hemebinding protein is sufficient to reduce the expression of HO-1 by at least about 40% when compared to the level of expression of the HO-1 in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the level of expression of the HO-1 in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the expression of HO-1 by at least about 45% when compared to the level of expression of the HO-1 in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the level of expression of the HO-1 in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the expression of HO-1 by at least about 50% when compared to the level of expression of the HO-1 in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the level of expression of the HO-1 in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a hemebinding protein is sufficient to reduce the expression of HO-1 by at least about 55% when compared to the level of expression of the HO-1 in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the level of expression of the HO-1 in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the expression of HO-1 by at least about 60% when compared to the level of expression of the HO-1 in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the level of expression of the HO-1 in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the expression of HO-1 by at least about 65% when compared to the level of expression of the HO-1 in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the level of expression of the HO-1 in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a hemebinding protein is sufficient to reduce the expression of HO-1 by at least about 70% when compared to the level of expression of the HO-1 in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the level of expression of the HO-1 in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the expression of HO-1 by at least about 75% when compared to the level of expression of the HO-1 in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the level of expression of the HO-1 in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the expression of HO-1 by at least about 80% when compared to the level of expression of the HO-1 in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the level of expression of the HO-1 in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a hemebinding protein is sufficient to reduce the expression of HO-1 by at least about 85% when compared to the level of expression of the HO-1 in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the level of expression of the HO-1 in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the expression of HO-1 by at least about 90% when compared to the level of expression of the HO-1 in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the level of expression of the HO-1 in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the expression of HO-1 by at least about 95% when compared to the level of expression of the HO-1 in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the level of expression of the HO-1 in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a hemebinding protein is sufficient to reduce the expression of HO-1 by at least about 100% when compared to the level of expression of the HO-1 in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the level of expression of the HO-1 in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. Suitable methods of measuring the expression of pro- inflammatory genes would be familiar to persons skilled in the art, illustrative examples of which are disclosed herein.

The level of expression of pro-inflammatory genes can be assessed by measuring the amount of protein, for example (but not limited to), using western blot analysis or immunohistochemistry, and measuring the intensity of antibody staining. The expression of the one or more pro-inflammatory markers can also be assessed by measuring the level of the mRNA transcripts, for example (but not limited to) using reverse transcription quantitative realtime PCR, microarrays and / or RNA sequencing.

The reduction in the severity of ischaemia-reperfusion injury and/or inflammation in the affected tissue or organ may also be suitably represented as a percentage reduction in the number of circulating immune cells, for example, by at least about 5%, preferably from about 10% to about 20%, preferably from about 15% to about 25%, preferably from about 20% to about 30%, preferably from about 25% to about 35%, preferably from about 30% to about 40%, preferably from about 35% to about 45%, preferably from about 40% to about 50%, preferably from about 45% to about 55%, preferably from about 50% to about 60%, preferably from about 55% to about 65%, preferably from about 60% to about 70%, preferably from about 65% to about 75%, preferably from about 70% to about 80%, preferably from about 75% to about 85%, preferably from about 80% to about 90%, preferably from about 85% to about 95%, or preferably by at least 100%, when compared to the number of circulating immune cells in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating immune cells in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In an embodiment, the number of circulating immune cells comprises a number of circulating T lymphocytes, a number of circulating B lymphocytes, or a number of circulating T and B lymphocytes. In an embodiment, the number of circulating T lymphocytes comprises a number of circulating CD8+ T lymphocytes.

In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the number of circulating B lymphocytes by at least about 5% when compared to the number of circulating B lymphocytes in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating B lymphocytes in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the number of circulating B lymphocytes by at least about 10% when compared to the number of circulating B lymphocytes in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating B lymphocytes in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the number of circulating B lymphocytes by at least about 15% when compared to the number of circulating B lymphocytes in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating B lymphocytes in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the number of circulating B lymphocytes by at least about 20% when compared to the number of circulating B lymphocytes in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating B lymphocytes in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a hemebinding protein is sufficient to reduce the number of circulating B lymphocytes by at least about 25% when compared to the number of circulating B lymphocytes in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating B lymphocytes in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the number of circulating B lymphocytes by at least about 30% when compared to the number of circulating B lymphocytes in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating B lymphocytes in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the number of circulating B lymphocytes by at least about 35% when compared to the number of circulating B lymphocytes in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating B lymphocytes in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the number of circulating B lymphocytes by at least about 40% when compared to the number of circulating B lymphocytes in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating B lymphocytes in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a hemebinding protein is sufficient to reduce the number of circulating B lymphocytes by at least about 45% when compared to the number of circulating B lymphocytes in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating B lymphocytes in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the number of circulating B lymphocytes by at least about 50% when compared to the number of circulating B lymphocytes in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating B lymphocytes in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the number of circulating B lymphocytes by at least about 55% when compared to the number of circulating B lymphocytes in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating B lymphocytes in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the number of circulating B lymphocytes by at least about 60% when compared to the number of circulating B lymphocytes in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating B lymphocytes in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a hemebinding protein is sufficient to reduce the number of circulating B lymphocytes by at least about 65% when compared to the number of circulating B lymphocytes in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating B lymphocytes in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the number of circulating B lymphocytes by at least about 70% when compared to the number of circulating B lymphocytes in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating B lymphocytes in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the number of circulating B lymphocytes by at least about 75% when compared to the number of circulating B lymphocytes in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating B lymphocytes in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the number of circulating B lymphocytes by at least about 80% when compared to the number of circulating B lymphocytes in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating B lymphocytes in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a hemebinding protein is sufficient to reduce the number of circulating B lymphocytes by at least about 85% when compared to the number of circulating B lymphocytes in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating B lymphocytes in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the number of circulating B lymphocytes by at least about 90% when compared to the number of circulating B lymphocytes in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating B lymphocytes in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the number of circulating B lymphocytes by at least about 95% when compared to the number of circulating B lymphocytes in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating B lymphocytes in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the number of circulating B lymphocytes by at least about 100% when compared to the number of circulating B lymphocytes in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating B lymphocytes in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. Suitable methods of measuring the number of circulating B lymphocytes will be familiar to persons skilled in the art, illustrative examples of which are described herein (e.g. immunohistochemistry and flow cytometry).

In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the number of circulating T lymphocytes by at least about 5% when compared to the number of circulating T lymphocytes in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating T lymphocytes in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the number of circulating T lymphocytes by at least about 10% when compared to the number of circulating T lymphocytes in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating T lymphocytes in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the number of circulating B lymphocytes by at least about 15% when compared to the number of circulating T lymphocytes in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating T lymphocytes in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the number of circulating B lymphocytes by at least about 20% when compared to the number of circulating T lymphocytes in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating T lymphocytes in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a hemebinding protein is sufficient to reduce the number of circulating T lymphocytes by at least about 25% when compared to the number of circulating T lymphocytes in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating T lymphocytes in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the number of circulating T lymphocytes by at least about 30% when compared to the number of circulating T lymphocytes in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating T lymphocytes in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the number of circulating T lymphocytes by at least about 35% when compared to the number of circulating T lymphocytes in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating T lymphocytes in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the number of circulating T lymphocytes by at least about 40% when compared to the number of circulating T lymphocytes in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating T lymphocytes in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a hemebinding protein is sufficient to reduce the number of circulating T lymphocytes by at least about 45% when compared to the number of circulating T lymphocytes in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating T lymphocytes in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the number of circulating T lymphocytes by at least about 50% when compared to the number of circulating T lymphocytes in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating T lymphocytes in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the number of circulating T lymphocytes by at least about 55% when compared to the number of circulating T lymphocytes in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating T lymphocytes in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the number of circulating T lymphocytes by at least about 60% when compared to the number of circulating T lymphocytes in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating T lymphocytes in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a hemebinding protein is sufficient to reduce the number of circulating T lymphocytes by at least about 65% when compared to the number of circulating T lymphocytes in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating T lymphocytes in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the number of circulating T lymphocytes by at least about 70% when compared to the number of circulating T lymphocytes in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating T lymphocytes in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the number of circulating T lymphocytes by at least about 75% when compared to the number of circulating T lymphocytes in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating T lymphocytes in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the number of circulating T lymphocytes by at least about 80% when compared to the number of circulating T lymphocytes in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating T lymphocytes in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme- binding protein is sufficient to reduce the number of circulating T lymphocytes by at least about 85% when compared to the number of circulating T lymphocytes in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating T lymphocytes in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the number of circulating T lymphocytes by at least about 90% when compared to the number of circulating T lymphocytes in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating T lymphocytes in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the number of circulating T lymphocytes by at least about 95% when compared to the number of circulating T lymphocytes in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating T lymphocytes in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the number of circulating T lymphocytes by at least about 100% when compared to the number of circulating T lymphocytes in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating T lymphocytes in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. Suitable methods of measuring the number of circulating T lymphocytes will be familiar to persons skilled in the art, illustrative examples of which are described herein (e.g. immunohistochemistry and flow cytometry).

In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the number of circulating CD8+ T lymphocytes by at least about 5% when compared to the number of circulating CD8+ T lymphocytes in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating CD8+ T lymphocytes in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the number of circulating CD8+ T lymphocytes by at least about 10% when compared to the number of circulating CD8+ T lymphocytes in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating CD8+ T lymphocytes in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the number of circulating B lymphocytes by at least about 15% when compared to the number of circulating CD8+ T lymphocytes in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating CD8+ T lymphocytes in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the number of circulating B lymphocytes by at least about 20% when compared to the number of circulating CD8+ T lymphocytes in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating CD8+ T lymphocytes in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the number of circulating CD8+ T lymphocytes by at least about 25% when compared to the number of circulating CD8+ T lymphocytes in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating CD8+ T lymphocytes in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the number of circulating CD8+ T lymphocytes by at least about 30% when compared to the number of circulating CD8+ T lymphocytes in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating CD8+ T lymphocytes in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the number of circulating CD8+ T lymphocytes by at least about 35% when compared to the number of circulating CD8+ T lymphocytes in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating CD8+ T lymphocytes in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the number of circulating CD8+ T lymphocytes by at least about 40% when compared to the number of circulating CD8+ T lymphocytes in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating CD8+ T lymphocytes in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the number of circulating CD8+ T lymphocytes by at least about 45% when compared to the number of circulating CD8+ T lymphocytes in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating CD8+ T lymphocytes in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the number of circulating CD8+ T lymphocytes by at least about 50% when compared to the number of circulating CD8+ T lymphocytes in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating CD8+ T lymphocytes in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the number of circulating CD8+ T lymphocytes by at least about 55% when compared to the number of circulating CD8+ T lymphocytes in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating CD8+ T lymphocytes in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the number of circulating CD8+ T lymphocytes by at least about 60% when compared to the number of circulating CD8+ T lymphocytes in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating CD8+ T lymphocytes in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the number of circulating CD8+ T lymphocytes by at least about 65% when compared to the number of circulating CD8+ T lymphocytes in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating CD8+ T lymphocytes in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the number of circulating CD8+ T lymphocytes by at least about 70% when compared to the number of circulating CD8+ T lymphocytes in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating CD8+ T lymphocytes in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the number of circulating CD8+ T lymphocytes by at least about 75% when compared to the number of circulating CD8+ T lymphocytes in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating CD8+ T lymphocytes in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the number of circulating CD8+ T lymphocytes by at least about 80% when compared to the number of circulating CD8+ T lymphocytes in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating CD8+ T lymphocytes in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the number of circulating CD8+ T lymphocytes by at least about 85% when compared to the number of circulating CD8+ T lymphocytes in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating CD8+ T lymphocytes in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the number of circulating CD8+ T lymphocytes by at least about 90% when compared to the number of circulating CD8+ T lymphocytes in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating CD8+ T lymphocytes in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the number of circulating CD8+ T lymphocytes by at least about 95% when compared to the number of circulating CD8+ T lymphocytes in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating CD8+ T lymphocytes in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to reduce the number of circulating CD8+ T lymphocytes by at least about 100% when compared to the number of circulating CD8+ T lymphocytes in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating CD8+ T lymphocytes in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. Suitable methods of measuring the number of circulating CD8+ T lymphocytes will be familiar to persons skilled in the art, illustrative examples of which are described herein (e.g. immunohistochemistry and flow cytometry).

The reduction in the severity of severity of ischaemia-reperfusion injury and/or inflammation may also be suitably represented as a percentage increase in the number of circulating dendritic cells, for example, by at least about 5%, preferably from about 10% to about 20%, preferably from about 15% to about 25%, preferably from about 20% to about 30%, preferably from about 25% to about 35%, preferably from about 30% to about 40%, preferably from about 35% to about 45%, preferably from about 40% to about 50%, preferably from about 45% to about 55%, preferably from about 50% to about 60%, preferably from about 55% to about 65%, preferably from about 60% to about 70%, preferably from about 65% to about 75%, preferably from about 70% to about 80%, preferably from about 75% to about 85%, preferably from about 80% to about 90%, preferably from about 85% to about 95%, or preferably by at least about 100%, when compared to the number of circulating dendritic cells in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating dendritic cells in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to increase the levels of circulating dendritic cells by at least about 5% when compared to the number of circulating dendritic cells in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating dendritic cells in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to increase the levels of circulating dendritic cells by at least about 10% when compared to the number of circulating dendritic cells in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating dendritic cells in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to increase the levels of circulating dendritic cells by at least about 15% when compared to the number of circulating dendritic cells in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating dendritic cells in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to increase the levels of circulating dendritic cells by at least about 20% when compared to the number of circulating dendritic cells in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating dendritic cells in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to increase the levels of circulating dendritic cells by at least about 25% when compared to the number of circulating dendritic cells in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating dendritic cells in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to increase the levels of circulating dendritic cells by at least about 30% when compared to the number of circulating dendritic cells in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating dendritic cells in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to increase the levels of circulating dendritic cells by at least about 35% when compared to the number of circulating dendritic cells in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating dendritic cells in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to increase the levels of circulating dendritic cells by at least about 40% when compared to the number of circulating dendritic cells in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating dendritic cells in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to increase the levels of circulating dendritic cells by at least about 45% when compared to the number of circulating dendritic cells in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating dendritic cells in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to increase the levels of circulating dendritic cells by at least about 50% when compared to the number of circulating dendritic cells in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating dendritic cells in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to increase the levels of circulating dendritic cells by at least about 55% when compared to the number of circulating dendritic cells in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating dendritic cells in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to increase the levels of circulating dendritic cells by at least about 60% when compared to the number of circulating dendritic cells in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating dendritic cells in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to increase the levels of circulating dendritic cells by at least about 65% when compared to the number of circulating dendritic cells in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating dendritic cells in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to increase the levels of circulating dendritic cells by at least about 70% when compared to the number of circulating dendritic cells in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating dendritic cells in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to increase the levels of circulating dendritic cells by at least about 75% when compared to the number of circulating dendritic cells in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating dendritic cells in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding is sufficient to increase the levels of circulating dendritic cells by at least about 80% when compared to the number of circulating dendritic cells in a subject to whom a therapeutically effective amount of the heme- binding protein has not been administered, or when compared to the number of circulating dendritic cells in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to increase the levels of circulating dendritic cells by at least about 85% when compared to the number of circulating dendritic cells in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating dendritic cells in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to increase the levels of circulating dendritic cells by at least about 90% when compared to the number of circulating dendritic cells in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating dendritic cells in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to increase the levels of circulating dendritic cells by at least about 95% when compared to the number of circulating dendritic cells in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating dendritic cells in the subject prior to administration of the therapeutically effective amount of the heme-binding protein. In some embodiments, administration of a therapeutically effective amount of a heme-binding protein is sufficient to increase the levels of circulating dendritic cells by at least about 100% when compared to the number of circulating dendritic cells in a subject to whom a therapeutically effective amount of the heme-binding protein has not been administered, or when compared to the number of circulating dendritic cells in the subject prior to administration of the therapeutically effective amount of the heme-binding protein.

Methods of assessing and characterising the number of circulating immune cells will be familiar to persons skilled in the art, illustrative examples of which include immunohistochemistry, and flow cytometry.

Suitable methods by which a subjective, qualitative and / or quantitative measure of the number and / or severity of IRI can be measured, including symptoms thereof, will be familiar to persons skilled in the art and the choice of method(s) may suitably depend on the nature of the parameter to be measured. Illustrative examples of such methods are described elsewhere herein. The term "therapeutically effective amount", as used herein, typically means the amount or concentration of heme-binding protein that is sufficient to allow the heme-binding protein to bind to and sequester an amount of cell-free heme at the affected tissue or organ that is sufficient to relieve, minimise, reduce, alleviate, ameliorate or otherwise inhibit the ischemiareperfusion injury, including one or more symptoms thereof, at the affected tissue or organ, as described herein. It would be understood by persons skilled in the art that the therapeutically effective amount of heme-binding protein may vary depending upon several factors, illustrative examples of which include the type of heme-binding protein and their heme-binding affinity / capacity to bind or sequester cell-free heme, the health and physical condition of the subject to be treated, the taxonomic group of subject to be treated, the severity of the IRI (e.g., the level of inflammation at the affected tissue or organ), the route of administration, the concentration and / or amount of cell-free heme at the affected tissue or organ, and combinations of any of the foregoing.

The therapeutically effective amount of heme-binding protein will typically fall within a relatively broad range that can be determined by persons skilled in the art. Illustrative examples of suitable therapeutically effective amounts of heme-binding protein include from about 0.05 to 200 mg/kg body weight, from about 1 to 200 mg/kg body weight, from about 2 to 200 mg/kg body weight, from about 5 to 200 mg/kg body weight, from about 10 to 200 mg/kg body weight, from about 12 to 200 mg/kg body weight, from about 13 to 200 mg/kg body weight, from about 14 to 200 mg/kg body weight, from about 15 to 200 mg/kg body weight, from about 16 to 200 mg/kg body weight, from about 17 to 200 mg/kg body weight, from about 18 to 200 mg/kg body weight, from about 19 to 200 mg/kg body weight, from about 20 to 200 mg/kg body weight, from about 30 to 200 mg/kg body weight, from about 40 to 200 mg/kg body weight, from about 50 to 200 mg/kg body weight, from about 60 to 200 mg/kg body weight, from about 70 to 200 mg/kg body weight, from about 80 to 200 mg/kg body weight, from about 90 to 200 mg/kg body weight, from about 100 to 200 mg/kg body weight, from about 120 to 200 mg/kg body weight, and from about 150 to 200 mg/kg body weight. Further examples of suitable therapeutically effective amounts of heme-binding protein include from about 0.05 to 150 mg/kg body weight, from about 1 to 150 mg/kg body weight, from about 2 to 150 mg/kg body weight, from about 5 to 150 mg/kg body weight, from about 10 to 150 mg/kg body weight, from about 12 to 150 mg/kg body weight, from about 13 to 150 mg/kg body weight, from about 14 to 150 mg/kg body weight, from about 15 to 150 mg/kg body weight, from about 16 to 150 mg/kg body weight, from about 17 to 150 mg/kg body weight, from about 18 to 150 mg/kg body weight, from about 19 to 150 mg/kg body weight, from about 20 to 150 mg/kg body weight, from about 30 to 150 mg/kg body weight, from about 40 to 150 mg/kg body weight, from about 50 to 150 mg/kg body weight. In some embodiments, the therapeutically effective amount of hemebinding protein is from about 1.25 to 10 g (17 to 133 mg/kg for a 75 kg human). In some embodiments, the therapeutically effective amount of heme-binding protein is from 5 g to 30 g (67 to 400 mg/kg).

In some embodiments, a suitable therapeutically effective amount of Hx is from about 0.05 to 200 mg/kg body weight, from about 1 to 200 mg/kg body weight, from about 2 to 200 mg/kg body weight, from about 5 to 200 mg/kg body weight, from about 10 to 200 mg/kg body weight, from about 12 to 200 mg/kg body weight, from about 13 to 200 mg/kg body weight, from about 14 to 200 mg/kg body weight, from about 15 to 200 mg/kg body weight, from about 16 to 200 mg/kg body weight, from about 17 to 200 mg/kg body weight, from about 18 to 200 mg/kg body weight, from about 19 to 200 mg/kg body weight, from about 20 to 200 mg/kg body weight, from about 30 to 200 mg/kg body weight, from about 40 to 200 mg/kg body weight, from about 50 to 200 mg/kg body weight, from about 60 to 200 mg/kg body weight, from about 70 to 200 mg/kg body weight, from about 80 to 200 mg/kg body weight, from about 90 to 200 mg/kg body weight, from about 100 to 200 mg/kg body weight, from about 120 to 200 mg/kg body weight, and from about 150 to 200 mg/kg body weight. Further examples of a suitable therapeutically effective amounts of Hx include from about 0.05 to 150 mg/kg body weight, from about 1 to 150 mg/kg body weight, from about 2 to 150 mg/kg body weight, from about 5 to 150 mg/kg body weight, from about 10 to 150 mg/kg body weight, from about 12 to 150 mg/kg body weight, from about 13 to 150 mg/kg body weight, from about 14 to 150 mg/kg body weight, from about 15 to 150 mg/kg body weight, from about 16 to 150 mg/kg body weight, from about 17 to 150 mg/kg body weight, from about 18 to 150 mg/kg body weight, from about 19 to 150 mg/kg body weight, from about 20 to 150 mg/kg body weight, from about 30 to 150 mg/kg body weight, from about 40 to 150 mg/kg body weight, from about 50 to 150 mg/kg body weight. In some embodiments, the therapeutically effective amount of Hx is from about 1.25 to 10 g (17 to 133 mg/kg for a 75 kg human). In some embodiments, the therapeutically effective amount of Hx is from 5 g to 30 g (67 to 400 mg/kg).

Other illustrative examples of suitable therapeutically effective amounts of heme-binding include from about 2 pM to about 1 mM, preferably from about 2 pM to about 400 pM, preferably from about 5 pM to about 400 pM, preferably from about 5 pM to about 200 pM, or more preferably from about 10 pM to about 200 pM. In an embodiment, the therapeutically effective amount of heme-binding is from about 2 pM to about 1 mM. In an embodiment, the therapeutically effective amount of heme-binding is from about 2 pM to about 400 pM. In an embodiment, the therapeutically effective amount of heme-binding is from about 5 pM to about 200 pM. In an embodiment, the therapeutically effective amount of heme-binding is from about 10 pM to about 200 pM.

In an embodiment, the therapeutically effective amount of heme-binding is at least an equimolar amount to the concentration of cell-free heme at the affected tissue or organ of the subject. Suitable methods of measuring the concentration of cell-free heme in at the affected tissue or organ will be familiar to persons skilled in the art, illustrative examples of which include taking a sample of the affected tissue or organ (e.g., a biopsy sample) and measuring the amount of cell-free heme in the sample, for example, by the method described in Wang et al. Front Immunol. 2019; 10: 2975), the contents of which is incorporated herein by reference in its entirety. In another embodiment, the concentration of cell-free heme can be measured in a blood sample taken from the site of the affected tissue or organ. Suitable methods of measuring the amount of cell-free heme in the blood sample will be familiar to persons skilled in the art, illustrative examples of which are described in Atamna etal. (2015. Metallomics 7(2): 309-321 ), Yuan et al. (2016. Proc. Natl. Acad. Sci. USA 1 13(35): E5144-E5152) and Hopp et al. (2024. Analyt Chim Acta 131 : 342766), the contents of which is incorporated herein by reference in its entirety.

Dosages of the heme-binding protein may also be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily, weekly, or at other suitable timed intervals, or the dosages may be proportionally reduced as indicated by the exigencies of the situation.

The heme-binding protein may be formulated into any of many suitable dosage forms, such as injectable formulations. The formulations and their subsequent administration (dosing) are within the skill of those in the art. Dosing may depend on the responsiveness of the subject to treatment, but will invariably last for as long as the desirable therapeutic response is achieved. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rate.

For example, the timing of the dosing may suitably be adjusted to provide the optimum therapeutic response. In some embodiments, one or more doses may be administered prior to; during; and/or after induction of the IRI. In an embodiment, the heme-binding protein is administered prior to and after induction of the IRI. It is to be understood that the heme-binding protein can be administered to the subject via any by any suitable route. The heme-binding protein can be administered locally (i.e. at the site of the IRI) or systemically to the subject. The heme-binding protein can be administered enterally or parenterally to the subject. In an embodiment, the heme-binding protein described herein can be administered parenterally. In other embodiments, the heme-binding protein described herein can be administered using intravenous, subcutaneous, or by infusion administration routes. In an embodiment, the heme-binding protein is administered intravenously.

Pharmaceutical compositions

In another aspect disclosed herein, there is provided a pharmaceutical composition for treating ischaemia-reperfusion injury (IRI) in a subject, in accordance with the methods described herein, the composition comprising a therapeutically effective amount of heme-binding protein, as described herein, and a pharmaceutically acceptable carrier.

In another aspect disclosed herein, there is provided a pharmaceutical composition for use in treating ischaemia-reperfusion injury (IRI) in a subject in accordance with the methods described herein, the composition comprising a therapeutically effective amount of hemebinding protein, as described herein, and a pharmaceutically acceptable carrier.

In an embodiment, the composition comprises from about 2 pM to about 80 mM heme-binding protein. In an embodiment, the composition comprises from about 2 pM to about 20 mM hemebinding protein. In an embodiment, the composition comprises from about 100 pM to about 20 mM heme-binding protein, or a functional analogue thereof. In an embodiment, the composition comprises from about 2 pM to about 1 ,200 pM heme-binding protein. In an embodiment, the composition comprises from about 5 pM to about 200 pM heme-binding protein. In an embodiment, the composition comprises from about 10 pM to about 120 pM heme-binding protein.

In another aspect disclosed herein, there is provided use of a therapeutically effective amount of a heme-binding protein, as described herein, in the manufacture of a medicament for treating or preventing ischaemia-reperfusion injury in a subject in accordance with the methods described herein.

The present invention is further described by reference to the following non-limiting examples.

EXAMPLES Animals

C57BL/6J (B6) male mice were used (Jackson Laboratory, Sacramento, California, 25-30 g/ body). Mouse experiments were in accordance with the German Animal Welfare Act and the European directive for animal experiments. The study protocol was approved by the Lower Saxony State Office for Consumer Protection and Food Safety (No. 20-3577).

Model of unilateral pulmonary IRI in mouse

B6 mice were anesthetized with 1% isoflurane (Abbvie Inc. North Chicago, Illinois) and 1 L/min oxygen and then intubated with a 20 G venous catheter and placed under the ventilator (UNO micro-ventilator, UNO Rostvaststaal, Zevenaar, Netherlands; rate 120/min, pressure controlled 12 cm H2O, PEEP 2 cm H2O). Analgesia was administered subcutaneously with 150 mg/kg body weight metamizole (Ratiopharm GmbH, Ulm, Germany) after which the animal underwent a left-sided thoracotomy. A model of unilateral warm ischemia-reperfusion (IR) of the left lung was established in B6 mice by unilateral pulmonary clamping the left lung hilum for 1 , 1 .5 and 2 h after thoracotomy followed by 4 h of reperfusion, as previously demonstrated by others (Geudens, et al. 2006; J. Heart Lung Transplant. 25(7): 839-846). According to the following histological evaluation, the ischemia times of 1.5 h followed by 4 h of reperfusion were applied throughout this study. Mice were divided into two groups in which the treatment group received human plasma derived Hx (CSL Behring) (2 x 2 mg: 0.07-0.08 mg/g body weight, n=5) intraperitoneally before and after lung hilum clamping. The second group of mice was treated with 0.9% sodium chloride as vehicle (n=5). After sacrificing, blood was taken from the inferior vena cava, lungs were rinsed with phosphate-buffered saline (PBS) solution containing heparin (2 lU/ml) and the heart-lung package was removed (Figure S1 A).

Unilateral pulmonary IRI mouse model

The extent of acute lung injury (ALI) was determined according to the guidelines of the American Thoracic Society (Matute-Bello, et al. 201 1 , Am J. Respir. Cell Mol Biol. 44(5): 725- 738). For the evaluation, ten view fields per lung section were assessed with hematoxylin- eosin (HE)-staining (400-fold magnification) of left and right lungs, respectively. The criteria included accumulation of neutrophils in the alveolar space and interstitium, formation of protein debridement, formation of hyaline membranes in the alveolar space, and thickening of the alveolar walls. Individual criteria received a score of 0 to 2, respectively, and were summed up.

Basic histopathology The removed heart-lung block was left overnight in a 3.7% buffered formalin solution. The lung was sectioned transversely and embedded in paraffin. The paraffin sections were treated in the Institute of Pathology at Hannover Medical School with HE, Elastica-van-Gieson (EvG) and periodic acid Schiff (PAS) staining, respectively.

Histological assessment of vascular thrombosis in lung

Thrombus formation in pulmonary vessels was evaluated in HE stained lung sections by determining the number of thrombi relative to the number of vessels.

Evaluating the number of infiltrating neutrophils in lung tissue

Infiltrating neutrophils were counted as a quantitative evaluation of sterile inflammation of the lung. In the PAS staining, at 400-fold magnification by light microscopy, ten view fields per section of right and left lungs were evaluated, respectively. Infiltrating neutrophils were identified by the eosinophilic granular cytoplasm and the circular nucleus and neutrophils in vessels were excluded.

Detection of perivascular edema

The method for assessing perivascular edema has previously been described (Conhaim, et al., 1986, J. Appl. Physiol. 60(2): 513-20, Lowe, et al. 2010, Crit. Care Med. 38(6): 1458-66). A phase contrast microscope was applied at 200-fold magnification and five vessels in left and right lungs were assessed, respectively. Shortest distances from tunica intima to tunica intima were compared with the shortest diameter from tunica adventia to tunica adventia, respectively. The shortest distances were considered as true values to compensate for a distortion caused by different cutting planes. To compare different vessel sizes, the results were combined into one parameter using the following formula (Conhaim, et al., 1986, J. Appl. Physiol. 60(2): 513-20) (Figure S1 B). Comparisons were performed for right and left lungs, and groups of mice treated with or without Hx, respectively.

Immunohistochemistry

Expression levels of HO-1 were determined by immunohistochemistry, as previously described (Wang, et al. 2019, Front Immunol.. 10: 2975).

Blood sampling and isolation of immune cells

At the end of reperfusion, mice were sacrificed with whole blood collection. One drop of the blood was suspended in 1 ml PBS containing 2 U/ ml heparin. The rests were centrifuged and the sera were kept at -20 °C. Peripheral blood mononuclear cells (PBMCs) from the suspended blood samples were isolated by gradient separation using Ficoll solution (Biocoll Separating Solution; Biochrom GmbH, Berlin, Germany), as indicated by the manufacturer. The isolated cells were kept on ice, stained and used for flow cytometry.

Flow cytometry

The gating strategy was performed as previously described (Nakagiri, et.al. 2021 , Respir. Res. 22(1 ): 295). All antibodies were obtained from Biolegend (San Diego, CA, USA). PBMCs were stained with fluorescently labeled anti-mouse antibodies to CD45 (APC/Cy7, clone 30-F1 1 ) and CD19 (APC, clone 6D5) to identify B cells. They were detected in the lymphocyte area in the SSC-FSC window. CD45 (APC/Cy7, clone 30-F11 ), CD3 (PE/Cy7, clone 17A2), CD4 (FITC, clone RM4-5) and CD8 (PerCP/Cy5.5, clone 53-6.7) were used to identify T cells (CD45⁺CD3⁺CD4⁺CD8^_ as helper T cells and CD45⁺CD3⁺CD4 CD8⁺ as cytotoxic T cells) in the same lymphocyte area. Dendritic cells (DCs) were determined by CD45 (APC, clone 30-F1 1 ), CD1 1 b (PE/Cy7, clone M1/70) and CD1 1c (PE, clone N418) in the whole leukocytes' area. An Attune™ NxT flow cytometer (Thermo Fisher Scientific, Waltham, Massachusetts, USA) was used. The data were analyzed by FlowJo software (FlowJo X 10.0.7r2, Becton, Dickinson & Company, Franklin Lakes, New Jersey, USA).

EXAMPLE 1 - Hx alleviates sterile inflammation in pulmonary IRI

A preclinical mouse model of experimental lung IRI, achieved through unilateral pulmonary artery clamping, was used in the following studies. Assessment using the ALI scoring criteria of the American Thoracic Society revealed elevated levels of several histopathological marker’s indicative of ALI in the ischemic lung compared to control lungs (Figure 1, see also Table 1 ) (Matute-Bello, etal. 2011 , Am J. Respir. Cell Mol Biol. 44(5): 725-738).

Table 1 : Scoring of lung injury. Table of various criteria and weighting formula

Score = (20 A) + (14 B) + (7 C) + (7 D) + (2 E) ¹ The presence of even a single well-formed eosinophilic band of fibrin within the airspace earns a score of one, whereas multiple membranes visible in the field are scored as two.

² Pink proteinaceous debris filling the airspace is considered in an analogous manner to hyaline membranes.

³ Only septal thickening that is equal or greater than twice normal is considered in these criteria.

In the treatment group of mice, Hx was administered directly before and after left lung arterial clamping (Figure 2), while a second control group was treated with saline solution as vehicle. To assess whether pulmonary IRI induces sterile inflammation, neutrophils were counted in ischemic and control lungs, respectively (McDonald, et al. 2010, Science 330(6002): 362-366, Prakash, et al. 2012, Anesthesiology 117(4): 822-835, Chacon-Alberty, et al. 2023, Transplantation 107(8): 1687-1697). The number of infiltrating neutrophils in the left ischemic lung was higher relative to the right control lung (Figure 3). Neutrophil counts were reduced in the IRI lung of Hx-treated mice compared to the control group of vehicle-treated mice (Figure 3). Hx had only a minor effect on neutrophil counts in non-ischemic control lungs. Importantly, pulmonary inflammation was significantly mitigated in the group of mice receiving Hx prior to the onset of the reperfusion phase (Figure 2A).

An early indicator of vascular inflammation is the level of perivascular edema that can be determined by quantification of the cuff area-to-vessel area ratio (Conhaim, et al., 1986, J. Appl. Physiol. 60(2): 513-20, Lowe, et al. 2010, Crit. Care Med. 38(6): 1458-66) (Figure 2B). A comparison of the cuff area-to-vessel area ratios in IRI- and control lungs revealed higher levels of perivascular edema in IRI lungs (Figure 4). The perivascular edema in ischemic lungs was reduced in mice treated with Hx. No effect of Hx was observed in control lungs (Figure 4). Overall, these findings indicate that pulmonary IRI induces sterile inflammation in the lung that is alleviated by treatment with Hx.

EXAMPLE 2 - Hx alleviates vascular thrombosis in pulmonary IRI

Heme has been implicated in causing thrombotic vasoocclusion in blood vessels due to vascular endothelial inflammation (Belcher, et al. 2014, Blood 123(3): 377-390). To assess thrombotic vasoocclusion, the number of thrombotic vessels in both IRI and control murine lungs were counted. The number of thrombotic blood vessels was markedly reduced in the group of mice treated with Hx compared to the control group. No appreciable signs of vasoocclusion were observed in the right lung after experimental ischemia with or without Hx (Figure 5). Taken together, pulmonary IRI is associated with vascular occlusion of the lung that can be alleviated by treatment with Hx. EXAMPLE 3 - Hx blocks up-regulation of HO-1 expression in pulmonary IRI

To further investigate the potential role of heme in IRI-dependent sterile pulmonary inflammation, expression of the heme-inducible enzyme HO-1 was determined via immunohistochemical studies. HO-1 expression was found to be up-regulated in the left ischemic lung, but not in the right control lung (Figure 6A, left panel). In mice treated with Hx, up-regulation of HO-1 expression was blocked in the left IRI lung (Figure 6, right panel). These data indicate that heme may play a critical role in mediating inflammation in pulmonary IRI.

EXAMPLE 4 - Hx regulates levels of circulating immune cells in the peripheral blood

To assess activation of the innate immune system and adaptive immune responses during IRI, specific immune cell populations in the peripheral blood of IRI mice with or without Hx treatment were assessed. Lower levels of circulating B- and CD8+ T-lymphocytes was observed in mice with pulmonary IRI when treated with Hx (Figure 6A and B). Conversely, the numbers of dendritic cells were higher in IRI mice after treatment with Hx (Figure 6C). Notably, there were no significant differences in CD4+ T-cells or natural killer cells after Hx treatment (data not shown). Thus, administration of Hx exerts regulatory effects on the levels of various immune cells in the peripheral blood of mice with experimental lung IRI.

Discussion

Increased levels of perivascular edema and vascular thrombosis were observed in the left IRI lung (Figures 4 and 5), corroborating the presence of sterile inflammation and implicating the inflammatory activation of macrophages and endothelial cells (Chiu and Bharat 2016, Curr. Opin. Ogan Transplant 21 (3): 239-245, Chacon-Alberty, et al. 2023, Transplantation 107(8): 1687-1697). Importantly, pulmonary inflammation was significantly mitigated in the group of mice receiving Hx prior to the onset of the reperfusion phase (Figure 2A).

The findings disclosed herein show, for the first time, that cell-free heme plays a key role in the pathophysiology of IRI-induced sterile inflammation, and that this inflammation can be counteracted by heme scavenger proteins, such as Hx. This is supported by the substantial upregulation of HO-1 , a surrogate marker of inflammation that is induced by heme. The upregulation of HO-1 was significantly attenuated by treatment with the heme-scavenger protein Hx (Figure 6). These finding confirm that cell-free heme likely plays a role in orchestrating inflammation in response to IRI, including in lung tissue. The findings disclosed herein also demonstrate the dynamic regulation of distinct immune cell populations, notably dendritic cells (DCs), in the peripheral blood following lung IRI (Figure 7). The role of DCs in the lungs is multifaceted, performing functions such as antigen presentation, immune activation, tolerance induction, and inflammation resolution. DCs serve as an orchestrator of both innate and adaptive immune responses within the pulmonary microenvironment (Condon, et al. 201 1 , J. Leukoc. Biol. 90(5): 883-985). Specifically, pulmonary DCs contribute to immune tolerance and homeostasis by promoting the development of regulatory T cells (Tregs), and participate in the resolution of inflammation by producing anti-inflammatory cytokines and inducing apoptosis of activated T cells (Peters, et al. 2019, Innate Immun. 25(6): 326-336). The observed effect of Hx on the regulation of circulating numbers of B- and T -cells , both of which play critical roles in transplantation settings (Naderi, et al. 2023, Curr. Opin. Immunol. 81 : 102284), is in line with a crosstalk of the innate and adaptive immune systems.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

Claims

Claims

1. A heme-binding protein for use in the treatment of ischemia-reperfusion injury (IRI) in a subject in need thereof.

2. The heme-binding protein for use according to claim 1 , wherein the IRI is to a tissue or organ.

3. The heme-binding protein for use according to claim 1 or claim 2, wherein the IRI is to a transplanted tissue or organ.

4. The heme-binding protein for use according to claim 3, wherein the tissue or organ is selected from the group consisting of lung, heart, kidney, liver, pancreas, intestine, stomach, thymus, uterus, bone marrow, skin, tendon and cornea.

5. The heme-binding protein for use according to claim 4, wherein the tissue or organ is lung.

6. The heme-binding protein for use according to any one of claims 1 to 5, wherein the hemebinding protein is hemopexin or a functional analogue thereof.

7. The heme-binding protein for use according to claim 6, wherein the hemopexin is plasma- derived hemopexin.

8. The heme-binding protein for use according to claim 6 or claim 7, wherein the hemopexin is human hemopexin or a functional analogue thereof.

9. The heme-binding protein for use according to any one of claims 1 to 8, wherein the hemebinding protein thereof is formulated for administration in a therapeutically effective amount of from about 0.05 mg/kg body weight to about 200 mg/kg body weight.

10. The heme-binding protein for use according to claim 9, wherein the heme-binding protein is formulated for administration in a therapeutically effective amount of from about 0.05 mg/kg body weight to about 150 mg/kg body weight.

1 1 . The heme-binding protein for use according to claim 9, wherein the heme-binding protein is formulated for administration in a therapeutically effective amount of from about 0.05 mg/kg body weight to about 1 mg/kg body weight.

12. The heme-binding protein for use according to any one of claims 1 to 11 , wherein the heme-binding protein is administered i) prior to; ii) during; and/or iii) after induction of the IRI.

13. The heme-binding protein for use according to any one of claims 1 to 12, wherein the heme-binding protein is administered prior to and after induction of the IRI.

14. The heme-binding protein for use according to any one of claims 1 to 13, wherein the heme-binding protein is administered parenterally.

15. The heme-binding protein for use according to any one of claims 1 to 14, wherein said treatment is characterised by one or more of: a. reduced inflammation; b. reduced number of infiltrating neutrophils; c. reduced perivascular edema; d. reduced levels of heme oxygenase-1 ; and e. reduced number of thrombotic blood vessels.

16. The heme-binding protein for use of any one of claims 1 to 15, wherein: the IRI is to a lung tissue or to a lung; and the heme-binding protein is hemopexin or a functional analogue thereof; optionally wherein the heme-binding protein is administered parenterally, preferably intravenously.

17. The heme-binding protein for use of claim 16, wherein: the lung tissue or lung is a transplanted lung tissue or transplanted lung; and/or the hemopexin or a functional analogue thereof is human hemopexin or a functional analogue thereof.

18. A method of treating ischaemia-reperfusion injury (IRI) in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a hemebinding protein.

19. The method of claim 18, wherein the heme-binding protein is hemopexin or a functional analogue thereof.

20. The method of claim 19, wherein the hemopexin is human hemopexin or a functional analogue thereof.

21 . The method of any one of claims 18-20, wherein the IRI is to a tissue or organ, optionally a transplanted tissue or organ, optionally wherein the tissue or organ is selected from the group consisting of lung, heart, kidney, liver, pancreas, intestine, stomach, thymus, uterus, bone marrow, skin, tendon and cornea.

22. The method of any one of claims 18-21 , wherein: the IRI is to a lung tissue or to a lung, optionally a transplanted lung tissue or transplanted lung; and the heme-binding protein is hemopexin or a functional analogue thereof, optionally a human hemopexin or a functional analogue thereof; optionally wherein the heme-binding protein is administered parenterally, preferably intravenously.