WO2022191759A1

WO2022191759A1 - A polymer-based carbonyl scavenger for detection and treatment of ischemic injuries

Info

Publication number: WO2022191759A1
Application number: PCT/SE2022/050231
Authority: WO
Inventors: Felix SELLBERG; Ola Mattias ÅBERG; Erik Berglund; David Berglund; Fanny FREDRIKSSON
Original assignee: Cremed AB
Current assignee: Cremed AB
Priority date: 2021-03-09
Filing date: 2022-03-09
Publication date: 2022-09-15
Anticipated expiration: 2023-09-09

Abstract

The present invention provides a polymer, a complex and a pharmaceutical composition comprising a carbazate-modified polyvinyl alcohol (PVAC) according to Formula (I) for use in preventing, diagnosing, detecting, monitoring, alleviating, restoring and/or treating ischemic injuries in an organ. A kit comprising said complex is also provided.

Description

Title

A polymer-based carbonyl scavenger for detection and treatment of ischemic injuries.

Technical field

The present invention relates to the field of ischemia, detection and treatment of the same.

Background of the invention

Polyvinylalchol-carbazate (PVAC) is a soluble functionalized polymer acting as a carbonyl scavenger. PVAC consists of a PVA backbone, functionalized with carbazate moieties that molecularly targets biological electrophiles such as carbonyls, and PVAC is capable of binding multiple carbonyls. Carbazate contains a neutrophilic nitrogen atom with similar properties to an amine. Compared to an amine carbazate allows for increased stabilization of electrons making it more reactive and the formed bonds more stable. Normally, the excess of such substances is taken care of by reducing agents such as glutathione, or enzymes such as aldehyde dehydrogenase.

In recent years it has been found that PVAC may be effective in prevention or treatment of bacteria-induced infections and diseases (WO 19016189 A1), stabilizing erythrocytes (WO 2018172422 A1), and treating or preventing inflammatory-related conditions (WO 2012105887 A1).

Ischemia results from an insufficient supply of blood to tissues or organs, usually caused by a blocked artery. For example, during kidney ischemia, the endothelium and tubular cells are injured, which lead to acute renal dysfunction. In critical limb ischemia, adducts accumulate and lead to progressive myofiber degeneration. Both represent major clinical problems with high mortality and morbidity. Insufficient blood supply creates insufficiency of oxygen and reduces the availability of nutrients and increases metabolic waste products. Restoration of blood supply to previously ischemic tissues causes additional damage known as ischemia-reperfusion injury (IRI) that compounds the initial ischemic insult's damage [1]

Reestablishment of blood flow is essential to salvage ischaemic tissues. IRI occurs in a wide range of organs including the heart, lung, kidney, gut, skeletal muscle and brain and may involve not only the ischaemic organ itself but may also induce systemic damage to distant organs, potentially leading to multi-system organ failure. Reperfusion injury is a multi-factorial process resulting in extensive tissue destruction [Fitridge R, Thompson M, editors. Adelaide (AU): University of Adelaide Press; 2011]

Specifically, the reintroduction of blood flow re-oxygenates the tissues, causing excess production of reactive oxygen species (ROS). As ROS reacts with fatty polyunsaturated fatty acids in the cell membrane reactive carbonyl species (RCS) are formed, i.e. , aldehydes including malondialdehyde (MDA), 4-hydroxy-2-nonenal (4- HNE) and acrolein. They are highly reactive molecules that react with proteins and form adducts, ultimately disrupting the protein's structure and function. Increased RCS levels have been demonstrated both in kidney and critical limb ischemia in animal models and humans.

Lung transplant is an established therapy for patients suffering from end stage lung disease. However, lung transplant recipient survival is poor compared to recipients of other solid organs, due largely to the development of graft dysfunction from ischemic reperfusion injury (IRI). Some transplanted recipients develop life threatening cause of acute respiratory failure (ARDS) due to IRI.

Apart from lung transplants over 190,000 Americans per year develop ARDS due to sepsis, severe injury, pneumonia viral infection such as influenzas and more recently the coronavirus disease pandemic (COVID 19). The active COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) primarily targets the respiratory system, with many patients rapidly developing ARDS. Although COVID- 19 pathogenesis is still not fully understood, therapeutic approaches have been similar to those for treatment of IRI/ARDS.

In view of the problems discussed above it is a continuous unmet need for alternative treatment strategies with novel modes of action. Hence, there is a need for new compositions for scavenging known mediators during for example IRI and ARDS.

Therefore, there is an urgent need to discover new therapeutic targets.

Summary of the invention

The present disclosure provides a polymer compound, a complex or composition, and a pharmaceutical composition comprising said polymer compound. The polymer compound is a carbazate-modified polyvinyl alcohol (PVAC) as represented by Formula I below. In a first aspect, there is provided a polymer compound (PVAC) for use in preventing, alleviating, restoring and/or treating ischemic injuries in a subject. The polymer compound comprising a carbazate-modified polyvinyl alcohol (PVAC) is represented by Formula I

Formula I wherein unmodified repeat units of PVA are denoted with n and carbazate groups conjugated to repeat units are denoted by m, as defined in present claim 1. Further, the degree of polymerization (DP) of the polymer is in the range of 100 < DP < 2000. In another embodiment the degree of polymerization (DP) of the polymer is in the range of 200 < DP < 1800. In yet another embodiment the degree of polymerization (DP) of the polymer is in the range of 300 < DP < 1500, or 400 < DP < 1200, or 500 < DP < 1000, or 600 < DP < 800. Further, the degree of substitution (DS) (m) of carbazate may be in the range of 0.5-50 %. In another embodiment, the degree of substitution (DS) (m) of carbazate may be in the range of range of 2-40 %. In yet another embodiment the degree of substitution (DS) (m) of carbazate may be in the range of range of 2.5-20 %. In yet another embodiment the degree of substitution (DS) (m) of carbazate may be in the range of range of 5-15 %, and in yet another embodiment the degree of substitution (DS) (m) of carbazate may be in the range of range of 7.5-10 %, or in yet another embodiment the degree of substitution (DS) (m) of carbazate may be 7.5 % or in another embodiment the degree of substitution (DS) (m) of carbazate may be 5 %. Further, the carbazate content of the polymer may be in the range of 0.1 -10 mmol/g polyvinyl alcohol carbazate, or preferably in the range of 0.5-8 mmol/g polyvinyl alcohol carbazate, or more preferably in the range of 1-6 mmol/g polyvinyl alcohol carbazate, or even more preferably in the range of 2 mmol/g polyvinyl alcohol carbazate. Further, the polymer compound (PVAC) represented by Formula I described above may preferably be in the form of a lyophilizate.

In a second aspect there is provided a complex or composition comprising the polymer compound (PVAC) described above, and an agent. The agent may be a labelling molecule or agent. Further, the labelling molecule or agent may be a radiolabel, for example [¹⁸F]4-fluorobenzaldehyde, or a fluorescent agent for example, fluorescein isothiocyanate (FITC). Further, non-radioactive contrast agents used for improving pictures of the inside of the body may also be used. Further, contrast agents may be for example iodine, barium or gadolinium based. The complex or composition may further be useful in diagnosing an ischemic injury or to monitor a treatment. The complex/composition may also be in the form of a lyophilizate. PVAC accumulates in ischemic tissue, thereby enabling using labelled PVAC for diagnosing and monitoring ischemic injuries/events.

In a third aspect there is provided a pharmaceutical composition comprising a polymer compound (PVAC) as defined in present claim 1 and Formula I. The pharmaceutical composition is for use in preventing, alleviating, restoring and/or treating an ischemic injury or event in a subject in need thereof. The pharmaceutical composition may further comprise a pharmaceutically acceptable excipient. Further, the polymer (PVAC), composition, complex and pharmaceutical composition described above may be dissolved in a suitable solvent before administration. The solvent may be aqueous such as water, sodium chloride, buffer, i.e. , any suitable solvent for the purpose may be used. Further, the pharmaceutical composition may also be in the form of a lyophilizate. Further, there is provided that the polymer (PVAC), complex or composition (label + PVAC), and the pharmaceutical composition comprising PVAC described above is provided for use in the treatment of an ischemic injury or event. The ischemic injury or event may be such as for example ischemia-reperfusion injury (IRI), acute myocardial infarction (Ml), stroke, critical limb ischemia (CLI) and/or acute respiratory distress syndrome (ARDS). Further, the polymer, complex/composition and/or the pharmaceutical composition may be used in combination with other common therapies for ischemia- reperfusion injury (IRI), acute myocardial infarction (Ml), stroke, critical limb ischemia (CLI) and acute respiratory distress syndrome (ARDS), such as supportive intensive care, corticosteroids or other anti-inflammatory therapies. Further, the administration of PVAC, complex/composition comprising PVAC + label, and the pharmaceutical composition described above is for example performed by intravenous, intramuscular, intraperitoneal, or subcutaneous injection or infusion.

In a fourth aspect there is provided a method for diagnosing, visualizing, detecting, and monitoring an ischemic injury or event in a subject. The method comprising administrating a complex or composition comprising a polymer such as PVAC and a labelling agent or molecule to a subject, allowing the complex or composition to reach the site, and thereafter detecting said complex or composition comprising the polymer and labelling agent or molecule. The detection may be performed by PET, MR, IR, CT and/or other X-ray based diagnostics.

In fifth aspect, there is provided a kit comprising a polymer complex/composition as described above, i.e. , PVAC and a labelling agent or molecule, for detection and/or monitoring an ischemic injury in a subject and instructions for use. Further there is provided a method for detection of ischemia or an ischemic injury in a subject, wherein the method comprises the steps of injecting a polymer complex (PVAC + Label) as described above, and thereafter detecting said polymer complex/composition by PET, ultrasound, magnetic resonance, MR, IR, x-ray computed tomography (CT), and/or other X-ray based diagnostics.

In a sixth aspect there is also provided a method for preventing, alleviating, restoring and/or treating an ischemic injury or event in an organ. The method comprising administrating a pharmaceutically effective dose of a polymer comprising a carbazate-modified polyvinyl alcohol (PVAC) according to Formula I

Formula I.

Further the unmodified repeat units of PVA are denoted with n and carbazate groups conjugated to repeat units are denoted by m, wherein the degree of polymerization (DP) is in the range of 100 < DP < 2000, and the degree of substitution (DS) (m) of carbazide is in the range of 0.5-50 %, preferably 2-40 %, or more preferably 2.5-20 % or even more preferably 5-15 %, or 7.5-10 %. Further, the carbazate content is in the range of 0.1-10 mmol/g polyvinyl alcohol carbazate, preferably 0.5-8 mmol/g polyvinyl alcohol carbazate, more preferably 1-6 mmol/g polyvinyl alcohol carbazate, and even more preferably 2 mmol/g polyvinyl alcohol carbazate. Further, said organ may be for example lung, liver, heart, pancreas and/or kidney. Further, the ischemic injury may be selected from reperfusion, IRS and ARDS, and similar conditions, injuries. Further, the administration may be performed by intravenous, intramuscular, intraperitoneal, or subcutaneous injection or infusion. Further, the method above may be performed in combination with another treatment, of standard care in the transplant area.

In a seventh aspect there is provided a method for preventing, alleviating, restoring and/or treating an ischemic injury or event in a subject. The method comprising administrating a therapeutically effective amount of the polymer PVAC defined in Formula I, or the pharmaceutical composition comprising PVAC defined in Formula I to a subject in need of such treatment. Further, the ischemic injury may for example be caused by anyone of reperfusion, ischemia-reperfusion injury (IRI), acute myocardial infarction (Ml), stroke, critical limb ischemia (CLI), acute respiratory distress syndrome (ARDS) and/or a corona virus infection. The corona virus infection may be corona virus disease 2019 (CGV!D-19). Further, the administration of said PVAC or pharmaceutical composition comprising PVAC may for example be performed by intravenous, intramuscular, intraperitoneal, or subcutaneous injection or infusion. Moreover, the method for preventing, alleviating, restoring and/or treating ischemia, may be performed in combination with other common therapies for ischemia-reperfusion injury (IRI), acute myocardial infarction (Ml), stroke, critical limb ischemia (CLI) and acute respiratory distress syndrome (ARDS), such as for example supportive intensive care, corticosteroids or other anti-inflammatory therapies.

Further, the method may be used in combination with another treatment wherein ischemic injury is a risk, i.e. , to monitor and detect if early signs of an ischemic injury occur.

In an eight aspect there is also provided a method for visualizing ischemia, i.e., diagnosing, detecting, and monitoring an ischemic injury or event, said method comprising the steps of administrating a polymer complex (PVAC and an agent) as described above, or a pharmaceutical composition comprising the polymer complex/composition into a subject, and detecting said polymer by PET, MR, IR, CT, and other X-ray based diagnostics. In a ninth aspect there is also provided a method for preventing, restoring and/or treating an ischemic injury in an organ, ex vivo. The method keeps and/or restores the oxygen at a good level in said organ. The aim of the organ may for example be to be transplanted to a subject. The method comprising administrating a polymer comprising a carbazate-modified polyvinyl alcohol (PVAC) according to Formula I

Formula I.

Further the unmodified repeat units of PVA are denoted with n and carbazate groups conjugated to repeat units are denoted by m, wherein the degree of polymerization (DP) is in the range of 100 < DP < 2000, and the degree of substitution (DS) (m) of carbazide is in the range of 0.5-50 %, preferably 2-40 %, or more preferably 2.5-20 % or even more preferably 5-15 %, or 7.5-10 %. Further, the carbazate content is in the range of 0.1-10 mmol/g polyvinyl alcohol carbazate, preferably 0.5-8 mmol/g polyvinyl alcohol carbazate, more preferably 1-6 mmol/g polyvinyl alcohol carbazate, and even more preferably 2 mmol/g polyvinyl alcohol carbazate. Further said organ may be lung, liver, pancreas, heart and/or kidney.

In a tenth aspect there is provided a method for preventing, alleviating, restoring and/or treating an ischemic injury in a subject. Said method comprising administrating a therapeutically effective amount of the polymer PVAC defined in Formula I above, or the pharmaceutical composition comprising PVAC as defined in Formula I above to a subject in need of such treatment. The Ischemic Injury may be caused by reperfusion, ischemia-reperfusion injury (IRI), acute myocardial infarction (Ml), stroke, critical limb ischemia (CLI), acute respiratory distress syndrome (ARDS) and/or a corona virus infection. The corona virus infection may be corona virus disease 2019 (COVID-19). Further, administration of PVAC or the pharmaceutical composition comprising PVAC as defined above may be performed by intravenous, intramuscular, intraperitoneal, or subcutaneous injection or infusion. The method may also be performed in combination with other common therapies for ischemia reperfusion injury (IRI), acute myocardial infarction (Ml), stroke, critical limb ischemia (CLI) and/or acute respiratory distress syndrome (ARDS), such as supportive intensive care, corticosteroids or other anti-inflammatory therapies.

All aspect may be combined in different embodiments unless expressly stated otherwise. Additional advantages of the disclosed subject matter will be set forth in part in the description that follows and the Figures, and will be obvious from the description, or can be learned by practice of the aspects described below.

Brief description of drawings

Fig. 1 Shows the synthesis of the PVAC-molecule (a) and labelling of PVAC with [18F]FBA (b) and FITC (c), respectively.

Fig. 2 Shows the elution profiles of [¹⁸F]FBA-PVAC and [¹⁸F]FBA, respectively.

Fig. 3 Shows the different reactive mediators (RCS) formed during IRI tested for their affinity towards PVAC. Figs. 4 A-C Show the pharmacokinetics of PVAC in PET scans of rats injected

(I.V or I.J) with either [18F]FBA-PVAC or FITC-PVAC.

Fig. 5 Shows the biodistribution of [¹⁸F]FBA-PVAC for different organs, ex vivo.

Figs. 6 A-C Show uptake of labelled PVAC in ischemic kidney tissue compared to non-ischemic kidney tissue.

Figs. 7 A-C Show uptake of labelled PVAC in an ischemic and non-ischemic limb model.

Figs. 8 A-C Show that PVAC treatment of IRI is potent in a model of lung IRI.

Detailed description of the invention Before the invention is described by examples it is to be understood that this invention is not limited to the particular configurations, process steps, and materials disclosed herein as such configurations, process steps, and materials may vary somewhat.

It is also to be understood that the terminology employed herein is used for the purpose of describing particular embodiments only and is not intended to be limiting since the scope of the present invention will be limited only by the appended claims and equivalents thereof.

All references cited are incorporated herein by reference in their entirely and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

The present invention is best understood by reference to the following definitions, the figures and exemplary disclosures provided herein.

In this specification the term PVAC means carbazate activated Polyvinyl alcohol, Carbazate modified polyvinyl alcohol, Polyvinyl alcohol carbazate, having the chemical name Poly[1 -hydroxyethylene)-ran-(1 -hydrazinecarboxylatoethylene)].

In this specification the term degree of polymerization (DP) means the average numberof monomeric units in a macromolecule or polymer.

In this specification the term degree of substitution (DS) means the average number (%) of substituent groups attached per base unit per monomeric unit.

In this specification, unless otherwise stated, the term "pharmaceutically acceptable excipient" means a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

In this specification, unless otherwise stated, the term "pharmaceutically active compound" encompasses any substance that will produce a therapeutically beneficial pharmacological response when administered to a host, including both humans and animals.

In this specification the term “targeted therapy” means that a pharmacologically active compound is bound to PVAC or in the case were PVAC is the pharmacologically active compound, PVAC is bound to a targeting compound, for example an antibody.

In this specification PVAC itself may be the pharmaceutically active compound and/or the targeting compound.

Visualized therapy or monitored therapy means that PVAC is bound to a label, labelling agent, for example a radiolabel, which enables monitoring and/or visualization of the journey, stops and end point(s) inside the body of a subject. In this specification the term “detection agent”, “detection compound”, “label” or “detection molecule” means an agent, a compound and/or molecule that can be detected in vivo and/or in vitro. The detection agent can also enable that the agent can be monitored (traced, followed) when administered into a subject’s body (blood stream). Fluorescein derivatives may be used as labels, for example Fluorescein isothiocyanate (FITC) which also was used in the present study. FITC is a derivative of fluorescein used in wide-ranging applications. Another possibility is to visualize an agent or a pharmacologically active compound by using a radiolabel, i.e. , to label PVAC with a radioactive atom or substance, in this study [¹⁸F] was used to radiolabel PVAC. The radiolabel makes it possible to monitor distribution, uptake, deposit, excretion, as well as metabolism of PVAC in vivo.

In this specification the term "administering”, or "administration" means providing a drug to a subject in a manner that is pharmacologically useful.

In this specification the term Ischemia or ischaemia, ischemic injury and ischemic event means a restriction in blood supply to tissues, causing a shortage of oxygen that is needed for cellular metabolism to keep tissue alive. Ischemia is generally caused by problems with blood vessels, with resultant damage to or dysfunction of tissue. It also means local anemia in a given part of a body sometimes resulting from constriction (such as vasoconstriction, thrombosis or embolism). Ischemia comprises not only insufficiency of oxygen, but also reduced availability of nutrients and inadequate removal of metabolic wastes. Ischemia can be partial (poor perfusion) or total.

In this specification the term reperfusion injury, ischemia-reperfusion injury (IRI) and reoxygenation injury, means the tissue damage caused when blood supply returns to tissue after a period of ischemia or lack of oxygen (anoxia or hypoxia). The absence of oxygen and nutrients from blood during the ischemic period creates a condition in which the restoration of circulation results in inflammation and oxidative damage through the induction of oxidative stress rather than (or along with) restoration of normal function.

In the present disclosure Positron emission tomography is abbreviated (PET), magnetic resonance is abbreviated (MR), Infra-red is abbreviated ((IR), and computer tomography is abbreviated (CT). Material and methods

Synthesis of carbazate-modified polyvinyl alcohol (PVAC)

All PVAC used in the experiments was synthesized at Angstrom Laboratory in Uppsala [1]

Labelling of PVAC

Synthesis of the labelling agent 4-[¹⁸F]fluorobenzaldehyde

All chemicals used in radiolabeling were bought from Sigma-Aldrich and used as received unless otherwise stated. For radiolabelling of PVAC, 4- [¹⁸F]fluorobenzaldehyde ([¹⁸F]FBA) was used as the labelling agent. (4- Formylphenyl)trimethylammonium trifluoromethane sulfonate was synthesized as described in the literature with minor modifications.

Labelling of PVAC utilizes the carbazate moiety, scavenging of RCS is also mediated by the carbazate. To minimize our influence on the scavenging capabilities of PVAC, we only utilized 5 % of carbazate when performing radiolabeling. When 5 % of carbazate groups are labelled, around 80 % of its scavenging properties remain [2]

Water used was MillieQ water 18.2 MW. For buffers pH was measured with a calibrated Mettler Toledo SevenEasy pH meter. Acetate buffers, 50 mM, were prepared by dissolving 0.205 g sodium acetate in 50 mL of water. The solution was fractionated, and acetic acid was added until the desired pH was achieved. Radioactivity was measured in an ion chamber (Veenstra Instruments). Glass reactor vials were heated in a React-Therm™ I aluminium heating block (Thermo Fisher Scientific) equipped with an external temperature probe immersed in a capped reference vial filled with DMSO placed next to the reaction vial. Illustra NAP™-5 column (G.E. Healthcare Life Sciences) was preconditioned with PBS (2 ml).

Freeze-dried PVAC (2.8 mg) was dissolved in sodium acetate buffer (pH 4.0, 200 pi). To the solution was added 4-[¹⁸F]fluorobenzaldehyde as a solution in ethanol (100 mI) and the reaction was allowed to react in room temperature for 15 min before it was quenched with PBS (200 mI). The solution was added to a NAP™-5 column and eluted in 250 mI fractions with PBS. Radioactivity and pH of the resulting carbazate hydrazine conjugate product solution was measured, Fig 1b. FITC-PVAC was produced via conjugation of PVAC to fluorescein isothiocyanate (FITC). In brief, PVAC was dissolved in Dl water (100 mg, 5 ml) and FITC dissolved in DMSO was added (1 mg, 0.1 ml), the reaction was stirred for 2 h, figure 1c. Purification was carried out via size exclusion with a dialysis membrane (ThermoFisher, SnakeSkin dialysis tubing, 3500 MWCO). FITC-PVAC has been used previously for flow cytometry and microscopy [2]

Investigation of affinity towards IRI mediators

PVACs ability to scavenge and neutralize mediators formed during IRI was investigated in vitro (Fig. 3). Megazyme Acetaldehyde Assay Kit (Bray, Ireland) was used to measure free RCS. The RCS assayed were oxidized proteins, methylglyoxal, malondialdehyde and acrolein (all obtained from Sigma). The kit is designed to measure acetaldehyde levels by using the physiological reaction with the enzyme aldehyde dehydrogenase (ALDFI). A side product NADFI is formed in the reaction where aldehydes are reduced to carboxylic acids, which correlates to the present amount of free acetaldehyde. NADFI can be quantified by measuring absorbance at 340 nm. ALDFI is reactive towards other aldehydes and RCS in general. The kit was used to the manufacturer's instructions with the addition of several targets for the enzyme and a preincubation step with or without PVAC.

To produce oxidized protein albumin (Sigma) was dissolved in PBS (1 ml, 100 mM) acrolein was added to the mixture to a final concentration of 200 mM. The mixture was incubated for 30 min under stirring and then purified via dialysis (ThermoFisher, SnakeSkin dialysis tubing, 3500 MWCO). PVAC was dissolved in PBS (5 ml, 2.5 mg/ml) the mediators were dissolved in PBS (1 ml per mediator, 2.2 mM). Mediators and were mixed with PVAC or vehicle (PBS) in a 96-well microplate (100 pi mediator, 100 mI of PVAC/PBS) the plate was placed on a shaker (450 rpm) for 30 min. The mixture was then added to wells containing reaction buffers, and baseline absorbance was measured. ALDFI was added and, the plate was incubated at 37 °C until absorbance stabilized. The plate was read on a Synergy FITX plate reader (BioTek).

Animals

All animals were treated following the recommendations from the National Guide for the Care and Use of Laboratory Animals [3] The animals were housed for one week before surgery and maintained under standard laboratory conditions (+ 22°C, with a 12-h light/dark cycle and fed pellet food and water ad libitum). The regional animal ethics committee approved the studies in Uppsala (C56/16) and Stockholm north (N81/14).

Experimental design

In order to determine the kinetics and biodistribution of PVAC eleven Sprague- Dawley rats, 8 female and 3 male, were after sedation injected either I.V. (tail vein, n=9) or I.J. (hind limb knee, n=2) with [18F]FBA-PVAC. Both I.J animals and five of the I.V. animals were then placed in a PET camera. For 90 min, a dynamic scan took place, followed by a C.T. scan, and finally the animals were euthanized. The remaining four animals, 2 female 2 male, were sedated for 90 min. They were then euthanized, and organs recovered for the study of biodistribution.

Nine Sprague-Dawley rats, all female, were used in the ischemic models for kidney (n=6) and hind limb (n=3). There was no need for sham-operations since each animal could serve as their internal control by having both a naive kidney and hind limb. Instead, in the ischemic kidney model, three animals were blocked by injecting PVAC before injecting [18F]FBA-PVAC. All animals were then placed in a PET camera and underwent a 60 (kidney) or 90 (hind limb) min dynamic scan, followed by a C.T. or MRI scan. They were then euthanized, and organs recovered for the study of biodistribution.

Radioactivity in the organs was measured using Nal well counter to determine the biodistribution of [18F]FBA-PVAC. These studies were performed at the preclinical PET-MRI platform, Uppsala.

The kinetic study of fluorochrome labelled (FITC-PVAC) PVAC was performed at Adlego, Stockholm where six male Sprague-Dawley rats were injected either I.V. (n=3, 2.5 mg/kg bw) or I.J. (n=3, 2.5 mg/animal) with FITC-PVAC. Blood was drawn via tail vein before injection and at multiple time-points (5, 15, 30, 60 min, 2, 4, 6, 24, 48, 72, 96,168 h) post-injection, to later be stored frozen as serum. Urine was collected during 0-6, 6-24, 24-48, 48-72 and, 72-96 h to later be stored frozen. The animals were euthanized at the end of the experiment.

To study the kinetics of FITC-PVAC, a 7-point standard curve was made by dissolving FITC-PVAC in water. Frozen serum and urine samples were thawed and diluted 1:1 in water. Standards and samples were placed in a 96-well plate in duplicates. The samples were then exited at 485 nm, and emission was registered at 528/20 nm using a Synergy HTX plate reader (BioTek). The data were exported and analyzed in GraphPad prism, and the standard curve was calculated using a 4- parameter logistic curve fit. Blank was subtracted for the standard curve, and urine samples wells containing water was used for serum samples the value obtained before injection was used, the values were then interpolated and reported as concentration (pg/ml). For urine samples, the total amount was also calculated by taking the concentration * the urine volume.

Surgical procedure

Anaesthesia was induced in a sealed chamber with inhaled 4 % isoflurane and maintained using a facemask delivering 2.5 % isoflurane. All procedures were performed under clean but nonsterile conditions. Both a heating lamp and a blanket were used to prevent heat loss during surgery and later ischemia. For the renal ischemia model, an incision was placed under the left rib arc, through the skin, muscle and peritoneum, approximately 3 cm in length and a cotton swab was used to visualize the left kidney and the left renal artery gently. Ischemia was induced by placing a micro-clip on the left renal artery for 30-45 min. The clip was then removed, and reperfusion of the kidney was observed before wound closure using continuous resorbable 5-0 Vicryl® sutures (Johnson & Johnson AB, Sollentuna, Sweden) for the fascia, and continuous non-resorbable 4-0 Ethilon® sutures (Johnson & Johnson AB) for the skin. The surgical procedure took ~ 60-75 min.

For the hind limb ischemic model, a 2 cm long incision was made through the femoral triangle's skin to visualize the femoral artery. Ischemia was achieved by placing a micro-clip on the femoral artery resulting in a whitening of the rat's left foot. Ischemia was manifest for 45 - 60 min before the clip was removed and reperfusion occurred. The wound was closed using continuous resorbable 5-0 Vicryl® sutures (Johnson & Johnson AB, Sollentuna, Sweden) for the muscles, and continuous non-resorbable 4- 0 Ethilon® sutures (Johnson & Johnson AB) for the skin. The surgical procedure took ~75-90 min.

For the hind limb ischemic model, a 2 cm long incision was made through the femoral triangle's skin to visualize the femoral artery. Ischemia was achieved by placing a micro-clip on the femoral artery resulting in a whitening of the rat's left foot. Ischemia was manifest for 45-60 min before the clip was removed and reperfusion occurred. The wound was closed using continuous resorbable 5-0 Vicryl® sutures (Johnson & Johnson AB, Sollentuna, Sweden) for the muscles, and continuous non-resorbable 4- 0 Ethilon® sutures (Johnson & Johnson AB) for the skin. The surgical procedure took ~75-90 min.

Imaging

The ischemia and reperfusion injury was followed by single bolus injection (max volume 500 mI) of [¹⁸F]FBA-PVAC via a tail vein catheter, and the animal underwent small-animal PET examination of the organ area of interest for 90 min in list mode, followed by a C.T. examination for 3 min. The rats were kept sedated during the whole procedure by 3.0 % isoflurane, blended with 450 ml/min air/0₂ (controlled by an anaesthesia vaporizer) which were delivered through a face mask and placed on a heated bed of the PET-SPECT-CT system (TriumphTMTrimodality System, TriFoil Imaging, Inc., Northridge, CA, USA) to prevent hypothermia. An integrated physiologic monitoring system monitored breathing rate and body temperature. A whole-body scan was performed by multiple bed positioning that lasted for 15 min. The dynamic datasets were reconstructed into 26 timeframes (12 frames of 10 sec, 3 frames of 1 min, 5 frames of 5 min, 6 frames of 10 min) using a maximum- likelihood expectation maximization 3-dimensional algorithm (10 iterations). Small- animal PET data were analyzed using PMOD (version 3.510; PMOD Technologies Ltd. Switzerland) and Image J (Fiji, 2.0.0).

PET measurement was followed by M.R. examinations (in n= 2 rats from the renal ischemia model) with a 3 Tesla scanner (nanoScan, Mediso, Medical Imaging Systems, Budapest, Hungary). Images were acquired in the coronal and axial planes over the region of interest using a T1 -weighted spin-echo sequence and whole-body transmit/receiver coil. FOV 64x64 mm, acquisition matrix 256x192, slice thickness 1.3 mm, intersection gap 0.2 mm, resolution in-plane 0.25 x 0.33 mm², bandwidth per pixel 156.25 Hz, number of signal averages 2 or 3, and echo time (T.E.) 11 ms. Time repetition (T.R.) was between 540 and 880 ms, depending on the number of slices.

Non-human primate (NHP) Lung IRI model Animal models provide an important bridge between the laboratory bench and the hospital bed and help to validate therapeutic strategies directly prior to human clinical trials.

Rodent studies have proven useful for understanding the immunological basis of disease and molecular mechanisms of treatment for many human disorders.

However, rodent studies of lung injury frequently cannot be successfully extrapolated to humans, due to significant differences in anatomy, physiology and signaling factors. It is therefore essential to study non-human primate (NHP) pre-clinical models for IRI/ARDS research.

NHP IRI/ARDS models have the clear and necessary advantage of similarity to humans. The direct translational relevance and ready availability of human clinical reagents render NHP models essential for effective studies of IRI/ARDS. In this study, the cynomolgus macaque model was used.

Method for induction of ischemia-reperfusion injury

Animals underwent left thoracotomy and clamping of pulmonary hilum for 30 min to induce ischemia phase and unclamp the hilum to induce reperfusion injury. Serial blood samples, lung biopsies, muscle and fat tissue samples and urine were collected.

After ischemia-reperfusion injury it was expected to see signs of acute lung injury and ARDS. Main clinical sign is progressive development of pulmonary edema where clinical signs include tachypnea and frothy discharge from endotracheal tube with EtC0₂>65mmHG/P0₂<65mmHg. Acute lung injury can be developed within 1 h after reperfusion or can take 4 hrs or more. If animal develop signs earlier, effects of cytokines in the lung is observed and other organs such as severe hypotension and acidosis. Acidosis is managed with Na bicarbonate and address hypotension with appropriate pressor (Dopamine, Dobutamine, phenylephrine).

Animals used in this study was transferred from resident terminal anesthesia training protocol just prior to euthanasia. Accordingly, these animals are anesthetized and under ventilation with appropriate anesthetic agents, along with peripheral venous, arterial and urinary catheters.

Animal was placed on right lateral position and a 2-4 cm incision was made at 1 cm cranial to the cervical inlet, lateral to the trachea after injection of a 0.5 % bupivacaine local anesthesia given pre-incision. Blunt dissection allows access to the external or internal jugular vein and to the carotid artery. Indwelling central venous catheter was inserted into the left external/internal jugular vein and advanced 4-6 cm, and another catheter was inserted into the left common carotid artery and advanced 3 cm. Drawback of blood confirmed proper placement. The catheter will be adjusted as needed. The tubes were then sutured in place using appropriate 2-0 and 3-0 suture. However, it might be decided to place one line in the femoral artery if available. For this procedure, a 2 cm transverse incision was made in the groin and dissection carried out through the skin, fat, and fascia until the femoral vein and artery are exposed. Each was encircled with a vessel loop during dissection and ligation of collaterals. Appropriate suture (2-0 or 3-0) was placed around the vessels proximally and distally and the distal one was tied. Sequentially, a small incision was made in the vein and artery and a catheter was inserted approximately 4-6 cm. The upper tie is then tied down, securing the line in place. Animals was placed on this protocol when they are ready to undergo thoracotomy. Clinical Veterinarians and/or VCS was managing anesthesia in concordance with VCS SOPs and/or current veterinary standards.

Each animal was placed in a right lateral decubitus position with the left entire chest exposed from the neck to the umbilicus. Intradermal injection of bupivacaine local anesthesia was given pre-incision. A left lateral thoracotomy in fifth intercostal space was made using a #10 blade, and each layer was dissected down to the pleura. Incision was about 10 cm long.

Left pulmonary hilum was identified by dissecting the subcutaneous tissues. 5 min after heparinization of the animal, pulmonary hilum was clamped using a vascular clamp to study the ischemic phase.

After the ischemic phase, the vascular clamp was removed to study the lung reperfusion phase.

Blood pressure was maintained at 60 mmHg +/- 10 mmHg throughout the study. F1O2 will be maintained at 1. The animal received buprenorphine IV orfentanyl CRI before the thoracotomy. Intercoastal nerve block was performed using bupivicaine (total ~5ml) at the left intercostal space of either side by direct visualization after thoracotomy to provide additional analgesia. Initially, it was proposed to keep the animal under anesthesia for maximum of 8 h or until the development of pulmonary edema. Therefore, total procedure length from sedation to euthanasia may vary from about 4 to about 8 h.

The experimental end point is reached when the animal develops pulmonary edema (i.e. , P0₂<65mmHg on 20 % oxygen). Four animals received PVAC IV just prior to reperfusion at a dose of 5 mg/kg/bw, four animals received NaCI as a vehicle control.

Hemostasis was ensured by cauterizing the site. Experimental procedures and intervals were the same for all animals. Blood was collected from the pulmonary vein and/or peripheral catheters for in vitro assays.

IRI injury is a stepwise process where the thrombo inflammatory, oxidative and then cytokine mediated damages are initiated in successive manner. Therefore, it is essential to collect blood samples for serum and peripheral blood monocytes (PBMC) during early time points to catch oxidative damage, and during later time points to monitor the inflammatory changes.

Collection of 3-12.5 ml of blood during each time point was planned, not to exceed 15 % of the total blood volume over the proposed course of study.

Cynomolgus macaques tolerate 15 % blood volume removal without adverse effect. Total collected blood volume was calculated as 6 % of the body weight. Blood was collected before clamping, 5 min after reperfusion, 10 min after reperfusion, 30 min after reperfusion, 1 h after reperfusion, 2 h after reperfusion and 4 h after reperfusion and/or at the end of the study. The animal received intravenous fluid throughout the procedure. Digital pressure was applied to ensure hemostasis.

The amount of blood collected, and collection intervals was the same for all animals. Urine was collected from the urinary catheter of the anesthetized animal during the procedure. Analysis of blood gases was carried out using an IDEXX Vet Stat Analyzer.

Statistics

Graphpad prism version 8.4.3 (GraphPad Software, Inc, La Jolla, CA, USA) was used for all data handling and statistical analysis. Figure data are presented as averages ± standard error of the mean (SEM), significant differences (p-value < 0.05) are denoted with *. Statistics in the text are reported as p-value, mean difference between groups tested, 95% Cl (when possible to calculate), in the format of, p = 0.05, z% (x - y), where z is the mean difference between the groups and x and y are the bounds of the 95% Cl. Multiple group analyses were carried out using one parameter one-way ANOVA for parametric data and Kruskal-Wallis for nonparametric data. Two-way ANOVA was used to analyze two-parameter data sets (e. g. SUV overtime). ANOVA was followed with multiple comparisons testing between groups using Tukey's in the case of one-way ANOVA and Dunnett's in two-way ANOVA. When possible, technical replicates were nested in the analysis; when this was not possible, the replicates' mean was used instead. Results

Example 1

Fig. 1 shows the synthesis and structures of PVAC, [¹⁸F]FBA-PVAC and FITC- PVAC.

Example 2 Eluation profiles of [₁₈FBA-PVAC and [FJFBA

Elution profiles of [¹⁸F]FBA-PVAC (filled circles) and [¹⁸F]FBA (non-filled circles) on an lllustra NAP™ -5 column (equilibrated and eluted with PBS pH 7.4) is shown in Fig. 2. Reaction conditions were optimized, and the maximum reaction rate was seen at pH 4.0. Isolation of [¹⁸F]FBA-PVAC (filled circles) from [¹⁸F]FBA (non-filled circles) was achieved by size exclusion chromatography, an exclusion limit of 5 kDa was used. Mean radioactivity in the final solution was 7.48 MBq for animals undergoing dynamic scan after I.V. administration.

Example 3

Investigation of affinity towards IRI mediators PVACs ability to scavenge and neutralize mediators formed during IRI was investigated in vitro (Fig. 3).

Different RCS formed during IRI (oxidized albumin, methylglyoxal, malondialdehyde, acrolein) were oxidized using ALDFI. All RCS tested resulted in an increased NADPFI production when they were not pre-incubated with PVAC showing that ALDFI reduces them. When RCS were pre-incubated with PVAC they showed a reduced absorbance: 79.23 % (p = 0.0001) for free oxidized albumin, 100% (p = 0.0015) for methylglyoxal, 88.68 % (p = 0.0022) for malondialdehyde and 99.33 % (p = 0.0073) for acrolein, (Fig. 3). A reduced absorbance at 340 nm equals a lower concentration of free RCS, corresponding to neutralization by PVAC.

Examples 4 & 5

Pharmacokinetics & Biodistribution

This study aimed to create a radiolabeled PVAC and investigate the pharmacokinetics and biodistribution of PVAC in naive animals and ischemia models.

PVAC was labelled using radionuclide [¹⁸F]FBA to track the substance with PET. Sprague Dawley rats underwent an ischemic injury, either to the hind limb or to the kidney, while others served as controls.

Example 4

Pharmacokinetics

Figs. 4A-C show the pharmacokinetics of PVAC studied in PET scans of rats injected (I.V or I.J) with either [¹⁸F]FBA-PVAC or FITC-PVAC.

The pharmacokinetics of PVAC was studied in PET scans of rats injected (I.V. or I.J.) with either [¹⁸F]FBA-PVAC or FITC-PVAC.

[¹⁸F]FBA-PVAC showed an uptake in the blood, the half-life (T-1/2) in aorta was 10.2 minutes (1.9- 40.8) and an increased uptake was seen in the bladder, T1/2 for the bladder was 10.3 minutes (5.6- 19.2) (B).

For FITC-PVAC the elimination phase was split into a fast phase and a slow phase. The fast phase (50 % of the elimination) had a T1/2 of 0.2 hours (0.11- 0.33). The slow phase had a r-1/2 of 10.73 hours (7.1- 15) (A).

I.J injection gave rapidly increased serum concentrations followed by a steady state at 2 hours between elimination and absorption and at six hours elimination was dominant leading to a r-1/2 of 34.90 hours (24.92-51.45). The dotted line marked the transition from fast to slow phase (A).

Post I.J injection instead showed localized uptake in the joint and low levels in the bladder and kidneys, (D, E). Total excreted FITC-PVAC in gathered urine (at 96 h) was 37.75 % (17.58- 57.91) after I.V injection and 20.30 % (13.39- 27.21) after I.J injection, (F). [¹⁸F]FBA-PVAC showed uptake in the blood, the half-life (t1/2) in the OFrta was 10.2 min (1.9 - 40.8), and an increased uptake was seen in the bladder, fi_/2forthe bladder was 10.3 min (5.6 - 19.2), Fig. 4b. For FITC-PVAC, the elimination phase was split into a fast phase and a slow phase. The last phase (50 % of the elimination) had a ii_/2 of 0.2 h (0.11 - 0.33). The slow phase had a ii_/2 of 10.73 h (7.1 - 15). I.J. injection gave rapidly increased serum concentrations followed by a steady-state at 2 h between elimination and absorption and at 6 h elimination was dominant leading to a T1/2 Of34.90 h (24.92 - 51.45), Fig. 4a. I.J. injection instead showed localized uptake in the joint and low levels in the bladder and kidneys, Fig.

4c. Injection of fluorochrome-labelled PVAC was followed by repeated blood sampling to measure the circulating concentration. In the 1980s and 1990s, several research groups studied the biodistribution of PVA using radiolabeled PVA, e.g. biodistribution of ¹⁴C-PVA in rats] and ¹²⁵I-PVA in rats and mice [26]

Molecular imaging with single-photon emission computed tomography (SPECT) as a biodistribution tool has been explored with ^99mTc-PVA for monitoring embolization. [27]

Radiolabeling of PVA with a PET radionuclide such as ¹¹C, ¹⁸F or ⁶⁸Ga has not been reported. Conjugation of [¹⁸F]FBA has been extensively used to label aminooxy functionalized (to form an oxime) [28] or hydrazino functionalized (to form hydrozone) [29] peptides for PET studies. [¹⁸F]FBA has not been reported for conjugation with a carbazate moiety to form carbazate hydrazine.

Monitoring of the reaction with the radiolabelled was achieved indirectly by tracking consumption of [¹⁸F]FBA at a wavelength of 254 nm. Reaction conditions were optimized and the maximum reaction rate was seen at pH 4.00. Isolation of [¹⁸F]FBA- PVAC from [¹⁸F]FBA was achieved by size exclusion chromatography, an exclusion limit of 5 kDa was used, Fig 2.

Example 5

Biodistribution

Fig. 5 illustrates the ex vivo biodistribution after injection of [¹⁸F]FBA-PVAC in different organs on x-axis and standardized uptake value (SUV) on y-axis. The numbers above the bars are average SUV for each organ from nine different animals (3 males, 6 females). The highest uptake was in urine (60.5 mean SUV ± 15.5) followed by blood (6.7 mean SUV ± 1.1) and kidneys (2.8 mean SUV ± 0.3). Well perfused organs such as the lungs (2.1 mean SUV ± 0.3), liver (1.7 mean SUV ± 0.3) and spleen (0.7 mean SUV ± 0.1) had a higher uptake than low perfusion organs such as muscle (0.1 mean SUV ± 0) and brain (0.2 mean SUV ± 0). The numbers above the bars are average SUV for each organ from nine different animals (3 male, 6 female).

The differences between sexes was also studied and there was no difference between any organ system apart from the reproduction organs (p = 0.0446), with a higher uptake in ovaries compared to testis.

Ischemic PET models

The ischemic kidney model had higher uptake of [18F]FBA-PVAC in the ischemic kidney than the non-ischemic kidney. Instead of an early peak during initial high circulating concentrations, the levels increased over time. The mean difference in standardized uptake value (SUV) at time point 60 min post-injection was 10.85 (8.1 - 15.8, p =<0.0001 ). The three ischemic kidney animals that were blocked by injecting PVAC before [18F]FBA-PVAC had no difference in the SUV (p = 0.6432) when comparing the ischemic kidney to the non-ischemic kidney, tested at the same time point (60 min post-injection of [18F]FBA-PVAC). The graph showed no differences (p = 0.6432) between the ischemic kidney and the non-ischemic kidney over 60 min or later during the dynamic PET scan, Fig 6G. The ex vivo measurements confirmed a difference between ischemic and non-ischemic kidneys (3.86, 1.5 - 6.2, p = 0.0095) and no difference (p = 0.9454) when the animals were blocked, Fig. 4H. Representative examples of PET scan showed increased uptake in the ischemic kidney than the non-ischemic kidney but not when the animal was blocked, Fig. 6.

Example 6

Fig. 6b shows a representative example of PET scan results from the Ischemic kidney model injected with [¹⁸F]FBA-PVAC or blocking with an infusion of PVAC before injecting [¹⁸F]FBA-PVAC and dynamic PET scan for 90 min

The ischemic kidney model had higher uptake of [18F]FBA-PVAC in the ischemic kidney than the non-ischemic kidney. Instead of an early peak during initial high circulating concentrations, the levels increased over time. The mean difference in standardized uptake value (SUV) at time point 60 min post-injection was 10.85 (8.1 - 15.8, p =<0.0001 ). The three ischemic kidney animals that were blocked by injecting PVAC before [18F]FBA-PVAC had no difference in the SUV (p = 0.6432) when comparing the ischemic kidney to the non-ischemic kidney, tested at the same time point (60 min post-injection of [18F]FBA-PVAC).

The graph showed no differences (p = 0.6432) between the ischemic kidney and the non-ischemic kidney over 60 min or later during the dynamic PET scan, Fig. 6a.

The ex vivo measurements confirmed a difference between ischemic and non ischemic kidneys (3.86, 1.5 - 6.2, p = 0.0095) and no difference (p = 0.9454) when the animals were blocked, Fig. 6c.

Example 7

Uptake of [¹⁸F]FBA-PVAC in non-ischemic and Ischemic limb models

Fig. 7 A-C The Ischemic limb model with injection of [¹⁸F]FBA-PVAC followed by dynamic PET scan for 90 minutes.

Data from dynamic PET scans is shown in (a), and the ischemic limb model revealed no difference in uptake between the ischemic and non-ischemic limb during the dynamic scan.

The ex vivo measurements showed an increased SUV in the ischemic limb muscle compared to a non-ischemic limb, 0.134 (0.075-0.193, p = 0.0003). There was no difference (p = 0.0699) when comparing an ischemic limb muscle with a paired non ischemic limb in the same animal (c).

Coronary section of ischemic limb (I.L) and non-ischemic limb (Ni.L) is shown in (b).

Example 8

IRI damage and ARDS progression in non-human primates (NHP)

The objective for this study was to develop a cynomolgus macaque model to study various stages of IRI damage and ARDS progression and to study drugs attempting to halt this progression.

Lung transplant is an established therapy for patients suffering from end stage lung disease. However, lung transplant recipient survival is poor compared to recipients of other solid organs, due largely to the development of graft dysfunction from ischemic reperfusion injury. Some transplanted recipients develop life threatening cause of acute respiratoryfailure (ARDS) due to IRI.

Fig. 8A shows that the pH in arterial blood decreased after induction of lung IRI in the control animals while pH remained stable in animals receiving PVAC prior to reperfusion.

The anion gap indicated that metabolic acidosis was the cause for the acidemia in arterial blood in the control group, all control animals developed metabolic acidosis while none of the PVAC treated animals did (Fig. 8B).

The oxygen tension remained stable in PVAC treated animals when comparing the values prior to surgery with the last recorded timepoint for the respective animals. In the control group oxygen tension was difficult to maintain and the animals had more than a 50 % reduction in oxygen tension when comparing post- and pre-surgery (Fig. 8C).

These results indicate that scavenging of reactive mediators of IRI can be a powerful tool for reducing the impact of lung IRI and ARDS. Oxygen tension was higher in treated animals, and they did not develop acidemia which are hallmarks of lung IRI.

Several known agents or methods have been used to reduce IRI, such as antioxidants to reduce oxidative stress, free radical scavengers to remove metabolic waste, and hypoxic preconditioning. PVAC is acting as a carbonyl scavenger and is reported to be effective in prevention or treatment of bacteria-induced infections and diseases (WO19016189A1), stabilizing erythrocytes (WO2018172422 A1), and treating or preventing inflammatory-related conditions (WO2012105887).

Here we also provide a method for visualizing ischemia. In this example, PVAC was labelled with a radionuclide thereby enable to trace the distribution in ischemic tissues and to investigate this potential.

Treatment of IRI and ARD by administering PVAC alone or in combination with other common therapies for IRI and ARD, such as supportive intensive care, corticosteroids or other anti-inflammatory therapies.

Conclusion

Acute trauma is one of the leading causes of morbidity and mortality in the world. An inappropriate response leads to a cytokine, immunological and genomic storms that contribute to the deleterious effects of acute trauma. If not resolve promptly and adequately, the lesion will become an ischemia-reperfusion injury (IRI) and eventually lead to tissue loss and/or death.

It appears that IRI is the common tissue response to acute insults and relates to its morbidity and mortality. It has been described to participate in several conditions like ischemic stroke, acute myocardium infarction, cardiac arrest, burns, and trauma. The acute interruption of blood and/or oxygen supply creates an imbalance between demand and supply. After some time, the oxygen re-entry upon reperfusion, may lead to a burst of oxidative damage mediated by the production of reactive oxygen species (ROS) and reduction of antioxidant reserves of the cells, tissues, organs or body. Oxidative stress starts in the hypoxic stage but is augmented in the reperfusion injury. If sustained for a period of hours, oxidative stress will generate an oxidative damage that may lead into multiple organ failure (MOF) and eventually death. IRI could also be named as hypoxic re-oxygenation injury. It is referred as the oxygen paradox in which reoxygenation of an ischemic tissue produces injury that greatly exceeds the one created by ischemia alone.

The results in the present disclosure clearly demonstrate that [¹⁸F]FBA-PVAC accumulates selectively in ischemic tissues, as illustrated by the ischemic models of both kidney and hind limb. The ischemic kidney model had a high uptake of [¹⁸F]FBA- PVAC readily detectable by both PET scans and ex vivo biodistribution, while the ischemic limb model only showed a statistically significant increase ex vivo. Still, PET images of the hind limb displayed SUVs about twice the level of the control signifying that qualitative visual assessment could also be useful in tissues with low blood flow.

The elimination of FITC-PVAC through the kidneys followed a two-phase elimination curve with a rapid phase lasting approximately 2 h followed by a slower phase with an accumulation in the bladder. The two-phase elimination could possibly be explained by the size range (15-35 kDa) of the PVAC molecules where the elimination through the kidneys would be more rapid in the smaller size range.

Indeed, a similar correlation between size and half-life has been observed with PVA [33] Alternatively, PVAC may bind to circulating molecules, e.g., carrier proteins, which could slow down the elimination of the bound fraction. Still, after 48 h circulating levels were low, suggesting a near-complete elimination from the circulation. When administered locally into the joint kinetics was slower with less PVAC excreted in the urine, signifying that a macro-molecule such as PVAC is retained in tissues.

Ex vivo biodistribution demonstrated that most PVAC is excreted in urine with the remaining fraction confined to the vascular system. Among solid organs, the uptake was highest in well-perfused organs such as kidneys, liver and lungs. In contrast, uptake in low-perfused organs such as skeletal muscle was substantially lower, indicating that the signal more directly correlates to blood content ex vivo rather than specific uptake. The only specific uptake that was not correlated to blood content level was observed in the ovaries, which displayed a notable uptake that increased over time. A similar pattern was not seen in the male sex organs. In all other organs the uptake diminished over time, apart from urinary tract were elimination leads to accumulation. Administration of PVAC during the kinetic studies was without any notable adverse injuries, and our unpublished toxicity data in rats and rabbits have not shown any adverse events/injuries even after very high doses (50 mg/kg bw).

PVAC effectively and almost completely scavenged RCS associated with IRI giving reasonable cause for the affinity of ischemic tissues. In ischemic models of both the kidney and hind limb, [¹⁸F]FBA-PVAC accumulated selectively in ischemic tissues. The ischemic kidney model had a high uptake of [¹⁸F]FBA-PVAC readily detectable by both PET scans and ex vivo biodistribution, while the ischemic limb model only showed a significant increase ex vivo. Still, PET images of the ischemic hind limb displayed twice as high SUVs as the control signifying that qualitative visual assessment could also be useful in tissues with low blood flow.

Interestingly, the increased uptake in ischemic kidneys was effectively prevented by a previous administration of PVAC. It is likely that the uptake of [¹⁸F]FBA-PVAC was saturated by injecting PVAC before administering [¹⁸F]FBA-PVAC, which indicates that [¹⁸F]FBA-PVACs binding to ischemic tissues was both specific and saturable.

Other tracers such as Na[¹⁸F]F, which has been investigated in humans and rats for myocardial IRI has shown promising results with the possibility to monitor treatment response. Compared with [¹⁸F]FBA-PVAC the Na[¹⁸F] uptake was measured longer after IRI (24 h) and the increase in SUV was less. Other tracers such as, [¹¹C]Choline and ⁶⁸Ga-DOTATATE have been explored to visualize ischemia in stroke, but were originally developed to detect tumours (prostate cancer and neuroendocrine tumours, respectively). The exact mechanism for accumulation of these substances in ischemic tissues is not known. The uptake was low, around 2 SUVs, yet visualization of the area was still clear. [¹⁸F]FDG has been investigated more thoroughly but with conflicting results. The current understanding of the distribution is a decreased uptake in the ischemic core combined with increased uptake in the border to the ischemic area. To summarize, many tracers have been explored to visualize ischemia, but none of these tracers are specifically designed for this task. Furthermore, the rapid accumulation observed with [¹⁸F]FBA-PVAC appear unique, with differentiation between ischemic and non-ischemic tissue 30 minutes after the insult, even in a mild model such as the ischemic hind limb.

In summary, in control animals, PVAC was mainly confined to the bloodstream followed by elimination via kidneys and accumulation in the bladder. Ex vivo biodistribution of PVAC confirmed the highest uptake in urine followed by blood, kidneys and other well-perfused organs. The elimination of I.V. administered PVAC was split into a fast phase (t1/2 = 0.2 h) followed by a slow phase (t1/2 = 10.73 h), with near-complete elimination from blood after 48 h. Both the ischemic kidney (fourfold increase, p = <0.001 ) and limb models (threefold increase, p = <0.001) demonstrated a higher uptake of PVAC in ischemic tissues, ex vivo radioactivity detection.

Moreover, the inventors show surprisingly effects when treating primates suffering from IRI with PVAC prior to reperfusion restored pH, did not develop metabolic acidosis (AGAP), and remained stable oxygen tension measured in blood, whereas the control group exhibited drop in pH, acidosis and a drop in oxygen tension.

PVAC, an aldehyde-carbonyl scavenger provides a novel way of detecting ischemic events and injuries. Flow scavenging of IRI mediators affects the clinical course of IRI remains to be investigated, but there is a potential for a dual-mode of action with visualization and therapeutic benefits.

The present invention provides methods for use in detecting and/or treating ischemic tissue in a subject, and for use in alleviating reperfusion injury.

This disclosure opens up for a novel method for treating all kind of acute responses to an ischemic injury, as a first line treatment, second or further line treatment, and in combination with treatments used today, such as angioplasty. Moreover, a labelled agent/compound providing the possibility to monitor and identify sites for different events and/or injuries during the journey to the target tissue in vivo provides new information of and insights.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive.

The advantages described herein will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims.

References Collard CD, Gelman S. Pathophysiology, clinical manifestations, and prevention of ischemia-reperfusion injury. Anesthesiology. 2001;94:1133-8. Sellberg F, Fredriksson F, Engstrand T, Bowden TM, Nilsson B, Flong J, et al. Polyvinylalcohol-carbazate (PVAC) reduces red blood cell hemolysis. Chalmers J, editor. PLOS ONE. 2019;14:e0225777.

Claims

1. A polymer comprising a carbazate-modified polyvinyl alcohol (PVAC) according to Formula I

O .,,NH

OH Q N 2 k H n m

Formula I for use in preventing, alleviating, restoring and/or treating an ischemic injury in a subject, wherein unmodified repeat units of PVA are denoted with n and carbazate groups conjugated to repeat units are denoted by m.

2. The polymer for use according to claim 1 , wherein the degree of polymerization (DP) is in the range of 100 < DP < 2000.

3. The polymer for use according to claim 1 or 2, wherein the degree of substitution (DS) (m) of carbazide is in the range of 0.5 - 50 %, preferably 2 -40 %, or more preferably 2.5-20 % or even more preferably 5 -15 %, 7.5 - 10 %.

4. The polymer for use according to any one of claims 1 to 3, wherein the carbazate content is in the range of 0.1 -10 mmol/g polyvinyl alcohol carbazate, preferably 0.5-8 mmol/g polyvinyl alcohol carbazate, more preferably 1-6 mmol/g polyvinyl alcohol carbazate, and even more preferably 2 mmol/g polyvinyl alcohol carbazate.

5. The polymer for use according to any one of the preceding claims in the form of a lyophilizate.

6. A complex or composition comprising a polymer according to any one of claims 1 to 5 and an agent, wherein the agent is a labelling agent or molecule.

7. The complex or composition according to claim 6, wherein the labelling molecule is a radiolabel, such as for example [¹⁸F]4-fluorobenzaldehyde, or a fluorescent agent such as for example fluorescein isothiocyanate (FITC).

8. A pharmaceutical composition comprising a polymer according to any one of claims 1 to 5, for use in preventing, alleviating, restoring and/or treating an ischemic event/injury in a subject.

9. The pharmaceutical composition according to claim 8, further comprising a pharmaceutically acceptable excipient.

10. The pharmaceutical composition for use according to claim 9, wherein the pharmaceutical acceptable carrier is an aqueous media.

11. The pharmaceutical composition for use according to any one of claims 8 to 10, in the form of a lyophilizate.

12. The polymer according to any of claims 1 to 5, complex or composition according to any one of claims 6 or 7, or the pharmaceutical composition according to any one of claims 8 to 11 for use in the treatment of an ischemic injury such as ischemia-reperfusion injury (IRI), acute myocardial infarction (Ml), stroke, critical limb ischemia (CLI) and acute respiratory distress syndrome (ARDS).

13. The polymer or the pharmaceutical composition for use according to claim 12, in combination with other common therapies for ischemia- reperfusion injury (IRI), acute myocardial infarction (Ml), stroke, critical limb ischemia (CLI) and acute respiratory distress syndrome (ARDS), such as supportive intensive care, corticosteroids or other anti-inflammatory therapies.

14. The polymer according to any one of claims 1-5, complex or composition according to any of claims 6 to 7, and/or the pharmaceutical composition according to any one of claims 8 to 11 for use in treating an ischemic injury, wherein the administration is performed by intravenous, intramuscular, intraperitoneal, or subcutaneous injection or infusion.

15. A method for diagnosing, visualizing, detecting, and monitoring an ischemic injury or event in a subject, said method comprising administrating a complex or composition comprising a polymer such as PVAC and a labelling agent or molecule according to any one of claims 6 to 7 to a subject, allowing the complex or composition to reach the site, and detecting said complex or composition comprising the polymer and labelling agent or molecule by PET, MR, IR, CT and/or other X-ray based diagnostics.

16. A kit for detecting and monitoring an ischemic injury in a subject, said kit comprising the complex or composition according to any one of claims 6 or 7, and instructions for use.

17. A method for preventing, alleviating, restoring and/or treating an ischemic injury in an organ to be transplanted, said method comprising administrating a pharmaceutically effective dose of a polymer comprising a carbazate-modified polyvinyl alcohol (PVAC) according to Formula I

Formula I wherein unmodified repeat units of PVA are denoted with n and carbazate groups conjugated to repeat units are denoted by m, wherein the degree of polymerization (DP) is in the range of 100 < DP < 2000, and the degree of substitution (DS) (m) of carbazide is in the range of 0.5-50 %, preferably 2-40 %, or more preferably 2.5-20 % or even more preferably 5-15 %, or 7.5-10 %.

18. The polymer according to claim 17, wherein the carbazate content is in the range of 0.1-10 mmol/g polyvinyl alcohol carbazate, preferably 0.5-8 mmol/g polyvinyl alcohol carbazate, more preferably 1-6 mmol/g polyvinyl alcohol carbazate, and even more preferably 2 mmol/g polyvinyl alcohol carbazate.

19. The method according to any one of claims 17 or 18, wherein said organ is lung, liver, heart, pancreas and/or kidney.

20. A method for preventing, alleviating, restoring and/or treating an ischemic injury in a subject, comprising administrating a therapeutically effective amount of the polymer according to any one of claims 1 to 5, or the pharmaceutical composition according to any one of claims 8 to 11 to a subject in need of such treatment.

21. The method according to claim 20, wherein the ischemic injury is caused by reperfusion, ischemia-reperfusion injury (IRI), acute myocardial infarction (Ml), stroke, critical limb ischemia (CLI) and acute respiratory distress syndrome

(ARDS)andZor a corona virus infection.

22. The method according to claim 21 , wherein the corona virus infection is corona virus disease 2019 (COVID-19).

23. The method for preventing, alleviating, and/or treating ischemic injury according to any one of claims 20 to 22, wherein the administration is performed by intravenous, intramuscular, intraperitoneal, or subcutaneous injection or infusion.

24. The method for preventing, alleviating, restoring and/or treating ischemia 5 according to any of claim 20 to 23, in combination with other common therapies for ischemia–reperfusion injury (IRI), acute myocardial infarction (MI), stroke, critical limb ischemia (CLI) and acute respiratory distress syndrome (ARDS), such as supportive intensive care, corticosteroids or other anti-inflammatory therapies. --- 10 33