Methods for treating or preventing adverse secondary neurological outcomes following hemorrhagic stroke - Patents.com

Abstract

The present disclosure relates generally to a method of treating or preventing extravascular hemolysis and adverse secondary neurological outcomes in a subject following a hemorrhagic stroke involving release of cell-free heme and / or cell-free hemoglobin (Hb) into the cerebrospinal fluid (CSF), comprising exposing the CSF of a subject in need thereof to a therapeutically effective amount of hemopexin (Hx) for a period of time sufficient to allow Hx to complex with, and thereby neutralize, the cell-free heme, and optionally exposing the CSF of the subject to a therapeutically effective amount of haptoglobin (Hp) for a period of time sufficient to allow Hp to complex with, and thereby neutralize, the cell-free Hb.

Description

[Technical field]

[0001] The present invention relates generally to methods and compositions for treating and / or preventing adverse secondary neurological outcomes in subjects following hemorrhagic stroke into the cerebrospinal fluid (CSF) compartment, particularly following subarachnoid hemorrhage (SAH). [Background technology]

[0002] All references cited herein, including any patents or patent applications, are hereby incorporated by reference to enable a complete understanding of the present invention. Nevertheless, such references should not be read as constituting an admission that any of those documents form part of the common general knowledge in the art in Australia or any other country.

[0003] Hemorrhagic stroke is associated with the rupture of blood vessels in or on the surface of the brain, with bleeding into surrounding tissue. Examples of hemorrhagic stroke include: i) intracerebral hemorrhage (herein referred to as ICH), which is associated with the rupture of blood vessels in the brain; ii) intraventricular hemorrhage (herein referred to as IVH), which is bleeding into the ventricular system; and iii) subarachnoid hemorrhage (herein referred to as SAH), which is associated with bleeding in the space between the brain and the tissues covering it (known as the subarachnoid space). The most common SAH is caused by aneurysm rupture (herein referred to as aSAH). Other causes of SAH include head injury, bleeding disorders, and the use of blood thinners.

[0004] Aneurysmal subarachnoid hemorrhage (aSAH) is the most common cause of SAH and is associated with the highest mortality and long-term neurological disability. Despite advances in aneurysm repair and neurocritical care, the median in-hospital mortality rate in Europe is 44.4% and in the United States is 32.2%. Thirty-five percent of survivors report poor overall quality of life one year after the hemorrhagic event, and 83-94% are unable to return to work. The estimated incidence of aSAH due to ruptured intracranial aneurysms in the United States is 1 case per 10,000 people, with approximately 27,000 new cases occurring each year.

[0005] Furthermore, aSAH is more common in women than men (2:1); the peak incidence is between 55 and 60 years of age.

[0006] In addition to the initial brain injury within the first 72 hours (Sehba et al., 2012), patient outcome after aSAH is determined by delayed secondary brain injury that occurs between 4 and 10 days after aneurysm rupture (Macdonald, 2014). Delayed secondary brain injury is considered multifactorial, involving cerebral macrovascular and microvascular dysfunction, neuritis, neuronal apoptosis, and pathological electrical activity (Macdonald, 2014). Two-thirds of patients after aSAH develop angiographic vasospasm of the cerebral arteries (aVSP) (Dorsch and King, 1994). Delayed cerebral ischemia (DCI) with radiological borders of the ischemic brain area and clinically evident delayed ischemic neurological deficit (DIND) are found in one-third of patients (Rowland et al., 2012). The occurrence of at least one of these secondary symptoms defines secondary subarachnoid hemorrhage-associated brain injury (SAH-SBI).

[0007] The lag time between aneurysm rupture and the development of SAH-SBI provides a great opportunity for preventive and therapeutic interventions, defining patients at high risk for SAH-SBI, as well as an unmet need to identify novel drug targets. To date, the only preventive intervention shown to moderately improve neurological outcome after aSAH is oral nimodipine (Class I, Level A) (Diringer et al., 2011; Connolly et al., 2012). In symptomatic patients, treatment options are currently limited to rescue therapy with induction of systemic arterial hypertension and, in selected patients, mechanical or chemical angioplasty to resolve aVSP (Diringer et al., 2011; Connolly et al., 2012). Thus, there is an urgent and unmet need for specific therapies to treat and / or prevent SAH-SBI in patients after aSAH.

[0008] Despite extensive research efforts, there are still no clinically established biomarkers to reliably monitor SAH-SBI. Although widely used, clinical scores (Hunt and Hess, 1968; Teasdale et al., 1988), radiological scores (Fisher et al., 1980; Frontera et al., 2006; Wilson et al., 2012), and routine assessments with transcranial doppler sonography (TCD) (Diringer et al., 2011; Connolly et al., 2012) show limited accuracy in detecting patients at risk (de Rooij et al., 2013).

[0009] Cell-free hemoglobin (Hb) accumulates in patients' CSF and has been considered an upstream driver of SAH-SBI (Pluta et al., 2009; Hugelshofer et al., 2019; Buehler et al., 2020). Data from a small pilot study of 18 patients using daily CSF spectrophotometry suggested that patients exposed to high cumulative CSF-Hb for 2 weeks after aneurysm rupture may be at increased risk for SAH-SBI (Hugelshofer et al., 2018). However, to date, no correlation has been clinically demonstrated between the concentration of cell-free Hb in patients' CSF (CSF-Hb) and the occurrence of secondary brain injury associated with subarachnoid hemorrhage (SAH-SBI). In particular, the clinically significant CSF-Hb concentration that could be used as a relevant biomarker for SAH-SBI is unknown. Summary of the Invention [Problem to be solved by the invention]

[0010] Overall, there is an urgent clinical need for diagnostic tools to predict and monitor SAH-SBI, as well as targeted therapeutic approaches to prevent and treat SAH-SBI. [Means for solving the problem]

[0011] The present inventors conducted a prospective study on a cohort of patients with aSAH and unexpectedly found a significant correlation between the concentration of cell-free Hb in the CSF (CSF-Hb) and the occurrence of secondary brain injury associated with subarachnoid hemorrhage (SAH-SBI) in these patients. The diagnostic accuracy of the correlation between CSF-Hb and SAH-SBI was identified within a narrow range of clinically significant CSF-Hb concentrations, and it was found to significantly surpass other physiological and biochemical biomarkers of aSAH, as well as radiological and clinical scores. Notably, a significant correlation was observed between CSF-Hb and the incidence of SAH-SBI for all three symptoms of SAH-SBI; angiographic vasospasm of the cerebral arteries (aVSP), delayed cerebral ischemia (DCI), and delayed ischemic neurological deficit (DIND). We also performed detailed CSF proteomic analysis, as well as ex vivo functional assays that identify CSF-Hb as a pathophysiological driver and therapeutic target.

[0012] The inventors have also surprisingly found that hemopexin (Hx) can reduce or otherwise prevent acellular heme-mediated adverse secondary neurological outcomes (e.g., oxidative tissue injury, neuritis), including functional and radiological neurological impairment. It has been found that Hx has the ability to selectively neutralize lipid oxidation activity of patient CSF-Hb when CSF-Hb is within the clinically relevant concentration ranges identified herein. The inventors have further surprisingly found that haptoglobin (Hp) exerts its antivasospastic and antioxidant effects within the clinically relevant CSF-Hb concentration ranges identified herein. In addition, the inventors have further surprisingly found that haptoglobin (Hp) and hemopexin (Hx) can exert their protective functions synergistically.

[0013] Thus, in one aspect of the invention, a method is provided for treating or preventing adverse secondary neurological outcomes in a subject following a hemorrhagic stroke involving extravascular hemolysis and release of acellular heme and / or acellular hemoglobin (Hb) into the cerebrospinal fluid (CSF), comprising exposing the CSF of a subject in need thereof to a therapeutically effective amount of hemopexin (Hx) for a period of time sufficient to allow Hx to complex with, and thereby neutralize, the acellular heme. In one embodiment, the method further comprises exposing the CSF of the subject to a therapeutically effective amount of haptoglobin (Hp) for a period of time sufficient to allow Hp to complex with, and thereby neutralize, the acellular Hb.

[0014] In another aspect disclosed herein, a pharmaceutical composition for treating or preventing adverse secondary neurological outcomes in a subject after hemorrhagic stroke is provided according to the methods described herein, the pharmaceutical composition comprising a therapeutically effective amount of hemopexin (Hx) and a pharma- ceutical acceptable carrier. In one embodiment, the composition further comprises a therapeutically effective amount of haptoglobin (Hp).

[0015] In another aspect disclosed herein, there is provided a pharmaceutical composition for use in treating or preventing adverse secondary neurological outcomes in a subject following hemorrhagic stroke, comprising a therapeutically effective amount of hemopexin (Hx) and a pharma- ceutical acceptable carrier, according to the methods described herein. In one embodiment, the composition further comprises a therapeutically effective amount of haptoglobin (Hp).

[0016] In another aspect disclosed herein, there is provided a use of a therapeutically effective amount of hemopexin (Hx) in the manufacture of a medicament for treating or preventing adverse secondary neurological outcomes in a subject following hemorrhagic stroke, according to the methods described herein. In one embodiment, the therapeutically effective amount of Hx is formulated for co-administration with a therapeutically effective amount of haptoglobin (Hp).

[0017] In another aspect disclosed herein, a therapeutically effective amount of hemopexin (Hx) is provided for use in treating or preventing adverse secondary neurological outcomes in a subject following hemorrhagic stroke, according to the methods described herein. In one embodiment, the therapeutically effective amount of Hx is formulated for co-administration with a therapeutically effective amount of haptoglobin (Hp).

[0018] In another aspect disclosed herein, there is provided an artificial CSF comprising Hx and optionally Hp as described herein. The disclosure also extends to a kit comprising an artificial CSF or composition as described herein.

[0019] In another aspect disclosed herein, there is provided a method for determining whether a subject is at risk for developing an adverse secondary neurological outcome following a hemorrhagic stroke involving extravascular hemolysis and release of cell-free heme and / or cell-free hemoglobin (Hb) into cerebrospinal fluid (CSF), comprising the steps of: (i) obtaining a CSF sample from the subject following a hemorrhagic stroke; (ii) measuring the amount of cell-free Hb in the CSF sample obtained in step (i); and (iii) comparing the amount of cell-free Hb in the CSF sample determined in step (ii) with a reference value, wherein the subject's risk of developing an adverse secondary neurological outcome is determined based on the comparison in step (iii).

[0020] In some embodiments, the method further comprises treating a subject determined to have a risk of an adverse secondary neurological outcome, the treatment comprising exposing the subject's CSF to (i) a therapeutically effective amount of hemopexin (Hx) for a sufficient period of time to allow Hx to complex with acellular heme and thereby neutralize it, as described herein; and / or (ii) a therapeutically effective amount of haptoglobin (Hp) for a sufficient period of time to allow Hp to complex with acellular Hb and thereby neutralize it, as described herein. Thus, in another aspect disclosed herein, a method of stratifying a subject for treatment of an adverse secondary neurological outcome in a subject following a hemorrhagic stroke involving extravascular hemolysis and release of acellular heme and / or acellular hemoglobin (Hb) into the cerebrospinal fluid (CSF) is provided, the method comprising (a) determining whether the subject is at risk of developing an adverse secondary neurological outcome, as described herein, and (b) treating the subject determined to have a risk of an adverse secondary neurological outcome based on the methods described herein. [Brief description of the drawings]

[0021] [Figure 1-1]Figure 2. Changes in the cerebrospinal fluid proteome after aneurysmal subarachnoid hemorrhage; A. Temporal profile of cerebrospinal fluid (CSF) oxyhemoglobin (oxyHb), bilirubin, biliverdin, and methemoglobin (metHb). A. k-means clustering of proteins identified in CSF after aSAH by LC-MS / MS. B. Volcano plot with global fold change and combined p-value in the CSF proteome. Color represents cluster and dot size represents raw average intensity of protein (unnormalized). C. Enrichment plot of the top negatively enriched hallmark gene set (coagulation) identified in GSEA of CSF proteome after aSAH. D. Time course of normalized protein intensity of haptoglobin (HP), haptoglobin-related protein (HPR), hemopexin (HPX), and albumin (ALB) in CSF after aSAH. E. Enrichment plot of the top positively enriched hallmark gene set (glycolysis) identified in GSEA of the CSF proteome after aSAH. F. Enrichment plot of the hallmark gene set (heme metabolism) identified in GSEA of the CSF proteome after aSAH. G. Time course of normalized protein intensities of carboanhydrase 1 (CA1), carboanhydrase 2 (CA2), catalase (CAT), and aldolase A (ALDOA) ​​in CSF. H. Time course of normalized protein intensities of CD163, colony stimulating factor 1 receptor (CSF1R), CD14, and inhibitor of metalloproteinase 1 (TIMP) in CSF. [Figure 1-2] Continued from Figure 1-1. [Figure 1-3] Continued from Figure 1-2. [Figure 1-4] Continued from Figure 1-3. [Figure 1-5] Continued from Figure 1-4. [Figure 1-6] Continued from Figure 1-5. [Figure 2-1]Determinants of CSF-Hb. A. Example of a subarachnoid hematoma in 3D representation. B. Partial dependence of CSF Hb levels on aneurysm size, aneurysm location, hematoma volume, presence of intraventricular hemorrhage (IVH), and days after aSAH based on a general additive model. C. Time course of CSF hemoglobin after aSAH stratified for the presence of IVH. D. Schematic showing subarachnoid hemorrhage originating from rupture of an anterior communicating artery aneurysm, comparing situations in which IVH is absent (blue inset) or present (red inset). [Figure 2-2] Continued from Figure 2-1. [Figure 2-3] Continued from Figure 2-2. [Figure 2-4] Continued from Figure 2-3. [Figure 2-5] Continued from Figure 2-4. [Figure 3-1] Association of cerebrospinal fluid hemoglobin with secondary brain damage after aneurysmal subarachnoid hemorrhage; A. CSF hemoglobin (Hb) in patients after aneurysmal subarachnoid hemorrhage stratified by angiographic vasospasm (aVSP), delayed cerebral ischemia (DCI), delayed ischemic neurological impairment (DIND), and composite outcome secondary brain injury per day (SAH-SBI). B. Partial dependence of CSF-Hb (log scale) and SAH-SBI on days after aSAH based on a general additive model. C. Receiver operating characteristic (ROC) curves and area under the curve (AUC) for Hb in the presence of DIND, DCI, and aVSP (left). ROC curve for Hb with values ​​for occurrence of SAH-SBI. D. Time course of CSF Hb (good / bad) stratified by GOSE at 3 months follow-up. [Figure 3-2] Continued from Figure 3-1. [Figure 3-3] Continued from Figure 3-2. [Diagram 3-4] Continued from Figure 3-3. [Figure 4]Illustrates the vasoconstrictor and oxidative capacities of hemoglobin; A. Vascular tension dose response to hemoglobin (Hb). B. Transcranial Doppler (TCD) velocities (scaled by maximum TCD) measured in patients correlated with CSF Hb levels. The presence of aVSPs is shown as a color overlay. C. Malondialdehyde formation in response to different concentrations of Hb in the TBARS assay. D. Correlation of measured CSF Hb concentrations to the occurrence of secondary brain injury (SBI). [Diagram 5] Protective effect of haptoglobin against vascular and oxidative Hb effects; A. Hb depletion of CSF using Hp affinity column. B. Vascular tension obtained from vessels bathed in Hb-depleted CSF and Hb-containing CSF. C. Dose response with haptoglobin (Hp) (data from FIG. 4A) or without Hb. D. Time course of oxidative capacity of post-aSAH patient CSF with and without added Hp or hemopexin (Hpx) assessed by malondialdehyde using the thiobarbituric acid reactive substances (TBARS) assay. [Figure 6] Figure 1. Pathophysiology of cerebrospinal fluid hemoglobin and heme metabolites after aneurysmal subarachnoid hemorrhage; A. Schematic of pathophysiological processes in the subarachnoid microenvironment after aneurysmal subarachnoid hemorrhage. The successive intracellular processes of erythrocyte phagocytosis and hemoglobin modification and heme metabolism are shown at the bottom. Saturation of biliverdin reductase and erythrocyte phagocytosis is shown by the red bars. Hemolysis in the subarachnoid CSF space is shown at the top of the schematic. Heme is released after released oxyhemoglobin is oxidized to methemoglobin. B. CSF concentrations of Hb, bilirubin, biliverdin, and metHb over time. The estimated time of saturation of biliverdin reductase and phagocytosis is shown by the vertical bars. [Figure 7-1]FIG. 7A shows a flow diagram of the clinical observational study. FIG. 7B shows a schematic of daily CSF sampling by EVD, centrifugation of CSF, and proteomic analysis of the supernatant using optical spectroscopy and LC-MS / MS. FIG. 7C shows the steps of subarachnoid hemorrhage segmentation for volumetric analysis. FIG. 7D shows an elbow plot to determine the optimal number of protein clusters. FIG. 7E shows a schematic of the neurovascular function experimental setup. FIG. 7F shows how the recorded vascular tension is normalized to the individual NO reserve capacity of each vessel. FIG. 7G shows a schematic of the steps in the TBARS assay method. [Figure 7-2] Continued from Figure 7-1. [Figure 7-3] Continued from Figure 7-2. [Figure 7-4] Continued from Figure 7-3. [Figure 7-5] Continued from Figure 7-4. [Figure 8-1] FIG. 1 shows individual profiles of CSF-Hb and heme metabolites; A. Individual temporal profile of oxyhemoglobin (oxyHb). B. Individual temporal profile of bilirubin. C. Individual temporal profile of biliverdin. D. Individual temporal profile of methemoglobin (metHb). [Figure 8-2] Continued from Figure 8-1. [Figure 9] FIG. 1 shows temporal profiles of oxyhemoglobin stratified by angiographic vasospasm (aVSP), delayed cerebral ischemia (DCI), and delayed ischemic neurological impairment (DIND); A. Time course of oxyhemoglobin (oxyHb) stratified by aVSP. B. Time course of oxyHb stratified by DCI. C. Time course of oxyHb stratified by DIND. [Figure 10-1]Figure 1. Association of cerebrospinal fluid hemoglobin, clinical and radiological scores with secondary brain injury after aneurysmal subarachnoid hemorrhage; A-C. Receiver operating characteristic (ROC) curves and area under the curve (AUC) of CSF-Hb for the occurrence of angiographic vasospasm (aVSP, A), delayed ischemic neurological deficit (DIND, B), and delayed ischemic neurological deficit (DIND, C), including each value. The optimal Youden-Index and its 95% confidence interval are shown in red. D. ROC curves and AUC of CSF-Hb for the occurrence of aVSP, DCI, DIND, and SAH-SBI, including data limited to the high-risk period (days 4-14). E-G. ROC curves and AUC of CSF-bilirubin (E), CSF-biliverdin (F), and CSF-methemoglobin (metHb) (G) for the occurrence of aVSP, DCI, DIND, and SAH-SBI. H-K. ROC curves and AUC of WFNS (H), Hunt and Hess (I), BNI (J), and Fisher (K) grades for the occurrence of aVSP, DCI, DIND, and SAH-SBI. L. Time course of CSF-Hb stratified by modified Rankin Scale (mRS) (good / poor) at 3-month follow-up. [Figure 10-2] Continued from Figure 10-1. [Figure 10-3] Continued from Figure 10-2. [Figure 10-4] Continued from Figure 10-3. [Figure 11-1] FIG. 1 shows quantification of hemoglobin and heme scavenger proteins in patient CSF samples. The top panel shows, from left to right, quantification of cell-free Hb, haptoglobin, hemopexin, and albumin in patient CSF at each collection time point expressed in weeks, compared to a healthy control sample (ctrl). The bottom panel shows, from left to right, scavenger proteins complexed with hemoglobin (Hb:Hp complex) or complexed with heme (heme:hemopexin or heme binding protein; heme protein). Data are shown as box plots representing mean ± min-max values, with individual data points (overlapped). Dashed lines indicate the lower limit of quantification (LLOQ) of the corresponding assay. [Figure 11-2] Continued from Figure 11-1. [Figure 11-3] Continued from Figure 11-2. [Figure 12-1] Neurological function 24 h after intrastriatal injection of haptoglobin, heme, or heme:hemopexin. Scoring of the open field test (A), rotarod test (B), and beam walking test (C) after injection of 10 μL each of haptoglobin (5 mM Hp, protein control) (black), 1 mM heme (dark gray), or 1 mM heme:1Hx (light gray) (n=6 per group). Mice after injection of heme but not heme:Hx show neurobehavioral dysfunction after 24 h. [Figure 12-2] Continued from Figure 12-1. [Figure 12-3] Continued from Figure 12-2. [Figure 13] Radiological changes 24 hours after intrastriatal injection of heme or heme:hemopexin. Illustrative case of radiological changes observed 24 hours after intrastriatal injection of heme (A) or heme:Hpx (B), respectively. After heme injection, perilesional edema is noted on T2-weighted sequences (left), accompanied by diffusion restriction on ADC maps (center) and focal perfusion defects on arterial spin labelling (ASL) images (right). After heme:Hpx injection, only minor radiological changes are observed on all three sequences. [Figure 14]Quantification of edema size and perfusion 24, 48, and 72 hours after intrastriatal injection of heme or heme:hemopexin. (A) Semi-automated quantification of edema size on T2-weighted images 24, 48, and 72 hours after injection of heme (black) and heme:Hpx (dark grey), respectively. ns: p>0.05; *: p≦0.05, **: p≦0.01; ***: p≦0.001, ****: p≦0.0001. (B) Normalized perfusion on arterial spin labeling images 24, 48, and 72 hours after injection of heme (circles) and heme:Hpx (filled squares) (n=4 per group). Intrastriatal injection of heme is associated with the formation of cerebral edema and a regional decrease in perfusion (not present after injection of heme:Hpx). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] Throughout this specification, unless the context otherwise requires, the word "comprise" or variations such as "comprises" or "comprising" are understood to imply the inclusion of a stated element or integer, or group of elements or integers, but not to exclude any other element or integer, or group of elements or integers.

[0023] Reference herein to any prior publication (or information derived therefrom), or to any matter in the public domain, is not, and should not be taken as, an admission or acknowledgment, or any form of suggestion, that the prior publication (or information derived therefrom) or matter in the public domain forms part of the common general knowledge in the field of endeavour to which this specification pertains.

[0024] It should be noted that, as used in the subject specification, the singular forms "a," "an," and "the" include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to "an agent" includes a single resin, as well as two or more agents; reference to "the composition" includes a single composition, as well as two or more compositions; and so forth.

[0025] In the absence of any indication to the contrary, "%" content is taken throughout this specification to mean % w / w (weight / weight). For example, a solution containing a haptoglobin content that is at least 80% of the total protein is taken to mean a composition containing a haptoglobin content that is at least 80% w / w of the total protein.

[0026] The present invention is based, at least in part, on the inventors' surprising discovery that hemopexin (Hx) can reduce or otherwise prevent acellular heme-mediated adverse secondary neurological outcomes in vivo, such as cerebral vasospasm. Accordingly, in one embodiment disclosed herein, there is provided a method of treating or preventing adverse secondary neurological outcomes in a subject following a hemorrhagic stroke involving extravascular hemolysis and release of acellular heme and / or acellular hemoglobin (Hb) into the cerebrospinal fluid (CSF), comprising exposing the CSF of a subject in need thereof to a therapeutically effective amount of hemopexin (Hx) for a period of time sufficient to allow Hx to complex with, and thereby neutralize, the acellular heme.

[0027] Hemorrhagic stroke Hemorrhagic stroke or bleeding into the CSF compartment is also referred to herein interchangeably as intracerebral hemorrhage, cerebral hemorrhage, or intracranial hemorrhage. Such hemorrhage is generally characterized by blood vessel rupture in the brain, causing localized bleeding. The location of hemorrhage can vary. For example, bleeding into the CSF compartment can result from intraventricular hemorrhage, intraparenchymal hemorrhage, and / or subarachnoid hemorrhage.

[0028] Hemorrhagic stroke consists of a spectrum of pathologies with different natural histories, evaluations, and management, as will be familiar to those skilled in the art. Hemorrhagic stroke is commonly classified as primary or secondary depending on the etiology.

[0029] In one embodiment, the hemorrhagic stroke is an intraventricular hemorrhage (IVH) or a subarachnoid hemorrhage (SAH). In one embodiment, the hemorrhagic stroke is an aneurysmal subarachnoid hemorrhage (aSAH).

[0030] Methods for diagnosing hemorrhagic stroke, particularly SAH, in a subject are familiar to those of skill in the art, illustrative examples of which include cerebral angiography, computed tomography (CT), and spectrophotometric analysis of oxyHb and bilirubin in the subject's CSF (see, e.g., Cruickshank AM., 2001, ACP Best Practice No 166, J. Clin. Path., 54(11):827-830).

[0031] It is understood by those skilled in the art that hemorrhagic stroke can be naturally occurring hemorrhage (e.g., as a result of a ruptured aneurysm) or traumatic hemorrhage (e.g., as a result of trauma to the head). In one embodiment, hemorrhagic stroke is naturally occurring hemorrhage, also known as non-traumatic hemorrhage. In one embodiment, hemorrhagic stroke is traumatic hemorrhage.

[0032] The term "cerebrospinal fluid" or CSF is understood to mean the fluid in the ventricles, as well as the cranial and spinal subarachnoid spaces. The ventricles, cranial and spinal subarachnoid spaces are collectively referred to herein as "CSF compartments". Thus, in one embodiment disclosed herein, the method comprises exposing the CSF compartment of a subject in need thereof to a therapeutically effective amount of Hp.

[0033] CSF is secreted primarily, but not exclusively, by the choroid plexus, which is composed of granular projections of the meninges that project into the ventricular lumen and whose epithelial surface is continuous with the ventricular epithelium. Studies suggest that brain interstitial fluid, ventricular epithelium, and capillaries may also play a role in CSF secretion. CSF volume is estimated to be about 150 mL in human adults, and although it varies significantly between individuals, it is commonly distributed between the cranial and spinal subarachnoid spaces, with 125 mL and 25 mL in the ventricles. CSF secretion in human adults can vary between 400 and 600 mL per day, with approximately 60-75% of CSF produced by the choroid plexus of the lateral ventricles and the choroids of the third and fourth ventricles. Choroidal secretion of CSF generally involves two steps: (i) passive filtration of plasma from the choroidal capillaries into the choroidal interstitial compartment due to a pressure gradient, and (ii) active transport from the interstitial compartment across the choroidal epithelium into the ventricular space, involving carbonic anhydrase and membrane ion carrier proteins. CSF plays an essential role in the homeostasis of the brain interstitial fluid and neuronal environment by controlling electrolyte balance, circulating active molecules, and removing catabolites. CSF transports choroid plexus secretion products to their sites of action, thereby modulating the activity of specific brain regions by impregnation, while synaptic transmission produces more rapid changes in activity. Waste products of cerebral metabolism, peroxidation products and glycosylated proteins, accumulate with age-related decreased CSF turnover (Sakka et al., 2011, European Annals of Otorhinolaryngology, Head and Neck Diseases, 128(6):309-316).

[0034] Subarachnoid Hemorrhage-Associated Secondary Brain Injury (SAH-SBI) In addition to the initial brain injury occurring within the first 72 hours, patient outcomes in aSAH may be determined by delayed secondary brain injury, which typically occurs between 4 and 10 days after aneurysm rupture. Delayed secondary brain injury is presumed to be multifactorial, including macrovascular and microvascular dysfunction, neuritis, neuronal apoptosis, and pathological electrical activity in the brain. Approximately two-thirds of patients after aSAH develop angiographic vasospasm of the cerebral arteries (aVSP). Delayed cerebral ischemia (DCI) with radiological borders of the ischemic brain area, and clinically evident delayed ischemic neurological deficit (DIND) are often found in approximately one-third of patients after aSAH. The occurrence of at least one of these secondary symptoms defines subarachnoid hemorrhage-associated secondary brain injury in the study described herein (SAH-SBI).

[0035] Adverse secondary neurological outcomes Even if they survive hemorrhagic stroke such as SAH, such patients are at significant risk of developing one or more adverse secondary neurological outcomes or complications.The term "adverse secondary neurological outcomes" as used herein refers to the adverse neurological events (secondary damage to brain tissue) that follow hemorrhagic stroke.Secondary damage after hemorrhagic stroke is believed to be caused by a cascade of events that are initiated by the primary injury (e.g., mass effect and physical disruption), by physiological response to hematoma (e.g., inflammation), and / or by the release of blood and blood components. Adverse secondary neurological outcomes are familiar to those skilled in the art, and illustrative examples include delayed ischemic neuropathy (DIND), delayed cerebral ischemia (DCI), neurotoxicity, apoptosis, inflammation, nitric oxide depletion, oxidative tissue injury, cerebral vasospasm, cerebrovascular reactivity, edema, and spreading depolarisation (see, e.g., Al-Tamimi et al., World Neurosurgery, 73(6):654-667 (2010); Macdonald et al., Neurocrit. Care, 13:416-424 (2010); and Macdonald et al., J. Neurosurg. 99:644-652 (2003)).

[0036] The terms "treating", "treatment", "treat" and the like are used interchangeably herein to mean alleviating, minimizing, reducing, ameliorating, or otherwise inhibiting an adverse secondary neurological outcome (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 adverse secondary neurological outcome from occurring or delaying the onset or subsequent progression of an adverse secondary neurological outcome in a subject who may be susceptible to or at risk of developing, but has not yet been diagnosed as having, an adverse secondary neurological outcome. In that context, the terms "treating", "treatment" and the like are used interchangeably with terms such as "prophylaxis", "prophylactic", and "preventative". However, it is understood that the methods disclosed herein do not require complete prevention of an adverse secondary neurological outcome from occurring in the subject being treated. The methods disclosed herein may be sufficient to merely palliate, reduce, alleviate, ameliorate, or otherwise inhibit adverse secondary neurological outcomes in a subject, so long as the number and severity of the adverse secondary neurological outcomes are less than would be observed in the absence of treatment. Thus, the methods described herein may reduce the number and / or severity of adverse secondary neurological outcomes in a subject following a hemorrhagic stroke.

[0037] When referring to a subject herein, it is not intended that the subject has had a hemorrhagic stroke, but is understood to include subjects at risk of hemorrhagic stroke. In one embodiment, the subject has (i.e., has experienced) a hemorrhagic stroke or symptoms thereof. In another embodiment, the subject does not already have a hemorrhagic stroke at the time of treatment, but is at risk of hemorrhagic stroke. As an illustrative example, the subject has an aneurysm that has not yet ruptured, but is at risk of rupturing. In this case, the subject may undergo surgical intervention (e.g., by clipping or endovascular coiling) to minimize the risk of the aneurysm rupturing. Thus, in cases where an aneurysm ruptures before, during, or after a surgical intervention, the methods described herein may be suitably specified as a preventive measure for the subject to minimize, reduce, negate, or otherwise inhibit adverse secondary neurological outcomes. In that context, the methods described herein may also be employed as a preventive measure performed before, during, or after a surgical intervention.

[0038] The extent to which the methods disclosed herein result in a subjective, qualitative, and / or quantitative reduction in the number and / or severity of adverse secondary neurological outcomes following hemorrhagic stroke, as compared to the number and / or severity of adverse secondary neurological outcomes prior to exposure of the CSF to a therapeutically effective amount of Hp, is, for example, at least 10%, preferably about 10% to about 20%, preferably about 15% to about 25%, preferably about 20% to about 30%, preferably about 25% to about 35%, preferably about This can be expressed as a decrease of 30% to about 40%, preferably about 35% to about 45%, preferably about 40% to about 50%, preferably about 45% to about 55%, preferably about 50% to about 60%, preferably about 55% to about 65%, preferably about 60% to about 70%, preferably about 65% to about 75%, preferably about 70% to about 80%, preferably about 75% to about 85%, preferably about 80% to about 90%, preferably about 85% to about 95%, or most preferably about 90% to 100%.

[0039] Suitable methods by which a subjective, qualitative, and / or quantitative decline in the number and / or severity of adverse secondary neurological outcomes following hemorrhagic stroke can be measured will be familiar to those skilled in the art and will generally depend on the nature of the adverse secondary neurological outcomes being measured. Illustrative examples are described elsewhere herein.

[0040] In one embodiment, the adverse secondary neurological outcome is selected from the group consisting of delayed ischemic neuropathy (DIND), delayed cerebral ischemia (DCI), neurotoxicity, inflammation, nitric oxide depletion, oxidative tissue injury, cerebral vasospasm, cerebrovascular hyporeactivity, edema, and widespread depolarization.

[0041] In one embodiment, the adverse secondary neurological outcome is delayed ischemic neurological injury (DIND). DIND after SAH is a severe, poorly understood syndrome of cerebral ischemia characterized by headache, worsening meninges, and / or elevated temperature, typically followed by a fluctuating decline in consciousness and the emergence of focal neurological symptoms. DIND is characteristically defined as a deterioration in neurological function at least 3-4 days after a hemorrhagic insult. DIND is also called clinical / symptomatic vasospasm. DIND remains a major cause of morbidity and mortality in survivors of the initial hemorrhage. The reported prevalence of DIND is about 20%-35%, but even in those with higher blood burdens, the prevalence can be as high as 40%. DIND is responsible for cerebral infarction in approximately 20% of patients and for about 13% of all deaths and disability following aSAH. Suitable methods for determining DIND are familiar to those skilled in the art, and illustrative examples thereof are described in Dreier et al., Brain, 2006;129(12):3224-3237, the contents of which are incorporated herein by reference in their entirety. In one embodiment, DIND is determined by spreading mass depolarization, as evidenced, for example, by spreading negative slow voltage variation by electrocorticography. In one embodiment, DIND is associated with a delayed decrease in consciousness of at least 2 GCS levels and / or new focal neurological deficits.

[0042] In one embodiment, the adverse secondary neurological outcome is cerebral vasospasm. Cerebral vasospasm or CV (also called "angiographic cerebral vasospasm") is one of the most common causes of focal ischemia after hemorrhagic stroke and may account for up to about 23% of SAH-related disability and death. CV is generally characterized by narrowing of blood vessels caused by persistent vasoconstriction after hemorrhagic stroke into the subarachnoid space, especially in the large capacitance arteries (i.e., cerebral arteries) at the base of the brain. The term "vasospasm" is therefore commonly used in the context of angiographically determined arterial narrowing. The persistent constriction of blood vessels reduces perfusion of distal brain regions and increases cerebrovascular resistance. If left untreated, CV can eventually lead to neurotoxicity (brain cell damage) in the form of cerebral ischemia and cerebral infarction, mainly due to limited blood supply to brain tissue. CV can be detected by any suitable means known to those skilled in the art, illustrative examples of which include digital subtraction angiography (DSA), computed tomography (CT) angiography (CTA), magnetic resonance (MR) angiography (MRA), transcranial Doppler ultrasound, and catheter (cerebral) angiography (CA). In one embodiment, CV is detected by digital subtraction angiography (DSA). Without being bound by theory or particular application, cerebral artery vasospasm typically begins about 3 days after SAH, peaks about 7-8 days later, and resolves by about 14 days (see, e.g., Weir et al., J.Neurosurg., 48:173-178 (1978)), although at least two-thirds of patients who have angiograms 4-12 days after SAH have some degree of angiographic stenosis.

[0043] The incidence of CV depends on the time after SAH. As noted elsewhere herein, the peak incidence generally occurs at about 7-8 days (range 3-12 days) after SAH. In addition to the time after SAH, other major factors that affect the prevalence of vasospasm are the volume, density, temporal persistence, and distribution of subarachnoid blood. Prognostic factors for CV include the amount of subarachnoid blood on CT scan, hypertension, anatomical and systemic factors, clinical grade, and whether the patient has been administered antifibrinolytic drugs.

[0044] Symptoms of CV are typically subacute in onset and may fluctuate, and may include excessive sleepiness, somnolence, coma, hemiparesis or hemiplegia, lack of willpower, speech disorders, visual field defects, gaze impairment, and cranial nerve palsies. Some symptoms are focal, but are generally not diagnostic of any particular pathological process. Cerebral angiography is commonly utilized as the gold standard for visualizing and examining the cerebral arteries, although transcranial Doppler ultrasound can also be used.

[0045] In one embodiment, the adverse secondary neurological outcome is delayed cerebral ischemia (DCI). DCI commonly occurs in about 1 / 3 of patients with aSAH, and causes death or permanent disability in half of these patients (Dorsch and King, Journal of Clinical Neuroscience, 1:19-26 (1994)). DCI is generally defined as radiologically detected cerebral infarction in patients with aSAH without other identifiable reasons (e.g., postoperative intervention).

[0046] A unified definition of "clinical regression caused by delayed cerebral ischemia" and "cerebral infarction" as reported by Vergouwen et al. (Stroke. 2010;41:2391-2395) should capture the most meaningful elements in terms of morphological and clinical characteristics without making assumptions about its etiology. Given that CT / MRI infarction has a strong correlation with functional outcome 3 months after SAH, and is expected to have a high interobserver agreement rate, DCI can be detected in sedated and comatose patients, and the results of DCI can be objectively quantified, neuroimaging infarction seems to be a better outcome indicator than clinical regression caused by DCI alone. Previous definitions of DCI often combined clinical features of DCI with angiographic / transcranial Doppler findings or neuroimaging or autopsy infarction, but the authors suggest that these should be reported separately. The authors also suggest that clinical regression caused by DCI should not be considered more than a secondary indicator of outcome, due to a suspected low rate of interobserver agreement. According to Vergouwen et al., the proposed definition of clinical regression caused by DCI is "the development of focal neurological deficits (e.g., hemiparesis, aphasia, apraxia, hemianopia, or idiopathic) or a decrease of at least 2 points on the Glasgow Coma Scale (in the total score or in one of its individual components [eyes, movements on either side, speech])." This should last at least 1 hour, not appear immediately after aneurysm occlusion, and cannot be attributed to other causes by clinical evaluation, CT or MRI scanning of the brain, and applicable laboratory tests.

[0047] Adverse secondary neurological outcomes after hemorrhagic stroke, including SAH, have been shown to be associated with inflammation, including immune cell activation and / or infiltration into the CSF compartment and release of inflammatory cytokines. As discussed by Miller et al. (Biomed Res Int. 2014;2014:384342), studies have revealed that inflammation is a direct mediator of neurological injury after SAH and also a causative factor in post-SAH vasospasm. The major inflammatory molecules involved in the pathophysiology of SAH will be familiar to those skilled in the art, and illustrative examples include selectins (L-selectin and P-selectin), integrins (e.g., lymphocyte function-associated antigen 1 (LFA-1) and Mac-1 integrin (CD11b / CD18)), TNFα, monocyte chemoattractant protein 1 (MCP-1), intercellular adhesion molecule 1 (ICAM-1), pro-inflammatory interleukins (e.g., IL-1, IL-6, IL-1B, IL-8), and endothelin 1 (ET-1). In one embodiment disclosed herein, the adverse secondary neurological outcome is associated with differential expression of one or more inflammatory markers selected from the group consisting of selectins (e.g., L-selectin and P-selectin), integrins (e.g., lymphocyte function-associated antigen 1 (LFA-1) and Mac-1 integrin (CD11b / CD18)), TNFα, monocyte chemoattractant protein 1 (MCP-1), intercellular adhesion molecule 1 (ICAM-1), proinflammatory interleukins, and endothelin 1 (ET-1). In one embodiment, the proinflammatory interleukins are selected from the group consisting of IL-1, IL-6, IL-1B, and IL-8.

[0048] Nissen et al. previously found that serum concentrations of P-selectin are significantly higher in patients with DIND compared to patients without DIND (J Neurol Neurosurg Psychiatry 2001;71:329-333). The authors also found that serum concentrations of L-selectin are significantly lower in patients with DIND compared to patients without DIND. Thus, in one embodiment, the extent to which the methods described herein reduce the number and / or severity of adverse secondary neurological outcomes following hemorrhagic stroke may be achieved by reducing the concentration of P-selectin in the serum or CSF of a subject, e.g., by at least 10%, preferably about 10% to about 20%, preferably about 15% to about 25%, preferably about 20% to about 30%, preferably about 25% to about 35%, preferably about 30%, compared to the concentration of P-selectin in the subject prior to treatment. % to about 40%, preferably about 35% to about 45%, preferably about 40% to about 50%, preferably about 45% to about 55%, preferably about 50% to about 60%, preferably about 55% to about 65%, preferably about 60% to about 70%, preferably about 65% to about 75%, preferably about 70% to about 80%, preferably about 75% to about 85%, preferably about 80% to about 90%, preferably about 85% to about 95%, or most preferably about 90% to 100%.In another embodiment, the extent to which the methods described herein reduce the number and / or severity of adverse secondary neurological outcomes following hemorrhagic stroke is determined by a decrease in the concentration of L-selectin in the serum or CSF of a subject, e.g., by at least 10%, preferably about 10% to about 20%, preferably about 15% to about 25%, preferably about 20% to about 30%, preferably about 25% to about 35%, preferably about 30% to about 40%, more preferably about 40% to about 50%, more preferably about 50% to about 60%, more preferably about 60% to about 70%, more preferably about 70% to about 80%, more preferably about 80% to about 90%, more preferably about 90% to about 100%, more preferably about 90% to about 100%, more preferably about 10% to about 120%, more preferably about 10% to about 150%, more preferably about 15% to about 25%, more preferably about 20% to about 30%, more preferably about 25% to about 35%, more preferably about 3 ...50%, more preferably about 15% to about 25%, more preferably about 25% to about 35%, more preferably about 30% to about 150%, more preferably about 15% to about 25%, more preferably about 25% to about 35%, more preferably about 30% to about 150%, more preferably about 15% to about 25%, more preferably about 25% to about 35%, more preferably about 30% to about 35%, more preferably about 30% to about 35%, more preferably about 30% to about 3 It is determined by an increase of about 40%, preferably about 35% to about 45%, preferably about 40% to about 50%, preferably about 45% to about 55%, preferably about 50% to about 60%, preferably about 55% to about 65%, preferably about 60% to about 70%, preferably about 65% to about 75%, preferably about 70% to about 80%, preferably about 75% to about 85%, preferably about 80% to about 90%, preferably about 85% to about 95%, or most preferably about 90% to 100%. Methods by which concentrations of P-selectin and L-selectin can be measured are familiar to those skilled in the art, and illustrative examples thereof are described in Nissen et al. (J Neurol Neurosurg Psychiatry 2001;71:329-333), the contents of which are incorporated herein by reference in their entirety.

[0049] Levels of the pro-inflammatory cytokines IL-1B, IL-6, IL-8, TNFα, and MCP-1, as well as endothelin-1, have also been found to be elevated in patients following SAH (Miller et al. Biomed Res Int. 2014;2014:384342). Thus, in one embodiment disclosed herein, the extent to which the methods described herein reduce the number and / or severity of adverse secondary neurological outcomes following hemorrhagic stroke may be determined by a decrease in the concentration of pro-inflammatory cytokines in the serum or CSF of a subject, e.g., at least 10%, preferably about 10% to about 20%, preferably about 15% to about 25%, preferably about 20% to about 30%, preferably about 25% to about 35%, preferably about 30% to about 40%, preferably about 35% to about 45%, in the concentration of pro-inflammatory cytokines in the serum or CSF of the subject, as compared to the concentration of pro-inflammatory cytokines in the subject prior to treatment. Preferably, the proinflammatory molecule is determined by a decrease of about 40% to about 50%, preferably about 45% to about 55%, preferably about 50% to about 60%, preferably about 55% to about 65%, preferably about 60% to about 70%, preferably about 65% to about 75%, preferably about 70% to about 80%, preferably about 75% to about 85%, preferably about 80% to about 90%, preferably about 85% to about 95%, or most preferably about 90% to 100%, where the proinflammatory molecule is selected from the group consisting of IL-1B, IL-6, IL-8, TNFα, MCP-1, and endothelin-1. Methods by which the concentration of inflammatory mediators can be measured will be familiar to those of skill in the art, as described herein.

[0050] Nitric oxide (NO) depletion in the CSF has also been shown to contribute to the pathogenesis of adverse secondary neurological outcomes after hemorrhagic stroke (see Pluta et al. JAMA. 2005;293(12):1477-1484). NO levels are decreased in the CSF after SAH due to (1) toxicity of oxyhemoglobin to neuronal nitric oxide synthase (NOS)-containing neurons in the arterial adventitia; (2) endogenous inhibition of endothelial NOS; and (3) scavenging of nitric oxide by oxyhemoglobin released from the subarachnoid clot. Thus, in one embodiment disclosed herein, the extent to which the methods described herein reduce the number and / or severity of adverse secondary neurological outcomes following hemorrhagic stroke may be achieved by, for example, a decrease in the concentration of NO in the subject's CSF by at least 10%, preferably about 10% to about 20%, preferably about 15% to about 25%, preferably about 20% to about 30%, preferably about 25% to about 35%, preferably about 30% to about 35%, more ... The increase is determined by an increase of 0% to about 40%, preferably about 35% to about 45%, preferably about 40% to about 50%, preferably about 45% to about 55%, preferably about 50% to about 60%, preferably about 55% to about 65%, preferably about 60% to about 70%, preferably about 65% to about 75%, preferably about 70% to about 80%, preferably about 75% to about 85%, preferably about 80% to about 90%, preferably about 85% to about 95%, or most preferably about 90% to 100%. Methods by which NO concentrations in CSF can be measured are familiar to those of skill in the art, and illustrative examples thereof are described in Pluta et al. (JAMA. 2005; 293(12): 1477-1484), the contents of which are incorporated herein by reference in their entirety.

[0051] In one embodiment, the adverse secondary neurological outcome is oxidative tissue injury. As noted elsewhere herein, the inventors have for the first time demonstrated that a therapeutically effective amount of Hx can reduce the oxidative potential of acellular heme in the CSF. Thus, in one embodiment, exposing the CSF of a subject in need thereof to Hx reduces oxidative tissue injury as described herein. Suitable methods for determining oxidative tissue injury are familiar to those skilled in the art, and illustrative examples thereof are described in Katerji et al. (2019; Oxid. Med. Cell Longey; 1279250), including measuring the level of malondialdehyde (MDA), an end product of lipid peroxidation. In one embodiment, exposing the CSF of a subject in need thereof to Hx reduces lipid peroxidation as described herein.

[0052] In some embodiments, therapeutic Hx may prevent acellular heme from penetrating from the CSF compartment into the interstitial space of the brain, thereby inhibiting the toxic effects of acellular heme on the cerebral vasculature and brain parenchyma. Thus, in one embodiment, the adverse secondary neurological outcome is an adverse secondary neurological outcome in the brain parenchyma.

[0053] Hemopexin Hemopexin is a 61 kDa plasma β-1B-glycoprotein composed of a single peptide chain 439 amino acids long formed by two four-bladed β-propeller domains (resembling two thickened disks fixed together at a 90° angle and connected by an interdomain linker peptide). Heme released into the blood as a result of intravascular and extravascular hemolysis binds between the two four-bladed β-propeller domains in a pocket formed by the interdomain linker peptide. Residues His213 and His266 determine the position of the heme iron atom, resulting in a stable bis-histidyl complex similar to that of hemoglobin.

[0054] Hemopexin contains approximately 20% carbohydrate, including sialic acid, mannose, galactose, and glucosamine. Twelve cysteine ​​residues are found within the protein sequence, likely accounting for six disulfide bridges. Hemopexin represents the first line of defense against heme toxicity due to its ability to bind heme with high affinity (Kd<1 pM) and to function as a heme-specific carrier from the bloodstream to the liver. Hemopexin binds heme in equimolar ratios, but there is no evidence that heme is covalently bound to proteins.

[0055] In addition to heme binding, hemopexin preparations have also been reported to retain serine protease activity (Lin et.al., 2016; Molecular Medicine 22:22-31), as well as several other functions, such as exerting anti- and pro-inflammatory activities, inhibiting cell adhesion and binding of certain divalent metal ions.

[0056] Although endogenous hemopexin can control the side effects of free heme at physiological steady state, it has little effect on maintaining steady heme levels under pathophysiological conditions (e.g., effects associated with hemolysis), and high levels of heme cause depletion of endogenous hemopexin and contribute to heme-mediated oxidative tissue damage.

[0057] It is understood that naturally occurring and recombinant forms of Hx are suitable for the methods described herein, so long as they have the ability to form a complex with cell-free heme and thereby neutralize the biological activity of cell-free heme. Suitable naturally occurring forms of Hx are known to those of skill in the art, and illustrative examples 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.

[0058] In one embodiment, Hx comprises, consists or consists essentially of plasma-derived Hx. In one embodiment, Hx is human Hx. Illustrative examples of human Hx will be familiar to those skilled in the art, and are described in UniProtKB P02790, reproduced below: MARVLGAPVA LGLWSLCWSL AIATPLPPTS AHGNVAEGET KPPDDVTERC 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 TPHGIILDSV DAAFICPGSS RLHIMAGRRL WWLDLKSGAQ ATWTELPWPH EKVDGALCME KSLGPNSCSA NGPGLYLIHG PNLYCYSDVE KLNAAKALPQ PQNVTSLLGC TH

[0059] In one embodiment, Hx comprises, consists or consists essentially of the sequence of human Hx as previously described.

[0060] Various protocols for isolating Hx from its natural source (e.g., plasma) will be familiar to those 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.

[0061] The term "hemopexin" (Hx), as used herein, is understood to include all phenotypes of Hx, including all isoforms. Hx can be homogeneous (where it is substantially composed of the same isoform of Hx) or heterogeneous (where it contains a combination of different Hx isoforms, including human and non-human isoforms of Hx). Suitable methods for determining the Hx isoforms present in an isolate are familiar to those skilled in the art, illustrative examples of which include high performance particle size exclusion chromatography (HPLC-SEC assay) and Hx ELISA.

[0062] Hx may be naturally occurring Hx (e.g., plasma derived) or may be produced as a recombinant protein. In one embodiment, Hx is plasma derived. In one embodiment, Hx comprises, consists of, or consists essentially of recombinant Hx.

[0063] Unless otherwise stated, the term Hx, as used herein, includes functional analogs of native or naturally occurring Hx. The term "functional analog" is understood to mean a substance that shares substantially the same biological activity as that of naturally occurring (native) Hx, so long as the biological activity is at least the ability of the analog to form a complex with cell-free heme, thereby neutralizing its biological activity. "Substantially the same biological activity" generally means that the functional analog has a binding affinity for cell-free heme that is at least 40% (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, etc.) of the binding affinity of naturally occurring Hx, including naturally occurring Hx isoforms (e.g., human and / or non-human isoforms of Hx). Suitable methods for determining whether a substance is a functional analog of Hx are familiar to those of skill in the art, and illustrative examples thereof (e.g., the ability of a functional analog to reduce cell-free heme-induced cerebral vasospasm) are described elsewhere herein.

[0064] In one embodiment disclosed herein, the functional analog of Hx is a functional fragment of native Hx. The functional fragment of native Hx can be of any suitable length, so long as the fragment retains the ability to form a complex with cell-free heme, thereby neutralizing its biological activity.

[0065] In another embodiment, a functional analog is a peptide (i.e., a comparator) that has an amino acid sequence that differs from that of the naturally occurring (native) Hx molecule. A functional analog can include a molecule that has an amino acid sequence that differs from that of the α-chain and / or β-chain of native Hx by one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or more) amino acid substitutions, provided that the differences do not abolish or completely abolish the ability of the analog to form a complex with cell-free heme and thereby neutralize its biological activity. In some embodiments, a functional analog contains an amino acid substitution that enhances the ability of the analog to form a complex with cell-free heme compared to native Hx. In one embodiment, a functional analog has an amino acid sequence that differs from that of the α-chain and / or β-chain of native Hp by one or more conservative amino acid substitutions. As used herein, the term "conservative amino acid substitution" refers to changing the identity of an amino acid at a given position to replace it with an amino acid of approximately equivalent size, charge, and / or polarity. Examples of naturally occurring conservative substitutions for amino acids include the following eight substitution groups (represented by the conventional one-letter codes): (1) M, I, L, V; (2) F, Y, W; (3) K, R, (4) A, G; (5) S, T; (6) Q, N; (7) D; and (8) C, S.

[0066] In one embodiment, a functional analog has at least 85% sequence identity to the amino acid sequence of the α and / or β chain of native Hx. "At least 85%" includes, for example, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or similarity after optimal alignment or best fit analysis. Thus, in one embodiment the sequence has at least 85%, preferably at least 86%, preferably at least 87%, preferably at least 88%, preferably at least 89%, preferably at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99%, or preferably 100% sequence identity or homology with a sequence identified herein, e.g. after performing an optimal alignment or best fit analysis.

[0067] 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 the aligned sequences, the amino acid residue is identical between the aligned sequences. The terms "similarity" or "sequence similarity", as used herein, refer to the similarity of the type of amino acid residue between the sequences at any particular position in the aligned sequences. For example, leucine can be substituted for an isoleucine or valine residue. As otherwise noted herein, this may be referred to as a conservative substitution. In one embodiment, the amino acid sequence may be modified by conservatively substituting any of the amino acid residues contained therein such that the modification does not affect the binding specificity or functional activity of the modified polypeptide when compared to the unmodified (native) Hx polypeptide.

[0068] In some embodiments, sequence identity in peptide sequences refers to the percentage of amino acid residues in a candidate sequence that are identical to the corresponding peptide sequence residues after aligning the sequences to maximize the percentage of homology, but without considering any conservative substitutions as part of sequence identity, and introducing gaps if necessary.Neither N- or C-terminal extensions nor insertions should be considered to reduce sequence identity or homology.Methods and computer programs for aligning two or more amino acid sequences and determining their sequence identity or homology are well known to those skilled in the art.For example, the percentage of identity or similarity of two amino acid sequences can be easily calculated using algorithms such as BLAST, FASTA, or Smith-Waterman algorithms.

[0069] Techniques for determining the "similarity" of amino acid sequences are well known to those skilled in the art. In general, "similarity" refers to an exact comparison of two or more peptide sequences, or amino acid-to-amino acid in suitable places, in which the amino acids are identical or have similar chemical and / or physical properties (such as charge or hydrophobicity). A so-called "percentage of similarity" can then be determined between the compared peptide sequences. In general, "identity" refers to the exact amino acid-to-amino acid correspondence of two peptide sequences.

[0070] Two or more peptide sequences can also be compared by determining their "percent identity". The percent identity of two sequences can be described as the number of exact matches between the two aligned sequences divided by the length of the shorter sequence multiplied by 100. An approximate alignment method 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 be used for peptide sequences using a scoring matrix developed by Dayhoff (Atlas of Protein Sequences and Structure, MO Dayhoff ed., 5 suppl. 3:353-358, National Biomedical Research Foundation, Washington, DC, USA) and standardized by Gribskov (Nucl. Acids Res. 14(6):6745-6763, 1986). Suitable programs for calculating the percentage of identity or similarity between sequences are generally known in the art.

[0071] The optimal sequence alignment method for aligning the comparison window can be performed by computer implementation of algorithms (GAP, BESTFIT, FASTA, and TFASTA, included in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA), or by selecting the method and the best alignment method (i.e., the highest percentage of homology in the comparison window) generated by any of the various methods. Reference can also be made to the BLAST family of programs, as disclosed, for example, by Altschul et al. (1997, Nucl. Acids Res. 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).

[0072] In one embodiment, the functional analog contains amino acid substitutions and / or other modifications compared to native Hx to increase the stability of the analog or to increase the solubility of the analog.

[0073] Functional analogs can be naturally occurring compounds / peptides or can be synthetically produced by chemical synthesis using methods known to those of skill in the art.

[0074] Hx may be suitably produced in a microorganism as a recombinant protein that can be isolated and, if desired, further purified. Suitable microorganisms for producing recombinant Hx are familiar to those skilled in the art, illustrative examples of which include bacteria, yeast, or fungi, eukaryotic cells (e.g., mammalian or insect cells), or in the form of a recombinant viral vector (e.g., adenovirus, poxvirus, herpesvirus, Semliki Forest virus, baculovirus, bacteriophage, Sindbis virus, or Sendai virus). Suitable bacteria for producing recombinant peptides are familiar to those skilled in the art, illustrative examples of which include E. coli, B. subtilis, or any other bacteria capable of expressing peptide sequences. Illustrative examples of suitable yeast species 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. Methods for isolating and purifying recombinantly produced peptide sequences are also well known in the art and include, for example, gel filtration, affinity chromatography, and ion exchange chromatography.

[0075] To facilitate isolation of recombinant Hx, as described herein, fusion polypeptides may be generated in which a peptide sequence of Hx or a functional analog thereof is translationally fused (covalently linked) to a heterologous polypeptide that allows for isolation by affinity chromatography. Illustrative examples of suitable heterologous polypeptides are His-tags (e.g., (His)6-6 histidine residues), GST-tags (glutathione-S-transferase), etc.

[0076] When preparing recombinant Hx, phage and / or peptide libraries, for example produced by combinatorial chemistry or, for the most diverse structures, obtained by high-throughput screening techniques, are also suitable (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).

[0077] Illustrative examples of recombinant Hx include the peptide having accession number NP_005134 (described by Morishita et al., 2018, Clin. Chim. Acta 487, 84-89), and accession number P00738.

[0078] Hx may be fused, coupled, or otherwise linked to one or more heterologous moieties as part of a fusion protein. The one or more heterologous moieties may improve, enhance, or otherwise extend the activity or stability of Hx. In one embodiment, Hx is suitably linked to a heterologous moiety to extend the in vivo half-life of Hx, as described herein. Suitable half-life extending heterologous moieties will be familiar to those skilled in the art, and illustrative examples thereof include polyethylene glycol (PEGylated), glycosylated PEG, hydroxyethyl starch (HESylated), polysialic acid, elastin-like polypeptides, heparosan polymers, and hyaluronic acid. Thus, in one embodiment disclosed herein, the heterologous moiety is selected from the group consisting of polyethylene glycol (PEGylated), glycosylated PEG, hydroxyethyl starch (HESylated), polysialic acid, elastin-like polypeptides, heparosan polymers, and hyaluronic acid. In other embodiments, the heterologous moiety may be a heterologous amino acid sequence fused to Hx.

[0079] Alternatively or additionally, the heterologous moiety may be chemically conjugated to Hx, for example, covalently. The half-life extending heterologous moiety may be fused, conjugated or otherwise linked to Hx by any suitable means known to those skilled in the art, an illustrative example being by a chemical linker. The principles of this conjugation technology are illustratively described by Conjuchem LLC (see, for example, U.S. Patent No. 7,256,253), the entire contents of which are incorporated herein by reference.

[0080] In other embodiments, the heterologous moiety is a half-life enhancing protein (HLEP). Suitable half-life enhancing proteins are familiar to those skilled in the art, and an illustrative example thereof is albumin or a fragment thereof. Thus, in one embodiment, the HLEP is albumin or a fragment thereof. The N-terminus of albumin or a fragment thereof may be fused to the C-terminus of the α-chain and / or β-chain of Hx. Alternatively or additionally, the C-terminus of albumin or a fragment thereof may be fused to the N-terminus of the α-chain and / or β-chain of Hx. One or more HLEPs may be fused to the N- or C-terminal portion(s) of the α-chain and / or β-chain of Hx, provided that such HLEPs do not abolish the binding of Hx to cell-free heme. However, it is understood that some reduction in the binding of Hx to cell-free heme may be tolerated, as long as the Hx component of the fusion protein still has the ability to form a complex with and thereby neutralize cell-free heme.

[0081] The fusion protein may further comprise a chemical bond or linker sequence disposed between the Hx and the heterologous moiety. The linker sequence may be a peptide linker consisting of one or more amino acids, in particular 1-50, preferably 1-30, preferably 1-20, preferably 1-15, preferably 1-10, preferably 1-5, or more preferably 1-3 (e.g., 1, 2, or 3) amino acids, although the linker sequences may be equal to or different from each other. Preferably, the linker sequence is not present at the corresponding position in wild-type Hx. Preferred amino acids present in said linker sequence include Gly and Ser. In a preferred embodiment, the linker sequence is substantially non-immunogenic to a subject treated according to the methods disclosed herein. By substantially non-immunogenic, it is meant that the linker sequence does not give rise to a detectable antibody response against the linker sequence in the subject to which it is administered. A preferred linker would be composed of alternating repeats of glycine and serine residues. Suitable linkers are familiar to those skilled in the art, and illustrative examples are described in WO2007 / 090584. In one embodiment, the peptide linker between Hx and the heterologous moiety comprises, consists of or consists essentially of a peptide sequence that acts as a natural interdomain linker in human proteins. Such a peptide sequence may be located close to the protein surface in its natural environment, but is also accessible to the immune system, so that natural tolerance to this sequence can be assumed. Illustrative examples are provided in WO2007 / 090584. Suitable cleavable linker sequences are described, for example, in WO2013 / 120939A1.

[0082] Illustrative examples of suitable HLEP sequences are described below. Fusions to the very "N-terminal amino acid" or the very "C-terminal amino acid" of each HLEP, or to the "N-terminal portion" or "C-terminal portion" of each HLEP are also disclosed herein, including N-terminal deletions of one or more amino acids of the HLEP. A fusion protein may contain more than one HLEP sequence, for example two or three HLEP sequences. These multiple HLEP sequences may be fused in tandem, for example as consecutive repeats, to the C-terminal portion of the α-chain and / or β-chain of Hp.

[0083] In one embodiment, the heterologous moiety is a half-life extending polypeptide. In one embodiment, the half-life extending polypeptide is selected from the group consisting of albumin, members of the albumin family or fragments thereof, solvated random chains with large hydrodynamic volumes (e.g., XTEN (see Schellenberger et al. 2009; Nature Biotechnol. 27:1186-1190), homoamino acid repeats (HAPs), or proline-alanine-serine repeats (PASs), afamin, alpha-fetoprotein, vitamin D binding protein, transferrin or variants or fragments thereof, the carboxyl terminal peptide of human chorionic gonadotropin-β subunit (CTP), polypeptides capable of binding to the neonatal Fc receptor (FcRn), particularly immunoglobulin constant regions and portions thereof, e.g., Fc fragments, polypeptides capable of binding to albumin under physiological conditions, e.g., ... homoamino acid repeats (HAPs), or proline-alanine-serine repeats (PASs), homoamino acid repeats (HAPs), or proline-alanine-serine repeats (PASs), homoamino acid repeats (HAPs), or proline-alanine-serine repeats (PASs), homoamino acid repeats (HAPs), homoamino acid repeats (HAPs), homoamino acid repeats (HAPs), homoamino acid repeat The half-life enhancing polypeptide, as used herein, is selected from the group consisting of a polypeptide or lipid having the ability to bind to a member of the albumin family or a fragment thereof, or to an immunoglobulin constant region or a portion thereof. The immunoglobulin constant region or a portion thereof is preferably an Fc fragment of immunoglobulin G1 (IgG1), an Fc fragment of immunoglobulin G2 (IgG2), or an Fc fragment of immunoglobulin A (IgA). A half-life enhancing polypeptide, as used herein, is a polypeptide having the ability to stabilize or extend the therapeutic or biological activity of Hx, particularly the in vivo half-life enhancing polypeptide of Hp. The HLEP fragment may be a full-length half-life enhancing protein capable of increasing half-life in vivo, or one or more fragments thereof. Such fragments may be 10 or more amino acids in length, or may contain 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 contain some or all of a particular domain of each HLEP, so long as the HLEP fragment provides a functional half-life extension of at least 10%, preferably at least 20%, or more preferably at least 25% compared to the respective Hx without the HLEP.Methods for determining whether a heterologous moiety confers a functional half-life extension to Hx (either in vivo or in vitro) are familiar to those of skill in the art, and illustrative examples thereof are described elsewhere herein.

[0084] The HLEP portion of the fusion protein may be a variant of wild-type HELP, as described herein. The term "variant" includes insertions, deletions and / or substitutions, whether conservative or non-conservative, where such changes do not substantially alter the ability of Hx to complex with and thereby neutralize cell-free heme. The HLEP may suitably be derived from any vertebrate, particularly any mammal, such as human, monkey, cow, sheep, or pig. Non-mammalian HLEPs include, but are not limited to, hen and salmon.

[0085] A fusion protein can be created by in-frame joining of at least two DNA sequences encoding heterologous moieties, such as Hx and HLEP, as described herein. Those skilled in the art will understand that the fusion protein DNA sequence, when translated, results in a single protein sequence. According to one embodiment disclosed herein, a fusion protein comprising Hp, a suitable linker, and a heterologous moiety can be obtained as a result of in-frame insertion of a DNA sequence encoding a peptide linker.

[0086] In one embodiment disclosed herein, Hx is fused to a heterologous moiety. In one embodiment, the heterologous moiety comprises, consists of, or consists essentially of a polypeptide selected from the group consisting of albumin or a fragment thereof, transferrin or a fragment thereof, the C-terminal peptide of human chorionic gonadotropin, XTEN sequence, homoamino acid repeat (HAP), proline-alanine-serine repeat (PAS), afamin, alpha-fetoprotein, vitamin D binding protein, a polypeptide capable of binding to albumin or to an immunoglobulin constant region under physiological conditions, a polypeptide capable of binding to neonatal Fc receptor (FcRn), in particular an immunoglobulin constant region and a portion thereof, preferably the Fc portion of an immunoglobulin, and any combination of the above. In another embodiment, the heterologous moiety is selected from the group consisting of hydroxyethyl starch (HES), polyethylene glycol (PEG), polysialic acid (PSA), elastin-like polypeptides, heparosan polymers, hyaluronic acid, and albumin binding ligands, such as fatty acid chains, and any combination of the above.

[0087] 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 include human serum albumin (and fragments and variants thereof), as well as albumin (and fragments and variants thereof) from other species.

[0088] As used herein, "albumin" collectively refers to an albumin polypeptide or amino acid sequence having one or more functional activities (e.g., biological activities) of albumin, or an albumin fragment or variant. In particular, "albumin" refers to human albumin or a fragment thereof, including the mature form of human albumin or albumin from other vertebrates, or a fragment thereof, or an analog or variant of such molecule or fragment thereof. In some embodiments disclosed herein, the alternative term "FP" is used to identify HLEP, and in particular to define albumin as HLEP.

[0089] The fusion proteins described herein may also suitably comprise naturally occurring polymorphic variants of human albumin and / or fragments of human albumin. Generally speaking, albumin fragments or variants are at least 10, preferably at least 40, or most preferably more than 70 amino acids in length.

[0090] In one embodiment, HLEP is an albumin variant that has enhanced binding to FcRn receptor. Such albumin variants may provide longer plasma half-life for Hx or its functional analogues compared to Hx or its functional fragments fused to wild-type albumin. The albumin portion of the fusion protein described herein may suitably comprise at least one subdomain or domain of human albumin, or a conservative modification thereof.

[0091] In one embodiment, the heterologous moiety is an immunoglobulin molecule or a functional fragment thereof. The immunoglobulin G (IgG) constant region (Fc) is known in the art to increase the half-life of therapeutic proteins (see, for example, Dumont JA et al. 2006. BioDrugs 20:151-160). The IgG constant region of the heavy chain consists of three domains (CH1-CH3) and a hinge region. The immunoglobulin sequence can be from any mammal or from each of the subclasses IgG1, IgG2, IgG3, or IgG4. IgG and IgG fragments without antigen-binding domains can also be used as heterologous moieties, including HLEP. Hp or its functional analogs may be suitably connected to the IgG or IgG fragments via the hinge region of the antibody or a peptide linker (which may be cleavable). Several patents and patent applications describe fusing therapeutic proteins to immunoglobulin constant regions to enhance the in vivo half-life of the therapeutic proteins. For example, US 2004 / 0087778 and WO 2005 / 001025 describe fusion proteins consisting of at least a portion of an Fc domain or immunoglobulin constant region and a biologically active peptide that increases the half-life of a peptide that would otherwise be rapidly cleared in vivo. Fc-IFN-β fusion proteins have been described that achieve enhanced biological activity, extended circulating half-life, and improved solubility (WO 2006 / 000448A2). Fc-EPO proteins with extended serum half-lives and enhanced potency in vivo have been 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), coagulation factors (WO 2004 / 101740 A2), and interleukin-10 (U.S. Pat. No. 6,403,077), all of which have half-life enhancing properties.

[0092] Illustrative examples of suitable HLEPs that can be used according to the present invention are also described in WO 2013 / 120939 A1, the contents of which are incorporated herein by reference in their entirety.

[0093] The term "therapeutically effective amount," as used herein, refers to an amount or concentration of Hx in the CSF sufficient to allow Hx to bind and complex with the cell-free heme present in the CSF, thereby neutralizing the otherwise deleterious biological effects of the cell-free heme. It will be understood by those skilled in the art that a therapeutically effective amount of a peptide can vary depending on several factors, illustrative examples of which include whether Hx is administered directly to the subject and how it is administered (e.g., intrathecally, intracranially, or intracerebroventricularly), the health and physical condition of the subject being treated, the taxonomic group of the subject being treated, the severity of the hemorrhage (e.g., the extent of the hemorrhage), the route of administration, the concentration and / or amount of cell-free heme in the CSF compartment, and any combination of the above.

[0094] The therapeutically effective amount of Hx falls within a relatively broad range that can be determined by one of skill in the art. Illustrative examples of suitable therapeutically effective amounts of Hx include about 2 μM to about 1 mM, preferably about 2 μM to about 400 mM, preferably about 5 μM to about 400 μM, preferably about 5 μM to about 200 μM, or more preferably about 10 μM to about 200 μM.

[0095] In one embodiment, the therapeutically effective amount of Hx is about 2 μM to about 1 mM. In one embodiment, the therapeutically effective amount of Hx is about 2 μM to about 400 μM. In one embodiment, the therapeutically effective amount of Hx is about 5 μM to about 200 mM. In one embodiment, the therapeutically effective amount of Hx is about 10 μM to about 200 μM.

[0096] In one embodiment, the therapeutically effective amount of Hx is at least equimolar to the cell-free heme concentration in the CSF of a subject following hemorrhage. In another embodiment, the therapeutically effective amount of Hx is an amount sufficient to complex with about 3 μM to about 1,200 μM of cell-free Hb in the CSF. In one embodiment, the therapeutically effective amount of Hx is at least four times the amount of cell-free hemoglobin (Hb) present in the CSF of a subject following hemorrhage. Suitable methods for measuring the concentration of cell-free Hb and heme in CSF are known to those skilled in the art, illustrative examples of which are described in Nyakundi et al. (2020, Oxid Med Cell Longev, 2020, 8929020), Righy et al. (2018, Rev Bras Ter Intestiva, 30(1):21-27), Cruickshank AM. (2001, ACP Best Practice No 166, J. Clin. Path., 54(11):827-830), Hugelshofer M. et al. (2018. World Neurosurg.; 120:e660-e666), and Hugelshofer M. et al. (J Clin Invest. 2019; 129(12):5219-5235), the contents of which are incorporated herein by reference in their entirety.

[0097] It is understood that the realization of a therapeutically effective amount of Hx, as described herein, may depend on the final volume of CSF to which Hx or its functional analogue is exposed. For example, when Hx is administered to an adult human subject (e.g., intrathecally), considering that the average volume of CSF in an adult human subject is about 150mL, a therapeutically effective amount of about 10μM Hx can be achieved by administering 5mL of about 310μM Hx solution to the subject. In another illustrative example, the method described herein includes removing 50mL of CSF from a subject and replacing it with 50mL of artificial CSF containing about 30μM Hx, thereby realizing a therapeutically effective amount of about 10μM Hx in the CSF compartment of the subject.

[0098] The dosage of Hx may be adjusted to achieve the optimal therapeutic response, for example, several divided doses may be administered daily, weekly or at other suitable time intervals, or the dosage may be proportionally reduced as the exigencies of the situation indicate.

[0099] The term "exposing" as used herein means contacting the CSF with Hx such that Hx binds to and complexes with cell-free heme present in the CSF, such that formation of the heme:Hx complex substantially neutralizes the otherwise deleterious biological effects of cell-free heme on brain tissue. "Substantially neutralize" refers to a biological effect that is at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 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, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 12 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 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 "A reduction in the deleterious biological effects of acellular heme on brain tissue" refers to a reduction in the deleterious biological effects of acellular heme on brain tissue, as subjectively or qualitatively expressed as a reduction of at least 10%, preferably about 10% to about 20%, preferably about 15% to about 25%, preferably about 20% to about 30%, preferably about 25% to about 35%, preferably about 30% to about 40%, preferably about 35% to about 45%, preferably about 40% to about 50%, preferably about 45% to about 55%, preferably about 50% to about 60%, preferably about 55% to about 65%, preferably about 60% to about 70%, preferably about 65% to about 75%, preferably about 70% to about 80%, preferably about 75% to about 85%, preferably about 80% to about 90%, preferably about 85% to about 95%, or most preferably about 90% to 100%, including 100%. Methods by which a reduction in the deleterious biological effects of cell-free Hx is measurable or determinable (either qualitatively or quantitatively) will be familiar to those of skill in the art, and illustrative examples thereof are described elsewhere herein.

[0100] As also noted herein, the inventors also demonstrated for the first time that a therapeutically effective amount of Hx can block and reduce the oxidative capacity of cell-free heme in CSF, as assessed by malondialdehyde using the thiobarbituric acid reactive substances (TBARS) assay.

[0101] It is understood that the manner in which the CSF of a subject in need thereof is exposed to a therapeutically effective amount of Hx as described herein can vary depending, for example, on whether the exposure is performed in the CSF compartment of a subject in need thereof (i.e., in vivo) or on CSF obtained from a subject externally (i.e., ex vivo). If the exposure is performed in the CSF compartment of a subject in need thereof (i.e., in vivo), the route of administration of Hx is selected to allow Hx to contact acellular heme in the CSF compartment. Suitable routes of administration are familiar to those skilled in the art, and illustrative examples include intrathecal, intracranial, and intraventricular. In one embodiment, a therapeutically effective amount of Hx is administered, for example, in a subject after a hemorrhagic stroke, via an external ventricular drain placed to temporarily drain CSF and reduce intracranial pressure.

[0102] In one embodiment, Hx is administered intracranially to the subject.

[0103] In one embodiment, Hx is administered intrathecally to the subject.

[0104] In one embodiment, Hx is administered intrathecally into the spinal canal.

[0105] In one embodiment, Hx is administered intrathecally into the subarachnoid space.

[0106] In one embodiment, Hx is administered intracerebroventricularly to the subject.

[0107] In one embodiment, the method comprises administering a therapeutically effective amount of Hx intrathecally to a subject. In one embodiment, the method comprises administering a therapeutically effective amount of Hx intrathecally to a subject into its spinal canal. In one embodiment, the method comprises administering a therapeutically effective amount of Hx intrathecally to a subject into its subarachnoid space. In one embodiment, the method comprises administering a therapeutically effective amount of Hx intracerebroventricularly to a subject.

[0108] Alternatively or additionally, the methods described herein include removing CSF from a subject in need thereof, exposing the CSF to a therapeutically effective amount of Hx for a period of time sufficient to allow Hx to form complexes with acellular heme in the CSF, thereby neutralizing the acellular heme, removing the heme:Hx complexes formed in the CSF to generate a heme-reduced CSF, and administering the heme-reduced CSF to the subject (e.g., intrathecally or intracerebroventricularly). It is understood that removing CSF from the CSF compartment does not generally result in a CSF compartment completely free of CSF, and it is noted that at least some CSF will persist in the CSF compartment. Optionally, once the CSF has been removed, the CSF compartment can be rinsed with a pharma- ceutically acceptable wash solution to remove at least some residual Hx that may be present in the CSF compartment. The wash solution can optionally include Hx to further form complexes, thereby neutralizing at least some residual acellular heme that may be present in the CSF compartment. In one embodiment, the irrigation solution is an artificial CSF as described elsewhere herein.

[0109] In one embodiment, the method described herein includes the steps of: removing a sample of CSF from the CSF compartment of a subject in need thereof; adding Hx to the CSF sample to obtain a Hx-enriched CSF sample; administering the Hx-enriched CSF sample to the CSF compartment of the subject, thereby exposing the CSF compartment to a therapeutically effective amount of Hx for a period of time (a period of time sufficient to allow Hx to form a complex with acellular heme and thereby neutralize the acellular heme in the CSF of the subject); and optionally repeating the steps.Preferably, the amount of Hx added to the CSF sample is determined so that, when administered to a subject, it results in a therapeutically effective amount of Hx in the CSF of the subject.Thus, the amount of Hx added to the CSF sample depends on the volume of the CSF sample removed and re-administered to the subject.

[0110] In one embodiment, the method comprises: (i) obtaining a CSF sample from the CSF compartment of a subject following bleeding; (ii) adding Hx to the CSF sample of step (i) to obtain a Hx-enriched CSF sample; (iii) administering the Hx-enriched CSF sample to a subject, thereby exposing the CSF compartment of the subject to a therapeutically effective amount of Hx for a period of time sufficient to allow Hx to form a complex with cell-free heme in the CSF compartment of the subject; and (iv) optionally repeating steps (i) to (iii); Includes.

[0111] It is desirable that the volume of CSF that is removed and re-administered to the subject is substantially the same. For example, if a 50 mL CSF sample is removed from the subject's CSF compartment, the entire 50 mL volume of CSF containing Hx is re-administered to the subject. However, it is understood that the volumes may differ, so long as any difference in volume does not cause significant adverse clinical outcomes. In some embodiments, the volume of CSF that is re-administered to the subject is less than the volume of CSF that was removed from the subject. In other embodiments, the volume of CSF that is re-administered to the subject is greater than the volume of CSF that was removed from the subject, due to the extra volume that is generated by the addition of Hx alone or in combination with any other therapeutic agent.

[0112] In another embodiment, the method described herein comprises the steps of removing a volume of CSF from the CSF compartment of a subject in need thereof, replacing the volume of CSF removed from the subject with a volume of artificial CSF containing Hx, exposing the subject's CSF to a therapeutically effective amount of Hx for a period of time sufficient to allow Hx to form a complex with acellular heme, thereby neutralizing the acellular heme in the subject's CSF. Preferably, the amount of Hx in the artificial CSF is determined to provide a therapeutically effective amount of Hx in the subject's CSF when administered to the subject. Thus, the amount of Hx added to the artificial CSF depends on the volume of artificial CSF administered to the subject.

[0113] In one embodiment, the method comprises: (i) removing a volume of CSF from the CSF compartment of a subject following hemorrhage; (ii) providing an artificial CSF containing Hx; (iii) administering the artificial CSF of (ii) to a subject, thereby exposing the CSF compartment of the subject to a therapeutically effective amount of Hx for a period of time sufficient to allow Hx to form a complex with cell-free heme in the CSF compartment of the subject; and (iv) optionally repeating steps (i) to (iii); Includes.

[0114] It is desirable that the volume of artificial CSF administered to a subject is substantially the same as the volume of CSF removed from the subject. For example, if a 50 mL CSF sample is removed from the subject's CSF compartment, a volume of about 50 mL of artificial CSF containing a therapeutically effective amount of Hx is used to replace the volume of CSF removed. However, it is understood that the volumes may differ, so long as any difference in volume does not cause significant adverse clinical outcomes. In some embodiments, the volume of artificial CSF administered to a subject is less than the volume of CSF removed from the subject. In other embodiments, the volume of artificial CSF administered to a subject is greater than the volume of CSF removed from the subject.

[0115] In one embodiment, the method described herein comprises exposing the CSF to Hx within about 21 days after the hemorrhagic stroke. In another embodiment disclosed herein, the method comprises exposing the CSF to Hx for about 2 days to about 4 days after the hemorrhagic stroke. In yet another embodiment, the method comprises exposing the CSF to Hx for about 5 days to about 14 days after the hemorrhagic stroke.

[0116] The present inventors have surprisingly found that exposure of acellular heme in the subarachnoid space to a therapeutically effective amount of Hx for at least about 2 minutes is sufficient to form detectable heme:Hx complexes in the CSF. Thus, in one embodiment, the period during which the CSF is exposed to a therapeutically effective amount of Hx is at least about 2 minutes (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 minutes, etc.). In one embodiment, the period during which the CSF is exposed to a therapeutically effective amount of Hx is at least about 4 minutes. In another embodiment, the period during which the CSF is exposed to a therapeutically effective amount of Hx is at least about 5 minutes. In yet another embodiment, the period during which the CSF is exposed to a therapeutically effective amount of Hx is at least about 10 minutes. In one embodiment, the period during which the CSF is exposed to a therapeutically effective amount of Hx is about 2 minutes to about 45 minutes, preferably about 2 minutes to about 20 minutes, or more preferably about 4 minutes to about 10 minutes.

[0117] The term "subject" as used herein refers to a mammalian subject for whom treatment or prevention is desired. Illustrative examples of suitable subjects include primates, particularly humans, companion animals such as cats and dogs, working animals such as horses, donkeys, livestock animals such as sheep, cows, goats, pigs, research test animals such as rabbits, mice, rats, guinea pigs, hamsters, and animals in zoos and wildlife parks, wild caught animals such as deer, dingoes, and the like. In one embodiment, the subject is a human. In further embodiments, the subject is a pediatric patient (i) from birth to about 2 years of age, (ii) from about 2 to about 12 years of age, or (iii) from about 12 to about 21 years of age.

[0118] In one embodiment, the methods described herein comprise exposing the CSF to a therapeutically effective amount of Hx exogenously.

[0119] When CSF is exposed to Hx ex vivo, the therapeutically effective amount will depend on the volume of CSF exposed to Hx, whether the CSF is exposed to Hx in solution or immobilized on a substrate (e.g., affinity chromatography), and any combination of the above. For example, when CSF is exposed to Hx to complex with and remove cell-free heme by affinity chromatography, the amount of Hx immobilized on the substrate need not cause complete complexation of the cell-free heme during the first pass period, and multiple passes over the substrate may be required to allow complexation, thereby removing substantially all of the cell-free heme from the CSF.

[0120] In one embodiment, the method comprises the steps of (i) obtaining a CSF sample from a subject after a hemorrhagic stroke and prior to exposing the CSF to Hx; (ii) measuring the amount of cell-free Hb or cell-free heme in the CSF sample obtained in step (i); and (iii) determining at least an equimolar amount of Hx based on the concentration of cell-free Hb or cell-free heme obtained from step (ii). Suitable methods for measuring the amount of cell-free Hb and cell-free heme in a CSF sample are known to those of skill in the art, and illustrative examples thereof are provided elsewhere herein.

[0121] As noted elsewhere herein, the complexation of acellular heme with Hx in the CSF neutralizes the otherwise deleterious biological activity of acellular heme on brain tissue. Although it is generally not necessary to extract or remove the acellular heme:Hx complex formed according to the methods disclosed herein, in some cases it may be desirable to remove the complex. For example, when the methods described herein include removing CSF from a subject and then exogenously exposing the CSF to Hx, it may be desirable to remove the acellular heme:Hx complex formed in the CSF before re-administering the CSF to the subject. Thus, in some embodiments, the methods include removing the Hx:acellular heme complex formed in the CSF.

[0122] In one embodiment, the method comprises: (i) obtaining CSF from a subject following bleeding; (ii) exposing the CSF resulting from step (i) to Hx under conditions that allow Hx to form a complex with cell-free heme within the CSF; (iii) after step (ii), extracting Hx:cell-free heme complexes from the CSF to obtain a heme-depleted CSF having a lower amount of cell-free heme when compared to the CSF obtained from step (i); (iv) optionally repeating steps (ii) and (iii) to obtain heme-depleted CSF that is substantially free of cell-free heme; and (v) administering the heme-depleted CSF obtained from step (iii) or step (iv) to the CSF compartment of the subject. Includes.

[0123] As used herein, "heme-reduced CSF" means CSF from which an amount of cell-free heme has been removed, such that the CSF has a lower amount of cell-free heme when compared to the amount of cell-free heme before step (iii). The term "heme-reduced CSF" is not intended to suggest that all of the cell-free heme has been removed from the CSF, and thus is understood to include embodiments in which at least some cell-free heme is present. In one embodiment, heme-depleted CSF contains at least about 5%, preferably at least about 10%, preferably at least about 15%, preferably at least about 20%, preferably at least about 25%, preferably at least about 30%, preferably at least about 35%, preferably at least about 40%, preferably at least about 45%, preferably 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%, or more preferably at least about 95% less cell-free heme compared to the amount of cell-free heme in CSF obtained from a subject prior to extraction of the Hx:cell-free heme complex from the CSF. In one embodiment, heme-depleted CSF contains about 5% to about 10%, preferably about 10% to about 20%, preferably about 20% to about 30%, preferably about 30% to about 40%, preferably about 40% to about 50%, preferably about 50% to about 60%, preferably about 60% to about 70%, preferably about 70% to about 80%, preferably about 80% to about 90%, or more preferably about 90% to about 99% less cell-free heme compared to the amount of cell-free heme in CSF obtained from a subject prior to extraction of the Hx:cell-free heme complex from the CSF.

[0124] As described elsewhere herein, the heme-reduced CSF may optionally be further treated by repeating steps (ii) and (iii) to remove additional cell-free heme from the CSF, preferably to obtain a heme-reduced CSF that is substantially free of cell-free heme. By "substantially free of cell-free heme" is meant that the Hb-reduced CSF contains about 70% to about 80%, preferably about 80% to about 90%, or more preferably about 90% to about 99% less cell-free heme than the amount of cell-free heme in the CSF obtained from the subject prior to extraction of Hx:cell-free heme complexes from the CSF.

[0125] In one embodiment, the method comprises repeating steps (ii) and (iii) at least once (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 times, etc.) In one embodiment, the method comprises repeating steps (ii) and (iii) at least once, preferably 2, preferably 3, preferably 4, preferably 5, preferably 6, preferably 7, preferably 8, preferably 9, or more preferably 10 times, as necessary, to obtain Hb-depleted CSF that is substantially free of cell-free heme.

[0126] It will be appreciated that the number of times steps (ii) and (iii) need to be repeated to obtain heme-depleted CSF that is substantially free of cell-free heme can depend on a number of factors, including, but not limited to, the concentration of cell-free heme in the CSF from the subject, the concentration of heme employed, the extraction method, etc. In some cases, it may be desirable to perform steps (ii) and (iii) only once, particularly where repeating steps (ii) and (iii) may expose the CSF to contaminants such as bacteria, yeast, fungi, viruses, and the like.

[0127] Suitable methods for extracting Hx:cell-free heme complexes from CSF are known to those skilled in the art, illustrative examples of which include size exclusion chromatography and / or affinity chromatography. Size exclusion chromatography allows for the identification and separation of Hx:cell-free heme complexes from other components in CSF due to the relatively large size of the complexes compared to free heme and Hx. Affinity chromatography allows for the identification and separation of Hx:cell-free heme complexes from other components in CSF by using a binding agent that specifically binds to the heme:Hx complex and negligible binding to free heme. Suitable binding agents include antibodies or anti-binding fragments thereof, as will be familiar to those skilled in the art.

[0128] In one embodiment, step (ii) comprises passing the CSF obtained from step (i) over a substrate on which Hx is immobilized. This advantageously allows the cell-free heme in the CSF to bind with Hx, thereby immobilizing the cell-free heme on the substrate and allowing the heme-depleted CSF to be conveniently eluted from the substrate. Thus, in one embodiment, the Hx in step (ii) is immobilized on a substrate. Suitable substrates will be familiar to those skilled in the art, and illustrative examples thereof include size exclusion chromatography resins, affinity chromatography resins, filters, or membranes. In one embodiment, the substrate is selected from the group consisting of size exclusion chromatography resins, affinity chromatography resins, filters, and membranes.

[0129] In one embodiment, step (ii) comprises passing the CSF obtained from step (i) over an affinity chromatography resin under conditions that allow cell-free heme in the CSF to bind to the resin; then step (iii) comprises eluting the CSF from the resin that has undergone step (ii); and then step (iv) comprises recovering the eluted CSF.

[0130] In step (vi), prior to administering the heme-depleted CSF resulting from step (iii) or step (iv) to the CSF compartment of the subject, it may be desirable to add a therapeutically effective amount of Hp to the heme-depleted CSF to remove residual cell-free Hb that may be present in the CSF compartment of the subject and that may cause adverse secondary neurological outcomes, as described elsewhere herein. Thus, in one embodiment, the method includes adding a therapeutically effective amount of Hx to the heme-depleted CSF prior to step (v), as described elsewhere herein.

[0131] Those skilled in the art will appreciate that even if CSF is removed from the CSF compartment of a subject following hemorrhagic stroke, there may still be residual cell-free Hb in the CSF compartment. Thus, it may be desirable to rinse the CSF compartment (once CSF is removed based on step (i) above) to remove at least some residual cell-free Hb, thereby eliminating or otherwise reducing adverse secondary neurological outcomes that such residual cell-free heme may cause. Thus, in one embodiment, the method further comprises, after step (i), washing the CSF compartment with a washing solution.

[0132] Suitable irrigation solutions are familiar to those skilled in the art. In one embodiment, the irrigation solution is an artificial CSF.

[0133] Artificial cerebrospinal fluid (aCSF) is a liquid that generally mimics natural CSF, including salt content. Suitable compositions of CSF are familiar to those skilled in the art, and illustrative examples are described in U.S. Patent No. 20060057065 and Matzneller et al. (Pharmacology, 2016; 97(5-6):233-44), the contents of which are incorporated herein by reference in their entirety. As familiar to those skilled in the art, aCSF may contain NaCl at a concentration similar to that found in natural CSF, but also generally contains a concentration within about 15%, more preferably within about 10%, of the concentration of NaCl in natural CSF. As familiar to those skilled in the art, aCSF may contain NaHCO3 at a concentration similar to that found in natural CSF, but also generally contains a concentration within about 15%, more preferably within about 10%, of the concentration of NaHCO3 in natural CSF. As will be familiar to those skilled in the art, aCSF may contain KCl at a concentration similar to that found in natural CSF, but generally contains a concentration within about 15%, more preferably within about 10%, of the concentration of KCl in natural CSF. As will be familiar to those skilled in the art, aCSF may contain NaH2PO4 at a concentration similar to that found in natural CSF, but generally contains a concentration within about 15%, more preferably within about 10%, of the concentration of NaH2PO4 in natural CSF. As will be familiar to those skilled in the art, aCSF may contain MgCl2 at a concentration similar to that found in natural CSF, but generally contains a concentration within about 15%, more preferably within about 10%, of the concentration of MgCl2 in natural CSF. As will be familiar to those skilled in the art, aCSF may contain glucose at a concentration similar to that found in natural CSF, but generally contains a concentration within about 15%, more preferably within about 10%, of the concentration of glucose in natural CSF. Alternatively, the artificial CSF may omit glucose to reduce the potential for bacterial growth in any catheter used to administer the aCSF to a subject.

[0134] In one embodiment, the artificial CSF / irrigation solution includes NaCl, KCl, KH2PO4, NaHCO3, MgCl6·H2O, CaCl2·H2O, and glucose.

[0135] In one embodiment, the cleaning solution comprises Hx.

[0136] In one embodiment, the wash solution contains about 2 μM to about 80 mM Hx. In one embodiment, the wash solution contains about 2 μM to about 20 mM Hx. In one embodiment, the wash solution contains about 100 μM to about 20 mM Hx. In one embodiment, the wash solution contains about 2 μM to about 1,200 μM Hx. In one embodiment, the wash solution contains about 5 μM to about 200 μM Hx. In one embodiment, the wash solution contains about 10 μM to about 120 μM Hx.

[0137] In one embodiment, the lavage solution comprises at least an equimolar amount of Hx relative to the concentration of cell-free heme in the CFS of a subject after hemorrhage. In another embodiment, the lavage solution comprises about 3 μM to about 1,200 μM Hx.

[0138] In some embodiments, it may be desirable to eliminate the step of extracorporeally removing acellular heme from the subject's CSF as described herein, and rather replace the subject's CSF with an artificial CSF as described elsewhere herein. Thus, in one embodiment, the method comprises: (i) removing CSF from a subject following bleeding; (ii) after step (i), rinsing the CSF compartment of the subject with a irrigation solution comprising a therapeutically effective amount of Hx; (iii) optionally repeating step (ii); and (iv) after step (ii) or step (iii), administering an artificial CSF to the CSF compartment of the subject. Includes.

[0139] By incorporating a therapeutically effective amount of Hx into the irrigation solution, residual cell-free heme in the CSF compartment may complex with Hx, thereby neutralizing the deleterious biological effects of cell-free heme on brain tissue.

[0140] In one embodiment, the artificial CSF includes NaCl, KCl, KH2PO4, NaHCO3, MgCl6·H2O, CaCl2·H2O, and glucose. In one embodiment, the artificial CSF includes Hx.

[0141] In one embodiment, the artificial CSF comprises about 2 μM to about 80 mM Hx. In one embodiment, the artificial CSF comprises about 2 μM to about 20 mM Hx. In one embodiment, the artificial CSF comprises about 100 μM to about 20 mM Hx. In one embodiment, the artificial CSF comprises about 2 μM to about 1,200 μM Hx. In one embodiment, the artificial CSF comprises about 5 μM to about 200 μM Hx. In one embodiment, the artificial CSF comprises about 10 μM to about 120 μM Hx.

[0142] In one embodiment, the artificial CSF comprises at least an equimolar amount of Hx relative to the concentration of cell-free heme in the CSF of a subject after hemorrhage. In another embodiment, the artificial CSF comprises about 3 μM to about 1,300 μM Hx.

[0143] Adjuvant therapy The method of treating or preventing adverse secondary neurological outcomes in a subject after hemorrhagic stroke may be suitably carried out in conjunction with, sequentially with, or in combination with (e.g., simultaneously with) one or more other treatment strategies designed to reduce, inhibit, prevent, or otherwise alleviate one or more adverse secondary neurological outcomes in a subject after hemorrhagic stroke, as described herein.Thus, in one embodiment, the method further comprises administering to the subject a second agent for treating or preventing adverse secondary neurological outcomes after hemorrhagic stroke.Other suitable treatment strategies or second agents for treating or preventing adverse secondary neurological outcomes after hemorrhagic stroke will be familiar to those skilled in the art, and illustrative examples thereof include: (i) Correction of coagulation disorders - for example, using vitamin K antagonists (VKAs), novel oral anticoagulants (NOACs such as dabigatran, rivaroxaban, and apixaban), factor VIII inhibitor bypassing agents (FEIBAs) and activated recombinant factor VII (rFVIIa), prothrombin complex concentrates, activated charcoal, antiplatelet therapy (APT), and aspirin monotherapy; (ii) Blood Pressure Reduction - e.g., antihypertensive agents, illustrative examples of which include: (i) diuretics such as thiazide compounds including chlorthalidone, chlorthiazide, dichlorophenamide, hydroflumethiazide, indapamide, and hydrochlorothiazide; loop diuretics such as bumetanide, ethacrynic acid, furosemide, and torsemide; potassium-sparing diuretics such as amiloride and triamterene; and aldosterone antagonists such as spironolactone, epirenone, and the like. (ii) β-adrenergic receptor blockers such as acebutolol, atenolol, betaxolol, bevantolol, bisoprolol, bopindolol, carteolol, carvedilol, celiprolol, esmolol, indenolol, metaprolol, nadolol, nebivolol, penbutolol, pindolol, propanolol, sotalol, tertatolol, tilisolol, and timolol; (iii) amlodipine, aranidipine, azelnidipine, barnidipine, benidipine, bepridil, cinardipine, etc. (iv) calcium channel blockers such as cinaldipine, clevidipine, diltiazem, efonidipine, felodipine, gallopamil, isradipine, lacidipine, remildipine, lercanidipine, nicardipine, nifedipine, nilvadipine, nimodepine, nisoldipine, nitrendipine, manidipine, pranidipine, and verapamil; (iv) benazepril; captopril; cilazapril; delapril; enalapril; fosinopril; imidapril; rosinopril; moexipril; (v) angiotensin-converting enzyme (ACE) inhibitors such as omapatrilat, cadoxatril, and ecadotril, fosidotril, sampatrilat, AVE7688, ER4030, and the like;(vi) endothelin antagonists, such as tezosentan, A308165, and YM62899; (vii) vasodilators, such as hydralazine, clonidine, minoxidil, and nicotinyl alcohol; (viii) angiotensin II receptor antagonists, such as candesartan, eprosartan, irbesartan, losartan, pratosartan, tasosartan, telmisartan, valsartan, and EXP-3137, FI6828K, and RNH6270. (ix) alpha / beta adrenergic receptor blockers such as nipradilol, arotinolol, and amosulalol; (x) alpha 1 blockers such as terazosin, urapidil, prazosin, bunazosin, trimazosin, doxazosin, naftopidil, indoramin, WHIP164, and XENOIO; and (xi) alpha 2 agonists such as lofexidine, tiamenidine, moxonidine, rilmenidine, and guanobenz. (ii-b) Vasodilators - for example, hydralazine (Apresoline), clonidine (Catapress), minoxidil (Loniten), nicotinyl alcohol (Loniacol), Sydnon, and sodium nitroprusside. (iii) seizure, glucose, and temperature management - e.g., antiepileptic drugs, insulin infusions to control blood glucose levels, maintenance of normothermia, and therapeutic cooling; (iv) surgical procedures - e.g., hematoma evacuation (surgical thrombectomy), decompressive craniectomy (DC), minimally invasive surgery (MIS; such as fine needle aspiration of basal ganglia hemorrhage), MIS with recombinant tissue-type plasminogen activator (rtPA); (v) timing of surgery - e.g., 4–96 hours after symptom onset; (vi) thrombin inhibitors - e.g., hirudin, argatroban, serine protease inhibitors (e.g., nafamostat mesylate); (vii) Prevention of heme and iron toxicity - for example, nonspecific heme oxygenase (HO) inhibitors such as tin-mesoporphyrin, iron chelators such as deferoxamine; (viii) PPARg antagonists and agonists—e.g., rosiglitazone, 15d-PGJ2, and pioglitazone; (ix) inhibition of microglial activation - e.g., tuftsin fragment 1-3 (microglia / macrophage inhibitory factor) or minocycline (a tetracycline class antibiotic); (x) upregulation of NF erythroid 2-related factor 2 (Nrf2); (xi) cyclooxygenase (COX) inhibition - e.g., celecoxib (selective COX2 inhibitor); (xii) matrix metalloproteinase; (xiii) TNFα modulators - for example, adenosine receptor agonists such as CGS21680, TNFα-specific antisense oligodeoxynucleotides such as ORF4-PE; (xiv) elevated blood pressure - e.g., catecholamines; and (xv) inhibitors of TLR4 signaling - e.g., antibodies Mts510 and TAK-242 (cyclohexene derivatives); Examples include:

[0144] In one embodiment, the second drug is a vasodilator. Suitable vasodilators are familiar to those skilled in the art, and illustrative examples thereof include sydnone and sodium nitroprusside. Thus, in one embodiment disclosed herein, the second drug is selected from the group consisting of sydnone and sodium nitroprusside.

[0145] The second agent may include haptoglobin (Hp). As described elsewhere herein, the inventors have demonstrated that exogenous haptoglobin can be used to provide antivasospastic and antioxidant effects within the clinically relevant CSF-Hb concentration range, and hypothesize that a therapeutic combination of haptoglobin and hemopexin may provide synergistic protection against the vasospastic and oxidative effects of cell-free Hb and cell-free heme in the context of aSAH. Thus, in one embodiment, the method described herein further comprises exposing the subject's CSF to a therapeutically effective amount of haptoglobin (Hp) for a period of time sufficient to allow Hp to complex with cell-free Hb, thereby neutralizing it.

[0146] As noted elsewhere herein, the inventors have found that Hp can reduce or otherwise prevent acellular Hb-mediated CV in vivo. It is understood that the extent to which the methods disclosed herein reduce or otherwise prevent heme-mediated CV may depend on several factors, such as the degree of vasoconstriction (narrowing of blood vessels) induced by acellular Hb after hemorrhagic stroke, the concentration of acellular Hb in the CSF of the subject after hemorrhagic stroke, the duration of exposure of the CSF to Hp, and the presence or absence of any persistent bleeding. Means for assessing whether the methods disclosed herein reduce or otherwise prevent CV in a subject are familiar to those skilled in the art, illustrative examples of which include digital subtraction angiography (DSA), computed tomography (CT) angiography (CTA), magnetic resonance (MR) angiography (MRA), and catheter angiography (CA).

[0147] In one embodiment, exposing the CSF of a subject in need thereof to Hp restores the mean diameter of the constricted cerebral vasculature lumen by at least 10%, preferably about 10% to about 20%, preferably about 15% to about 25%, preferably about 20% to about 30%, preferably about 25% to about 35%, preferably about 30% to about 40%, preferably about 35% to about 45%, or more preferably about 40% to about 50% after 60 minutes of exposure as determined by DSA as described herein.

[0148] In one embodiment, exposing the CSF of a subject in need thereof to Hp restores the mean diameter of the lumen of the constricted anterior cerebral artery by at least 10%, preferably about 10% to about 20%, preferably about 15% to about 25%, preferably about 20% to about 30%, preferably about 25% to about 35%, preferably about 30% to about 40%, preferably about 35% to about 45%, or more preferably about 40% to about 50% after 60 minutes of exposure as determined by DSA as described herein.

[0149] In one embodiment, exposing the CSF of a subject in need thereof to Hp restores the mean diameter of the lumen of the constricted internal carotid artery by at least 10%, preferably about 10% to about 20%, preferably about 15% to about 25%, preferably about 20% to about 30%, preferably about 25% to about 35%, preferably about 30% to about 40%, preferably about 35% to about 45%, or more preferably about 40% to about 50% after 60 minutes of exposure as determined by DSA as described herein.

[0150] In one embodiment, exposing the CSF of a subject in need thereof to Hp restores the mean diameter of the lumen of the constricted middle cerebral artery by at least 10%, preferably about 10% to about 20%, preferably about 15% to about 25%, preferably about 20% to about 30%, preferably about 25% to about 35%, preferably about 30% to about 40%, preferably about 35% to about 45%, or more preferably about 40% to about 50% after 60 minutes of exposure as determined by DSA as described herein.

[0151] In one embodiment, exposing the CSF of a subject in need thereof to Hp restores the mean diameter of the constricted basilar artery lumen by at least 10%, preferably about 10% to about 20%, preferably about 15% to about 25%, preferably about 20% to about 30%, preferably about 25% to about 35%, preferably about 30% to about 40%, preferably about 35% to about 45%, or more preferably about 40% to about 50% after 60 minutes of exposure as determined by DSA as described herein.

[0152] In one embodiment, exposing the CSF of a subject in need thereof to Hp increases the average diameter of the lumen of a constricted small parenchymal vessel (e.g., cerebral arteriole) by at least 10%, preferably about 20%, preferably about 25%, preferably about 30%, preferably about 35%, preferably about 40%, preferably about 45%, preferably about 55%, preferably about 60%, preferably about 65%, preferably about 70%, preferably about 75%, or more preferably about 80% after 60 minutes of exposure as determined by in vivo perfusion imaging (e.g., MRI perfusion, CT perfusion) as described herein. In one embodiment, the small parenchymal vessel is a cerebral arteriole. In one embodiment, exposing the CSF of a subject in need thereof to Hp restores cerebral microperfusion by at least 10%, preferably about 20%, preferably about 25%, preferably about 30%, preferably about 35%, preferably about 40%, preferably about 45%, preferably about 55%, preferably about 60%, preferably about 65%, preferably about 70%, preferably about 75%, or more preferably about 80% by preventing the constriction of small parenchymal lumens after 60 minutes of exposure as determined by in vivo perfusion imaging (e.g., MRI perfusion, CT perfusion) as described herein. In one embodiment, the small parenchymal lumens are cerebral arterioles.

[0153] As noted elsewhere herein, the present inventors have demonstrated for the first time that a therapeutically effective amount of Hp can reduce the oxidative capacity of cell-free Hb in the CSF. Thus, in one embodiment, exposing the CSF of a subject in need thereof to Hp reduces oxidative tissue damage as described herein. Suitable methods for determining oxidative tissue damage are familiar to those skilled in the art, and illustrative examples thereof are described in Katerji et al. (2019; Oxid. Med. Cell Longey; 1279250), including measuring the level of malondialdehyde (MDA), an end product of lipid peroxidation. Thus, in one embodiment, exposing the CSF of a subject in need thereof to Hp reduces lipid peroxidation as described herein.

[0154] Haptoglobin Haptoglobin (Hp) has a tetrameric structure containing two α-chains and two β-chains linked by disulfide bonds. The β-chains (245 amino acids) have a mass of about 40 kDa (of which approximately 30% w / w is carbohydrate) and are shared by all phenotypes. The α-chains exist in at least two forms: α1 (83 amino acids, 9 kDa) and α2 (142 amino acids, 17.3 kDa). Thus, Hp occurs in three phenotypes, termed Hp1-1, Hp2-1, and Hp2-2. Hp1-1 contains two α1 chains, Hp2-2 contains two α2 chains, and Hp2-1 contains one α1 chain and one α2 chain. Hp1-1 has a molecular mass of 100 kDa, or 165 kDa when complexed with Hb. Hp1-1 exists as a single isoform and is also called the Hp dimer. Hp2-1 has an average molecular mass of 220 kDa and forms a linear polymer. Hp2-2 has an average molecular mass of 400 kDa and forms a circular polymer. Each different polymer form is a different isoform. A PCR method has been devised to study Hp polymorphisms (Koch et al. 2002, Clin. Chem. 48:1377-1382).

[0155] Two important alleles, Hp1 and Hp2, exist for the Hp gene found on chromosome 16. The two alleles are responsible for three different possible genotypes: homozygous (1-1 or 2-2) and heterozygous 2-1, which are structural polymorphisms. In Western populations, the distribution of Hp1-1 is estimated to be about 16%, Hp2-1 about 48%, and Hp2-2 about 36%. Hp is cleaved into two subunits, the α-chain and the β-chain, which are linked by disulfide bonds. Both alleles share an identical β-chain. The β-chain is involved in binding to Hb, and therefore both genotypes have similar Hb-binding affinities.

[0156] It is understood that naturally occurring and recombinant Hp are suitable for the methods described herein, so long as they have the ability to form complexes with cell-free Hb and thereby neutralize the biological activity of cell-free Hb. Suitable naturally occurring forms of Hp are known to those of skill in the art, and illustrative examples are described in Koch et al. (2002, Clin. Chem. 48:1377-1382) and Kasvosve et al. (2010, Chapter 2-Haptoglobin Polymorphism and Infection; Advances in Clinical Chemistry, 50:23-46), the entire contents of which are incorporated herein by reference.

[0157] In one embodiment, Hp comprises, consists of or consists essentially of plasma-derived Hp.Hp is preferably human Hp.Various protocols for isolating Hp from natural sources (e.g., plasma) are familiar to those skilled in the art, and illustrative examples thereof are described in U.S. Patent Nos. 4,061,735 and 4,137,307 (Funakoshi et al.) and U.S. Patent Publication No. 20140094411 (Brinkman), the entire contents of which are incorporated herein by reference. Other suitable methods for isolating Hp from natural sources are described by Katnik and Jadach (1993, Arch. Immunol. Ther. Exp. (Warz) 41:303-308), Tseng et al. (2004, Protein Expr Purif 33:265-273), Katnik et al. (1995, Eur J. Clin. Chem. Clin. Biochem. 33:727-732), Yang and Mao (1999, J. Chromatogr. B. Biomed. Sci. Appl., 731:395-402), and Basler and Burrel (1983 Inflammation 7(4):387-400).

[0158] The term "haptoglobin" as used herein is understood to include all phenotypes of Hp (including all isoforms). Hp can be homogeneous (if it is substantially composed of Hp of the same isoform) or heterogeneous (if it contains a combination of different Hp isoforms, including Hp1-1, Hp1-2, and Hp2-2). It is understood that the composition of Hp ultimately depends on the phenotype of the original Hp. For example, if a pooled plasma sample is used to extract / purify Hp, more than one isoform of Hp may be isolated. Suitable methods for determining the Hp isoforms present in an isolate are familiar to those skilled in the art, and illustrative examples are discussed in Shih et al. (2014, Hematology, 89(4):443-447), the entire contents of which are incorporated herein by reference. Other suitable methods for determining the Hp isoforms present in an isolate include high performance particle size exclusion chromatography (HPLC-SEC assay), Hp ELISA, and turbimetric reading, with different isoforms of Hp generally resulting in different signals in different assays.

[0159] In one embodiment, Hp is selected from the group consisting of Hp1-1 homodimer, Hp1-2 multimer, Hp2-2 multimer, and any combination of the above. In a preferred embodiment, Hp comprises, consists of, or substantially consists of Hp1-1 homodimer. Hp can be naturally occurring Hp (e.g., plasma-derived) or can be produced as a recombinant protein, illustrative examples of which are described elsewhere herein. In one embodiment, plasma-derived Hp comprises, consists of, or substantially consists of Hp1-1 homodimer. In one embodiment, Hp comprises, consists of, or substantially consists of recombinant Hp.

[0160] Unless otherwise indicated, the term Hp, as used herein, includes functional analogs of native or naturally occurring Hp. The term "functional analog" is understood to mean a substance that shares substantially the same biological activity as that of naturally occurring (native) Hp, so long as the biological activity is at least the ability of the analog to form a complex with cell-free Hb, thereby neutralizing its biological activity. By "substantially the same biological activity" it is generally meant that the functional analog has a binding affinity for cell-free Hb that is at least 40% (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, etc.) of the binding affinity of naturally occurring Hp, including naturally occurring Hp isoforms (e.g., Hp1-1, Hp1-2, and Hp2-2). Suitable methods for determining whether a substance is a functional analog of Hp are familiar to those of skill in the art, and illustrative examples thereof (e.g., the ability of a functional analog to reduce cell-free Hb-induced cerebral vasospasm) are described elsewhere herein.

[0161] In one embodiment disclosed herein, the functional analog of Hp is a functional fragment of native Hp. The functional fragment of native Hp can be of any suitable length, so long as the fragment retains the ability to form a complex with cell-free Hb, thereby neutralizing its biological activity.

[0162] In another embodiment, a functional analog is a peptide (i.e., a comparator) that has an amino acid sequence that differs from that of the naturally occurring (native) Hp molecule. A functional analog can include a molecule that has an amino acid sequence that differs from that of the α-chain and / or β-chain of native Hp by one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or more) amino acid substitutions, provided that the differences do not abolish or completely abolish the ability of the analog to form a complex with cell-free Hb and thereby neutralize its biological activity. In some embodiments, a functional analog contains an amino acid substitution that enhances the ability of the analog to form a complex with cell-free Hb compared to native Hp. In one embodiment, a functional analog has an amino acid sequence that differs from that of the α-chain and / or β-chain of native Hp by one or more conservative amino acid substitutions. As used herein, the term "conservative amino acid substitution" refers to changing the identity of an amino acid at a given position to replace it with an amino acid of approximately equivalent size, charge, and / or polarity. Examples of naturally occurring conservative substitutions for amino acids include the following eight substitution groups (represented by the conventional one-letter codes): (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.

[0163] In one embodiment, a functional analog has at least 85% sequence identity to the amino acid sequence of the α and / or β chain of native Hp. "At least 85%" includes, for example, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or similarity after optimal alignment or best fit analysis. Thus, in one embodiment the sequence has at least 85%, preferably at least 86%, preferably at least 87%, preferably at least 88%, preferably at least 89%, preferably at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99%, or preferably 100% sequence identity or homology with a sequence identified herein, e.g. after performing an optimal alignment or best fit analysis.

[0164] 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 the aligned sequences, the amino acid residue is identical between the aligned sequences, as described elsewhere herein. The terms "similarity" or "sequence similarity," as used herein, refer to the type of amino acid residue at any particular position in the aligned sequences being similar between the sequences, as described elsewhere herein. In one embodiment, the amino acid sequence is modified by conservatively substituting any of the amino acid residues contained therein, such that the modification does not affect the binding specificity or functional activity of the modified polypeptide when compared to the unmodified (native) Hp polypeptide.

[0165] In one embodiment, the functional analog contains amino acid substitutions and / or other modifications compared to native Hp to increase the stability of the analog or to increase the solubility of the analog. Functional analogs can be naturally occurring compounds / peptides or can be synthetically produced by chemical synthesis using methods known to those of skill in the art.

[0166] Hp may be suitably produced in a microorganism as a recombinant protein, which can be isolated and, if desired, further purified. Suitable microorganisms for producing recombinant Hp are familiar to those skilled in the art, and illustrative examples thereof include bacteria, yeast, or fungi, eukaryotic cells (e.g., mammalian or insect cells), or recombinant viral vectors (e.g., adenovirus, poxvirus, herpesvirus, Semliki Forest virus, baculovirus, bacteriophage, Sindbis virus, or Sendai virus). Suitable bacteria for producing recombinant peptides are familiar to those skilled in the art, and illustrative examples thereof include E. coli, B. subtilis, or any other bacteria capable of expressing 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. Methods for isolating and purifying recombinantly produced peptide sequences are also well known in the art, and include, for example, gel filtration, affinity chromatography, and ion exchange chromatography.

[0167] As described herein, to facilitate the isolation of recombinant Hp, fusion polypeptides can be created in which the peptide sequence of Hp or a functional analog thereof is translationally fused (covalently linked) to a heterologous polypeptide that allows for isolation by affinity chromatography. Illustrative examples of suitable heterologous polypeptides are His-Tag (e.g., His6, six histidine residues), GST-Tag (glutathione-S-transferase), and the like.

[0168] When producing recombinant Hp, phage and / or peptide libraries are also suitable, for example, for production by means of combinatorial chemistry or for obtaining highly variable structures by high-throughput screening techniques (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).

[0169] Illustrative examples of recombinant Hp include the peptide with accession number NP_005134 (described by Morishita et al., 2018, Clin. Chim. Acta 487, 84-89), and the peptide with accession number P00738.

[0170] Hp may be fused, coupled or otherwise linked as part of a fusion protein to one or more heterologous moieties, including those described elsewhere herein in the context of Hx.

[0171] In the context of Hp, the term "therapeutically effective amount" as used herein means an amount or concentration of Hp that is sufficient when administered to a subject in need thereof to allow Hp to bind to and form a complex with acellular Hb present in the subject's CSF, thereby neutralizing the otherwise deleterious biological effects of acellular Hb. It will be understood by those skilled in the art that a therapeutically effective amount of a peptide can vary depending on several factors, illustrative examples of which include whether Hp is administered directly to the subject or how it is administered (e.g., intrathecally, intracranially, or intraventricularly), the health and physical condition of the subject being treated, the taxonomic group of the subject being treated, the severity of the hemorrhage (e.g., the extent of the hemorrhage), the route of administration, the concentration and / or amount of acellular Hb in the CSF compartment, and any combination of the above.

[0172] The therapeutically effective amount of Hp falls within a relatively broad range that can be determined by one skilled in the art. Illustrative examples of suitable therapeutically effective amounts of Hp include about 2 μM to about 20 mM, preferably about 2 μM to about 5 mM, preferably about 100 μM to about 5 mM, preferably about 2 μM to about 300 μM, preferably about 5 μM to about 100 μM, preferably about 5 μM to about 50 μM, or more preferably about 10 μM to about 30 μM.

[0173] In one embodiment, the therapeutically effective amount of Hp is about 2 μM to about 20 mM. In one embodiment, the therapeutically effective amount of Hp is about 2 μM to about 5 mM. In one embodiment, the therapeutically effective amount of Hp is about 100 μM to about 5 mM. In one embodiment, the therapeutically effective amount of Hp is about 2 μM to about 300 μM. In one embodiment, the therapeutically effective amount of Hp is about 5 μM to about 50 μM. In one embodiment, the therapeutically effective amount of Hp is about 10 μM to about 30 μM.

[0174] In one embodiment, the therapeutically effective amount of Hp is at least equimolar to the cell-free Hb concentration in the CSF of the subject after hemorrhage. In another embodiment, the therapeutically effective amount of Hp is sufficient to complex with about 3 μM to about 300 μM of cell-free Hb in the CSF. Suitable methods for measuring the concentration of cell-free Hb in the CSF are known to those skilled in the art, and illustrative examples thereof are described in Cruickshank AM., 2001, ACP Best Practice No166, J. Clin. Path., 54(11):827-830), and Hugelshofer M. et al., 2018. World Neurosurg.; 120:e660-e666), the contents of which are incorporated herein by reference in their entirety.

[0175] It is understood that the realization of a therapeutically effective amount of Hp may depend on the final volume of CSF to which Hp or its functional analog is exposed, as described herein.For example, when Hp is administered to an adult human subject (e.g., intrathecally), considering that the average volume of CSF in an adult human subject is about 150mL, a therapeutically effective amount of Hp of about 10μM can be achieved by administering 5mL of about 310μM Hp solution to the subject.In another illustrative example, the method described herein includes removing 50mL of CSF from a subject and replacing it with 50mL of artificial CSF containing about 30μM Hp, thereby realizing a therapeutically effective amount of Hp of about 10μM in the CSF compartment of the subject.

[0176] The dosage of Hp may be adjusted to achieve the optimal therapeutic response, for example, several divided doses may be administered daily, weekly or at other suitable time intervals, or the dosage may be proportionally reduced as the exigencies of the situation indicate.

[0177] In the context of Hp, the term "therapeutically effective amount" is not understood to mean an amount or concentration of Hp that, when administered to a subject in need thereof, complexes with and neutralizes all acellular Hb present in the subject's CSF. In some embodiments, it is sufficient that a therapeutically effective amount of Hp, when administered to a subject in need thereof, complexes with and neutralizes a sufficient amount of acellular Hb present in the subject's CSF such that it minimizes, neutralizes, or otherwise reduces adverse secondary neurological outcomes in the subject mediated by acellular Hb after hemorrhagic stroke into the subject's CSF. The reduction in acellular Hb concentration in the subject's CSF after treatment required to minimize, neutralize, or otherwise reduce adverse secondary neurological outcomes after hemorrhagic stroke into the subject's CSF is familiar to those skilled in the art, as described herein. In one embodiment, the therapeutically effective amount of Hp is sufficient to reduce the amount of cell-free Hb in the subject's CSF to a level of about 8 μM or less, preferably about 7 μM or less, preferably about 6 μM or less, preferably about 5 μM or less, preferably about 4 μM or less, preferably about 3 μM or less, preferably about 2 μM or less, or more preferably about 1 μM or less during the treatment period. In one embodiment, the therapeutically effective amount of Hp is sufficient to reduce the amount of cell-free Hb in the subject's CSF to a level of about 7 μM or less during the treatment period. In one embodiment, the therapeutically effective amount of Hp is sufficient to reduce the amount of cell-free Hb in the subject's CSF to a level of about 6 μM or less during the treatment period.

[0178] In the context of Hp, the term "exposing" as used herein means contacting the CSF with Hp such that Hp binds to and complexes with cell-free Hb (the major form of which is oxyHb) present in the CSF, thereby enabling formation of the Hb:Hp complex to substantially neutralize the otherwise deleterious biological effects of cell-free Hb on brain tissue. "Substantially neutralize" refers to a biological effect that is at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 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, 100, 101, 102, 103, 104, 105, 106, 107, 10 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 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 "reduction in the deleterious biological effects of cell-free Hb on brain tissue" refers to a reduction in the deleterious biological effects of cell-free Hb on brain tissue, as subjectively or qualitatively expressed as a reduction of at least 10%, preferably about 10% to about 20%, preferably about 15% to about 25%, preferably about 20% to about 30%, preferably about 25% to about 35%, preferably about 30% to about 40%, preferably about 35% to about 45%, preferably about 40% to about 50%, preferably about 45% to about 55%, preferably about 50% to about 60%, preferably about 55% to about 65%, preferably about 60% to about 70%, preferably about 65% to about 75%, preferably about 70% to about 80%, preferably about 75% to about 85%, preferably about 80% to about 90%, preferably about 85% to about 95%, or most preferably about 90% to 100%, including 100%. Methods by which the reduction in the deleterious biological effects of cell-free Hb is measurable or determinable (either qualitatively or quantitatively) will be familiar to those of skill in the art, and illustrative examples thereof are described elsewhere herein.

[0179] It is understood that the manner in which the CSF of a subject in need thereof is exposed to a therapeutically effective amount of Hp may vary, for example, depending on whether the exposure is performed in the CSF compartment of a subject in need thereof (i.e., in vivo) or whether the exposure is performed externally in CSF obtained from a subject (i.e., ex vivo). If the exposure is performed in the CSF compartment of a subject in need thereof (i.e., in vivo), the route of administration of Hp is selected to allow Hp to contact with acellular Hb in the CSF compartment. Suitable routes of administration are familiar to those skilled in the art, and illustrative examples include intrathecal, intracranial, and intraventricular. In one embodiment, a therapeutically effective amount of Hp is administered, for example, in a subject after a hemorrhagic stroke, via an external ventricular drain placed to temporarily drain CSF and reduce intracranial pressure.

[0180] In one embodiment, the method comprises administering a therapeutically effective amount of Hp intracranially to the subject.

[0181] In one embodiment, the method comprises administering a therapeutically effective amount of Hp intrathecally to a subject. In one embodiment, the method comprises administering a therapeutically effective amount of Hp intrathecally to a subject into its spinal canal. In one embodiment, the method comprises administering a therapeutically effective amount of Hp intrathecally to a subject into its subarachnoid space. In one embodiment, the method comprises administering a therapeutically effective amount of Hp intracerebroventricularly to a subject.

[0182] Alternatively or additionally, the method described herein includes removing CSF from a subject in need thereof, exposing the CSF to a therapeutically effective amount of Hp for a period of time sufficient to allow Hp to complex with and thereby neutralize acellular Hb in the CSF, removing the Hb:Hp complex formed in the CSF to generate a Hb-reduced CSF, and administering the Hb-reduced CSF to the subject (e.g., intrathecally or intracerebroventricularly). It is understood that removing CSF from the CSF compartment does not generally result in a CSF compartment completely free of CSF, and it is noted that at least some CSF will remain in the CSF compartment. Optionally, once the CSF has been removed, the CSF compartment can be rinsed with a pharma- ceutically acceptable washing solution to remove at least some residual Hb that may be present in the CSF compartment. The washing solution can optionally include Hp to further complex and thereby neutralize at least some residual acellular Hb that may be present in the CSF compartment. In one embodiment, the irrigation solution is an artificial CSF as described elsewhere herein.

[0183] In one embodiment, the method described herein comprises the steps of: taking a sample of CSF from the CSF compartment of a subject in need thereof; adding Hp to the CSF sample to obtain a Hp-enriched CSF sample; administering the Hp-enriched CSF sample to the CSF compartment of the subject, thereby exposing the CSF compartment to a therapeutically effective amount of Hp for a period of time sufficient to allow Hp to form a complex with acellular Hb in the CSF of the subject, thereby neutralizing the acellular Hb in the CSF of the subject; and optionally repeating the steps.Preferably, the amount of Hp added to the CSF sample is determined so that, when administered to a subject, it will result in a therapeutically effective amount of Hp in the CSF of the subject.Therefore, the amount of Hp added in the CSF sample depends on the volume of the CSF sample taken and re-administered to the subject.

[0184] In one embodiment, the method comprises: (i) obtaining a CSF sample from the CSF compartment of a subject following bleeding; (ii) adding Hp to the CSF sample of step (i) to obtain an Hp-enriched CSF sample; (iii) administering to the subject a Hp-enriched CSF sample, thereby exposing the CSF compartment of the subject to a therapeutically effective amount of Hp for a period of time sufficient to allow Hp to form a complex with cell-free Hb in the CSF compartment of the subject; and (iv) optionally repeating steps (i) to (iii); Includes.

[0185] It is desirable that the volume of CSF that is removed and re-administered to the subject is substantially the same. For example, if a 50 mL CSF sample is removed from the CSF compartment of the subject, the entire 50 mL volume of CSF containing Hp is re-administered to the subject. However, it is understood that the volumes may differ, so long as any difference in volume does not cause significant adverse clinical outcomes. In some embodiments, the volume of CSF that is re-administered to the subject is less than the volume of CSF that was removed from the subject. In other embodiments, the volume of CSF that is re-administered to the subject is greater than the volume of CSF that was removed from the subject, due to the extra volume that is generated by adding Hp alone or in combination with any other therapeutic agent.

[0186] In another embodiment, the method described herein comprises the steps of removing a volume of CSF from the CSF compartment of a subject in need thereof, replacing the volume of CSF removed from the subject with a volume of artificial CSF containing Hp, thereby exposing the CSF of the subject to a therapeutically effective amount of Hp for a period of time sufficient to allow Hp to form complexes with acellular Hb in the CSF of the subject, thereby neutralizing the acellular Hb in the CSF of the subject. Preferably, the amount of Hp in the artificial CSF is determined to provide a therapeutically effective amount of Hp in the CSF of the subject when administered to the subject. Thus, the amount of Hp added to the artificial CSF depends on the volume of artificial CSF administered to the subject.

[0187] In one embodiment, the method comprises: (i) removing a volume of CSF from the CSF compartment of a subject following hemorrhage; (ii) providing an artificial CSF containing Hp; (iii) administering the artificial CSF of (ii) to a subject, thereby exposing the CSF compartment of the subject to a therapeutically effective amount of Hp for a period of time sufficient to allow Hp to form a complex with cell-free Hb in the CSF compartment of the subject; and (iv) optionally repeating steps (i) to (iii); Includes.

[0188] It is desirable that the volume of artificial CSF administered to the subject is substantially the same as the volume of CSF removed from the subject. For example, if a 50 mL CSF sample is removed from the subject's CSF compartment, a volume of about 50 mL of artificial CSF containing a therapeutically effective amount of Hp is used to replace the volume of CSF removed. However, it is understood that the volumes may be different, so long as any difference in volume does not cause significant adverse clinical outcomes. In some embodiments, the volume of artificial CSF administered to the subject is less than the volume of CSF removed from the subject. In other embodiments, the volume of artificial CSF administered to the subject is greater than the volume of CSF removed from the subject.

[0189] In one embodiment, the method described herein comprises exposing the CSF to Hp within about 21 days after the hemorrhagic stroke. In another embodiment disclosed herein, the method comprises exposing the CSF to Hp for about 2 days to about 4 days after the hemorrhagic stroke. In yet another embodiment, the method comprises exposing the CSF to Hp for about 5 days to about 14 days after the hemorrhagic stroke.

[0190] In one embodiment, the period during which the CSF is exposed to a therapeutically effective amount of Hp is at least about 2 minutes (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 minutes, etc.). In one embodiment, the period during which the CSF is exposed to a therapeutically effective amount of Hp is at least about 4 minutes. In another embodiment, the period during which the CSF is exposed to a therapeutically effective amount of Hp is at least about 5 minutes. In yet another embodiment, the period during which the CSF is exposed to a therapeutically effective amount of Hp is at least about 10 minutes. In one embodiment, the period during which the CSF is exposed to a therapeutically effective amount of Hp is about 2 minutes to about 45 minutes, preferably about 2 minutes to about 20 minutes, or more preferably about 4 minutes to about 10 minutes.

[0191] In one embodiment, the methods described herein further comprise exogenously exposing the CSF to a therapeutically effective amount of Hp.

[0192] When CSF is exposed to Hp ex vivo, the therapeutically effective amount will depend on the volume of CSF exposed to Hp, whether the CSF is exposed to Hp in solution or immobilized on a substrate (e.g., affinity chromatography), and any combination of the above. For example, when CSF is exposed to Hp to form a complex with cell-free Hb and remove the cell-free Hb by affinity chromatography, the amount of Hp immobilized on the substrate need not cause complete complexation of cell-free Hb during the first pass period, and multiple passes over the substrate may be required to form the complex, which will remove substantially all of the cell-free Hb from the CSF.

[0193] In one embodiment, the method includes the steps of (i) obtaining a CSF sample from a subject after hemorrhagic stroke and before exposing the CSF to Hp; (ii) measuring the amount of cell-free Hb in the CSF sample obtained in step (i); and (iii) determining at least an equimolar amount of Hp based on the concentration of cell-free Hb obtained from step (ii). Suitable methods for measuring the amount of cell-free Hb in a CSF sample are known to those skilled in the art, an illustrative example of which is described by Oh et al. (2016, Redox Biology, 9:167-177), the contents of which are incorporated herein by reference in their entirety.

[0194] As noted elsewhere herein, the formation of a complex between Hb and cell-free Hb in the CSF neutralizes the otherwise deleterious biological activity of cell-free Hb on brain tissue. Although it is generally unnecessary to extract or remove the cell-free Hb:Hp complex formed according to the methods disclosed herein, in some cases it may be desirable to remove the complex. For example, when the methods described herein include removing CSF from a subject and then exposing the CSF to Hp ex vivo, it may be desirable to remove the cell-free Hb:Hp complex formed in the CSF before re-administering the CSF to the subject. Thus, in some embodiments, the methods include removing the Hp:cell-free Hb complex formed in the CSF.

[0195] In one embodiment, the method comprises: (i) obtaining CSF from a subject following bleeding; (ii) exposing the CSF resulting from step (i) to Hp under conditions that allow Hp to form a complex with cell-free Hb in the CSF; (iii) extracting Hp:cell-free Hb complexes from the CSF following step (ii) to obtain a Hb-depleted CSF having a lower amount of cell-free Hb when compared to the CSF obtained from step (i); (iv) optionally repeating steps (ii) and (iii) to obtain Hb-depleted CSF that is substantially free of cell-free Hb; and (v) administering the Hb-depleted CSF obtained from step (iii) or step (iv) to the CSF compartment of the subject. Further includes:

[0196] As used herein, "Hb-depleted CSF" means CSF from which an amount of cell-free Hb has been removed, such that the CSF has a lower amount of cell-free Hb when compared to the amount of cell-free Hb before step (iii). The term "Hb-depleted CSF" is not intended to suggest that all of the cell-free Hb has been removed from the CSF, and thus is understood to include embodiments in which at least some cell-free Hb is present. In one embodiment, Hb-depleted CSF contains at least about 5%, preferably at least about 10%, preferably at least about 15%, preferably at least about 20%, preferably at least about 25%, preferably at least about 30%, preferably at least about 35%, preferably at least about 40%, preferably at least about 45%, preferably 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%, or more preferably at least about 95% less cell-free Hb when compared to the amount of cell-free Hb in CSF obtained from the subject. In one embodiment, Hb-depleted CSF contains about 5% to about 10%, preferably about 10% to about 20%, preferably about 20% to about 30%, preferably about 30% to about 40%, preferably about 40% to about 50%, preferably about 50% to about 60%, preferably about 60% to about 70%, preferably about 70% to about 80%, preferably about 80% to about 90%, or more preferably about 90% to about 99% less cell-free Hb compared to the amount of cell-free Hb in CSF obtained from a subject.

[0197] As described elsewhere herein, the Hb-depleted CSF may optionally be further treated by repeating steps (ii) and (iii) to remove additional cell-free Hb from the CSF, preferably to obtain a Hb-depleted CSF that is substantially free of cell-free Hb. By "substantially free of cell-free Hb" it is meant that the Hb-depleted CSF contains about 70% to about 80%, preferably about 80% to about 90%, or more preferably about 90% to about 99% less cell-free Hb when compared to the amount of cell-free Hb in the CSF obtained from the subject.

[0198] In one embodiment, the method comprises repeating steps (ii) and (iii) at least once (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 times, etc.) In one embodiment, the method comprises repeating steps (ii) and (iii) at least once, preferably 2, preferably 3, preferably 4, preferably 5, preferably 6, preferably 7, preferably 8, preferably 9, or more preferably 10 times, as necessary, to obtain Hb-depleted CSF that is substantially free of cell-free Hb.

[0199] It will be appreciated that the number of times steps (ii) and (iii) need to be repeated to obtain Hb-depleted CSF that is substantially free of cell-free Hb may depend on several factors, including, but not limited to, the concentration of cell-free Hb in the CSF from the subject, the concentration of Hb employed, the extraction method, etc. In some cases, it may be desirable to perform steps (ii) and (iii) only once, especially when repeating steps (ii) and (iii) may expose the CSF to contaminants such as bacteria, yeast, fungi, viruses, and the like.

[0200] Suitable methods for extracting Hp:cell-free Hb complexes from CSF are known to those skilled in the art, and illustrative examples include size exclusion chromatography and / or affinity chromatography. Size exclusion chromatography allows the identification and separation of Hp:cell-free Hb complexes from other components in CSF due to the relatively large size of the complexes compared to free Hb and Hp. Affinity chromatography allows the identification and separation of Hp:cell-free Hb complexes from other components in CSF by using a binding agent that specifically binds to Hb:Hp complexes and negligible binding to free Hb. Suitable binding agents include antibodies or anti-binding fragments thereof, as will be familiar to those skilled in the art.

[0201] In one embodiment, step (ii) comprises passing the CSF obtained from step (i) over a substrate on which Hp is immobilized. This advantageously allows cell-free Hb in the CSF to bind to Hp, thereby immobilizing the cell-free Hb on the substrate and allowing the Hb-depleted CSF to be conveniently eluted from the substrate. Thus, in one embodiment, Hp in step (ii) is immobilized on a substrate. Suitable substrates are familiar to those skilled in the art, and illustrative examples thereof include size exclusion chromatography resins and affinity chromatography resins. In one embodiment, the substrate is an affinity chromatography resin.

[0202] In one embodiment, step (ii) comprises passing the CSF obtained from step (i) through an affinity chromatography resin under conditions that allow cell-free Hb in the CSF to bind to the resin, step (iii) comprises eluting the CSF from the resin after step (ii), and step (iv) comprises recovering the eluted CSF.

[0203] In step (vi), prior to administering the Hb-depleted CSF resulting from step (iii) or step (iv) to the CSF compartment of the subject, it may be desirable to add a therapeutically effective amount of Hp to the Hb-depleted CSF to remove residual cell-free Hb that may be present in the CSF compartment of the subject and that may cause adverse secondary neurological outcomes, as described elsewhere herein. Thus, in one embodiment, the method comprises adding a therapeutically effective amount of Hp to the Hb-depleted CSF prior to step (v), as described elsewhere herein.

[0204] Those skilled in the art will appreciate that even if CSF is removed from the CSF compartment of a subject after hemorrhagic stroke, there may still be residual cell-free Hb in the CSF compartment. Thus, it may be desirable to rinse the CSF compartment (once CSF is removed based on step (i) above) to remove at least some residual cell-free Hb, thereby eliminating or otherwise reducing adverse secondary neurological outcomes that such residual cell-free Hb may cause. Thus, in one embodiment, the method further comprises, after step (i), washing the CSF compartment with a washing solution.

[0205] Suitable irrigation solutions will be familiar to those of skill in the art. In one embodiment, the irrigation solution is an artificial CSF as described elsewhere herein.

[0206] In one embodiment, the washing solution further comprises Hp. In one embodiment, the washing solution comprises about 2 μM to about 20 mM Hp. In one embodiment, the washing solution comprises about 2 μM to about 5 mM Hp. In one embodiment, the washing solution comprises about 100 μM to about 5 mM Hp. In one embodiment, the washing solution comprises about 2 μM to about 300 μM Hp. In one embodiment, the washing solution comprises about 5 μM to about 50 μM Hp. In one embodiment, the washing solution comprises about 10 μM to about 30 μM Hp.

[0207] In one embodiment, the lavage solution comprises at least an equimolar amount of Hp relative to the concentration of cell-free Hb in the CSF of the subject after bleeding, hi another embodiment, the lavage solution comprises about 3 μM to about 300 μM Hp.

[0208] In some embodiments, it may be desirable to eliminate the step of extracorporeally removing cell-free Hb from the subject's CSF as described herein, and rather replace the subject's CSF with artificial CSF as described elsewhere herein. Thus, in one embodiment, the method comprises: (i) removing CSF from a subject following bleeding; (ii) after step (i), rinsing the CSF compartment of the subject with a irrigation solution containing a therapeutically effective amount of Hp; (iii) optionally repeating step (ii); and (iv) after step (ii) or step (iii), administering an artificial CSF to the CSF compartment of the subject. Includes.

[0209] By incorporating a therapeutically effective amount of Hp into the lavage solution, residual cell-free Hb in the CSF compartment may complex with Hp, thereby neutralizing the deleterious biological effects of cell-free Hb on brain tissue.

[0210] artificial CSF In another aspect disclosed herein, there is provided an artificial cerebrospinal fluid (CSF) comprising Hx as described herein.

[0211] Artificial cerebrospinal fluid (aCSF) is generally a liquid that mimics natural CSF by including salt content. Suitable compositions of aCSF are familiar to those skilled in the art, and illustrative examples thereof are described in U.S. Patent No. 20060057065 and Matzneller et al. (Pharmacology, 2016; 97(5-6):233-44, the contents of which are incorporated herein by reference in their entirety). As familiar to those skilled in the art, aCSF may contain NaCl at a concentration similar to that found in natural CSF, and generally contains a concentration within about 15%, more preferably within about 10%, of the concentration of NaCl in natural CSF. As familiar to those skilled in the art, aCSF may contain NaHCO3 at a concentration similar to that found in natural CSF, and generally contains a concentration within about 15%, more preferably within about 10%, of the concentration of NaHCO3 in natural CSF. As will be familiar to those skilled in the art, aCSF may contain KCl at a concentration similar to that found in natural CSF, and generally includes a concentration within about 15%, more preferably within about 10%, of the concentration of KCl in natural CSF. As will be familiar to those skilled in the art, aCSF may contain NaH2PO4 at a concentration similar to that found in natural CSF, and generally includes a concentration within about 15%, more preferably within about 10%, of the concentration of NaH2PO4 in natural CSF. As will be familiar to those skilled in the art, aCSF may contain MgCl2 at a concentration similar to that found in natural CSF, and generally includes a concentration within about 15%, more preferably within about 10%, of the concentration of MgCl2 in natural CSF. As will be familiar to those skilled in the art, aCSF may contain glucose at a concentration similar to that found in natural CSF, and generally includes a concentration within about 15%, more preferably within about 10%, of the concentration of glucose in natural CSF. Alternatively, the artificial CSF may omit glucose to reduce the potential for bacterial growth in any catheter used to administer the aCSF to a subject.

[0212] In one embodiment, the artificial CSF comprises about 2 μM to about 80 mM Hx. In one embodiment, the artificial CSF comprises about 2 μM to about 20 mM Hx. In one embodiment, the artificial CSF comprises about 100 μM to about 20 mM Hx. In one embodiment, the artificial CSF comprises about 2 μM to about 1,200 μM Hx. In one embodiment, the artificial CSF comprises about 5 μM to about 200 μM Hx. In one embodiment, the artificial CSF comprises about 10 μM to about 120 μM Hx.

[0213] In one embodiment, the artificial CSF comprises at least an equimolar amount of Hx relative to the concentration of cell-free heme in the CSF of a subject following hemorrhage. In another embodiment, the artificial CSF comprises about 3 μM to about 1,200 μM Hx.

[0214] In one embodiment, the artificial CSF further comprises Hp. In one embodiment, the artificial CSF comprises about 2 μM to about 20 mM Hp. In one embodiment, the artificial CSF comprises about 2 μM to about 5 mM Hp. In one embodiment, the artificial CSF comprises about 100 μM to about 5 mM Hp. In one embodiment, the artificial CSF comprises about 2 μM to about 300 μM Hp. In one embodiment, the artificial CSF comprises about 5 μM to about 50 μM Hp. In one embodiment, the artificial CSF comprises about 10 μM to about 30 μM Hp.

[0215] In one embodiment, the artificial CSF comprises at least an equimolar amount of Hp relative to the concentration of acellular Hp in the CSF of a subject after hemorrhage, hi another embodiment, the artificial CSF comprises about 3 μM to about 300 μM Hp.

[0216] In one embodiment, the artificial CSF comprises about 2 μM to about 80 mM Hx, and about 2 μM to about 20 mM Hp. In one embodiment, the artificial CSF comprises about 2 μM to about 20 mM Hx, and about 2 μM to about 5 mM Hp. In one embodiment, the artificial CSF comprises about 100 μM to about 20 mM Hx, and about 2 μM to about 5 mM Hp. In one embodiment, the artificial CSF comprises about 2 μM to about 1,200 μM Hx, and about 2 μM to about 300 mM Hp. In one embodiment, the artificial CSF comprises about 5 μM to about 200 μM Hx, and about 2 μM to about 50 mM Hp. In one embodiment, the artificial CSF comprises about 10 μM to about 120 μM Hx, and about 2 μM to about 30 mM Hp.

[0217] Pharmaceutical Compositions In another aspect disclosed herein, there is provided a pharmaceutical composition for treating or preventing adverse secondary neurological outcomes in a subject following hemorrhagic stroke, based on the methods described herein, comprising a therapeutically effective amount of Hx as described herein and a pharma- ceutical acceptable carrier.

[0218] In another aspect disclosed herein, there is provided a pharmaceutical composition for use in treating or preventing adverse secondary neurological outcomes in a subject following hemorrhagic stroke based on the methods described herein, the pharmaceutical composition comprising a therapeutically effective amount of Hx as described herein and a pharma- ceutical acceptable carrier.

[0219] In one embodiment, the composition comprises about 2 μM to about 80 mM Hx. In one embodiment, the composition comprises about 2 μM to about 20 mM Hx. In one embodiment, the composition comprises about 100 μM to about 20 mM Hx, or a functional analog thereof. In one embodiment, the composition comprises about 2 μM to about 1,200 μM Hx. In one embodiment, the composition comprises about 5 μM to about 200 μM Hx. In one embodiment, the composition comprises about 10 μM to about 120 μM Hx.

[0220] In one embodiment, the composition comprises at least an equimolar amount of Hx relative to the concentration of cell-free heme in the CSF of a subject after hemorrhage, hi another embodiment, the composition comprises about 3 μM to about 1,200 μM Hx.

[0221] In one embodiment, the composition further comprises a therapeutically effective amount of Hp, as described herein.

[0222] In one embodiment, the composition comprises about 2 μM to about 20 mM Hp. In one embodiment, the composition comprises about 2 μM to about 5 mM Hp. In one embodiment, the composition comprises about 100 μM to about 5 mM Hp. In one embodiment, the composition comprises about 2 μM to about 300 μM Hp. In one embodiment, the composition comprises about 5 μM to about 50 μM Hp. In one embodiment, the composition comprises about 10 μM to about 30 μM Hp.

[0223] In one embodiment, the composition comprises Hp in an amount at least equimolar to the concentration of cell-free Hb in the CSF of a subject after hemorrhage, hi another embodiment, the composition comprises about 3 μM to about 300 μM Hp.

[0224] In one embodiment, the composition comprises about 2 μM to about 80 mM Hx, and about 2 μM to about 20 mM Hp. In one embodiment, the composition comprises about 2 μM to about 20 mM Hx, and about 2 μM to about 5 mM Hp. In one embodiment, the composition comprises about 100 μM to about 20 mM Hx, and about 2 μM to about 5 mM Hp. In one embodiment, the composition comprises about 2 μM to about 1,200 μM Hx, and about 2 μM to about 300 mM Hp. In one embodiment, the composition comprises about 5 μM to about 200 μM Hx, and about 2 μM to about 50 mM Hp. In one embodiment, the composition comprises about 10 μM to about 120 μM Hx, and about 2 μM to about 30 mM Hp.

[0225] In another aspect disclosed herein, there is provided the use of a therapeutically effective amount of Hx as described herein in the manufacture of a medicament for treating or preventing an adverse secondary neurological outcome in a subject following hemorrhagic stroke, based on the methods described herein.

[0226] In one embodiment, the pharmaceutical composition disclosed herein is formulated for intrathecal administration.Suitable intrathecal delivery system is familiar to those skilled in the art, and its illustrative example is described by Kilburn et al. (2013, Intrathecal Administration.In:Rudek M., Chau C., Figg W., McLeod H. (eds) Handbook of Anticancer Pharmacokinetics and Pharmacodynamics.Cancer Drug Discovery and Development.Springer, New York, NY), the contents of which are incorporated herein by reference in their entirety.

[0227] In another embodiment, the pharmaceutical compositions disclosed herein are formulated for intracranial administration.Suitable intrathecal delivery systems are familiar to those skilled in the art, and an illustrative example thereof is described by Upadhyay et al. (2014, PNAS, 111(45):16071-16076), the contents of which are incorporated herein by reference in their entirety.

[0228] In another embodiment, the pharmaceutical compositions disclosed herein are formulated for intraventricular administration.Suitable intrathecal delivery systems are familiar to those skilled in the art, and illustrative examples thereof are described by Cook et al. (2009, Pharmacotherapy. 29(7):832-845), the contents of which are incorporated herein by reference in their entirety.

[0229] Suitable pharmaceutical compositions and unit dosage forms thereof may contain conventional ingredients in conventional proportions, with or without additional active compounds or principles, but such unit dosage forms may contain any suitable effective amount of the active ingredients commensurate with the adopted range for the intended daily dosage.

[0230] kit In another embodiment disclosed herein, a kit is provided that includes an artificial CSF as described herein or a pharmaceutical composition as described herein. The active agents as described herein (including Hx and optionally Hp) may be in the form of a kit of several components designed to allow simultaneous, separate or sequential administration of the active agents. Each carrier, diluent, adjuvant, and / or excipient must be "pharmaceutical acceptable" so as to be compatible with the other components of the composition and physiologically tolerated by the subject. The compositions are conveniently provided in unit dosage form and can be prepared by methods well known in the art of pharmacy. Such methods include the step of bringing the active ingredient into association with the carrier (which may constitute one or more accessory ingredients). In general, the compositions are prepared by uniformly and intimately bringing the active ingredient into association with the liquid carrier, diluent, adjuvant, and / or excipient, or finely divided solid carrier, or both, and then, if necessary, shaping the product.

[0231] Diagnostic methods As described elsewhere herein, the inventors have surprisingly discovered that the concentration of ventricular CSF-Hb can be used to determine whether a subject is at risk for developing an adverse secondary neurological outcome following hemorrhagic stroke.

[0232] Accordingly, in another embodiment disclosed herein, there is provided a method for determining whether a subject is at risk for developing an adverse secondary neurological outcome following a hemorrhagic stroke involving extravascular hemolysis and release of cell-free heme and / or cell-free hemoglobin (Hb) into cerebrospinal fluid (CSF), comprising: (i) obtaining a CSF sample from a subject following a hemorrhagic stroke; (ii) measuring the amount of cell-free Hb in the CSF sample obtained in step (i); and (iii) comparing the amount of cell-free Hb in the CSF sample determined in step (ii) with a reference value. wherein the subject's risk of developing an adverse secondary neurological outcome is determined based on the comparison in step (iii).

[0233] Suitable methods for measuring the amount of cell-free Hb in a CSF sample are known to those of skill in the art, illustrative examples of which are provided elsewhere herein.

[0234] In one embodiment, "reference value" is a value that represents the amount of acellular Hb in CSF of a healthy subject or a group of healthy subjects.In this context, if the amount of acellular Hb in CSF sample from test subject is higher than the reference value, it is suggested that the subject has a risk of developing adverse secondary neurological outcomes after hemorrhagic stroke.On the contrary, if the amount of acellular Hb in CSF sample from test subject is equal to or lower than the reference value, it is suggested that the subject does not have a risk of developing adverse secondary neurological outcomes after hemorrhagic stroke.

[0235] In one embodiment, "reference value" is a value that represents the amount of acellular Hb in CSF of a subject or a group of subjects who have had adverse secondary neurological outcomes after hemorrhagic stroke.In this context, if the amount of acellular Hb in CSF sample from test subject is equal to or exceeds the reference value, it is suggested that the subject has a risk of developing adverse secondary neurological outcomes after hemorrhagic stroke.On the contrary, if the amount of acellular Hb in CSF sample from test subject is lower than the reference value, it is suggested that the subject does not have a risk of developing adverse secondary neurological outcomes after hemorrhagic stroke.

[0236] In one embodiment, the reference value is substantially zero, meaning that there is no detectable physiological cell-free Hb in the CSF. In another embodiment, the reference value represents the level of cell-free Hb in the CSF of one or more patients who have had a hemorrhagic stroke but have no adverse neurological outcome. In this context, it is understood that in some cases, there may be a threshold level of cell-free Hb in the CSF that is acceptable without resulting in an adverse neurological outcome.

[0237] In one embodiment, the subject is determined to be at risk of developing an adverse secondary neurological outcome if the amount of cell-free Hb in the CSF sample determined in step (ii) is at least about 7 μM.

[0238] In one embodiment, the adverse secondary neurological outcome is angiographic vasospasm, and the subject is determined to be at risk for developing angiographic vasospasm if the amount of cell-free Hb in the CSF sample determined in step (ii) is at least about 3 μM.

[0239] In one embodiment, the adverse secondary neurological outcome is delayed cerebral ischemia, and the subject is determined to be at risk of developing delayed cerebral ischemia if the amount of cell-free Hb in the CSF sample determined in step (ii) is at least about 5 μM.

[0240] In one embodiment, the adverse secondary neurological outcome is delayed ischemic neuropathy, and the subject is determined to be at risk for developing delayed ischemic neuropathy if the amount of cell-free Hb in the CSF sample determined in step (ii) is at least about 7 μM.

[0241] "At least about 7 μM" includes at least about 7 μM, at least about 8 μM, at least about 9 μM, at least about 10 μM, at least about 15 μM, at least about 20 μM, at least about 25 μM, at least about 30 μM, at least about 35 μM, at least about 40 μM, at least about 45 μM, at least about 50 μM, etc. In one embodiment, the subject is determined to be at risk for developing an adverse secondary neurological outcome following hemorrhagic stroke if the amount of cell-free Hb in the CSF sample determined in step (ii) is at least about 7 μM, preferably at least about 8 μM, preferably at least about 9 μM, preferably at least about 10 μM, preferably at least about 15 μM, preferably at least about 20 μM, preferably at least about 25 μM, preferably at least about 30 μM, preferably at least about 35 μM, preferably at least about 40 μM, preferably at least about 45 μM, or more preferably at least about 50 μM.

[0242] Similarly, "at least about 3 μM" includes at least about 3 μM, at least about 4 μM, at least about 5 μM, at least about 6 μM, at least about 7 μM, at least about 8 μM, at least about 9 μM, at least about 10 μM, at least about 15 μM, at least about 20 μM, at least about 25 μM, at least about 30 μM, at least about 35 μM, at least about 40 μM, at least about 45 μM, at least about 50 μM, etc. In one embodiment, the subject is determined to be at risk of developing an adverse secondary neurological outcome following hemorrhagic stroke if the amount of cell free Hb in the CSF sample determined in step (ii) is at least about 3 μM, at least about 4 μM, at least about 5 μM, at least about 6 μM, at least about 7 μM, preferably at least about 8 μM, preferably at least about 9 μM, preferably at least about 10 μM, preferably at least about 15 μM, preferably at least about 20 μM, preferably at least about 25 μM, preferably at least about 30 μM, preferably at least about 35 μM, preferably at least about 40 μM, preferably at least about 45 μM, or more preferably at least about 50 μM.

[0243] The method of determining the risk of developing adverse secondary neurological outcomes after hemorrhagic stroke as described herein advantageously allows the subject to be stratified to the corresponding treatment regimen while considering its determined risk.For example, if the subject is determined to have a risk of developing adverse secondary neurological outcomes after hemorrhagic stroke, the subject is stratified to the treatment method described herein.On the other hand, if the subject is determined not to have a risk of developing adverse secondary neurological outcomes after hemorrhagic stroke, the subject may be stratified to no treatment or to an alternative and less aggressive treatment regimen.

[0244] The method of determining risk may suitably include using a receiver operating characteristic (ROC) curve (e.g., ROCR package (v1.0-11) (Sing et al., 2005), plotROC package (v2.2.1) (Sachs, 2017)), the corresponding bootstrap area under the curve (AUC) (pROC package (v1.16.2) (Robin et al., 2011)), and / or the optimal Youden index (ROCit package (v1.1.1) (Md Riaz Ahmed, 2019)). Illustrative examples of methods of determining risk of developing adverse secondary neurological outcomes after hemorrhagic stroke are also described elsewhere herein.

[0245] In one embodiment, the method further comprises treating the subject determined to be at risk for an adverse secondary neurological outcome, said treatment comprising exposing the subject's CSF to (i) a therapeutically effective amount of hemopexin (Hx), as described herein, for a period of time sufficient to allow Hx to complex with, and thereby neutralize, cell-free heme; and / or (ii) a therapeutically effective amount of haptoglobin (Hp), as described herein, for a period of time sufficient to allow Hp to complex with, and thereby neutralize, cell-free Hb.

[0246] In another aspect disclosed herein, there is provided a method of stratifying a subject for a treatment corresponding to an adverse secondary neurological outcome in a subject following a hemorrhagic stroke involving extravascular hemolysis and release of cell-free heme and / or cell-free hemoglobin (Hb) into cerebrospinal fluid (CSF), the method comprising: (a) determining whether the subject is at risk for developing an adverse secondary neurological outcome as described herein; and (b) treating a subject determined to be at risk for an adverse secondary neurological outcome based on the methods described herein.

[0247] Those skilled in the art will recognize that the invention described herein is prone to variations and modifications other than those specifically described. It is understood that the invention includes all such variations and modifications that fall within its spirit and scope. The invention also includes all of the steps, properties, compositions, and compounds referred to or shown in this specification, individually or as a whole, as well as any and all combinations of any two or more of said steps or properties.

[0248] Specific embodiments of the present invention will now be described with reference to the following examples, which are intended to be limiting for illustrative purposes and are not intended to limit the generality of what has been previously described herein. EXAMPLES

[0249] Ventricular CSF Hb as a biomarker for SAH-SBI Ethical approval was obtained prior to initiation of the study. Informed consent was obtained from all patients or their legal representatives. All experiments were performed in accordance with biosafety regulations and under suitable working conditions. Results are reported in accordance with the STROBE Declaration (von Elm et al., 2007).

[0250] Clinical trial population Clinical data and CSF samples were obtained from a prospective, consecutive cohort of patients with aSAH admitted to the Neurointensive Care Unit of an academic tertiary care center over a 3-year period (April 2017–March 2020).Eligibility criteria included: (i) age >18 years; (ii) radiological diagnosis of subarachnoid hemorrhage; (iii) exclusion of non-aneurysmal subarachnoid hemorrhage (e.g., trauma, perimesencephalic subarachnoid hemorrhage); and (iv) insertion of an external ventricular drain (EVD) as part of standard medical care.

[0251] Acquisition of clinical data Patient data collection was performed independently of and blinded to CSF ​​sampling. Baseline constructs including demographic characteristics (age, sex), clinical scores and indices (WFNS grade, Hunt & Hess grade, initial Glasgow Coma Scale (GCS)), and radiological assessments (aneurysm location, maximum aneurysm diameter, hematoma volume (see below), presence of intraventricular hemorrhage (IVH), Fisher grading, BNI grading) were evaluated at diagnosis (Figure 7A). Clinical data were collected prospectively for SAH-SBI, during the 14-day high-risk phase, and as part of the 3-month follow-up, based on standardized multimodal monitoring performed in a neurocritical care unit (Figure 7A). General patient management was in accordance with current international guidelines (Diringer et al., 2011; Connolly et al., 2012). The presence of DIND, DCI, or aVSP was assessed daily. DIND was defined as a decrease in GCS of at least 2 points for at least 2 hours that was not due to new focal neurological deficit or any other identifiable cause, such as hydrocephalus, seizures, or systemic factors. If the patient could not be clinically evaluated (e.g., sedated), clinical data were classified as non-available (NA). DCI was defined as new ischemia or new infarction on CT / CTP or MRI that could not be attributed to surgical intervention. The definition of aVSP included new narrowing of cerebral arteries based on digital subtraction angiography (DSA), magnetic resonance angiography (MRA) and computed tomography angiography (CTA). If there was no suitable imaging procedure on each day, this was noted as NA. SAH-SBI was defined as a composite outcome of DIND, DCI, and aVSP. In addition, flow velocities of the middle cerebral artery, both anterior and posterior cerebral arteries were assessed daily using TCD.

[0252] Complications (meningoencephalitis, central salt wasting, diabetes insipidus, pneumonia, takotsubo cardiomyopathy, nonconvulsive status epilepticus, chronic hydrocephalus, death) and functional status (Extended Glasgow Outcome Scale (GOSE) and modified Rankin Scale (mRS)) were assessed up to 3-month follow-up.

[0253] Subarachnoid hematoma segmentation and volumetric analysis Based on the initial CT scan, the volume of the subarachnoid hematoma was manually contoured using 3DSlicer (Kikinis et al., 2014). This was performed in 46 of the 47 subjects (one subject only underwent an initial MRI and was therefore excluded from this analysis). Preprocessing before contouring consisted of brain extraction. The largest cavity was extracted from a skull mask thresholded at 200 HU (Weidert et al., 2020). The brain mask was therefore multiplied by the original CT scan and median filtered with a 5 × 5 × 3 kernel (Figure 7B). The volume of the hemorrhage was determined by multiplying the voxel size by the number of segmented voxels (Figure 2C).

[0254] CSF sampling CSF samples were collected daily from the EVD starting after aneurysm repair. The sampling period was between day 1 (the day after bleeding) and day 14. CSF collection was performed blinded to clinical data. Immediately after sample collection, CSF was centrifuged at 1500G for 15 minutes (Capricorn CEP 2000 Benchtop centrifuge, Capricorn labs, UK) and the supernatant was stored at -80°C for further analysis.

[0255] Spectrophotometric measurement of CSF Hb and heme metabolites The absorption spectra in the visual range between 350 and 650 nm of all CSF samples were measured on a Shimadzu UV-1800 spectrophotometer (Shimadzu, Japan). Quantification of the different Hb species and their metabolites (oxyHb, metHb, bilirubin, biliverdin) was performed using spectral deconvolution. Therefore, the extinction curves of individual substances with known concentrations were fitted to the extinction curve of CSF using a non-negative least squares algorithm as previously described (Deuel et al., 2015).

[0256] LC-MSMS CSF proteome analysis Using a label-free proteomics quantification approach, the CSF proteome of 18 patients was characterized at five consecutive time points along the observation period (0, 0.5, 1, 1.5, and 2 weeks after aSAH) (Figure 7C). To improve generalizability, samples were processed in two batches, which were combined when analyzed after standardization (85 samples in total - 48 in batch 1 and 37 in batch 2). Hb is abundant in CSF after red blood cell lysis, but to avoid ion suppression and increase sensitivity, it was selected out using a haptoglobin affinity column before analysis (Hugelshofer et al., 2019).

[0257] Following digestion of Hb-depleted CSF samples with trypsin, mass spectrometry analysis was performed on a Q Exactive HF mass spectrometer (Thermo Scientific) equipped with a Digital PicoView source (New Objective) and interfaced to an M-Class UPLC (Waters).

[0258] A. Sample Preparation Total protein concentration was estimated using Qubit® Protein Assay Kit (Life technologies, Zurich, Switzerland). Samples were then prepared using the commercial iST Kit (PreOmics Phoenix, Germany) with the latest version of the protocol. Briefly, 20 μg protein was solubilized in "lysis" buffer, boiled at 95°C for 10 min, and treated with HIFU for 30 s with ultrasonic intensity set at 85%. Afterwards, samples were transferred to the cartridge and digested by adding 50 μl "digestion" solution. After 60 min of incubation at 37°C, digestion was stopped with 100 μl stop solution. The solution in the cartridge was removed by centrifugation at 3800 g, while the peptides were retained by the iST-filter. Finally, peptides were washed, eluted, dried, and resolubilized in 20 μl buffer (3% acetonitrile, 0.1% formic acid) for LC-MS analysis.

[0259] B. Liquid Chromatography-Mass Spectrometry Analysis Mass spectrometry analysis was performed on an Orbitrap Fusion Lumos (Thermo Scientific) equipped with a Digital PicoView source (New Objective) and connected to an M-Class UPLC (Waters). The solvent composition in the two channels was 0.1% formic acid for channel A and 0.1% formic acid, 99.9% acetonitrile for channel B. For each sample, 1 μL of diluted peptides was loaded onto a commercial MZ Symmetry C18 Trap Column (100 Å, 5 μm, 180 μm×20 mm, Waters) followed by a nanoEase MZ C18 HSS T3 Column (100 Å, 1.8 μm, 75 μm×250 mm, Waters). Peptides were eluted at a flow rate of 300 nL / min with a gradient of ∼22% B in 77 min, 32% B in 10 min, and 95% B over 10 min. Samples were acquired in random order. The mass spectrometer was operated in data-dependent mode (DDA) acquiring full scan MS spectra (300-1'500 m / z) at a resolution of 120'000 at 200 m / z after accumulation to a target value of 500'000. Data-dependent MS / MS were recorded in a linear ion trap using quadrupole isolation with a window of 0.8 Da and HCD fragmentation with 35% fragmentation energy. The ion trap was operated in fast scan mode with a target value of 10'000 and a maximum injection time of 50 ms. Only precursors with an intensity above 5'000 were selected for MS / MS and the maximum cycle time was set to 3 seconds. Charge state screening was enabled. Singlet, unassigned and charge states higher than 7 were rejected. Precursor masses previously selected for MS / MS measurements were excluded from further selection for 20 seconds and the exclusion window was set to 10 ppm. An internal lock mass calibration was used to acquire samples at m / z 371.1012 and 445.1200. A local laboratory information management system (LIMS) was used to handle mass spectrometry proteomics data (Turker et al., 2010).

[0260] C. Protein Identification and Label-Free Protein Quantification Acquired raw MS data were processed by MaxQuant (version 1.6.2.3) followed by protein identification using the integrated Andromeda search engine (Cox and Mann, 2008). Spectra were searched against the Swissprot Homo sapiens reference proteome (taxonmy 9606, version 9 July 2019 or later) and linked to its reversed decoyed fasta database and common protein contaminants. Cysteine ​​carbamidomethylation was set as fixed modification, while methionine oxidation and N-terminal protein acetylation were set as variables. Enzyme specificity was set to trypsin / P, with a minimum peptide length of 7 amino acids and a maximum of 2 missed cleavages allowed. Default search settings of MaxQuant Orbitrap were used. Maximum false positive rate (FDR) was set to 0.01 for peptides and 0.05 for proteins. Label-free quantification was enabled and a 2-minute window for match between runs was applied. In the MaxQuant experimental design template, each file is kept separate within the experimental design to obtain individual quantitative values. MaxQuant results were loaded into Scaffold (Proteome Software Inc.) and validated for peptide and protein identification. Only proteins identified with at least two peptides were considered for follow-up analysis.

[0261] Individual protein intensities of proteins were normalized to the mean intensity of each protein at baseline (week 0) and log2 transformed. Global fold changes were calculated as the sum of log ratios for each protein over the entire time course. Furthermore, for each time point, normalized protein intensities were compared to baseline (week 0) using Wilcoxon rank sum tests, and p-values ​​were integrated across all time points using the metap package (Dewey, 2020). A k-means analysis was used to cluster temporal changes in the CSF proteome, whereby the optimal number of clusters was visually determined using an elbow diagram (Figure 7D) using the Factoextra package (v1.0.7) (Kassambara and Mundt, 2020).

[0262] Hemoglobin, haptoglobin, hemopexin, and rLP Hb used in ex vivo experiments was purified from expired human blood concentrates as previously described (Elmer et al., 2009). Hb concentrations were determined by spectral deconvolution as described above and are expressed as the molar concentration of total heme (1 M Hb tetramer is equivalent to 4 M heme). Hx was used at equimolar concentrations relative to the concentration of heme measured in the test samples. For all Hb used in these studies, ferrous oxyHb (HbFe 2+ The percentage of HO) was always greater than 98% as determined by spectrophotometry. Purified haptoglobin (phenotype 1-1) and hemopexin from human plasma were obtained from CSL Behring.

[0263] Neurovascular function Vascular function assays (Figure 7E) were performed using fresh porcine basilar arteries obtained from a local slaughterhouse (n=12). Vessels were prestretched to reach an optimal passive pretension of 8, as previously published (Hugelshofer et al., 2020). Next, 10 μM prostaglandin F2α (PGF2α; Sigma, Buchs, Switzerland) was used as a precontractor. To determine the maximum possible tension in the absence of endogenous NO, a concentration of 10 μM L-N5-(1-iminoethyl)ornithine hydrochloride (L-NIO; Sigma-Aldrich, Saint Louis, US-MO) was added at the end of the experiment. Recorded tensions were normalized to the individual NO reserve capacity of each vessel, i.e., baseline after PGF2α addition (0%) and tension after L-NIO addition at the end of the experiment (100%) (Figure 7F). Injured vessels that showed a continuous loss of tension during the experimental period, resulting in a ratio of PGF2α max / L-NIO max below 0.7, were excluded from the analysis (Figure 7F). A first series of experiments was performed to evaluate the vasoconstrictor potential of CSF from patients at high risk for SAH-SBI after aSAH and to evaluate the special role of oxyHb in this process. For this purpose, patient CSF from the high risk period (between days 4 and 14) was selectively depleted for Hb using a haptoglobin affinity column. In the initial phase of the experiment, vessels were immersed in Hb-depleted CSF. Hb was then added to the CSF in exactly the amount previously depleted in order to define its specific effect on vascular tension. In a second series of experiments, the effect on vascular tension of increasing exposure to Hb was evaluated. A half-log10 Hb dilution series (10 -4 M~10 -8 M) was made and vessels were bathed in increasing concentrations of Hb. For evaluation of haptoglobin effects, a third series of experiments was performed using the same Hb dose escalation, except for equimolar addition of haptoglobin.

[0264] TBARS The oxidative capacity of CSF samples was quantified by measuring the formation of malondialdehyde (MDA) as the final lipid peroxidation product after incubation with well-defined reconstituted lipoproteins (rLP, obtained from CSL Behring, Bern, Switzerland) (Figure 7G). Using a 96-well plate, 25 μL of CSF was mixed with 5 μL of rLP (3 mg / mL) and incubated at 37°C for 24 h. The concentration of MDA was then measured using the thiobarbituric acid reactive substances (TBARS) assay. Briefly, 125 μL of 1 M HCl containing 750 mM trichloroacetic acid was added to the samples, followed by the addition of 100 μL of 1 M NaOH containing 25 mM 2-thiobarbituric acid after brief vortexing.

[0265] After a 60 min incubation period at 80° C., TBARS was quantified in the supernatant. For absolute quantification of MDA, the absorbance at 600 nm was subtracted from the absorbance at 532 nm, and the molar extinction coefficient was 0.156 mM. -1 ·cm -1 Concentrations were calculated using the formula: To achieve a more sensitive but relative quantification, the fluorescence emission at 550 nm was measured while using 510 nm as the excitation wavelength.

[0266] statistical analysis Statistical analysis was performed using R3.6.3 (R Core Team, 2020). Categorical data are provided as absolute numbers (n) and percentages (%), and continuous data as means and standard deviations (SD). For factorial experiments, repeated measures ANOVA was performed using the ez package (v4.4-0) (Lawrence, 2016). Mixed linear model analysis was performed with the lme4 package (v1.1-23) (Bates et al., 2015). Gene set enrichment analysis used the Gene Set Enrichment Analysis GSEA software from the Broad Institute (Subramanian et al., 2005), and the Hallmark gene set from the Molecular Signature Database (MSigDB) (Liberzon et al., 2015). As input, a ranked list of identified proteins was used, and rank scores were calculated as the product of the protein's -log10 (p-value) and the sum (log ratio). Associations between baseline measurements (aneurysm location, aneurysm size, hematoma volume, presence of IVH) and CSF-Hb, as well as between CSF-Hb and SAH-SBI, were analyzed using general additive models (GAMs) with spline approximations for random effects for days after aSAH and patient ID, using the mgcv package (v1.8-31) ( Wood, 2017 ). The sensitivity and specificity of CSF oxyHb for SAH-SBI were analyzed using receiver operating characteristic (ROC) curves (ROCR package (v1.0-11) (Sing et al., 2005), plotROC package (v2.2.1) (Sachs, 2017), the corresponding bootstrap area under the curve (AUC) (pROC package (v1.16.2) (Robin et al., 2011), and the optimal Youden Index (ROCit package (v1.1.1) (Md Riaz Ahmed, 2019). TCD flow velocity was used first categorically according to its clinical assessment (pathological or non-pathological), which serves to determine sensitivity and specificity.Second, TCD scaling used continuous TCD flow velocity to correlate pathophysiologically with measured CSF Hb, with each value scaled relative to the mean flow rate over the corresponding vessel (anterior, middle, or posterior cerebral artery) in the cohort. Maximum scaled TCD represented the maximum value after scaling the daily measured values. Due to the exploratory nature of the study, we did not define a level of statistical significance, instead interpreting the results based on the level of evidence: p<0.001: very strong evidence; p<0.01: strong evidence; p<0.05: evidence; p<0.1: weak evidence; p>0.1: no evidence (Bland, 2015).

[0267] result A. Clinical Trial Population Of 52 consecutively screened patients who met the eligibility criteria, 47 were included.

[0268] Five patients or their legal representatives refused informed consent. Table 1 summarizes the baseline composition of the patient cohort.

[0269] [Table 1]

[0270] B. Spectrophotometric Measurement of CSF-Hb and Heme Metabolites Spectrophotometric measurements using spectral deconvolution were performed to determine the amount of cell-free oxyHb (ferrous HbFe 2+ ), and its downstream metabolites biliverdin and bilirubin, as well as the major Hb oxidation product metHb (ferric HbFe 3+) was used to quantify the oxyHb concentration. The individual temporal profiles and peak concentrations (0.6 μM to 242.2 μM) of oxyHb were highly variable (Fig. 8). In most patients, oxyHb remained very low for the first 2–3 days after aSAH, then increased significantly, plateaued between days 9–12, and then decreased (Fig. 1A). Bilirubin was already elevated on day 1, showed the most significant increase during the first 3–5 days, and then plateaued (Fig. 1A). This time course reflects phagocytosis and heme metabolism by macrophages in the subarachnoid space. The intermediate metabolite biliverdin began to increase significantly from day 4 onwards and reached its peak on day 12 (Fig. 1A). Again, metHb followed a delayed increase, peaking 11 days after aSAH, consistent with the slow autoxidation of cell-free oxyHb in the CSF (Fig. 1A).

[0271] C. LC-MSMS CSF Proteome Analysis Quantitative LC-MS / MS analysis was performed on 85 CSF samples from 18 patients collected at 5 time points after hemorrhage. Figure 1B illustrates the time course of 757 CSF proteins, which were assigned to 4 groups by k-means clustering. Figure 1C shows a Volcano plot of normalized signal intensity summed over all samples per patient, providing an overview of protein accumulation or depletion within 2 weeks after hemorrhage. Cluster 1 (green) included proteins that remained unchanged over time. Cluster 4 (yellow) represents proteins that decreased in intensity over time. These were mainly plasma proteins that entered the subarachnoid space with the initial hemorrhage and were subsequently cleared from the CSF. Ranked proteins were subjected to gene set enrichment analysis (GSEA) using hallmark gene sets from the Molecular Signature Database (MSigDB) (Liberzon et al., 2015) and the top negative enrichment was assigned to coagulation (ES=-0.66, FDR=0.007, Figure 1D). Figure 1E shows the time course of four selected plasma proteins (HP, HPR, HPX, and ALB). Cluster 2 (purple) contained proteins with 12 examples that significantly accumulated over time, and cluster 3 (blue) contained proteins that were moderately increased.

[0272] GSEA identified glycolysis (ES=0.78, FDR=0.016, Figure 1F) and heme metabolism (ES=0.74, FDR=0.096, Figure 1H) as the top enriched hallmarks representing red blood cell (RBC) lysis with release of cytoplasmic proteins and degradation of Hb by macrophages, respectively. Figure 1G shows the time course of RBC proteins carboanhydrase 1 (CA1) and 2 (CA2), catalase (CAT), and aldolase A (ALDOA). Figure 1I shows the macrophage proteins CD163, CSF1R, CD14, and TIMP-1. Accumulation of these proteins points to a prominent influx of macrophages into the subarachnoid space within the first few days after hemorrhage. Overall, the proteomic dynamics within patient CSF after aSAH confirmed that hemolysis as well as the adaptive macrophage response are the two predominant processes occurring within the CSF space after aSAH.

[0273] D. Determinants of CSF-Hb levels To estimate the determinants of CSF-Hb concentrations, a general additive model (GAM) was applied with spline approximations for random effects for days after aSAH and patient ID. Partial dependence plots are shown in Figure 2B. Aneurysm size did not appear to be associated with the course of CSF-Hb. Among several different aneurysm locations, only middle cerebral artery aneurysms were negatively associated with Hb levels (p=0.0018). Hematoma volume showed a strong association with CSF-Hb levels. Days after aSAH had a negative association in the first 3-5 days, but then a weak positive association was observed in the subsequent days. This association most likely reflects a delayed onset of hemolysis and CSF-Hb generation in the subarachnoid space. The presence of IVH was associated with higher levels of CSF-Hb (p<0.0001). In the presence of IVH, CSF-Hb showed an initial increase followed by a plateau, whereas in the absence of IVH, the increase was slower and more gradual, but plateaued at comparable peak concentrations around day 12 (Fig. 2C), most likely due to a CSF compartment effect (Fig. 2D).

[0274] Increased E.CSF-Hb is associated with secondary brain injury Cohort outcome parameters (per patient and per day) representative of SAH-SBI, rescue therapy, complications, mortality, and functional status at 3-month follow-up are summarized in Table 3. CSF-Hb levels stratified by SAH-SBI are shown in Figure 3A and Figure 9. Based on GAM with spline approximation for random effects for days after aSAH and patient ID, very strong evidence (p = 0.00045) was found for an association between the occurrence of SAH-SBI and CSF-Hb concentrations. Figure 3B shows the partial dependence of SAH-SBI on CSF-Hb and days after SAH. To estimate the power of daily measured CSF-Hb as a monitoring marker for SAH-SBI, each receiver operating characteristic (ROC) curve and the corresponding area under the ROC curve (AUC) were calculated for DIND, DCI, aVSP (Figures 9A-C, Table 2) and the composite outcome SAH-SBI (Figure 3C, Table 2). By restricting the data to the high-risk period (days 4–14) and avoiding potential bias introduced by the generally low Hb levels in the first few days after bleeding, the high diagnostic accuracy remained unchanged in the recalculated model (Figure 9D). Bilirubin, biliverdin, and methemoglobin had lower diagnostic accuracy than oxyHb (Figures 9E–G). By calculating the optimal Youden Index, a value of 7.1 μM for CSF-Hb was obtained for SAH-SBI (Figure 3C). For the individual outcomes of aVSP, DCI, and DIND, the optimal Youden Index was 3.4, 5.5, and 7.1 μM, respectively (Figures 9A–C).

[0275] [Table 2]

[0276] [Table 3]

[0277] Given its frequent use in clinical practice (Diringer et al., 2011; Connolly et al., 2012), we evaluated the diagnostic accuracy of clinical and radiological scores as well as daily TCD measurements in our patient cohort. Clinical and radiological scores showed poor predictive quality (Table 2, Figures 9H-K). TCD measurements showed high specificity for SAH-SBI, aVSP, DCI, and DIND. However, the sensitivity of TCD was marginal (Table 2).

[0278] High CSF-Hb levels were also associated with poor clinical outcomes as expressed by GOSE scores (p=0.0056, Fig. 3D) and mRS scores (p=0.0016, Fig. 9L) at 3-month follow-up.

[0279] F. Basilar artery vasoconstriction and lipid peroxidation occur within the range of CSF-Hb concentrations in patients The data suggest that cell-free Hb is an upstream mediator of SAH-SBI. To support the validity of this association, ex vivo vasoconstriction and lipid peroxidation were examined using exposure to Hb that matched the CSF-Hb concentration range within the patient cohort. A novel vascular function model was established using porcine basilar arteries precontracted with PGF2α and dilated by an intrinsic endothelial NOS (eNOS) response at the end of the experiment. This configuration allowed investigation of the effect of Hb on endogenous NO reserve capacity. No supplemental NO donor was used.

[0280] Figure 4A shows the 10 -6.5 ~10 -6 M oxyHb, accompanied by a rapid increase in vascular tone, and -5 Figure 4B shows a sigmoidal dose-response curve reaching maximum contraction at 100 M oxyHb. Nearly identical curves were obtained when plotting maximum scaled patient TCD values ​​against the categories of CSF-Hb (Figure 4B). The CSF-Hb categories were chosen to match the concentration steps used in the ex vivo vasoconstriction assay. Patients with aVSP (red dots) showed approximately 10 -5M or higher. We also measured the generation of TBARS in mixtures of oxyHb and reconstituted lipoproteins containing unsaturated phosphatidylcholine, which is thought to be the major physiological lipid substrate for Hb peroxidation in vivo (Deuel et al., 2015). Dose-response experiments shown in Figure 4C show that the most rapid increase in MDA formation occurred at 10 -5.5 ~10 -5 Figure 4D plots the CSF-Hb concentrations of our patients on the same x-axis as the values ​​of the above parameters. Figure 4D shows that the calculated cutoff concentration for CSF-Hb (optimal Youden Index) that distinguishes between patients with and without SAH-SBI coincides with near-maximal basilar artery vasoconstriction, TCD values, and TBARS generation.

[0281] Overall, vascular function and lipid peroxidation tests suggest critical inflection points that overlap with relevant CSF-Hb concentrations in patients with SAH-SBI.

[0282] G. Depletion and neutralization of CSF-Hb alleviates pathological vasoconstriction and lipid peroxidation. To mechanistically link CSF-Hb in patients to SAH-SBI-related pathophysiology, a series of Hb depletion and neutralization experiments were performed. Porcine basilar artery segments bathed in CSF that had been depleted of CSF-Hb by a haptoglobin column (Figure 5A) had significantly lower contractile force than segments bathed in the same CSF sample supplemented with highly purified Hb to the original CSF-Hb concentration (Figure 5B). This experimental setup controls for potential nonspecific removal of vasoactive substances by the haptoglobin column. Hb dose-response experiments were also repeated to confirm the antivasospastic effect of soluble haptoglobin 1-1 (Hp1-1). Across the Hb concentration range, Hp1-1 significantly attenuated basilar artery contractile force (repeated measures ANOVA, p<0.001) (Figure 5C).

[0283] The oxidative capacity of CSF samples from patients after aSAH was also quantified. Compared to baseline CSF (day 0), we found increased variability in MDA products after lipoprotein exposure to CSF ​​samples collected during the high-risk phase (0.5–2 weeks; Figure 5D). Addition of Hp1-1 at equimolar concentrations relative to CSF-Hb effectively attenuated MDA formation. Notably, addition of hemopexin at equimolar concentrations showed an even stronger attenuation of MDA formation. EXAMPLES

[0284] Quantitative assessment of hemoglobin and heme scavenger proteins in patient CSF samples A. Quantification of human Hp, Hpx, and albumin in CSF samples CSF samples, 10 microliters, were placed in clean Eppendorf tubes, after which 80 μL of MeOH was added to precipitate proteins. Methanol was removed after centrifugation, and the pellet was air-dried and then resuspended in 50 mM NH4HCO3 / 0.16% proteaseMAX containing heavy isotope-labeled peptides (specific for human haptoglobin, hemopexin, and albumin, and used as internal standards). After 45 min incubation at 56°C / 550 rpm, samples were reduced by adding 0.5 M DTT (56°C / 550 rpm, 20 min). Samples were then alkylated by adding 0.5 M IAA and incubating for 20 min at RT, protected from light. Trypsin digestion was performed at 37°C / 550 rpm and stopped after 3 h by adding formic acid. After centrifugation, the samples were rapidly separated on a C18 column (AdvanceBio Peptide Mapping, 2.1×150 mM). Measurements were performed using an Agilent 1290 Infinity II-6550 iFunnel QTOF LC-MS system.

[0285] Data was analyzed by calculating the peak areas of the analytes and internal standards using Agilent MassHunter Quant software. Standard curves were generated for each protein in Agilent MassHunter Quant by plotting the average response ratio of the analyte to the internal standard versus concentration for each standard sample. Analyte concentrations in CSF samples were back-calculated using the standard curve equation.

[0286] B. Determination of free hemoglobin and haptoglobin-bound hemoglobin in CSF Hp-bound and unbound fractions of Hb (cell-free Hb) were determined by SEC-HPLC using an Ultimate3000SD HPLC (ThermoFisher) coupled to an LPG-3400SD quaternary pump and a photodiode array detector (DAD). CSF samples and Hb standards were separated on a Diol-120 (3 μm, 300 × 8.0 mm) column (YMC CO Ltd.) at a flow rate of 1 mL / min using PBS, pH 7.4 (Bichsel) as the mobile phase. For all samples, two wavelengths were recorded (λ = 280 nm and λ = 414 nm). Bound and unbound Hb in CSF were determined by calculating the peak area of ​​both peaks (Hb:Hp, retention time 7-8 min depending on Hp phenotype; for cell-free Hb, retention time 10 min). Values ​​obtained from CSF samples were interpolated by generating a standard curve based on peak areas plotted against concentration.

[0287] C. Determination of heme-bound heme proteins in CSF Heme-bound hemoproteins were determined by SEC-high performance liquid chromatography (SEC-HPLC) using an Ultimate 3000SD HPLC (ThermoFisher) coupled to an LPG-3400SD quaternary pump and a photodiode array detector (DAD). CSF samples and heme standards were separated on a Diol-120 (3 μm, 300 × 8.0 mm) column (YMC CO Ltd.) at a flow rate of 1 mL / min using PBS, pH 7.4 (Bichsel) as the mobile phase. For all samples, two wavelengths were recorded (λ = 280 nm and λ = 414 nm). Hemoproteins in CSF were determined by calculating the peak area of ​​both peaks (retention time 9 min for heme-bound hemoproteins; retention time 10 min for cell-free Hb). Values ​​obtained from CSF samples were interpolated by generating a standard curve based on peak areas plotted against concentration.

[0288] D. Detection of hemopexin:heme complexes in human CSF samples CSF samples, 50 microliters, were placed in clean Eppendorf tubes, followed by the addition of 150 μL of Buffer A (Multiple Affinity Removal Systems, Agilent). The first chromatographic step, which depleted highly abundant human proteins, was performed according to the manufacturer's protocol on an Ultimate 3000SD HPLC (ThermoFisher) coupled to two LPG-3400SD quaternary pumps and a photodiode array detector (DAD). Briefly, diluted plasma samples were injected onto a multiple affinity removal column that depleted human albumin and IgG (human IgG / HSA, 4.6×50 mM, Agilent), and separated at a flow rate of 0.25 mL / min using Buffer A (Multiple Affinity Removal Systems, Agilent) as the mobile phase. Depleted CSF was collected in a fresh HPLC vial, reinjected, and separated on a Diol-300 (3 μm, 300×8.0 mm) column (YMC CO Ltd.) at a flow rate of 1 mL / min using PBS, pH 7.4 (Bichsel) as the mobile phase. For all samples, two wavelengths were recorded (λ=280 nm and λ=414 nm). The amount of hemopexin:heme complex was determined by calculating the peak area of ​​the complex (retention time 9 min). Values ​​obtained from depleted CSF samples were interpolated by generating a standard curve based on peak areas plotted against concentration.

[0289] result Cell-free hemoglobin was assessed in CSF samples from 28 aSAH patients collected over a 14-day period (FIG. 11). Although the methodology for quantifying cell-free Hb in CSF samples differed from that applied to the cohort described in Example 1 above (the cohort was the same), the results were entirely consistent (correlation coefficient (r)=0.96). Again, the data demonstrated that cell-free Hb levels increased over time and peaked at approximately day 10 (week 1.5). In contrast, haptoglobin and hemopexin concentrations increased at very early time points compared to healthy controls due to blood seeping into the CSF. Both scavenger proteins showed inverse kinetics to hemoglobin levels, suggesting depletion due to prolonged hemolysis. Albumin concentrations remained similar throughout the time course, but were significantly increased when compared to healthy control CSF samples.

[0290] In the next step, the scavenger proteins haptoglobin and hemopexin were investigated for their presence as complexes with hemoglobin or heme. Overall, as expected based on the kinetics of both proteins, the values ​​were very low and most data points interpolated outside the standard curve. Taken together with the cell-free Hb data, this suggests that at early time points when both proteins are clearly detected in the patient CSF, the targets are not yet present, and at later time points when Hb levels are increasing, both scavengers are already depleted and cleared from the CSF. Thus, these data suggest that the native Hb detoxification pathway is completely saturated and the scavenger proteins are depleted. Albumin, an intermediate heme storage protein, was found to be elevated throughout the study, and additional investigation of heme proteins, heme binding proteins other than cell-free Hb or Hp-bound Hb, revealed a pattern similar to that of total albumin in the CSF. Because heme bound to hemopexin was identified as very low or even absent, these data suggest that the majority of detected heme proteins is in fact heme bound to albumin. In contrast to hemopexin, heme bound to albumin can still participate in iron-mediated toxicity and is therefore considered one of the major contributors to secondary brain injury in patients with aSAH. These data support the use of hemopexin to further reduce heme-mediated oxidative damage in patients with aSAH. EXAMPLES

[0291] Heme Toxicity / Hemopexin Protection A. Intrastriatal injection model Wild-type male C57BL / 6J mice (10-12 weeks old) were obtained from Charles River (Sulzfeld, Germany), maintained in the animal facility of the University of Zurich (LASC) and treated according to the guidelines of the Swiss Federal Veterinary Office.

[0292] Mice were anesthetized with vaporized isoflurane (4-5% for induction, 1-2% for maintenance; Baxter Healthcare Co., Deerfield, IL, USA) in 100% oxygen. Body temperature was monitored with a rectal probe and maintained constant throughout the surgical procedure using an electronic thermostatically controlled heating blanket. Anesthetized mice were placed in a motorized stereotaxic frame (Stoelting, Dublin, Ireland) and a 1-cm long midline skin incision was made. After removing the soft tissue overlying the skull, the frame was aligned to the bregma and a small skull burr hole was drilled above the entry point for the target. The 33G needle was then slowly advanced (0.1 mm / sec) into the striatum (bregma coordinates: 2 mm ML, 0.5 mm AP, 3.5 mm DV) followed by injection of 10 μL of heme-albumin (1 mM), haptoglobin (5 mM) (protein control group), or heme-hemopexin (1 mM) at an infusion rate of 100 nl / sec. The needle was left in place for 5 minutes, then slowly removed, and the skin incision was closed with sutures. After the surgical procedure, the animals were placed in a heated wake box and monitored until they had fully recovered and could be returned to their home cage.

[0293] B. Behavioral Testing The open field exploration test was carried out in four equal square arenas (40x40x35cm (height)) made of opaque acrylic glass located in the test room under diffuse lighting (approximately 35lx measured in the center of the arena). Digital cameras were attached directly on top of the four arenas. Images were captured at a rate of 5Hz and transmitted to a PC running an Ethovision (Noldus, The Netherlands) tracking system. For measuring basal locomotor activity, animals were placed quietly in the center of the arena and allowed to explore for 15 minutes. The total distance traveled (in cm) was measured for 10 minutes, starting after a 5 minute acclimation period.

[0294] In the rotarod behavioral test, mice are placed on a rotating cylinder and thus forced to walk. This allows the assessment of forced locomotor activity. The rotation of the cylinder is accelerated from 5 rotations per minute (at the beginning) to a maximum of 40 rotations per minute (at the end) over a period of 300 seconds. The time it takes for the mouse to fall off the cylinder is measured.

[0295] For each neurological disorder, mice were subjected to the beam walking test, in which they had to cross an elevated beam to reach a safe location (a small shelter). The beam had a length of 100 cm and a width of 13 mm. During the assessment period, motor function was scored by two investigators blinded to the treatment.

[0296] C.MRI Protocol Mice were anesthetized with vaporized isoflurane (4–5% for induction, 1–2% for maintenance; Baxter Healthcare Co., Deerfield, IL, USA) in 100% oxygen. Throughout the imaging procedure, body temperature was monitored with a rectal probe and kept constant using a heating blanket, and respiratory rate was recorded by plethysmography. Mice were placed in a 7T small animal MRI scanner (Brucker, Germany). T2, ASL, and DWI images were recorded according to a standardized protocol.

[0297] result As shown in Figure 12, heme impaired neurological function in mice 24 hours after intrastriatal injection, as evidenced by impaired performance in the open field test (Figure 12A), rotarod test (Figure 12B), and beam walking test (Figure 12C). In contrast, mice intrastriatally injected with heme:Hx complexes showed no impairment in neurobehavioral activity at the same time point (24 hours). The impairment in neurological function was associated with radiological changes in mice intrastriatally injected with heme, including perilesional edema, diffusion restriction in ADC maps, and focal perfusion impairment on arterial spin labeling (ASL) images (Figure 13). Mice intrastriatally injected with heme:Hpx showed only minor radiological changes. As shown in Figure 14, quantification of radiological changes confirmed that intrastriatal injection of heme was associated with the formation of cerebral edema and a focal reduction in perfusion (not present in mice injected with heme:Hpx).

[0298] Consideration We have identified ventricular CSF-Hb as a biomarker for SAH-SBI, which surprisingly surpasses the diagnostic accuracy of other established methods. Within clinically relevant concentration ranges, CSF-Hb induces vasoconstriction and oxidizes unsaturated lipids ex vivo, suggesting that CSF-Hb plays a role as an upstream mediator of adverse secondary neurological outcomes after hemorrhagic stroke with extravascular hemolysis and release of cell-free heme and / or cell-free hemoglobin (Hb) into the cerebrospinal fluid (CSF). The strong association of high CSF-Hb with adverse clinical outcomes, the underlying pathophysiological basis, and the favorable effects of hemopexin and haptoglobin position CSF-Hb as a suitable biomarker and target in patients at risk for SAH-SBI.

[0299] In addition to its high diagnostic accuracy for adverse secondary neurological outcomes after hemorrhagic stroke, such as DIND, DCI, and aVSP, an additional advantage of CSF-Hb is its simple and reliable analytical. Precise CSF-Hb values ​​can be determined in the ICU with a simple two-step procedure consisting of a centrifugation step to remove cells and debris from the CSF, followed by spectrophotometric measurement with automated spectral deconvolution. Moreover, the physical laws underlying spectrophotometric measurement allow absolute quantification of CSF-Hb without the need for a standard curve or parallel measurement of calibration samples. Overall, this procedure may significantly lower analytical costs and operating times, and increase widespread applicability and acceptability.

[0300] We also demonstrated that CSF-Hb is the major vasoconstrictor in patient CSF. Moreover, the active dynamic range of Hb concentrations in an ex vivo vasospasm model overlaps with the TCD flow velocity in cerebral arteries and the critical inflection point determining the presence or absence of SAH-SBD in our patient cohort. Within the same clinically relevant concentration range, CSF-Hb was found to be highly active as a lipid oxidant, suggesting that oxidative Hb toxicity contributes to nonischemic neuronal injury. Lipid oxidative activity in patient CSF was attributable, at least in part, to heme, a downstream Hb metabolite. Its selective neutralization by hemopexin substantially blocked the formation of MDA, whereas haptoglobin provided partial inhibition.

[0301] The data disclosed herein demonstrate that CSF-Hb is a suitable drug target for preventing and treating SAH-SBI. This is supported by the strong association of CSF-Hb with adverse clinical outcomes, the rational pathophysiology, and the favorable functional effects of CSF-Hb neutralization with hemopexin and haptoglobin in the clinically important toxicity range. To date, the only drug that reduces the occurrence of SAH-SBI after aSAH is the calcium channel blocker nimodipine (class I, level A). Although nimodipine has a positive effect on clinical outcomes, it does not prevent the occurrence of aVSP. Previous trials of the preventive effects of various drugs, CSF drainage, subarachnoid lavage, endplate fenestration, or head concussion have yet to show a significant effect on the risk of SAH-SBI. In already symptomatic patients, treatment options are limited to non-causal rescue therapy to improve regional cerebral blood flow.

[0302] In the CSF analysis described herein, it was found that endogenous haptoglobin is cleared from the CSF within the first few days after aSAH, before the associated production of cell-free Hb. Thus, the protective function of endogenous haptoglobin appears negligible, while exogenous haptoglobin provides antivasospastic and antioxidant effects within the clinically relevant CSF-Hb concentration range. Unexpectedly, the heme scavenger protein hemopexin surpassed the antioxidant function of haptoglobin, suggesting that a portion of heme may already be released from oxidized or degraded CSF-Hb, forming a pool of oxidizable free heme that remains associated with low affinity heme-binding proteins. These data suggest that the therapeutic combination of haptoglobin and hemopexin in the context of aSAH is expected to provide synergistic protection against adverse secondary neurological outcomes after hemorrhagic stroke.

[0303] We also demonstrated that hemopexin neutralized the heme-mediated impairment of neurological function following intrastriatal injection, and note that this effect correlated with radiological changes, with mice intrastriatally injected with heme exhibiting perilesional edema, diffusion restriction in ADC maps, and focal perfusion defects on arterial spin labeling (ASL) images, whereas these adverse radiological changes were largely absent in mice intrastriatally injected with heme:Hpx complexes.

[0304] References 1. Bates D, Machler M, Bolker B, Walker S. Fitting Linear Mixed-Effects Models Usinglme4 [Internet]. Journal of Statistical Software 2015; Available from: http: / / dx.doi.org / 10.18637 / jss.v067.i01 2. Behrouz R. The Rise and Fall of Transcranial Doppler Ultrasonography for the Diagnosis of Vasospasm in Aneurysmal Subarachnoid Hemorrhage. J Neurosurg Anesthesiol 2019; 31: 79-80. 3. Bland M. An Introduction to Medical Statistics. Oxford University Press; 2015 4. Connolly ES Jr, Rabinstein AA, Carhuapoma JR, Derdeyn CP, Dion J, Higashida RT, et al. Guidelines for the management of aneurysmal subarachnoid hemorrhage: a guideline for healthcare professionals from the American Heart Association / american Stroke Association. Stroke 2012; 43: 1711-37. 5. Deuel JW, Vallelian F, Schaer CA, Puglia M, Buehler PW, Schaer DJ. Different target specificities of haptoglobin and hemopexin define a sequential protection system against vascular haemoglobin toxicity. Free Radic Biol Med 2015; 89: 931-43. 6. Dewey M. metap: meta-analysis of significance values. 2020 7. Diringer MN, Bleck TP, Claude Hemphill J 3rd, Menon D, Shutter L, Vespa P, et al. Critical care management of patients following aneurysmal subarachnoid hemorrhage: recommendations from the Neurocritical Care Society’s Multidisciplinary Consensus Conference. Neurocrit Care 2011; 15: 211-40. 8. Elmer J, Harris DR, Sun G, Palmer AF. Purification of hemoglobin by tangential flow filtration with diafiltration. Biotechnol Prog 2009; 25: 1402-10. 9. Hugelshofer M, Buzzi RM, Schaer CA, Richter H, Akeret K, Anagnostakou V, et al. Haptoglobin administration into the subarachnoid space prevents hemoglobin-induced cerebral vasospasm. J Clin Invest 2019; 129: 5219-35. 10. Kassambara A, Mundt F. factoextra: Extract and Visualize the Results of Multivariate Data Analyses [Internet]. 2020; Available from: https: / / CRAN.R-project.org / package=factoextra 11. Kikinis R, Pieper SD, Vosburgh KG. 3D Slicer: A Platform for Subject-Specific Image Analysis, Visualization, and Clinical Support. Intraoperative Imaging and Image-Guided Therapy 2014: 277-89. 12. Lawrence MA. ez: Easy Analysis and Visualization of Factorial Experiments [Internet]. 2016; Available from: https: / / CRAN.R-project.org / package=ez 13. Liberzon A, Birger C, Thorvaldsdottir H, Ghandi M, Mesirov JP, Tamayo P. The Molecular Signatures Database (MSigDB) hallmark gene set collection. Cell Syst 2015; 1: 417-25. 14. Md Riaz Ahmed. ROCit- An R Package for Performance Assessment of Binary Classifier with Visualization [Internet]. 2019 [cited 2020 Sep 17] Available from: https: / / openprairie.sdstate.edu / datascience_symposium / 2019 / posters / 6 / 15. Robin X, Turck N, Hainard A, Tiberti N, Lisacek F, Sanchez J-C, et al. pROC: an open-source package for R and S to analyze and compare ROC curves [Internet]. BMC Bioinformatics 2011; Available from: http: / / dx.doi.org / 10.1186 / 1471-2105-12-77 16. Sachs MC. plotROC: A Tool for Plotting ROC Curves [Internet]. J Stat Softw 2017; Available from: http: / / dx.doi.org / 10.18637 / jss.v079.c02 17. Sing T, Sander O, Beerenwinkel N, Lengauer T. ROCR: visualizing classifier performance in R.Bioinformatics 2005; 21: 3940-1. 18. Sloan MA, Haley EC Jr, Kassell NF, Henry ML, Stewart SR, Beskin RR, et al. Sensitivity and specificity of transcranial Doppler ultrasonography in the diagnosis of vasospasm following subarachnoid hemorrhage. Neurology 1989; 39: 1514-8. 19. Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A 2005; 102: 15545-50. 20. Weidert S, Andress S, Linhart C, Suero EM, Greiner A, Bocker W, et al. 3D printing method for next-day acetabular fracture surgery using a surface filtering pipeline: feasibility and 1-year clinical results. International Journal of Computer Assisted Radiology and Surgery 2020; 15: 565-75. 21. Wood SN. Generalized Additive Models: An Introduction with R, Second Edition. CRC Press; 2017

Claims

1. A pharmaceutical composition for treating or preventing adverse secondary neurological outcomes in a subject following a hemorrhagic stroke involving extravascular hemolysis and release of cell-free heme and / or cell-free hemoglobin (Hb) into the cerebrospinal fluid (CSF), the composition comprising a therapeutically effective amount of hemopexin (Hx) and a pharma- ceutical acceptable carrier, wherein the treatment or prevention comprises exposing the CSF of a subject in need thereof to a therapeutically effective amount of Hx for a period of time sufficient to allow Hx to complex with the cell-free heme, thereby permitting its neutralization.

2. 2. The pharmaceutical composition according to claim 1, wherein the hemorrhagic stroke is spontaneous or traumatic hemorrhage, intraventricular or subarachnoid hemorrhage, preferably aneurysmal subarachnoid hemorrhage.

3. 2. The pharmaceutical composition of claim 1, wherein the adverse secondary neurological outcome is selected from the group consisting of delayed ischemic neuropathy (DIND), delayed cerebral ischemia (DCI), neurotoxicity, inflammation, nitric oxide depletion, oxidative tissue injury, cerebral vasospasm, cerebrovascular reactivity, and edema, preferably an adverse secondary neurological outcome in the brain parenchyma.

4. Exposing the CSF to Hx within about 21 days after the onset of bleeding, preferably for about 2 to about 14 days. The pharmaceutical composition according to claim 1, comprising the step of:

5. 2. The pharmaceutical composition of claim 1, wherein the Hx is for intracranial, intraventricular or intrathecal administration to a subject, preferably intrathecally into the spinal canal or into the subarachnoid space.

6. The treatment or prevention may include: (i) obtaining a CSF sample from the subject after bleeding and prior to exposing the CSF to Hx; (ii) measuring the amount of cell-free Hb or cell-free heme in the CSF sample obtained in step (i); and (iii) determining at least an equimolar amount of Hx based on the concentration of cell-free Hb, expressed as heme equivalents, or cell-free heme obtained from step (ii); 2. The pharmaceutical composition of claim 1, further comprising:

7. 2. The pharmaceutical composition of claim 1, wherein the therapeutically effective amount of Hx is from about 2 μM to about 1 mM, preferably from about 2 μM to about 400 μM, more preferably from about 5 μM to about 200 μM, and most preferably from about 10 μM to about 200 μM.

8. The pharmaceutical composition of claim 1 , wherein the treatment or prevention further comprises removing Hx:cell-free heme complexes formed in the CSF.

9. The treatment or prevention may further comprise the step of exposing the CSF to a therapeutically effective amount of Hx exogenously, preferably (i) obtaining a sample of CSF from the CSF compartment of a subject following bleeding; (ii) adding Hx to the CSF sample of step (i) to obtain an Hx-enriched CSF sample; (iii) administering to the subject an Hx-enriched CSF sample, thereby exposing the CSF compartment of the subject to a therapeutically effective amount of Hx for a period of time sufficient to allow Hx to form a complex with cell-free heme in the CSF compartment of the subject; and (iv) optionally repeating steps (i) to (iii).

2. The pharmaceutical composition of claim 1, further comprising:

10. The treatment or prevention may further comprise the step of exposing the CSF to a therapeutically effective amount of Hx exogenously, preferably (i) removing a volume of CSF from the CSF compartment of a subject following hemorrhage; (ii) providing an artificial CSF comprising Hx; (iii) administering the artificial CSF of (ii) to a subject, thereby exposing the CSF compartment of the subject to a therapeutically effective amount of Hx for a period of time sufficient to allow Hx to form a complex with cell-free heme in the CSF compartment of the subject; and (iv) optionally repeating steps (i) to (iii).

2. The pharmaceutical composition of claim 1, further comprising:

11. The treatment or prevention may further comprise the step of exposing the CSF to a therapeutically effective amount of Hx exogenously, preferably (i) obtaining CSF from a subject following a hemorrhage; (ii) exposing the CSF resulting from step (i) to Hx under conditions that allow Hx, or a functional analogue thereof, to form a complex with cell-free heme in the CSF; (iii) extracting Hx:cell-free heme complexes from the CSF following step (ii) to obtain a heme-depleted CSF having a reduced amount of cell-free heme when compared to the CSF obtained from step (i); (iv) optionally repeating steps (ii) and (iii) to obtain heme-depleted CSF that is substantially free of cell-free heme; and (v) administering the heme-depleted CSF resulting from step (iii) or step (iv) to the CSF compartment of the subject.

2. The pharmaceutical composition of claim 1, further comprising:

12. 12. The pharmaceutical composition of claim 11, wherein the treatment or prevention further comprises the step of adding a therapeutically effective amount of Hx to the heme-depleted CSF prior to step (vi).

13. The pharmaceutical composition of claim 1 , wherein Hx is a recombinant protein and / or derived from plasma.

14. 2. The pharmaceutical composition of claim 1, wherein the treatment or prevention further comprises exposing the subject's CSF to a therapeutically effective amount of haptoglobin (Hp) for a period of time sufficient to allow Hp to form a complex with cell-free Hb, thereby permitting its neutralization.

15. The treatment or prevention may include: (i) obtaining a CSF sample from the subject after bleeding and prior to exposing the CSF to Hp; (ii) measuring the amount of cell-free Hb in the CSF sample obtained in step (i); and (iii) determining at least an equimolar amount of Hp based on the concentration of cell-free Hb obtained from step (ii); 15. The pharmaceutical composition of claim 14, further comprising:

16. The pharmaceutical composition of claim 14, wherein the treatment or prevention further comprises removing Hp:cell-free Hb complexes formed in the CSF.

17. 2. The pharmaceutical composition of claim 1, wherein the treatment or prevention further comprises administering to the subject a second agent for treating or preventing adverse secondary neurological outcomes after hemorrhagic stroke, preferably the second agent is a vasodilator, more preferably sydnon or sodium nitroprusside.

18. 1. Use of a therapeutically effective amount of hemopexin (Hx) in the manufacture of a medicament for treating or preventing adverse secondary neurological outcomes in a subject following a hemorrhagic stroke involving extravascular hemolysis and release of cell-free heme and / or cell-free hemoglobin (Hb) into cerebrospinal fluid (CSF), wherein Hx forms a complex with and is thereby neutralized by the cell-free heme.

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